Research ArticleAmelioration of Colitis in Mouse Model by ExploringAntioxidative Potentials of an Indigenous Probiotic Strain ofLactobacillus fermentum Lf1

Ritu Chauhan1 Aparna Sudhakaran Vasanthakumari1 Harsh Panwar1

Rashmi H Mallapa1 Raj Kumar Duary2 Virender Kumar Batish1 and Sunita Grover1

1 Molecular Biology Unit Department of Dairy Microbiology National Dairy Research Institute Karnal Haryana 132001 India2Department of Food Engineering and Technology Tezpur University Napaam Assam 784028 India

Correspondence should be addressed to Sunita Grover sungrogmailcom

Received 24 February 2014 Revised 26 May 2014 Accepted 27 May 2014 Published 1 July 2014

Academic Editor Borja Sanchez

Copyright copy 2014 Ritu Chauhan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Based on the preliminary screening of eight indigenous putative probiotic Lactobacilli Lactobacillus fermentum Lf1 was selectedfor assessing its antioxidative efficacy in DSS colitis mouse model based on its ability to enhance the expression of ldquoNrf 2rdquo by643-fold and malondialdehyde (MDA) inhibition by 781 plusmn 024 in HT-29 cells under H

2O2stress The Disease Activity Index

and histological scores of Lf1-treated mice were lower than the control group However expression of ldquoNrf 2rdquo was not observedin Lf1-treated mice A significant increase in the expression of antioxidative enzymes such as SOD2 and TrxR-1 was recorded inboth of the groups The expression of SOD2 was significantly downregulated in colitis-induced mice by minus10000-fold relative tocontrol group and the downregulation was considerably reduced to minus3704-fold in colitis Lf1 treatment group Almost a similartrend was recorded in case of ldquothioredoxinrdquo expression though ldquoCATrdquo was refractile to expression The Lf1-treated group haddecreasedmalondialdehyde level as compared to colitis control (3792 plusmn 631 versus 9113 plusmn 576 120583Mg)These results point towardsLf1-induced activation of the antioxidant enzyme system in the mouse model and its prospects to be explored as a new strategy forIBD management

1 Introduction

Inflammatory bowel diseases (IBDs) including Crohnrsquos dis-ease (CD) and ulcerative colitis (UC) have been recognizedas the chronic inflammatory disorders of the gastrointestinaltract whose pathogenesis is not completely understood [1]However high-grade oxidative stress induced as a resultof generation of excessive level of reactive oxygen species(ROS) has been commonly implicated in the pathogenesisof some chronic human disorders including autoimmunediseases and also happens to be the hall mark of IBDs [2ndash4]The production of free radicals at high levels in the gut canexert cytotoxic effects on the membrane phospholipids of theintestinal epithelial cells resulting in the formation of toxicproducts such as malondialdehyde (MDA) Similarly theoccurrence of severe peroxidative changes due to lipids andfree radicals reaction resulting in enhanced lipid peroxidation

has been found to be commonly associated with the onsetof IBDs [5] However human body has evolved genetic pro-grammes through intervention of antioxidative enzymes toprotect itself from such oxidative stresses by maintaining cel-lular homeostasis and functionThe key antioxidant enzymeswhich include superoxide dismutase (ldquoSODrdquo) glutathioneperoxidase (ldquoGPxrdquo) and catalase (ldquoCATrdquo) form the backboneof the enzymatic antioxidant cascade and offer protectionto cells and tissues against oxidative injury [6] In thiscontext nuclear factor erythroid 2-related factor 2 (ldquoNrf 2rdquo)has been recognized as one of the key transcriptional factorsthat can play a significant protective role by controllingthe antioxidant response element- (ARE-) dependent generegulation in response to oxidative stress [7ndash9]

Recently food supplementation with antioxidants hasbeen the major focus of attention amongst the health pro-fessionals across the world to explore it as a strategy to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 206732 12 pageshttpdxdoiorg1011552014206732

2 BioMed Research International

protect against the injurious effects of oxidative stress Oneof the dietary based strategies currently in vogue exploresprobiotics for amelioration of oxidative stress-related diseasesby augmentation of antioxidant defense systems operatingin the human body Probiotics have recently emerged as thepowerful microbial tools for novel therapeutic applicationsspecifically targeted against IBD by virtue of expressingseveral physiologically important functions under both invitro and in vivo conditions Besides displaying a plethoraof novel health promoting functions which are highlystrain specific probiotic bacteria including Lactobacilli alsodemonstrated strong antioxidative potentials as reportedpreviously [11ndash13] In this context one such study meritsspecial attention wherein L fermentum ME-3 a potentialprobiotic strain of proven efficacy possesses antagonisticand antioxidative properties when used in conjunction withofloxacin decreased lipid peroxide value in the mucosa of thesmall intestine and liver of Salmonella typhimurium infectedmurine model [14 15] As probiotics showed a positivefunctionality on oxidative stress-related indices they can helpboth to stabilize and to promote the potency of the wholebody antioxidative defense system and thus in turn mayhave an impact on lowering the risk of several inflammatorymetabolic disorders including IBDs Besides this a large bodyof evidence also suggests that probiotics like VSL3 and Lrhamnosus GG could serve as the promising candidates forthe prevention and control of IBD although some conflictingresults have also been reported [16ndash20]

Several animal models of IBD have been designed andamongst them dextran sulfate sodium (DSS) induced colitismodel appears to represent the most accurate model ofIBD since it provides human IBD-like symptoms [21] Datafrom animal models of colitis have indicated that specificprobiotic Lactobacillus and Bifidobacterium strains couldprevent and treat intestinal inflammation [22ndash25]This studywas specifically undertaken to showcase the antioxidativepotentials of L fermentum Lf1 a promising indigenous pro-biotic Lactobacillus strain tomanage oxidative stress throughldquoNrf 2rdquo activation and modulation of lipid peroxidation byimpactingMDA level under both in vitro (HT-29) and in vivoconditions in DSS colitis mouse model

2 Materials and Methods

21 Ethical Statement Before setting up the animal studyin colitis mouse model prior approval of the InstitutersquosAnimal Ethics Committee (IAEC) of the National DairyResearch Institute (NDRI Karnal India) was obtained Theexperimental animals used in this study were maintained asper National Institute of Nutrition (NIN) India guidelinesfor the care and use of laboratory animals (date of approval301010) Surgery was performed at anesthesia conditions tominimize suffering of the animals

22 Bacterial Strains A total of ten bacterial cultures whichincluded eight indigenous Lactobacillus isolates comprisingof seven strains of Lactobacillus plantarum (L plantarum9 10 42 55 78 91 and S3) and a strain of Lactobacillus

fermentum (Lf1 an indigenous isolate of Indian gut origindeposited in International Depository Budapest Treaty atMicrobial Type Culture Collection MTCC 5689) besidestwo reference probiotic strains L plantarum CSCC5276 (alsodesignated as CSCC5276 NCDO82 or VTTE-71034) whichwas procured from Dr N P Shah from Victoria UniversityAustralia [26 27] and L acidophilus NCFM (also knownas Howaru Dophilus LA-1 NCK56 NCK45 N2 RL8KRRL8KS and RL8K) [28ndash30] formed the subject of this studyThey were procured from the probiotic culture collectionmaintained at Molecular Biology Unit Dairy MicrobiologyDivision National Dairy Research Institute Karnal Bacterialstrains were activated prior to use by subculturing in steriledeManRogosa Sharpe (MRS) broth (HiMedia India) at 37∘Cfor 18ndash24 h

23 Propagation of Human Colonic Epithelial Cell Lines (HT-29 Cells) and Treatments Human colonic epithelial cellline (HT-29) was procured from National Centre of CellSciences (NCCS Pune India) The cell line was cultured inDulbeccorsquos Modified Eagles Medium (DMEM Sigma USA)The propagation of HT-29 cells was done as reported in aprevious study and carried out in our lab [31] After attainingconfluency HT-29 cells were challenged with each of the1mL of live probiotic strains (sim1 times 109 cfumL resuspended inDMEM) and incubated at 37∘C in 5 CO

2for 4 h followed

by addition of fresh medium without antibiotic and foetalbovine serum After incubation cells were challenged with1mMH

2O2(Sigma USA) and again incubated for additional

30min After incubation H2O2was completely removed and

trypsinization (with Trypsin-EDTA solution Sigma USA)was done Cells were again resuspended in DMEM mediumand pelleted (centrifugation at 1700 rpm for 5min at RT) forRNA isolation The supernatant was stored at minus80∘C for totalantioxidant activity and lipid peroxidation determination

231 ldquoNrf2rdquo Gene Expression Study in HT-29 Cells RNA wasisolated from the cell pellet by TRIzol method (TRI ReagentSigma USA) and cDNA was prepared using ImProm-IIreverse transcriptase kit (Promega USA) and random hex-amer primers (100120583M) provided with the same as describedpreviously [32] The expression of target genes ldquoNrf 2rdquo alongwith housekeeping gene ldquo120573-actinrdquo for HT-29 cells and fromHT-29 cells (after H

2O2challenge) was studied by reverse

transcriptase-quantitative PCR (RT-qPCR) Untreated HT-29 cells grown in DMEM served as controlThe primers usedin this study are listed in Table 1

232 Lipid Peroxidation The extent of lipid peroxidation anindex of oxidative stress was measured as thiobarbituric acidreactive substances (TBARS) formed Lipid hydroperoxideswere measured by standard TBA test method [33]

24 Experimental Design of an In Vivo Study Using DSSColitis MouseModel A total of thirty-two adult male (seven-to-eight weeks old) Swiss Albino mice weighing 25ndash30 gon average were used in this study The animals were fednormal diet (Bengal gram crushed 58 wheat starch 15

BioMed Research International 3

Table 1 Sequences of the primers used for RT-qPCR

Genes Primer sequence (51015840-31015840) Ampliconsize (bp)

Nrf 2 HFNrf 2 HR

gcg acg gaa aga gta tga cgtt ggc aga tcc act ggt tt 181

Nrf 2 MFNrf 2 MR

ttc agc aca aca ctg gga agtgt tgc tgg ggt tta tag gc 102

SOD2 MFSOD2 MR

gct ggc ttg gct tca ata agtaa ggc ctg ttg ttc ctt gc 87

CAT1 MFCAT1 MR

gct gag aag cct aag aac gcacct tcg cag cca tgt gag a 64

Trx-1 MF1Trx-1 MR1

tcc att tcc atc tgg ttc tgcttc acc att ttg gct gtt gc 65

120573-Actin F120573-Actin R

tgg ctg ggg tgt tga agg tctagc acg gca tcg tca cca act

238[10]

120573-Actin mus2F120573-Actin mus2R

agt gtg acg ttg aca tcc gtagcc aga gca gta atc tcc ttc t 112

groundnut cake 10 casein 4 groundnut oil 4 saltmixture 4 vitamin mixture 02 and choline chloride02) and water ad libitum during the entire course of theexperiment The animals were divided into four homoge-neous groups (noncolitis control NC-PBS noncolitis (Lf1)control NC-Lf1 colitis control C-PBS and colitis-Lf1 (C-Lf1) treatment groups) comprising of eight animals eachhoused in individual cages and maintained under a constant12 h light-12 h dark cycle The temperature was controlled at22ndash25∘C with about 56ndash60 relative humidity There wasno significant difference in the body weight of mice amongthe four groups on day zero The body weight of mice ineach group on the first day was taken as the basal level Thesubsequent changes in their body weight were monitoredperiodically during the entire 13-day experimental periodThe body weight of mice each day minus the basal bodyweight was expressed as the bodyweight changeThenegativevalue indicated the decreased weight while the positive valuerepresented the increased weight

25 Feeding of Mice and Experimental Design The micewere fed for twelve days by oral intubation with 15mL ofovernight grown Lactobacillus culture (Lf1) at 109 cfumLafter centrifugation at 7000 rpm for 10min and resuspendingthe cell pellet in 200 120583L of sterile PBS solution with the helpof 1mL syringe and silicon tube The mice fed with PBSalone served as the control group The experimental designhas been illustrated in Figure 1(S) in Supplementary Mate-rial available online at httpdxdoiorg1011552014206732Noncolitis control group was treated with PBS alone for12 days Noncolitis (Lf1) treatment group was administeredwith 200120583L Lactobacillus culture (Lf1) orally by orogastrictube Colitis (Lf1) mice were fed with 200 120583L Lactobacillusculture (108-109 cfuml) orally by orogastric tube for 7 daysbefore starting dextran sodium sulfate (DSS) and continuedtill 12th day after DSS induction PBS was administered incolitis control group Colitis was induced by 5 (wv) DSS(MW = 40000ndash50000 USB Corporation Cleveland ICN

Biomedicals OH USA) dissolved in drinking water after 7days All the animals in different groups were monitoredon every alternate day for physical parameters like weight-lossgain faecal matter consistency and blood in stoolsFaecal samples were collected every three days and plated onMRS media to determine Lactobacillus counts in faeces aswell as to assess the persistence of the viable probiotic cellsin mice gut Disease Activity Index (DAI) scores were alsorecorded every alternate day Length of colon was measuredand histological scores were assessed on the 13th day Colontissue was used for performing lipid peroxidation assay aswell as for RNA isolation for gene expression studies withregard to ldquoNrf 2rdquo and antioxidant genes by RT-qPCR

26 Evaluation of Colonic Damage and Inflammation Sever-ity of colitis was assessed every alternate day using a DiseaseActivity Index (DAI) DAI is known to be a valid measurefor the acute colitis produced by DSS [34] The scores wereassigned on 0ndash4 scale taking into account weight loss stoolconsistency and blood in stool on scales of 0ndash4 (Table 1(S) inSupplementary Material) [35] For each mouse a daily DAIscore was calculated as the aggregate of the weight lossscore stool consistency score and rectal bleeding divided bythree The DAI values for the groups were then recorded asmean plusmn standard deviation for each day of the experimentSubsequently the daily DAI score for each experimentalgroup was calculated as the mean plusmn standard error of theindividualDAI scores of eachmouse in the group (Table 1(S))The DAI clinical parameters used here are comprehensivefunctional measures that are somewhat analogous to clinicalsymptoms observed in human IBD and the scoring methodthat has been validated by repeated studies Besides thisweight of mice was recorded at every alternate day as it is alsocorrelated with severity of induced colitis Scoring system forthe comparative analysis of intestinal bleeding is given in theform of Table 1(S)

The animals were sacrificed on the thirteenth day Bycarefully opening the mouse by a ventral midline incisionthe colon was collected from the colon-cecal junction to theanus and its length was measured After removal of faecalmatter colon was rinsed with DEPC water A small part(02-03 cm) of colon was kept in 10 buffered formalin forhistopathological examination (ie Hematoxylin and Eosinstaining) and the remaining tissue was immediately wrappedin aluminium foil and snap frozen in liquid nitrogen for RNAisolation and lipid peroxidation analysis Colonic damageand inflammation were recorded by the intensity of inflam-mation monitored through histopathological examinationPersistence of probiotic Lactobacilli was also monitored bytaking faecal counts

27 RAPD Profiles of Lactobacillus Faecal Isolates Faecalsamples were collected every alternate day of probioticadministrationPBS (control group) Faecalmaterial (100mg)was homogenized in one mL of PBS Serial dilutions weremade and plating for Lactobacillus counts was done onMRS-BCP agar The plates were incubated at 37∘C for 24ndash48 hColonies were counted and recorded Colonies from different

4 BioMed Research International

faecal count plates were randomly picked and grown for16 to 18 h at 37∘C after inoculation into MRS broth Thegenomic DNA from faecal isolates was then isolated byPospiech and Neumann method [36] PCR with randomoligo primer named 275 (51015840-ccg ggc aag c-31015840) was carried outfor RAPD profiling PCR reaction was performed in 25 120583Lreaction volume containing PCR buffer (25 120583L) oligo primer(20 120583L) 200 120583M dNTP (20 120583L) 10 U Taq DNA polymerase(05 120583L) and template (20 120583L) The PCR cycling parametersincluded an initial denaturation of 95∘C5min followedby 45 cycles each of denaturation (95∘C30 sec) annealing(40∘C30 sec) extension (72∘C2min) and final extensionof 72∘C10min The PCR products were electrophoresedon 18ndash2 agarose gel with ethidium bromide (05 120583gmL)(Amersham Biosciences USA) at 100V using 1X TAE bufferThe RAPD patterns were compared with the patterns of thepure cultures of the administered probiotic strain

28 Histopathological Examination The intestinal tissuesfrom the colon of the probiotic and control groups wereset apart for histopathological examination Intestines (02-03 cm of tissue) of different groups were kept in 10 bufferedformalin solution for at least 24 h prior to cassetting Thecolon was sliced mounted stained with hematoxylin (HiMe-dia India) and eosin (HiMedia India) and then scored bya blinded pathologist who rated the tissues based on thegrade of the disease 0ndash4 the percentage of the disease 0ndash4and the severity of the inflammation 0ndash3 (Table 2(S) andashc)[35] Histopathological examination of the affected tissuesfrom the mice groups analyzed in this study was done by ablinded histologist who rated the tissues based on the gradeof the disease 0ndash4 the percentage of the disease 0ndash4 andthe severity of the inflammation 0ndash3Microscopic evaluationof all the sections of tissue samples was done at 4x and 10xmagnification and histological scores were graded on thebasis of the severity of inflammation infiltration ulcerationand crypt damage

29 Lipid Peroxidation For detection of lipid peroxides(malondialdehyde) in colon homogenate the methodof Uchiyama and Mihara was followed [37] The colonhomogenate was prepared in 1X PBS An aliquot of 3mLof 1 phosphoric acid and one mL of 06 TBA solutionwere added to 05mL of colon homogenate The mixturewas heated for 45min on a boiling water bath After cooling4mL of n-butanol was added and mixed vigorously Thebutanol phase was separated by centrifugation at 5000 rpmfor 10min and absorbance was measured at 535 and 520 nmThe difference was used as the TBA value A standard curveof malondialdehyde was drawn by taking TBA value atvarious concentration of malondialdehyde MDA standardcurve was prepared by carrying out overnight digestionof different concentrations of 1133-tetraethoxypropane(01mM) in presence of 02N HCl

210 Gene Expression Studies RNAwas isolated frommousecolon by TRIzol method (TRI Reagent Sigma USA) cDNAwas synthesized and RT-qPCR in LightCycler 480 (Roche

Switzerland) was carried out as described previously [31]The thermal cycling conditions included initial denaturationat 95∘C for 5min followed by 40 cycles each of denat-uration (95∘C30 s) annealing (53∘C30 s) and extension(72∘C45 s) with a single fluorescence measurement a meltcurve program (60∘Cndash95∘C with a heating rate of 011∘Csand continuous fluorescence measurement) and finally acooling step at 40∘C RNA extraction and measurement ofgene expression by RT-qPCR were performed in triplicateand the mean of all these values was used for final analysis

The quantitative data generated by real-time PCR (thequantification cycle Cq value) were analyzed by relativeexpression software tool (REST 2009) (httpwwwgene-quantificationinfo) as described previously [32]

211 Statistical Analyses Thedata obtained from each animalexperiment were expressed as mean plusmn standard deviationvalues Analyses of variance (ANOVA) for estimation ofsignificance of differences betweenmeans were automaticallydone with the Statistical Package for the Social Science (SPSSfor Windows version 101 SPSS Inc Chicago) softwareby Bonferroni post hoc test and considered significantlydifferent at 119875 le 005

3 Results

31 Screening of Probiotic Lactobacilli Initially during thispart of study some putative probiotic Lactobacilli werescreened on the basis of their ability to enhance ldquoNrf 2rdquoexpression in HT-29 cells along with lipid peroxidationinhibitory potential to short list the most promising strainfor further investigations to explore its in vivo efficacyin colitis mice model From the critical appraisal of thedata recorded in Figure 1(a) it can be inferred that therewas considerable variation in ldquoNrf 2rdquo expression induced inHT-29 cells by different probiotic strains as they behaveddifferently in eliciting ldquoNrf 2rdquo expression The expression ofldquoNrf 2rdquo in HT-29 cells was significantly downregulated (119875 lt0001) in the sample group (relative to control group) byminus188-fold when subjected to H

2O2stress alone (Figure 1(a))

Although some of the probiotic strains when used alone orin conjunction with H

2O2did not show any significant effect

on the ldquoNrf 2rdquo expression in HT-29 cells Lp5276 and S3 didinduce significant downregulation at the level of minus138- andminus164-fold respectively However it was quite interesting tonote that three of the strains namely Lp55 Lp91 and Lf1were able to induce a significant (119875 lt 0001) upregulation inthe expression of ldquoNrf 2rdquo in HT-29 cells at the levels of 748- 258- and 643-fold when used in conjunction with H

2O2

Hence the latter were rated as the prospective candidateprobiotic strains to explore their efficacy in the managementof oxidative stress induced diseases

The total antioxidative activity of the aforesaid Lacto-bacillus strains in terms of their lipid peroxidation inhibitionpotentials inHT-29 cells was also investigated after subjectingthe same to H

2O2treatment by measuring the level of

MDA the secondary product of lipid peroxidation From thedata presented in the form of a bar diagram illustrated in

BioMed Research International 5

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

) 10

8

6

4

2

0

minus2

minus4

lowastlowast

lowast

lowast

lowast

lowast lowastlowastlowast

Lf1+

H2O2

S3+

H2O2

H2O2

Lp9

Lp10

Lp10+

H2O2

Lp42

Lp42+

H2O2

Lp55+

H2O2

Lp78

Lp78+

H2O2

Lp91

Lp91+

H2O2

Lp9+

H2O2

Lp55

Lp5276+

H2O2

Lf1 S3

NCF

M

Lp5276

NCF

M+

H2O2

(a)

100

75

50

25

0

MD

A

inhi

bitio

n

lowastlowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

LP9+

H2O2

Lp10+

H2O2

Lp42+

H2O2

Lp55+

H2O2

Lp78+

H2O2

Lp91+

H2O2

Lf1+

H2O2

S3+

H2O2

Lp5276+

H2O2

NCF

M+

H2O2

(b)

Figure 1 Relative expression of ldquoNrf 2rdquo and MDA inhibitory effect by Lactobacilli strains in HT-29 cell lines under H2O2stress conditions

Data are represented as mean plusmn SD number of RT-qPCR experiments (119899) = 3 (a) Comparative analysis of relative expression of ldquoNrf 2rdquo inHT-29 on challenge with different strains of probiotic Lactobacilli and H

2O2stress lowastData are significantly different compared with control

(119875 lt 0001 Bonferroni post hoc test) (b) Inhibitory effect of various probiotic Lactobacillus cultures on lipid peroxidation in HT-29 cellsData are significantly different compared with control (H

2O2alone) (lowast119875 lt 0001 lowastlowast119875 lt 001 Bonferroni post hoc test)

Figure 1(b) it is clearly indicated that Lactobacillus spp S3showed the highest percent inhibition that is 832 plusmn 053followed by Lf1 (781 plusmn 024) as compared to the standardreference strains showing relatively poor response as can bereflected from the lower percent inhibition values that is556 plusmn 04 (Lp5276) to 552 plusmn 036 (LaNCFM) indicatingthat the indigenous probiotic strains were better equippedto neutralize the injurious effects of oxidative stress Hencebased on the overall combined effects of both of theseactivities inHT-29 cells we finally selected Lf1 as the potentialcandidate strain for in vivo study

32 InVivo Studies inDSSColitisMouseModel Feedingmicewith DSS via drinking water for five consecutive days ledto acute colitis characterized typically by bloody diarrhoeaulcerations and infiltrationswith granulocytes Furthermoresince DSS happens to be toxic to gut epithelial cells of thebasal crypts andhence can impact the integrity of themucosalbarrier all the experimental animals belonging to either thecontrol or the treatment groups were critically examined forthe following clinical parameters besides other importantphysicochemical changes recorded inmice groups during thecourse of this experiment

321 Weight Loss The gross changes in the body weightof mice in different treatment groups including probioticinterventions weremonitored during the entire 13-day exper-imental period of the study The body weight was foundto increase gradually in both the noncolitis control and Lf1groups Mice in the colitis group showed a gradual increasein weight for the first seven days but later after starting DSStreatment excessive weight loss was recorded that becamehighly significant starting from day eight to day thirteen

(119875 lt 005) due to severe experimental colitis The colitis-Lf1 treatment group showed a mixed response as there wasgradual increase in body weight in the first seven days butafter induction of colitis significant inhibition of weight losswas recorded (119875 lt 005) Quite interestingly some of themice in the same treatment group showed the opposite effectby recording appreciable weight gain The changes recordedwith regard to body weight in different mice groups duringthe course of this study have been illustrated in Figure 2However there was no significant difference in weight gainbetween both NC-PBS versus NC-Lf1 and C-PBS versus C-Lf1 groups (119875 gt 005)

322 Length of Colon The length of mice colon in thenoncolitis control with respect to noncolitis Lf1 fed groupswas 705 plusmn 021 and 815 plusmn 014 respectively The length ofcolon of colitis-Lf1 treatmentmice wasmuch longer than thatof mice in the colitis group (793 plusmn 038 versus 638 plusmn 039 cm119875 lt 005) There was no significant difference (119875 gt 005)between the length of colon of colitis-Lf1 treatment group andnoncolitis Lf1-treated group

323 Disease Activity Index (DAI) Scores As can be reflectedfrom Figure 3 the highest DAI score was recorded in colitiscontrol group DAI scores of noncolitis control group andnoncolitis probiotic group were however zero as weight losschanges observed in these groups were not significant to beconsidered as DAI and hence were rated as nonsignificant(NS) groups However DAI scores were improved signifi-cantly in colitis-Lf1 treatment group as shown in Figure 3On comparative evaluation there was significant difference(119875 lt 005) recorded between the DAI scores of colitis control

6 BioMed Research International

0 2 4 6 8 10 12 14

Days

Mea

n bo

dy w

eigh

t cha

nges

minus1

0

1

2

3

25

15

05

minus05

C-PBSC-Lf1

NC-PBSNC-Lf1

Before DSS colitis induction After DSS colitis induction

Figure 2 Bodyweight changes among different groups ofmiceNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups The data fordifferent groups were reported as mean plusmn standard deviation

0 2 4 6 8 10 12 140

05

1

15

2

25

3

35

DA

I sco

re

Days

NC-PBSNC-Lf1

C-PBSC-Lf1

lowast

lowastlowast

lowast

lowast

lowast

lowast

Figure 3 Comparative analysis of mean DAI scores in differentgroups in mice NC-PBS noncolitis control NC-Lf1 noncolitis(Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treat-ment groups Data showing asterisk mark (lowast) without line showssignificant difference compared with noncolitis control (NC-PBS)(Bonferroni post hoc test) lowast119875 lt 0001 The data for different groupswere reported as mean plusmn standard deviation

and colitis-Lf1 treatment groups due to probiotic treatmentin the latter (Figure 3)

324 Histological Scores Microscopic examination of theaffected tissues from the noncolitis control group revealednormal colonic epithelium with intact crypts and glands(Figures 4(a) and 4(b)) However amild lymphocytic infiltra-tion was also discernible although such minor changes werealso recorded even under normal conditions of inflammatoryresponse Almost a similar trend was also recorded in

biopsies of noncolitis Lf1 mice On the other hand biopsiesof the colitis mice revealed focally ulcerated epitheliumwithmoderate-to-excessive inflammatory infiltrate primarilycomposed of polymorphs and lymphocytes Crypt abscessesalongwith their shortening and branchingwere also recordedaccompanied by focal lymphoid follicle formation cryptitisand gland destruction These symptoms gave indication thatacute colitis was developed in mice Contrary to this biopsystudies of the colitis-Lf1 treatment group demonstratedconsiderable improvement in the tissue damage as can bereflected from the intact epithelium with mild acute andchronic inflammatory infiltration of lymphocytes Nonethe-less focal lymphoid follicle formation ulcerated epitheliumwithmoderate inflammatory infiltrate composed of polymor-phic lymphocytes focal cryptitis and gland destruction werealso recorded in two of the samples in this group Similarlythere was no evidence of granulomatous inflammation ormalignancy or cryptitis or crypt abscess

325 Persistence of L fermentum Lf1 in Colitis and NoncolitisModels The persistence and viability of Lf1 in mice gutwere confirmed by monitoring the log counts in the faecalsamples from different mice groups and also subjecting somerandomly selected colonies of faecal isolates recovered onMRS agar (Figure 5) by species specific PCR (data not shown)and RAPD to ascertain whether they belonged to Lf1 or notFrom the log count data presented in Figure 5 it is quiteclear that Lf1 not only persisted in the mice gut in all thetreatment groups but also proliferated as can be reflectedfrom the gradual increase in the faecal Lactobacillus logcounts during the treatment period registering more thanone log cycle increase towards the end of the experimentalperiod The preponderance of Lf1 in the faecal Lactobacillusisolates in the faecal samples of mice was demonstrated byPCR and RAPDThe typical RAPD banding patterns of pureLf1 along with some of the randomly selected representativefaecal Lactobacillus isolates obtained with primer 275 havebeen recorded in Figure 5 Since the typical RAPD bandingpatterns recorded with most of the faecal isolates matchedthose obtained from the pure culture of Lf1 it gives indicationthat most of the faecal isolates belonged to the clones of Lf1that was administered to the mice From these results it canbe inferred that Lf1 could survive and persist in the colon toexpress its antioxidative functions

33 MDA Level as an Index of Lipid Peroxidation The dataon the level of MDA estimated in each tissue sample fromeach mice group under different treatments is recorded inFigure 6 There was significant difference in the MDA con-tent of colitis control (9113 120583Mg) and colitis-Lf1 treatmentgroup (3792 120583Mg) thereby suggesting that our probioticstrain exhibited strong antioxidative property in colon andprevented lipid peroxidation by decreasing the level of MDAat significant level

34 Relative Expression Studies of ldquoNrf2rdquo and Antioxida-tive Enzyme Systems (ldquoSuperoxide Dismutase 2rdquo Thioredoxin

BioMed Research International 7

2

1

1

2

1

4

3

2

1 1

4

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

50120583m50120583m50120583m50120583m

50120583m 50120583m 50120583m 50120583m

50120583m50120583m50120583m50120583m

(a)

NC-PBS NC-Lf1 C-PBS C-Lf10

05

1

15

2

25

3

Hist

olog

ical

scor

e

lowastlowast

lowast

(b)

Figure 4 Histological examination and scores of different groups ofmice (a) Histological examination of noncolitis control group noncolitis(Lf1) control colitis group and Lf1-treated group of mice A-B NC-PBS (noncolitis control) showing normal mucosal and epithelial layeralong with intact glands with no sign of inflammation infiltration ulceration and cryptitis C-D NC-Lf1 (noncolitis (Lf1) control) showingnormal histology of noncolitis (Lf1) control similar to noncolitis control EndashH C-PBS (colitis control) showing severity of colitis in colitiscontrol group (1) Inflammation infiltrate (lymphocytes and neutrophils) (2) gland destruction (3) cryptitis (4) loss of entire crypt witherosion of surface epithelium and I-L C-Lf1 (colitis-Lf1 treatment groups) showing normalization of histological score in colitis- (Lf1) treatedgroup with no cryptitis no ulceration andminimal-to-moderate infiltration (b) Histological scoreschanges induced in different mice colonin different mice groups in different treatments NC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control andC-Lf1 colitis-Lf1 treatment groups lowastData are significantly different within the group (119875 lt 0001 Bonferroni post hoc test) The data fordifferent groups were reported as mean plusmn standard deviation

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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Evolutionary BiologyInternational Journal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

protect against the injurious effects of oxidative stress Oneof the dietary based strategies currently in vogue exploresprobiotics for amelioration of oxidative stress-related diseasesby augmentation of antioxidant defense systems operatingin the human body Probiotics have recently emerged as thepowerful microbial tools for novel therapeutic applicationsspecifically targeted against IBD by virtue of expressingseveral physiologically important functions under both invitro and in vivo conditions Besides displaying a plethoraof novel health promoting functions which are highlystrain specific probiotic bacteria including Lactobacilli alsodemonstrated strong antioxidative potentials as reportedpreviously [11ndash13] In this context one such study meritsspecial attention wherein L fermentum ME-3 a potentialprobiotic strain of proven efficacy possesses antagonisticand antioxidative properties when used in conjunction withofloxacin decreased lipid peroxide value in the mucosa of thesmall intestine and liver of Salmonella typhimurium infectedmurine model [14 15] As probiotics showed a positivefunctionality on oxidative stress-related indices they can helpboth to stabilize and to promote the potency of the wholebody antioxidative defense system and thus in turn mayhave an impact on lowering the risk of several inflammatorymetabolic disorders including IBDs Besides this a large bodyof evidence also suggests that probiotics like VSL3 and Lrhamnosus GG could serve as the promising candidates forthe prevention and control of IBD although some conflictingresults have also been reported [16ndash20]

Several animal models of IBD have been designed andamongst them dextran sulfate sodium (DSS) induced colitismodel appears to represent the most accurate model ofIBD since it provides human IBD-like symptoms [21] Datafrom animal models of colitis have indicated that specificprobiotic Lactobacillus and Bifidobacterium strains couldprevent and treat intestinal inflammation [22ndash25]This studywas specifically undertaken to showcase the antioxidativepotentials of L fermentum Lf1 a promising indigenous pro-biotic Lactobacillus strain tomanage oxidative stress throughldquoNrf 2rdquo activation and modulation of lipid peroxidation byimpactingMDA level under both in vitro (HT-29) and in vivoconditions in DSS colitis mouse model

2 Materials and Methods

21 Ethical Statement Before setting up the animal studyin colitis mouse model prior approval of the InstitutersquosAnimal Ethics Committee (IAEC) of the National DairyResearch Institute (NDRI Karnal India) was obtained Theexperimental animals used in this study were maintained asper National Institute of Nutrition (NIN) India guidelinesfor the care and use of laboratory animals (date of approval301010) Surgery was performed at anesthesia conditions tominimize suffering of the animals

22 Bacterial Strains A total of ten bacterial cultures whichincluded eight indigenous Lactobacillus isolates comprisingof seven strains of Lactobacillus plantarum (L plantarum9 10 42 55 78 91 and S3) and a strain of Lactobacillus

fermentum (Lf1 an indigenous isolate of Indian gut origindeposited in International Depository Budapest Treaty atMicrobial Type Culture Collection MTCC 5689) besidestwo reference probiotic strains L plantarum CSCC5276 (alsodesignated as CSCC5276 NCDO82 or VTTE-71034) whichwas procured from Dr N P Shah from Victoria UniversityAustralia [26 27] and L acidophilus NCFM (also knownas Howaru Dophilus LA-1 NCK56 NCK45 N2 RL8KRRL8KS and RL8K) [28ndash30] formed the subject of this studyThey were procured from the probiotic culture collectionmaintained at Molecular Biology Unit Dairy MicrobiologyDivision National Dairy Research Institute Karnal Bacterialstrains were activated prior to use by subculturing in steriledeManRogosa Sharpe (MRS) broth (HiMedia India) at 37∘Cfor 18ndash24 h

23 Propagation of Human Colonic Epithelial Cell Lines (HT-29 Cells) and Treatments Human colonic epithelial cellline (HT-29) was procured from National Centre of CellSciences (NCCS Pune India) The cell line was cultured inDulbeccorsquos Modified Eagles Medium (DMEM Sigma USA)The propagation of HT-29 cells was done as reported in aprevious study and carried out in our lab [31] After attainingconfluency HT-29 cells were challenged with each of the1mL of live probiotic strains (sim1 times 109 cfumL resuspended inDMEM) and incubated at 37∘C in 5 CO

2for 4 h followed

by addition of fresh medium without antibiotic and foetalbovine serum After incubation cells were challenged with1mMH

2O2(Sigma USA) and again incubated for additional

30min After incubation H2O2was completely removed and

trypsinization (with Trypsin-EDTA solution Sigma USA)was done Cells were again resuspended in DMEM mediumand pelleted (centrifugation at 1700 rpm for 5min at RT) forRNA isolation The supernatant was stored at minus80∘C for totalantioxidant activity and lipid peroxidation determination

231 ldquoNrf2rdquo Gene Expression Study in HT-29 Cells RNA wasisolated from the cell pellet by TRIzol method (TRI ReagentSigma USA) and cDNA was prepared using ImProm-IIreverse transcriptase kit (Promega USA) and random hex-amer primers (100120583M) provided with the same as describedpreviously [32] The expression of target genes ldquoNrf 2rdquo alongwith housekeeping gene ldquo120573-actinrdquo for HT-29 cells and fromHT-29 cells (after H

2O2challenge) was studied by reverse

transcriptase-quantitative PCR (RT-qPCR) Untreated HT-29 cells grown in DMEM served as controlThe primers usedin this study are listed in Table 1

232 Lipid Peroxidation The extent of lipid peroxidation anindex of oxidative stress was measured as thiobarbituric acidreactive substances (TBARS) formed Lipid hydroperoxideswere measured by standard TBA test method [33]

24 Experimental Design of an In Vivo Study Using DSSColitis MouseModel A total of thirty-two adult male (seven-to-eight weeks old) Swiss Albino mice weighing 25ndash30 gon average were used in this study The animals were fednormal diet (Bengal gram crushed 58 wheat starch 15

BioMed Research International 3

Table 1 Sequences of the primers used for RT-qPCR

Genes Primer sequence (51015840-31015840) Ampliconsize (bp)

Nrf 2 HFNrf 2 HR

gcg acg gaa aga gta tga cgtt ggc aga tcc act ggt tt 181

Nrf 2 MFNrf 2 MR

ttc agc aca aca ctg gga agtgt tgc tgg ggt tta tag gc 102

SOD2 MFSOD2 MR

gct ggc ttg gct tca ata agtaa ggc ctg ttg ttc ctt gc 87

CAT1 MFCAT1 MR

gct gag aag cct aag aac gcacct tcg cag cca tgt gag a 64

Trx-1 MF1Trx-1 MR1

tcc att tcc atc tgg ttc tgcttc acc att ttg gct gtt gc 65

120573-Actin F120573-Actin R

tgg ctg ggg tgt tga agg tctagc acg gca tcg tca cca act

238[10]

120573-Actin mus2F120573-Actin mus2R

agt gtg acg ttg aca tcc gtagcc aga gca gta atc tcc ttc t 112

groundnut cake 10 casein 4 groundnut oil 4 saltmixture 4 vitamin mixture 02 and choline chloride02) and water ad libitum during the entire course of theexperiment The animals were divided into four homoge-neous groups (noncolitis control NC-PBS noncolitis (Lf1)control NC-Lf1 colitis control C-PBS and colitis-Lf1 (C-Lf1) treatment groups) comprising of eight animals eachhoused in individual cages and maintained under a constant12 h light-12 h dark cycle The temperature was controlled at22ndash25∘C with about 56ndash60 relative humidity There wasno significant difference in the body weight of mice amongthe four groups on day zero The body weight of mice ineach group on the first day was taken as the basal level Thesubsequent changes in their body weight were monitoredperiodically during the entire 13-day experimental periodThe body weight of mice each day minus the basal bodyweight was expressed as the bodyweight changeThenegativevalue indicated the decreased weight while the positive valuerepresented the increased weight

25 Feeding of Mice and Experimental Design The micewere fed for twelve days by oral intubation with 15mL ofovernight grown Lactobacillus culture (Lf1) at 109 cfumLafter centrifugation at 7000 rpm for 10min and resuspendingthe cell pellet in 200 120583L of sterile PBS solution with the helpof 1mL syringe and silicon tube The mice fed with PBSalone served as the control group The experimental designhas been illustrated in Figure 1(S) in Supplementary Mate-rial available online at httpdxdoiorg1011552014206732Noncolitis control group was treated with PBS alone for12 days Noncolitis (Lf1) treatment group was administeredwith 200120583L Lactobacillus culture (Lf1) orally by orogastrictube Colitis (Lf1) mice were fed with 200 120583L Lactobacillusculture (108-109 cfuml) orally by orogastric tube for 7 daysbefore starting dextran sodium sulfate (DSS) and continuedtill 12th day after DSS induction PBS was administered incolitis control group Colitis was induced by 5 (wv) DSS(MW = 40000ndash50000 USB Corporation Cleveland ICN

Biomedicals OH USA) dissolved in drinking water after 7days All the animals in different groups were monitoredon every alternate day for physical parameters like weight-lossgain faecal matter consistency and blood in stoolsFaecal samples were collected every three days and plated onMRS media to determine Lactobacillus counts in faeces aswell as to assess the persistence of the viable probiotic cellsin mice gut Disease Activity Index (DAI) scores were alsorecorded every alternate day Length of colon was measuredand histological scores were assessed on the 13th day Colontissue was used for performing lipid peroxidation assay aswell as for RNA isolation for gene expression studies withregard to ldquoNrf 2rdquo and antioxidant genes by RT-qPCR

26 Evaluation of Colonic Damage and Inflammation Sever-ity of colitis was assessed every alternate day using a DiseaseActivity Index (DAI) DAI is known to be a valid measurefor the acute colitis produced by DSS [34] The scores wereassigned on 0ndash4 scale taking into account weight loss stoolconsistency and blood in stool on scales of 0ndash4 (Table 1(S) inSupplementary Material) [35] For each mouse a daily DAIscore was calculated as the aggregate of the weight lossscore stool consistency score and rectal bleeding divided bythree The DAI values for the groups were then recorded asmean plusmn standard deviation for each day of the experimentSubsequently the daily DAI score for each experimentalgroup was calculated as the mean plusmn standard error of theindividualDAI scores of eachmouse in the group (Table 1(S))The DAI clinical parameters used here are comprehensivefunctional measures that are somewhat analogous to clinicalsymptoms observed in human IBD and the scoring methodthat has been validated by repeated studies Besides thisweight of mice was recorded at every alternate day as it is alsocorrelated with severity of induced colitis Scoring system forthe comparative analysis of intestinal bleeding is given in theform of Table 1(S)

The animals were sacrificed on the thirteenth day Bycarefully opening the mouse by a ventral midline incisionthe colon was collected from the colon-cecal junction to theanus and its length was measured After removal of faecalmatter colon was rinsed with DEPC water A small part(02-03 cm) of colon was kept in 10 buffered formalin forhistopathological examination (ie Hematoxylin and Eosinstaining) and the remaining tissue was immediately wrappedin aluminium foil and snap frozen in liquid nitrogen for RNAisolation and lipid peroxidation analysis Colonic damageand inflammation were recorded by the intensity of inflam-mation monitored through histopathological examinationPersistence of probiotic Lactobacilli was also monitored bytaking faecal counts

27 RAPD Profiles of Lactobacillus Faecal Isolates Faecalsamples were collected every alternate day of probioticadministrationPBS (control group) Faecalmaterial (100mg)was homogenized in one mL of PBS Serial dilutions weremade and plating for Lactobacillus counts was done onMRS-BCP agar The plates were incubated at 37∘C for 24ndash48 hColonies were counted and recorded Colonies from different

4 BioMed Research International

faecal count plates were randomly picked and grown for16 to 18 h at 37∘C after inoculation into MRS broth Thegenomic DNA from faecal isolates was then isolated byPospiech and Neumann method [36] PCR with randomoligo primer named 275 (51015840-ccg ggc aag c-31015840) was carried outfor RAPD profiling PCR reaction was performed in 25 120583Lreaction volume containing PCR buffer (25 120583L) oligo primer(20 120583L) 200 120583M dNTP (20 120583L) 10 U Taq DNA polymerase(05 120583L) and template (20 120583L) The PCR cycling parametersincluded an initial denaturation of 95∘C5min followedby 45 cycles each of denaturation (95∘C30 sec) annealing(40∘C30 sec) extension (72∘C2min) and final extensionof 72∘C10min The PCR products were electrophoresedon 18ndash2 agarose gel with ethidium bromide (05 120583gmL)(Amersham Biosciences USA) at 100V using 1X TAE bufferThe RAPD patterns were compared with the patterns of thepure cultures of the administered probiotic strain

28 Histopathological Examination The intestinal tissuesfrom the colon of the probiotic and control groups wereset apart for histopathological examination Intestines (02-03 cm of tissue) of different groups were kept in 10 bufferedformalin solution for at least 24 h prior to cassetting Thecolon was sliced mounted stained with hematoxylin (HiMe-dia India) and eosin (HiMedia India) and then scored bya blinded pathologist who rated the tissues based on thegrade of the disease 0ndash4 the percentage of the disease 0ndash4and the severity of the inflammation 0ndash3 (Table 2(S) andashc)[35] Histopathological examination of the affected tissuesfrom the mice groups analyzed in this study was done by ablinded histologist who rated the tissues based on the gradeof the disease 0ndash4 the percentage of the disease 0ndash4 andthe severity of the inflammation 0ndash3Microscopic evaluationof all the sections of tissue samples was done at 4x and 10xmagnification and histological scores were graded on thebasis of the severity of inflammation infiltration ulcerationand crypt damage

29 Lipid Peroxidation For detection of lipid peroxides(malondialdehyde) in colon homogenate the methodof Uchiyama and Mihara was followed [37] The colonhomogenate was prepared in 1X PBS An aliquot of 3mLof 1 phosphoric acid and one mL of 06 TBA solutionwere added to 05mL of colon homogenate The mixturewas heated for 45min on a boiling water bath After cooling4mL of n-butanol was added and mixed vigorously Thebutanol phase was separated by centrifugation at 5000 rpmfor 10min and absorbance was measured at 535 and 520 nmThe difference was used as the TBA value A standard curveof malondialdehyde was drawn by taking TBA value atvarious concentration of malondialdehyde MDA standardcurve was prepared by carrying out overnight digestionof different concentrations of 1133-tetraethoxypropane(01mM) in presence of 02N HCl

210 Gene Expression Studies RNAwas isolated frommousecolon by TRIzol method (TRI Reagent Sigma USA) cDNAwas synthesized and RT-qPCR in LightCycler 480 (Roche

Switzerland) was carried out as described previously [31]The thermal cycling conditions included initial denaturationat 95∘C for 5min followed by 40 cycles each of denat-uration (95∘C30 s) annealing (53∘C30 s) and extension(72∘C45 s) with a single fluorescence measurement a meltcurve program (60∘Cndash95∘C with a heating rate of 011∘Csand continuous fluorescence measurement) and finally acooling step at 40∘C RNA extraction and measurement ofgene expression by RT-qPCR were performed in triplicateand the mean of all these values was used for final analysis

The quantitative data generated by real-time PCR (thequantification cycle Cq value) were analyzed by relativeexpression software tool (REST 2009) (httpwwwgene-quantificationinfo) as described previously [32]

211 Statistical Analyses Thedata obtained from each animalexperiment were expressed as mean plusmn standard deviationvalues Analyses of variance (ANOVA) for estimation ofsignificance of differences betweenmeans were automaticallydone with the Statistical Package for the Social Science (SPSSfor Windows version 101 SPSS Inc Chicago) softwareby Bonferroni post hoc test and considered significantlydifferent at 119875 le 005

3 Results

31 Screening of Probiotic Lactobacilli Initially during thispart of study some putative probiotic Lactobacilli werescreened on the basis of their ability to enhance ldquoNrf 2rdquoexpression in HT-29 cells along with lipid peroxidationinhibitory potential to short list the most promising strainfor further investigations to explore its in vivo efficacyin colitis mice model From the critical appraisal of thedata recorded in Figure 1(a) it can be inferred that therewas considerable variation in ldquoNrf 2rdquo expression induced inHT-29 cells by different probiotic strains as they behaveddifferently in eliciting ldquoNrf 2rdquo expression The expression ofldquoNrf 2rdquo in HT-29 cells was significantly downregulated (119875 lt0001) in the sample group (relative to control group) byminus188-fold when subjected to H

2O2stress alone (Figure 1(a))

Although some of the probiotic strains when used alone orin conjunction with H

2O2did not show any significant effect

on the ldquoNrf 2rdquo expression in HT-29 cells Lp5276 and S3 didinduce significant downregulation at the level of minus138- andminus164-fold respectively However it was quite interesting tonote that three of the strains namely Lp55 Lp91 and Lf1were able to induce a significant (119875 lt 0001) upregulation inthe expression of ldquoNrf 2rdquo in HT-29 cells at the levels of 748- 258- and 643-fold when used in conjunction with H

2O2

Hence the latter were rated as the prospective candidateprobiotic strains to explore their efficacy in the managementof oxidative stress induced diseases

The total antioxidative activity of the aforesaid Lacto-bacillus strains in terms of their lipid peroxidation inhibitionpotentials inHT-29 cells was also investigated after subjectingthe same to H

2O2treatment by measuring the level of

MDA the secondary product of lipid peroxidation From thedata presented in the form of a bar diagram illustrated in

BioMed Research International 5

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

) 10

8

6

4

2

0

minus2

minus4

lowastlowast

lowast

lowast

lowast

lowast lowastlowastlowast

Lf1+

H2O2

S3+

H2O2

H2O2

Lp9

Lp10

Lp10+

H2O2

Lp42

Lp42+

H2O2

Lp55+

H2O2

Lp78

Lp78+

H2O2

Lp91

Lp91+

H2O2

Lp9+

H2O2

Lp55

Lp5276+

H2O2

Lf1 S3

NCF

M

Lp5276

NCF

M+

H2O2

(a)

100

75

50

25

0

MD

A

inhi

bitio

n

lowastlowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

LP9+

H2O2

Lp10+

H2O2

Lp42+

H2O2

Lp55+

H2O2

Lp78+

H2O2

Lp91+

H2O2

Lf1+

H2O2

S3+

H2O2

Lp5276+

H2O2

NCF

M+

H2O2

(b)

Figure 1 Relative expression of ldquoNrf 2rdquo and MDA inhibitory effect by Lactobacilli strains in HT-29 cell lines under H2O2stress conditions

Data are represented as mean plusmn SD number of RT-qPCR experiments (119899) = 3 (a) Comparative analysis of relative expression of ldquoNrf 2rdquo inHT-29 on challenge with different strains of probiotic Lactobacilli and H

2O2stress lowastData are significantly different compared with control

(119875 lt 0001 Bonferroni post hoc test) (b) Inhibitory effect of various probiotic Lactobacillus cultures on lipid peroxidation in HT-29 cellsData are significantly different compared with control (H

2O2alone) (lowast119875 lt 0001 lowastlowast119875 lt 001 Bonferroni post hoc test)

Figure 1(b) it is clearly indicated that Lactobacillus spp S3showed the highest percent inhibition that is 832 plusmn 053followed by Lf1 (781 plusmn 024) as compared to the standardreference strains showing relatively poor response as can bereflected from the lower percent inhibition values that is556 plusmn 04 (Lp5276) to 552 plusmn 036 (LaNCFM) indicatingthat the indigenous probiotic strains were better equippedto neutralize the injurious effects of oxidative stress Hencebased on the overall combined effects of both of theseactivities inHT-29 cells we finally selected Lf1 as the potentialcandidate strain for in vivo study

32 InVivo Studies inDSSColitisMouseModel Feedingmicewith DSS via drinking water for five consecutive days ledto acute colitis characterized typically by bloody diarrhoeaulcerations and infiltrationswith granulocytes Furthermoresince DSS happens to be toxic to gut epithelial cells of thebasal crypts andhence can impact the integrity of themucosalbarrier all the experimental animals belonging to either thecontrol or the treatment groups were critically examined forthe following clinical parameters besides other importantphysicochemical changes recorded inmice groups during thecourse of this experiment

321 Weight Loss The gross changes in the body weightof mice in different treatment groups including probioticinterventions weremonitored during the entire 13-day exper-imental period of the study The body weight was foundto increase gradually in both the noncolitis control and Lf1groups Mice in the colitis group showed a gradual increasein weight for the first seven days but later after starting DSStreatment excessive weight loss was recorded that becamehighly significant starting from day eight to day thirteen

(119875 lt 005) due to severe experimental colitis The colitis-Lf1 treatment group showed a mixed response as there wasgradual increase in body weight in the first seven days butafter induction of colitis significant inhibition of weight losswas recorded (119875 lt 005) Quite interestingly some of themice in the same treatment group showed the opposite effectby recording appreciable weight gain The changes recordedwith regard to body weight in different mice groups duringthe course of this study have been illustrated in Figure 2However there was no significant difference in weight gainbetween both NC-PBS versus NC-Lf1 and C-PBS versus C-Lf1 groups (119875 gt 005)

322 Length of Colon The length of mice colon in thenoncolitis control with respect to noncolitis Lf1 fed groupswas 705 plusmn 021 and 815 plusmn 014 respectively The length ofcolon of colitis-Lf1 treatmentmice wasmuch longer than thatof mice in the colitis group (793 plusmn 038 versus 638 plusmn 039 cm119875 lt 005) There was no significant difference (119875 gt 005)between the length of colon of colitis-Lf1 treatment group andnoncolitis Lf1-treated group

323 Disease Activity Index (DAI) Scores As can be reflectedfrom Figure 3 the highest DAI score was recorded in colitiscontrol group DAI scores of noncolitis control group andnoncolitis probiotic group were however zero as weight losschanges observed in these groups were not significant to beconsidered as DAI and hence were rated as nonsignificant(NS) groups However DAI scores were improved signifi-cantly in colitis-Lf1 treatment group as shown in Figure 3On comparative evaluation there was significant difference(119875 lt 005) recorded between the DAI scores of colitis control

6 BioMed Research International

0 2 4 6 8 10 12 14

Days

Mea

n bo

dy w

eigh

t cha

nges

minus1

0

1

2

3

25

15

05

minus05

C-PBSC-Lf1

NC-PBSNC-Lf1

Before DSS colitis induction After DSS colitis induction

Figure 2 Bodyweight changes among different groups ofmiceNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups The data fordifferent groups were reported as mean plusmn standard deviation

0 2 4 6 8 10 12 140

05

1

15

2

25

3

35

DA

I sco

re

Days

NC-PBSNC-Lf1

C-PBSC-Lf1

lowast

lowastlowast

lowast

lowast

lowast

lowast

Figure 3 Comparative analysis of mean DAI scores in differentgroups in mice NC-PBS noncolitis control NC-Lf1 noncolitis(Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treat-ment groups Data showing asterisk mark (lowast) without line showssignificant difference compared with noncolitis control (NC-PBS)(Bonferroni post hoc test) lowast119875 lt 0001 The data for different groupswere reported as mean plusmn standard deviation

and colitis-Lf1 treatment groups due to probiotic treatmentin the latter (Figure 3)

324 Histological Scores Microscopic examination of theaffected tissues from the noncolitis control group revealednormal colonic epithelium with intact crypts and glands(Figures 4(a) and 4(b)) However amild lymphocytic infiltra-tion was also discernible although such minor changes werealso recorded even under normal conditions of inflammatoryresponse Almost a similar trend was also recorded in

biopsies of noncolitis Lf1 mice On the other hand biopsiesof the colitis mice revealed focally ulcerated epitheliumwithmoderate-to-excessive inflammatory infiltrate primarilycomposed of polymorphs and lymphocytes Crypt abscessesalongwith their shortening and branchingwere also recordedaccompanied by focal lymphoid follicle formation cryptitisand gland destruction These symptoms gave indication thatacute colitis was developed in mice Contrary to this biopsystudies of the colitis-Lf1 treatment group demonstratedconsiderable improvement in the tissue damage as can bereflected from the intact epithelium with mild acute andchronic inflammatory infiltration of lymphocytes Nonethe-less focal lymphoid follicle formation ulcerated epitheliumwithmoderate inflammatory infiltrate composed of polymor-phic lymphocytes focal cryptitis and gland destruction werealso recorded in two of the samples in this group Similarlythere was no evidence of granulomatous inflammation ormalignancy or cryptitis or crypt abscess

325 Persistence of L fermentum Lf1 in Colitis and NoncolitisModels The persistence and viability of Lf1 in mice gutwere confirmed by monitoring the log counts in the faecalsamples from different mice groups and also subjecting somerandomly selected colonies of faecal isolates recovered onMRS agar (Figure 5) by species specific PCR (data not shown)and RAPD to ascertain whether they belonged to Lf1 or notFrom the log count data presented in Figure 5 it is quiteclear that Lf1 not only persisted in the mice gut in all thetreatment groups but also proliferated as can be reflectedfrom the gradual increase in the faecal Lactobacillus logcounts during the treatment period registering more thanone log cycle increase towards the end of the experimentalperiod The preponderance of Lf1 in the faecal Lactobacillusisolates in the faecal samples of mice was demonstrated byPCR and RAPDThe typical RAPD banding patterns of pureLf1 along with some of the randomly selected representativefaecal Lactobacillus isolates obtained with primer 275 havebeen recorded in Figure 5 Since the typical RAPD bandingpatterns recorded with most of the faecal isolates matchedthose obtained from the pure culture of Lf1 it gives indicationthat most of the faecal isolates belonged to the clones of Lf1that was administered to the mice From these results it canbe inferred that Lf1 could survive and persist in the colon toexpress its antioxidative functions

33 MDA Level as an Index of Lipid Peroxidation The dataon the level of MDA estimated in each tissue sample fromeach mice group under different treatments is recorded inFigure 6 There was significant difference in the MDA con-tent of colitis control (9113 120583Mg) and colitis-Lf1 treatmentgroup (3792 120583Mg) thereby suggesting that our probioticstrain exhibited strong antioxidative property in colon andprevented lipid peroxidation by decreasing the level of MDAat significant level

34 Relative Expression Studies of ldquoNrf2rdquo and Antioxida-tive Enzyme Systems (ldquoSuperoxide Dismutase 2rdquo Thioredoxin

BioMed Research International 7

2

1

1

2

1

4

3

2

1 1

4

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

50120583m50120583m50120583m50120583m

50120583m 50120583m 50120583m 50120583m

50120583m50120583m50120583m50120583m

(a)

NC-PBS NC-Lf1 C-PBS C-Lf10

05

1

15

2

25

3

Hist

olog

ical

scor

e

lowastlowast

lowast

(b)

Figure 4 Histological examination and scores of different groups ofmice (a) Histological examination of noncolitis control group noncolitis(Lf1) control colitis group and Lf1-treated group of mice A-B NC-PBS (noncolitis control) showing normal mucosal and epithelial layeralong with intact glands with no sign of inflammation infiltration ulceration and cryptitis C-D NC-Lf1 (noncolitis (Lf1) control) showingnormal histology of noncolitis (Lf1) control similar to noncolitis control EndashH C-PBS (colitis control) showing severity of colitis in colitiscontrol group (1) Inflammation infiltrate (lymphocytes and neutrophils) (2) gland destruction (3) cryptitis (4) loss of entire crypt witherosion of surface epithelium and I-L C-Lf1 (colitis-Lf1 treatment groups) showing normalization of histological score in colitis- (Lf1) treatedgroup with no cryptitis no ulceration andminimal-to-moderate infiltration (b) Histological scoreschanges induced in different mice colonin different mice groups in different treatments NC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control andC-Lf1 colitis-Lf1 treatment groups lowastData are significantly different within the group (119875 lt 0001 Bonferroni post hoc test) The data fordifferent groups were reported as mean plusmn standard deviation

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

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Advances in

Virolog y

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International Journal of

Microbiology

BioMed Research International 3

Table 1 Sequences of the primers used for RT-qPCR

Genes Primer sequence (51015840-31015840) Ampliconsize (bp)

Nrf 2 HFNrf 2 HR

gcg acg gaa aga gta tga cgtt ggc aga tcc act ggt tt 181

Nrf 2 MFNrf 2 MR

ttc agc aca aca ctg gga agtgt tgc tgg ggt tta tag gc 102

SOD2 MFSOD2 MR

gct ggc ttg gct tca ata agtaa ggc ctg ttg ttc ctt gc 87

CAT1 MFCAT1 MR

gct gag aag cct aag aac gcacct tcg cag cca tgt gag a 64

Trx-1 MF1Trx-1 MR1

tcc att tcc atc tgg ttc tgcttc acc att ttg gct gtt gc 65

120573-Actin F120573-Actin R

tgg ctg ggg tgt tga agg tctagc acg gca tcg tca cca act

238[10]

120573-Actin mus2F120573-Actin mus2R

agt gtg acg ttg aca tcc gtagcc aga gca gta atc tcc ttc t 112

groundnut cake 10 casein 4 groundnut oil 4 saltmixture 4 vitamin mixture 02 and choline chloride02) and water ad libitum during the entire course of theexperiment The animals were divided into four homoge-neous groups (noncolitis control NC-PBS noncolitis (Lf1)control NC-Lf1 colitis control C-PBS and colitis-Lf1 (C-Lf1) treatment groups) comprising of eight animals eachhoused in individual cages and maintained under a constant12 h light-12 h dark cycle The temperature was controlled at22ndash25∘C with about 56ndash60 relative humidity There wasno significant difference in the body weight of mice amongthe four groups on day zero The body weight of mice ineach group on the first day was taken as the basal level Thesubsequent changes in their body weight were monitoredperiodically during the entire 13-day experimental periodThe body weight of mice each day minus the basal bodyweight was expressed as the bodyweight changeThenegativevalue indicated the decreased weight while the positive valuerepresented the increased weight

25 Feeding of Mice and Experimental Design The micewere fed for twelve days by oral intubation with 15mL ofovernight grown Lactobacillus culture (Lf1) at 109 cfumLafter centrifugation at 7000 rpm for 10min and resuspendingthe cell pellet in 200 120583L of sterile PBS solution with the helpof 1mL syringe and silicon tube The mice fed with PBSalone served as the control group The experimental designhas been illustrated in Figure 1(S) in Supplementary Mate-rial available online at httpdxdoiorg1011552014206732Noncolitis control group was treated with PBS alone for12 days Noncolitis (Lf1) treatment group was administeredwith 200120583L Lactobacillus culture (Lf1) orally by orogastrictube Colitis (Lf1) mice were fed with 200 120583L Lactobacillusculture (108-109 cfuml) orally by orogastric tube for 7 daysbefore starting dextran sodium sulfate (DSS) and continuedtill 12th day after DSS induction PBS was administered incolitis control group Colitis was induced by 5 (wv) DSS(MW = 40000ndash50000 USB Corporation Cleveland ICN

Biomedicals OH USA) dissolved in drinking water after 7days All the animals in different groups were monitoredon every alternate day for physical parameters like weight-lossgain faecal matter consistency and blood in stoolsFaecal samples were collected every three days and plated onMRS media to determine Lactobacillus counts in faeces aswell as to assess the persistence of the viable probiotic cellsin mice gut Disease Activity Index (DAI) scores were alsorecorded every alternate day Length of colon was measuredand histological scores were assessed on the 13th day Colontissue was used for performing lipid peroxidation assay aswell as for RNA isolation for gene expression studies withregard to ldquoNrf 2rdquo and antioxidant genes by RT-qPCR

26 Evaluation of Colonic Damage and Inflammation Sever-ity of colitis was assessed every alternate day using a DiseaseActivity Index (DAI) DAI is known to be a valid measurefor the acute colitis produced by DSS [34] The scores wereassigned on 0ndash4 scale taking into account weight loss stoolconsistency and blood in stool on scales of 0ndash4 (Table 1(S) inSupplementary Material) [35] For each mouse a daily DAIscore was calculated as the aggregate of the weight lossscore stool consistency score and rectal bleeding divided bythree The DAI values for the groups were then recorded asmean plusmn standard deviation for each day of the experimentSubsequently the daily DAI score for each experimentalgroup was calculated as the mean plusmn standard error of theindividualDAI scores of eachmouse in the group (Table 1(S))The DAI clinical parameters used here are comprehensivefunctional measures that are somewhat analogous to clinicalsymptoms observed in human IBD and the scoring methodthat has been validated by repeated studies Besides thisweight of mice was recorded at every alternate day as it is alsocorrelated with severity of induced colitis Scoring system forthe comparative analysis of intestinal bleeding is given in theform of Table 1(S)

The animals were sacrificed on the thirteenth day Bycarefully opening the mouse by a ventral midline incisionthe colon was collected from the colon-cecal junction to theanus and its length was measured After removal of faecalmatter colon was rinsed with DEPC water A small part(02-03 cm) of colon was kept in 10 buffered formalin forhistopathological examination (ie Hematoxylin and Eosinstaining) and the remaining tissue was immediately wrappedin aluminium foil and snap frozen in liquid nitrogen for RNAisolation and lipid peroxidation analysis Colonic damageand inflammation were recorded by the intensity of inflam-mation monitored through histopathological examinationPersistence of probiotic Lactobacilli was also monitored bytaking faecal counts

27 RAPD Profiles of Lactobacillus Faecal Isolates Faecalsamples were collected every alternate day of probioticadministrationPBS (control group) Faecalmaterial (100mg)was homogenized in one mL of PBS Serial dilutions weremade and plating for Lactobacillus counts was done onMRS-BCP agar The plates were incubated at 37∘C for 24ndash48 hColonies were counted and recorded Colonies from different

4 BioMed Research International

faecal count plates were randomly picked and grown for16 to 18 h at 37∘C after inoculation into MRS broth Thegenomic DNA from faecal isolates was then isolated byPospiech and Neumann method [36] PCR with randomoligo primer named 275 (51015840-ccg ggc aag c-31015840) was carried outfor RAPD profiling PCR reaction was performed in 25 120583Lreaction volume containing PCR buffer (25 120583L) oligo primer(20 120583L) 200 120583M dNTP (20 120583L) 10 U Taq DNA polymerase(05 120583L) and template (20 120583L) The PCR cycling parametersincluded an initial denaturation of 95∘C5min followedby 45 cycles each of denaturation (95∘C30 sec) annealing(40∘C30 sec) extension (72∘C2min) and final extensionof 72∘C10min The PCR products were electrophoresedon 18ndash2 agarose gel with ethidium bromide (05 120583gmL)(Amersham Biosciences USA) at 100V using 1X TAE bufferThe RAPD patterns were compared with the patterns of thepure cultures of the administered probiotic strain

28 Histopathological Examination The intestinal tissuesfrom the colon of the probiotic and control groups wereset apart for histopathological examination Intestines (02-03 cm of tissue) of different groups were kept in 10 bufferedformalin solution for at least 24 h prior to cassetting Thecolon was sliced mounted stained with hematoxylin (HiMe-dia India) and eosin (HiMedia India) and then scored bya blinded pathologist who rated the tissues based on thegrade of the disease 0ndash4 the percentage of the disease 0ndash4and the severity of the inflammation 0ndash3 (Table 2(S) andashc)[35] Histopathological examination of the affected tissuesfrom the mice groups analyzed in this study was done by ablinded histologist who rated the tissues based on the gradeof the disease 0ndash4 the percentage of the disease 0ndash4 andthe severity of the inflammation 0ndash3Microscopic evaluationof all the sections of tissue samples was done at 4x and 10xmagnification and histological scores were graded on thebasis of the severity of inflammation infiltration ulcerationand crypt damage

29 Lipid Peroxidation For detection of lipid peroxides(malondialdehyde) in colon homogenate the methodof Uchiyama and Mihara was followed [37] The colonhomogenate was prepared in 1X PBS An aliquot of 3mLof 1 phosphoric acid and one mL of 06 TBA solutionwere added to 05mL of colon homogenate The mixturewas heated for 45min on a boiling water bath After cooling4mL of n-butanol was added and mixed vigorously Thebutanol phase was separated by centrifugation at 5000 rpmfor 10min and absorbance was measured at 535 and 520 nmThe difference was used as the TBA value A standard curveof malondialdehyde was drawn by taking TBA value atvarious concentration of malondialdehyde MDA standardcurve was prepared by carrying out overnight digestionof different concentrations of 1133-tetraethoxypropane(01mM) in presence of 02N HCl

210 Gene Expression Studies RNAwas isolated frommousecolon by TRIzol method (TRI Reagent Sigma USA) cDNAwas synthesized and RT-qPCR in LightCycler 480 (Roche

Switzerland) was carried out as described previously [31]The thermal cycling conditions included initial denaturationat 95∘C for 5min followed by 40 cycles each of denat-uration (95∘C30 s) annealing (53∘C30 s) and extension(72∘C45 s) with a single fluorescence measurement a meltcurve program (60∘Cndash95∘C with a heating rate of 011∘Csand continuous fluorescence measurement) and finally acooling step at 40∘C RNA extraction and measurement ofgene expression by RT-qPCR were performed in triplicateand the mean of all these values was used for final analysis

The quantitative data generated by real-time PCR (thequantification cycle Cq value) were analyzed by relativeexpression software tool (REST 2009) (httpwwwgene-quantificationinfo) as described previously [32]

211 Statistical Analyses Thedata obtained from each animalexperiment were expressed as mean plusmn standard deviationvalues Analyses of variance (ANOVA) for estimation ofsignificance of differences betweenmeans were automaticallydone with the Statistical Package for the Social Science (SPSSfor Windows version 101 SPSS Inc Chicago) softwareby Bonferroni post hoc test and considered significantlydifferent at 119875 le 005

3 Results

31 Screening of Probiotic Lactobacilli Initially during thispart of study some putative probiotic Lactobacilli werescreened on the basis of their ability to enhance ldquoNrf 2rdquoexpression in HT-29 cells along with lipid peroxidationinhibitory potential to short list the most promising strainfor further investigations to explore its in vivo efficacyin colitis mice model From the critical appraisal of thedata recorded in Figure 1(a) it can be inferred that therewas considerable variation in ldquoNrf 2rdquo expression induced inHT-29 cells by different probiotic strains as they behaveddifferently in eliciting ldquoNrf 2rdquo expression The expression ofldquoNrf 2rdquo in HT-29 cells was significantly downregulated (119875 lt0001) in the sample group (relative to control group) byminus188-fold when subjected to H

2O2stress alone (Figure 1(a))

Although some of the probiotic strains when used alone orin conjunction with H

2O2did not show any significant effect

on the ldquoNrf 2rdquo expression in HT-29 cells Lp5276 and S3 didinduce significant downregulation at the level of minus138- andminus164-fold respectively However it was quite interesting tonote that three of the strains namely Lp55 Lp91 and Lf1were able to induce a significant (119875 lt 0001) upregulation inthe expression of ldquoNrf 2rdquo in HT-29 cells at the levels of 748- 258- and 643-fold when used in conjunction with H

2O2

Hence the latter were rated as the prospective candidateprobiotic strains to explore their efficacy in the managementof oxidative stress induced diseases

The total antioxidative activity of the aforesaid Lacto-bacillus strains in terms of their lipid peroxidation inhibitionpotentials inHT-29 cells was also investigated after subjectingthe same to H

2O2treatment by measuring the level of

MDA the secondary product of lipid peroxidation From thedata presented in the form of a bar diagram illustrated in

BioMed Research International 5

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

) 10

8

6

4

2

0

minus2

minus4

lowastlowast

lowast

lowast

lowast

lowast lowastlowastlowast

Lf1+

H2O2

S3+

H2O2

H2O2

Lp9

Lp10

Lp10+

H2O2

Lp42

Lp42+

H2O2

Lp55+

H2O2

Lp78

Lp78+

H2O2

Lp91

Lp91+

H2O2

Lp9+

H2O2

Lp55

Lp5276+

H2O2

Lf1 S3

NCF

M

Lp5276

NCF

M+

H2O2

(a)

100

75

50

25

0

MD

A

inhi

bitio

n

lowastlowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

LP9+

H2O2

Lp10+

H2O2

Lp42+

H2O2

Lp55+

H2O2

Lp78+

H2O2

Lp91+

H2O2

Lf1+

H2O2

S3+

H2O2

Lp5276+

H2O2

NCF

M+

H2O2

(b)

Figure 1 Relative expression of ldquoNrf 2rdquo and MDA inhibitory effect by Lactobacilli strains in HT-29 cell lines under H2O2stress conditions

Data are represented as mean plusmn SD number of RT-qPCR experiments (119899) = 3 (a) Comparative analysis of relative expression of ldquoNrf 2rdquo inHT-29 on challenge with different strains of probiotic Lactobacilli and H

2O2stress lowastData are significantly different compared with control

(119875 lt 0001 Bonferroni post hoc test) (b) Inhibitory effect of various probiotic Lactobacillus cultures on lipid peroxidation in HT-29 cellsData are significantly different compared with control (H

2O2alone) (lowast119875 lt 0001 lowastlowast119875 lt 001 Bonferroni post hoc test)

Figure 1(b) it is clearly indicated that Lactobacillus spp S3showed the highest percent inhibition that is 832 plusmn 053followed by Lf1 (781 plusmn 024) as compared to the standardreference strains showing relatively poor response as can bereflected from the lower percent inhibition values that is556 plusmn 04 (Lp5276) to 552 plusmn 036 (LaNCFM) indicatingthat the indigenous probiotic strains were better equippedto neutralize the injurious effects of oxidative stress Hencebased on the overall combined effects of both of theseactivities inHT-29 cells we finally selected Lf1 as the potentialcandidate strain for in vivo study

32 InVivo Studies inDSSColitisMouseModel Feedingmicewith DSS via drinking water for five consecutive days ledto acute colitis characterized typically by bloody diarrhoeaulcerations and infiltrationswith granulocytes Furthermoresince DSS happens to be toxic to gut epithelial cells of thebasal crypts andhence can impact the integrity of themucosalbarrier all the experimental animals belonging to either thecontrol or the treatment groups were critically examined forthe following clinical parameters besides other importantphysicochemical changes recorded inmice groups during thecourse of this experiment

321 Weight Loss The gross changes in the body weightof mice in different treatment groups including probioticinterventions weremonitored during the entire 13-day exper-imental period of the study The body weight was foundto increase gradually in both the noncolitis control and Lf1groups Mice in the colitis group showed a gradual increasein weight for the first seven days but later after starting DSStreatment excessive weight loss was recorded that becamehighly significant starting from day eight to day thirteen

(119875 lt 005) due to severe experimental colitis The colitis-Lf1 treatment group showed a mixed response as there wasgradual increase in body weight in the first seven days butafter induction of colitis significant inhibition of weight losswas recorded (119875 lt 005) Quite interestingly some of themice in the same treatment group showed the opposite effectby recording appreciable weight gain The changes recordedwith regard to body weight in different mice groups duringthe course of this study have been illustrated in Figure 2However there was no significant difference in weight gainbetween both NC-PBS versus NC-Lf1 and C-PBS versus C-Lf1 groups (119875 gt 005)

322 Length of Colon The length of mice colon in thenoncolitis control with respect to noncolitis Lf1 fed groupswas 705 plusmn 021 and 815 plusmn 014 respectively The length ofcolon of colitis-Lf1 treatmentmice wasmuch longer than thatof mice in the colitis group (793 plusmn 038 versus 638 plusmn 039 cm119875 lt 005) There was no significant difference (119875 gt 005)between the length of colon of colitis-Lf1 treatment group andnoncolitis Lf1-treated group

323 Disease Activity Index (DAI) Scores As can be reflectedfrom Figure 3 the highest DAI score was recorded in colitiscontrol group DAI scores of noncolitis control group andnoncolitis probiotic group were however zero as weight losschanges observed in these groups were not significant to beconsidered as DAI and hence were rated as nonsignificant(NS) groups However DAI scores were improved signifi-cantly in colitis-Lf1 treatment group as shown in Figure 3On comparative evaluation there was significant difference(119875 lt 005) recorded between the DAI scores of colitis control

6 BioMed Research International

0 2 4 6 8 10 12 14

Days

Mea

n bo

dy w

eigh

t cha

nges

minus1

0

1

2

3

25

15

05

minus05

C-PBSC-Lf1

NC-PBSNC-Lf1

Before DSS colitis induction After DSS colitis induction

Figure 2 Bodyweight changes among different groups ofmiceNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups The data fordifferent groups were reported as mean plusmn standard deviation

0 2 4 6 8 10 12 140

05

1

15

2

25

3

35

DA

I sco

re

Days

NC-PBSNC-Lf1

C-PBSC-Lf1

lowast

lowastlowast

lowast

lowast

lowast

lowast

Figure 3 Comparative analysis of mean DAI scores in differentgroups in mice NC-PBS noncolitis control NC-Lf1 noncolitis(Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treat-ment groups Data showing asterisk mark (lowast) without line showssignificant difference compared with noncolitis control (NC-PBS)(Bonferroni post hoc test) lowast119875 lt 0001 The data for different groupswere reported as mean plusmn standard deviation

and colitis-Lf1 treatment groups due to probiotic treatmentin the latter (Figure 3)

324 Histological Scores Microscopic examination of theaffected tissues from the noncolitis control group revealednormal colonic epithelium with intact crypts and glands(Figures 4(a) and 4(b)) However amild lymphocytic infiltra-tion was also discernible although such minor changes werealso recorded even under normal conditions of inflammatoryresponse Almost a similar trend was also recorded in

biopsies of noncolitis Lf1 mice On the other hand biopsiesof the colitis mice revealed focally ulcerated epitheliumwithmoderate-to-excessive inflammatory infiltrate primarilycomposed of polymorphs and lymphocytes Crypt abscessesalongwith their shortening and branchingwere also recordedaccompanied by focal lymphoid follicle formation cryptitisand gland destruction These symptoms gave indication thatacute colitis was developed in mice Contrary to this biopsystudies of the colitis-Lf1 treatment group demonstratedconsiderable improvement in the tissue damage as can bereflected from the intact epithelium with mild acute andchronic inflammatory infiltration of lymphocytes Nonethe-less focal lymphoid follicle formation ulcerated epitheliumwithmoderate inflammatory infiltrate composed of polymor-phic lymphocytes focal cryptitis and gland destruction werealso recorded in two of the samples in this group Similarlythere was no evidence of granulomatous inflammation ormalignancy or cryptitis or crypt abscess

325 Persistence of L fermentum Lf1 in Colitis and NoncolitisModels The persistence and viability of Lf1 in mice gutwere confirmed by monitoring the log counts in the faecalsamples from different mice groups and also subjecting somerandomly selected colonies of faecal isolates recovered onMRS agar (Figure 5) by species specific PCR (data not shown)and RAPD to ascertain whether they belonged to Lf1 or notFrom the log count data presented in Figure 5 it is quiteclear that Lf1 not only persisted in the mice gut in all thetreatment groups but also proliferated as can be reflectedfrom the gradual increase in the faecal Lactobacillus logcounts during the treatment period registering more thanone log cycle increase towards the end of the experimentalperiod The preponderance of Lf1 in the faecal Lactobacillusisolates in the faecal samples of mice was demonstrated byPCR and RAPDThe typical RAPD banding patterns of pureLf1 along with some of the randomly selected representativefaecal Lactobacillus isolates obtained with primer 275 havebeen recorded in Figure 5 Since the typical RAPD bandingpatterns recorded with most of the faecal isolates matchedthose obtained from the pure culture of Lf1 it gives indicationthat most of the faecal isolates belonged to the clones of Lf1that was administered to the mice From these results it canbe inferred that Lf1 could survive and persist in the colon toexpress its antioxidative functions

33 MDA Level as an Index of Lipid Peroxidation The dataon the level of MDA estimated in each tissue sample fromeach mice group under different treatments is recorded inFigure 6 There was significant difference in the MDA con-tent of colitis control (9113 120583Mg) and colitis-Lf1 treatmentgroup (3792 120583Mg) thereby suggesting that our probioticstrain exhibited strong antioxidative property in colon andprevented lipid peroxidation by decreasing the level of MDAat significant level

34 Relative Expression Studies of ldquoNrf2rdquo and Antioxida-tive Enzyme Systems (ldquoSuperoxide Dismutase 2rdquo Thioredoxin

BioMed Research International 7

2

1

1

2

1

4

3

2

1 1

4

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

50120583m50120583m50120583m50120583m

50120583m 50120583m 50120583m 50120583m

50120583m50120583m50120583m50120583m

(a)

NC-PBS NC-Lf1 C-PBS C-Lf10

05

1

15

2

25

3

Hist

olog

ical

scor

e

lowastlowast

lowast

(b)

Figure 4 Histological examination and scores of different groups ofmice (a) Histological examination of noncolitis control group noncolitis(Lf1) control colitis group and Lf1-treated group of mice A-B NC-PBS (noncolitis control) showing normal mucosal and epithelial layeralong with intact glands with no sign of inflammation infiltration ulceration and cryptitis C-D NC-Lf1 (noncolitis (Lf1) control) showingnormal histology of noncolitis (Lf1) control similar to noncolitis control EndashH C-PBS (colitis control) showing severity of colitis in colitiscontrol group (1) Inflammation infiltrate (lymphocytes and neutrophils) (2) gland destruction (3) cryptitis (4) loss of entire crypt witherosion of surface epithelium and I-L C-Lf1 (colitis-Lf1 treatment groups) showing normalization of histological score in colitis- (Lf1) treatedgroup with no cryptitis no ulceration andminimal-to-moderate infiltration (b) Histological scoreschanges induced in different mice colonin different mice groups in different treatments NC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control andC-Lf1 colitis-Lf1 treatment groups lowastData are significantly different within the group (119875 lt 0001 Bonferroni post hoc test) The data fordifferent groups were reported as mean plusmn standard deviation

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

faecal count plates were randomly picked and grown for16 to 18 h at 37∘C after inoculation into MRS broth Thegenomic DNA from faecal isolates was then isolated byPospiech and Neumann method [36] PCR with randomoligo primer named 275 (51015840-ccg ggc aag c-31015840) was carried outfor RAPD profiling PCR reaction was performed in 25 120583Lreaction volume containing PCR buffer (25 120583L) oligo primer(20 120583L) 200 120583M dNTP (20 120583L) 10 U Taq DNA polymerase(05 120583L) and template (20 120583L) The PCR cycling parametersincluded an initial denaturation of 95∘C5min followedby 45 cycles each of denaturation (95∘C30 sec) annealing(40∘C30 sec) extension (72∘C2min) and final extensionof 72∘C10min The PCR products were electrophoresedon 18ndash2 agarose gel with ethidium bromide (05 120583gmL)(Amersham Biosciences USA) at 100V using 1X TAE bufferThe RAPD patterns were compared with the patterns of thepure cultures of the administered probiotic strain

28 Histopathological Examination The intestinal tissuesfrom the colon of the probiotic and control groups wereset apart for histopathological examination Intestines (02-03 cm of tissue) of different groups were kept in 10 bufferedformalin solution for at least 24 h prior to cassetting Thecolon was sliced mounted stained with hematoxylin (HiMe-dia India) and eosin (HiMedia India) and then scored bya blinded pathologist who rated the tissues based on thegrade of the disease 0ndash4 the percentage of the disease 0ndash4and the severity of the inflammation 0ndash3 (Table 2(S) andashc)[35] Histopathological examination of the affected tissuesfrom the mice groups analyzed in this study was done by ablinded histologist who rated the tissues based on the gradeof the disease 0ndash4 the percentage of the disease 0ndash4 andthe severity of the inflammation 0ndash3Microscopic evaluationof all the sections of tissue samples was done at 4x and 10xmagnification and histological scores were graded on thebasis of the severity of inflammation infiltration ulcerationand crypt damage

29 Lipid Peroxidation For detection of lipid peroxides(malondialdehyde) in colon homogenate the methodof Uchiyama and Mihara was followed [37] The colonhomogenate was prepared in 1X PBS An aliquot of 3mLof 1 phosphoric acid and one mL of 06 TBA solutionwere added to 05mL of colon homogenate The mixturewas heated for 45min on a boiling water bath After cooling4mL of n-butanol was added and mixed vigorously Thebutanol phase was separated by centrifugation at 5000 rpmfor 10min and absorbance was measured at 535 and 520 nmThe difference was used as the TBA value A standard curveof malondialdehyde was drawn by taking TBA value atvarious concentration of malondialdehyde MDA standardcurve was prepared by carrying out overnight digestionof different concentrations of 1133-tetraethoxypropane(01mM) in presence of 02N HCl

210 Gene Expression Studies RNAwas isolated frommousecolon by TRIzol method (TRI Reagent Sigma USA) cDNAwas synthesized and RT-qPCR in LightCycler 480 (Roche

Switzerland) was carried out as described previously [31]The thermal cycling conditions included initial denaturationat 95∘C for 5min followed by 40 cycles each of denat-uration (95∘C30 s) annealing (53∘C30 s) and extension(72∘C45 s) with a single fluorescence measurement a meltcurve program (60∘Cndash95∘C with a heating rate of 011∘Csand continuous fluorescence measurement) and finally acooling step at 40∘C RNA extraction and measurement ofgene expression by RT-qPCR were performed in triplicateand the mean of all these values was used for final analysis

The quantitative data generated by real-time PCR (thequantification cycle Cq value) were analyzed by relativeexpression software tool (REST 2009) (httpwwwgene-quantificationinfo) as described previously [32]

211 Statistical Analyses Thedata obtained from each animalexperiment were expressed as mean plusmn standard deviationvalues Analyses of variance (ANOVA) for estimation ofsignificance of differences betweenmeans were automaticallydone with the Statistical Package for the Social Science (SPSSfor Windows version 101 SPSS Inc Chicago) softwareby Bonferroni post hoc test and considered significantlydifferent at 119875 le 005

3 Results

31 Screening of Probiotic Lactobacilli Initially during thispart of study some putative probiotic Lactobacilli werescreened on the basis of their ability to enhance ldquoNrf 2rdquoexpression in HT-29 cells along with lipid peroxidationinhibitory potential to short list the most promising strainfor further investigations to explore its in vivo efficacyin colitis mice model From the critical appraisal of thedata recorded in Figure 1(a) it can be inferred that therewas considerable variation in ldquoNrf 2rdquo expression induced inHT-29 cells by different probiotic strains as they behaveddifferently in eliciting ldquoNrf 2rdquo expression The expression ofldquoNrf 2rdquo in HT-29 cells was significantly downregulated (119875 lt0001) in the sample group (relative to control group) byminus188-fold when subjected to H

2O2stress alone (Figure 1(a))

Although some of the probiotic strains when used alone orin conjunction with H

2O2did not show any significant effect

on the ldquoNrf 2rdquo expression in HT-29 cells Lp5276 and S3 didinduce significant downregulation at the level of minus138- andminus164-fold respectively However it was quite interesting tonote that three of the strains namely Lp55 Lp91 and Lf1were able to induce a significant (119875 lt 0001) upregulation inthe expression of ldquoNrf 2rdquo in HT-29 cells at the levels of 748- 258- and 643-fold when used in conjunction with H

2O2

Hence the latter were rated as the prospective candidateprobiotic strains to explore their efficacy in the managementof oxidative stress induced diseases

The total antioxidative activity of the aforesaid Lacto-bacillus strains in terms of their lipid peroxidation inhibitionpotentials inHT-29 cells was also investigated after subjectingthe same to H

2O2treatment by measuring the level of

MDA the secondary product of lipid peroxidation From thedata presented in the form of a bar diagram illustrated in

BioMed Research International 5

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

) 10

8

6

4

2

0

minus2

minus4

lowastlowast

lowast

lowast

lowast

lowast lowastlowastlowast

Lf1+

H2O2

S3+

H2O2

H2O2

Lp9

Lp10

Lp10+

H2O2

Lp42

Lp42+

H2O2

Lp55+

H2O2

Lp78

Lp78+

H2O2

Lp91

Lp91+

H2O2

Lp9+

H2O2

Lp55

Lp5276+

H2O2

Lf1 S3

NCF

M

Lp5276

NCF

M+

H2O2

(a)

100

75

50

25

0

MD

A

inhi

bitio

n

lowastlowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

LP9+

H2O2

Lp10+

H2O2

Lp42+

H2O2

Lp55+

H2O2

Lp78+

H2O2

Lp91+

H2O2

Lf1+

H2O2

S3+

H2O2

Lp5276+

H2O2

NCF

M+

H2O2

(b)

Figure 1 Relative expression of ldquoNrf 2rdquo and MDA inhibitory effect by Lactobacilli strains in HT-29 cell lines under H2O2stress conditions

Data are represented as mean plusmn SD number of RT-qPCR experiments (119899) = 3 (a) Comparative analysis of relative expression of ldquoNrf 2rdquo inHT-29 on challenge with different strains of probiotic Lactobacilli and H

2O2stress lowastData are significantly different compared with control

(119875 lt 0001 Bonferroni post hoc test) (b) Inhibitory effect of various probiotic Lactobacillus cultures on lipid peroxidation in HT-29 cellsData are significantly different compared with control (H

2O2alone) (lowast119875 lt 0001 lowastlowast119875 lt 001 Bonferroni post hoc test)

Figure 1(b) it is clearly indicated that Lactobacillus spp S3showed the highest percent inhibition that is 832 plusmn 053followed by Lf1 (781 plusmn 024) as compared to the standardreference strains showing relatively poor response as can bereflected from the lower percent inhibition values that is556 plusmn 04 (Lp5276) to 552 plusmn 036 (LaNCFM) indicatingthat the indigenous probiotic strains were better equippedto neutralize the injurious effects of oxidative stress Hencebased on the overall combined effects of both of theseactivities inHT-29 cells we finally selected Lf1 as the potentialcandidate strain for in vivo study

32 InVivo Studies inDSSColitisMouseModel Feedingmicewith DSS via drinking water for five consecutive days ledto acute colitis characterized typically by bloody diarrhoeaulcerations and infiltrationswith granulocytes Furthermoresince DSS happens to be toxic to gut epithelial cells of thebasal crypts andhence can impact the integrity of themucosalbarrier all the experimental animals belonging to either thecontrol or the treatment groups were critically examined forthe following clinical parameters besides other importantphysicochemical changes recorded inmice groups during thecourse of this experiment

321 Weight Loss The gross changes in the body weightof mice in different treatment groups including probioticinterventions weremonitored during the entire 13-day exper-imental period of the study The body weight was foundto increase gradually in both the noncolitis control and Lf1groups Mice in the colitis group showed a gradual increasein weight for the first seven days but later after starting DSStreatment excessive weight loss was recorded that becamehighly significant starting from day eight to day thirteen

(119875 lt 005) due to severe experimental colitis The colitis-Lf1 treatment group showed a mixed response as there wasgradual increase in body weight in the first seven days butafter induction of colitis significant inhibition of weight losswas recorded (119875 lt 005) Quite interestingly some of themice in the same treatment group showed the opposite effectby recording appreciable weight gain The changes recordedwith regard to body weight in different mice groups duringthe course of this study have been illustrated in Figure 2However there was no significant difference in weight gainbetween both NC-PBS versus NC-Lf1 and C-PBS versus C-Lf1 groups (119875 gt 005)

322 Length of Colon The length of mice colon in thenoncolitis control with respect to noncolitis Lf1 fed groupswas 705 plusmn 021 and 815 plusmn 014 respectively The length ofcolon of colitis-Lf1 treatmentmice wasmuch longer than thatof mice in the colitis group (793 plusmn 038 versus 638 plusmn 039 cm119875 lt 005) There was no significant difference (119875 gt 005)between the length of colon of colitis-Lf1 treatment group andnoncolitis Lf1-treated group

323 Disease Activity Index (DAI) Scores As can be reflectedfrom Figure 3 the highest DAI score was recorded in colitiscontrol group DAI scores of noncolitis control group andnoncolitis probiotic group were however zero as weight losschanges observed in these groups were not significant to beconsidered as DAI and hence were rated as nonsignificant(NS) groups However DAI scores were improved signifi-cantly in colitis-Lf1 treatment group as shown in Figure 3On comparative evaluation there was significant difference(119875 lt 005) recorded between the DAI scores of colitis control

6 BioMed Research International

0 2 4 6 8 10 12 14

Days

Mea

n bo

dy w

eigh

t cha

nges

minus1

0

1

2

3

25

15

05

minus05

C-PBSC-Lf1

NC-PBSNC-Lf1

Before DSS colitis induction After DSS colitis induction

Figure 2 Bodyweight changes among different groups ofmiceNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups The data fordifferent groups were reported as mean plusmn standard deviation

0 2 4 6 8 10 12 140

05

1

15

2

25

3

35

DA

I sco

re

Days

NC-PBSNC-Lf1

C-PBSC-Lf1

lowast

lowastlowast

lowast

lowast

lowast

lowast

Figure 3 Comparative analysis of mean DAI scores in differentgroups in mice NC-PBS noncolitis control NC-Lf1 noncolitis(Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treat-ment groups Data showing asterisk mark (lowast) without line showssignificant difference compared with noncolitis control (NC-PBS)(Bonferroni post hoc test) lowast119875 lt 0001 The data for different groupswere reported as mean plusmn standard deviation

and colitis-Lf1 treatment groups due to probiotic treatmentin the latter (Figure 3)

324 Histological Scores Microscopic examination of theaffected tissues from the noncolitis control group revealednormal colonic epithelium with intact crypts and glands(Figures 4(a) and 4(b)) However amild lymphocytic infiltra-tion was also discernible although such minor changes werealso recorded even under normal conditions of inflammatoryresponse Almost a similar trend was also recorded in

biopsies of noncolitis Lf1 mice On the other hand biopsiesof the colitis mice revealed focally ulcerated epitheliumwithmoderate-to-excessive inflammatory infiltrate primarilycomposed of polymorphs and lymphocytes Crypt abscessesalongwith their shortening and branchingwere also recordedaccompanied by focal lymphoid follicle formation cryptitisand gland destruction These symptoms gave indication thatacute colitis was developed in mice Contrary to this biopsystudies of the colitis-Lf1 treatment group demonstratedconsiderable improvement in the tissue damage as can bereflected from the intact epithelium with mild acute andchronic inflammatory infiltration of lymphocytes Nonethe-less focal lymphoid follicle formation ulcerated epitheliumwithmoderate inflammatory infiltrate composed of polymor-phic lymphocytes focal cryptitis and gland destruction werealso recorded in two of the samples in this group Similarlythere was no evidence of granulomatous inflammation ormalignancy or cryptitis or crypt abscess

325 Persistence of L fermentum Lf1 in Colitis and NoncolitisModels The persistence and viability of Lf1 in mice gutwere confirmed by monitoring the log counts in the faecalsamples from different mice groups and also subjecting somerandomly selected colonies of faecal isolates recovered onMRS agar (Figure 5) by species specific PCR (data not shown)and RAPD to ascertain whether they belonged to Lf1 or notFrom the log count data presented in Figure 5 it is quiteclear that Lf1 not only persisted in the mice gut in all thetreatment groups but also proliferated as can be reflectedfrom the gradual increase in the faecal Lactobacillus logcounts during the treatment period registering more thanone log cycle increase towards the end of the experimentalperiod The preponderance of Lf1 in the faecal Lactobacillusisolates in the faecal samples of mice was demonstrated byPCR and RAPDThe typical RAPD banding patterns of pureLf1 along with some of the randomly selected representativefaecal Lactobacillus isolates obtained with primer 275 havebeen recorded in Figure 5 Since the typical RAPD bandingpatterns recorded with most of the faecal isolates matchedthose obtained from the pure culture of Lf1 it gives indicationthat most of the faecal isolates belonged to the clones of Lf1that was administered to the mice From these results it canbe inferred that Lf1 could survive and persist in the colon toexpress its antioxidative functions

33 MDA Level as an Index of Lipid Peroxidation The dataon the level of MDA estimated in each tissue sample fromeach mice group under different treatments is recorded inFigure 6 There was significant difference in the MDA con-tent of colitis control (9113 120583Mg) and colitis-Lf1 treatmentgroup (3792 120583Mg) thereby suggesting that our probioticstrain exhibited strong antioxidative property in colon andprevented lipid peroxidation by decreasing the level of MDAat significant level

34 Relative Expression Studies of ldquoNrf2rdquo and Antioxida-tive Enzyme Systems (ldquoSuperoxide Dismutase 2rdquo Thioredoxin

BioMed Research International 7

2

1

1

2

1

4

3

2

1 1

4

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

50120583m50120583m50120583m50120583m

50120583m 50120583m 50120583m 50120583m

50120583m50120583m50120583m50120583m

(a)

NC-PBS NC-Lf1 C-PBS C-Lf10

05

1

15

2

25

3

Hist

olog

ical

scor

e

lowastlowast

lowast

(b)

Figure 4 Histological examination and scores of different groups ofmice (a) Histological examination of noncolitis control group noncolitis(Lf1) control colitis group and Lf1-treated group of mice A-B NC-PBS (noncolitis control) showing normal mucosal and epithelial layeralong with intact glands with no sign of inflammation infiltration ulceration and cryptitis C-D NC-Lf1 (noncolitis (Lf1) control) showingnormal histology of noncolitis (Lf1) control similar to noncolitis control EndashH C-PBS (colitis control) showing severity of colitis in colitiscontrol group (1) Inflammation infiltrate (lymphocytes and neutrophils) (2) gland destruction (3) cryptitis (4) loss of entire crypt witherosion of surface epithelium and I-L C-Lf1 (colitis-Lf1 treatment groups) showing normalization of histological score in colitis- (Lf1) treatedgroup with no cryptitis no ulceration andminimal-to-moderate infiltration (b) Histological scoreschanges induced in different mice colonin different mice groups in different treatments NC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control andC-Lf1 colitis-Lf1 treatment groups lowastData are significantly different within the group (119875 lt 0001 Bonferroni post hoc test) The data fordifferent groups were reported as mean plusmn standard deviation

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

) 10

8

6

4

2

0

minus2

minus4

lowastlowast

lowast

lowast

lowast

lowast lowastlowastlowast

Lf1+

H2O2

S3+

H2O2

H2O2

Lp9

Lp10

Lp10+

H2O2

Lp42

Lp42+

H2O2

Lp55+

H2O2

Lp78

Lp78+

H2O2

Lp91

Lp91+

H2O2

Lp9+

H2O2

Lp55

Lp5276+

H2O2

Lf1 S3

NCF

M

Lp5276

NCF

M+

H2O2

(a)

100

75

50

25

0

MD

A

inhi

bitio

n

lowastlowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

LP9+

H2O2

Lp10+

H2O2

Lp42+

H2O2

Lp55+

H2O2

Lp78+

H2O2

Lp91+

H2O2

Lf1+

H2O2

S3+

H2O2

Lp5276+

H2O2

NCF

M+

H2O2

(b)

Figure 1 Relative expression of ldquoNrf 2rdquo and MDA inhibitory effect by Lactobacilli strains in HT-29 cell lines under H2O2stress conditions

Data are represented as mean plusmn SD number of RT-qPCR experiments (119899) = 3 (a) Comparative analysis of relative expression of ldquoNrf 2rdquo inHT-29 on challenge with different strains of probiotic Lactobacilli and H

2O2stress lowastData are significantly different compared with control

(119875 lt 0001 Bonferroni post hoc test) (b) Inhibitory effect of various probiotic Lactobacillus cultures on lipid peroxidation in HT-29 cellsData are significantly different compared with control (H

2O2alone) (lowast119875 lt 0001 lowastlowast119875 lt 001 Bonferroni post hoc test)

Figure 1(b) it is clearly indicated that Lactobacillus spp S3showed the highest percent inhibition that is 832 plusmn 053followed by Lf1 (781 plusmn 024) as compared to the standardreference strains showing relatively poor response as can bereflected from the lower percent inhibition values that is556 plusmn 04 (Lp5276) to 552 plusmn 036 (LaNCFM) indicatingthat the indigenous probiotic strains were better equippedto neutralize the injurious effects of oxidative stress Hencebased on the overall combined effects of both of theseactivities inHT-29 cells we finally selected Lf1 as the potentialcandidate strain for in vivo study

32 InVivo Studies inDSSColitisMouseModel Feedingmicewith DSS via drinking water for five consecutive days ledto acute colitis characterized typically by bloody diarrhoeaulcerations and infiltrationswith granulocytes Furthermoresince DSS happens to be toxic to gut epithelial cells of thebasal crypts andhence can impact the integrity of themucosalbarrier all the experimental animals belonging to either thecontrol or the treatment groups were critically examined forthe following clinical parameters besides other importantphysicochemical changes recorded inmice groups during thecourse of this experiment

321 Weight Loss The gross changes in the body weightof mice in different treatment groups including probioticinterventions weremonitored during the entire 13-day exper-imental period of the study The body weight was foundto increase gradually in both the noncolitis control and Lf1groups Mice in the colitis group showed a gradual increasein weight for the first seven days but later after starting DSStreatment excessive weight loss was recorded that becamehighly significant starting from day eight to day thirteen

(119875 lt 005) due to severe experimental colitis The colitis-Lf1 treatment group showed a mixed response as there wasgradual increase in body weight in the first seven days butafter induction of colitis significant inhibition of weight losswas recorded (119875 lt 005) Quite interestingly some of themice in the same treatment group showed the opposite effectby recording appreciable weight gain The changes recordedwith regard to body weight in different mice groups duringthe course of this study have been illustrated in Figure 2However there was no significant difference in weight gainbetween both NC-PBS versus NC-Lf1 and C-PBS versus C-Lf1 groups (119875 gt 005)

322 Length of Colon The length of mice colon in thenoncolitis control with respect to noncolitis Lf1 fed groupswas 705 plusmn 021 and 815 plusmn 014 respectively The length ofcolon of colitis-Lf1 treatmentmice wasmuch longer than thatof mice in the colitis group (793 plusmn 038 versus 638 plusmn 039 cm119875 lt 005) There was no significant difference (119875 gt 005)between the length of colon of colitis-Lf1 treatment group andnoncolitis Lf1-treated group

323 Disease Activity Index (DAI) Scores As can be reflectedfrom Figure 3 the highest DAI score was recorded in colitiscontrol group DAI scores of noncolitis control group andnoncolitis probiotic group were however zero as weight losschanges observed in these groups were not significant to beconsidered as DAI and hence were rated as nonsignificant(NS) groups However DAI scores were improved signifi-cantly in colitis-Lf1 treatment group as shown in Figure 3On comparative evaluation there was significant difference(119875 lt 005) recorded between the DAI scores of colitis control

6 BioMed Research International

0 2 4 6 8 10 12 14

Days

Mea

n bo

dy w

eigh

t cha

nges

minus1

0

1

2

3

25

15

05

minus05

C-PBSC-Lf1

NC-PBSNC-Lf1

Before DSS colitis induction After DSS colitis induction

Figure 2 Bodyweight changes among different groups ofmiceNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups The data fordifferent groups were reported as mean plusmn standard deviation

0 2 4 6 8 10 12 140

05

1

15

2

25

3

35

DA

I sco

re

Days

NC-PBSNC-Lf1

C-PBSC-Lf1

lowast

lowastlowast

lowast

lowast

lowast

lowast

Figure 3 Comparative analysis of mean DAI scores in differentgroups in mice NC-PBS noncolitis control NC-Lf1 noncolitis(Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treat-ment groups Data showing asterisk mark (lowast) without line showssignificant difference compared with noncolitis control (NC-PBS)(Bonferroni post hoc test) lowast119875 lt 0001 The data for different groupswere reported as mean plusmn standard deviation

and colitis-Lf1 treatment groups due to probiotic treatmentin the latter (Figure 3)

324 Histological Scores Microscopic examination of theaffected tissues from the noncolitis control group revealednormal colonic epithelium with intact crypts and glands(Figures 4(a) and 4(b)) However amild lymphocytic infiltra-tion was also discernible although such minor changes werealso recorded even under normal conditions of inflammatoryresponse Almost a similar trend was also recorded in

biopsies of noncolitis Lf1 mice On the other hand biopsiesof the colitis mice revealed focally ulcerated epitheliumwithmoderate-to-excessive inflammatory infiltrate primarilycomposed of polymorphs and lymphocytes Crypt abscessesalongwith their shortening and branchingwere also recordedaccompanied by focal lymphoid follicle formation cryptitisand gland destruction These symptoms gave indication thatacute colitis was developed in mice Contrary to this biopsystudies of the colitis-Lf1 treatment group demonstratedconsiderable improvement in the tissue damage as can bereflected from the intact epithelium with mild acute andchronic inflammatory infiltration of lymphocytes Nonethe-less focal lymphoid follicle formation ulcerated epitheliumwithmoderate inflammatory infiltrate composed of polymor-phic lymphocytes focal cryptitis and gland destruction werealso recorded in two of the samples in this group Similarlythere was no evidence of granulomatous inflammation ormalignancy or cryptitis or crypt abscess

325 Persistence of L fermentum Lf1 in Colitis and NoncolitisModels The persistence and viability of Lf1 in mice gutwere confirmed by monitoring the log counts in the faecalsamples from different mice groups and also subjecting somerandomly selected colonies of faecal isolates recovered onMRS agar (Figure 5) by species specific PCR (data not shown)and RAPD to ascertain whether they belonged to Lf1 or notFrom the log count data presented in Figure 5 it is quiteclear that Lf1 not only persisted in the mice gut in all thetreatment groups but also proliferated as can be reflectedfrom the gradual increase in the faecal Lactobacillus logcounts during the treatment period registering more thanone log cycle increase towards the end of the experimentalperiod The preponderance of Lf1 in the faecal Lactobacillusisolates in the faecal samples of mice was demonstrated byPCR and RAPDThe typical RAPD banding patterns of pureLf1 along with some of the randomly selected representativefaecal Lactobacillus isolates obtained with primer 275 havebeen recorded in Figure 5 Since the typical RAPD bandingpatterns recorded with most of the faecal isolates matchedthose obtained from the pure culture of Lf1 it gives indicationthat most of the faecal isolates belonged to the clones of Lf1that was administered to the mice From these results it canbe inferred that Lf1 could survive and persist in the colon toexpress its antioxidative functions

33 MDA Level as an Index of Lipid Peroxidation The dataon the level of MDA estimated in each tissue sample fromeach mice group under different treatments is recorded inFigure 6 There was significant difference in the MDA con-tent of colitis control (9113 120583Mg) and colitis-Lf1 treatmentgroup (3792 120583Mg) thereby suggesting that our probioticstrain exhibited strong antioxidative property in colon andprevented lipid peroxidation by decreasing the level of MDAat significant level

34 Relative Expression Studies of ldquoNrf2rdquo and Antioxida-tive Enzyme Systems (ldquoSuperoxide Dismutase 2rdquo Thioredoxin

BioMed Research International 7

2

1

1

2

1

4

3

2

1 1

4

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

50120583m50120583m50120583m50120583m

50120583m 50120583m 50120583m 50120583m

50120583m50120583m50120583m50120583m

(a)

NC-PBS NC-Lf1 C-PBS C-Lf10

05

1

15

2

25

3

Hist

olog

ical

scor

e

lowastlowast

lowast

(b)

Figure 4 Histological examination and scores of different groups ofmice (a) Histological examination of noncolitis control group noncolitis(Lf1) control colitis group and Lf1-treated group of mice A-B NC-PBS (noncolitis control) showing normal mucosal and epithelial layeralong with intact glands with no sign of inflammation infiltration ulceration and cryptitis C-D NC-Lf1 (noncolitis (Lf1) control) showingnormal histology of noncolitis (Lf1) control similar to noncolitis control EndashH C-PBS (colitis control) showing severity of colitis in colitiscontrol group (1) Inflammation infiltrate (lymphocytes and neutrophils) (2) gland destruction (3) cryptitis (4) loss of entire crypt witherosion of surface epithelium and I-L C-Lf1 (colitis-Lf1 treatment groups) showing normalization of histological score in colitis- (Lf1) treatedgroup with no cryptitis no ulceration andminimal-to-moderate infiltration (b) Histological scoreschanges induced in different mice colonin different mice groups in different treatments NC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control andC-Lf1 colitis-Lf1 treatment groups lowastData are significantly different within the group (119875 lt 0001 Bonferroni post hoc test) The data fordifferent groups were reported as mean plusmn standard deviation

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

0 2 4 6 8 10 12 14

Days

Mea

n bo

dy w

eigh

t cha

nges

minus1

0

1

2

3

25

15

05

minus05

C-PBSC-Lf1

NC-PBSNC-Lf1

Before DSS colitis induction After DSS colitis induction

Figure 2 Bodyweight changes among different groups ofmiceNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups The data fordifferent groups were reported as mean plusmn standard deviation

0 2 4 6 8 10 12 140

05

1

15

2

25

3

35

DA

I sco

re

Days

NC-PBSNC-Lf1

C-PBSC-Lf1

lowast

lowastlowast

lowast

lowast

lowast

lowast

Figure 3 Comparative analysis of mean DAI scores in differentgroups in mice NC-PBS noncolitis control NC-Lf1 noncolitis(Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treat-ment groups Data showing asterisk mark (lowast) without line showssignificant difference compared with noncolitis control (NC-PBS)(Bonferroni post hoc test) lowast119875 lt 0001 The data for different groupswere reported as mean plusmn standard deviation

and colitis-Lf1 treatment groups due to probiotic treatmentin the latter (Figure 3)

324 Histological Scores Microscopic examination of theaffected tissues from the noncolitis control group revealednormal colonic epithelium with intact crypts and glands(Figures 4(a) and 4(b)) However amild lymphocytic infiltra-tion was also discernible although such minor changes werealso recorded even under normal conditions of inflammatoryresponse Almost a similar trend was also recorded in

biopsies of noncolitis Lf1 mice On the other hand biopsiesof the colitis mice revealed focally ulcerated epitheliumwithmoderate-to-excessive inflammatory infiltrate primarilycomposed of polymorphs and lymphocytes Crypt abscessesalongwith their shortening and branchingwere also recordedaccompanied by focal lymphoid follicle formation cryptitisand gland destruction These symptoms gave indication thatacute colitis was developed in mice Contrary to this biopsystudies of the colitis-Lf1 treatment group demonstratedconsiderable improvement in the tissue damage as can bereflected from the intact epithelium with mild acute andchronic inflammatory infiltration of lymphocytes Nonethe-less focal lymphoid follicle formation ulcerated epitheliumwithmoderate inflammatory infiltrate composed of polymor-phic lymphocytes focal cryptitis and gland destruction werealso recorded in two of the samples in this group Similarlythere was no evidence of granulomatous inflammation ormalignancy or cryptitis or crypt abscess

325 Persistence of L fermentum Lf1 in Colitis and NoncolitisModels The persistence and viability of Lf1 in mice gutwere confirmed by monitoring the log counts in the faecalsamples from different mice groups and also subjecting somerandomly selected colonies of faecal isolates recovered onMRS agar (Figure 5) by species specific PCR (data not shown)and RAPD to ascertain whether they belonged to Lf1 or notFrom the log count data presented in Figure 5 it is quiteclear that Lf1 not only persisted in the mice gut in all thetreatment groups but also proliferated as can be reflectedfrom the gradual increase in the faecal Lactobacillus logcounts during the treatment period registering more thanone log cycle increase towards the end of the experimentalperiod The preponderance of Lf1 in the faecal Lactobacillusisolates in the faecal samples of mice was demonstrated byPCR and RAPDThe typical RAPD banding patterns of pureLf1 along with some of the randomly selected representativefaecal Lactobacillus isolates obtained with primer 275 havebeen recorded in Figure 5 Since the typical RAPD bandingpatterns recorded with most of the faecal isolates matchedthose obtained from the pure culture of Lf1 it gives indicationthat most of the faecal isolates belonged to the clones of Lf1that was administered to the mice From these results it canbe inferred that Lf1 could survive and persist in the colon toexpress its antioxidative functions

33 MDA Level as an Index of Lipid Peroxidation The dataon the level of MDA estimated in each tissue sample fromeach mice group under different treatments is recorded inFigure 6 There was significant difference in the MDA con-tent of colitis control (9113 120583Mg) and colitis-Lf1 treatmentgroup (3792 120583Mg) thereby suggesting that our probioticstrain exhibited strong antioxidative property in colon andprevented lipid peroxidation by decreasing the level of MDAat significant level

34 Relative Expression Studies of ldquoNrf2rdquo and Antioxida-tive Enzyme Systems (ldquoSuperoxide Dismutase 2rdquo Thioredoxin

BioMed Research International 7

2

1

1

2

1

4

3

2

1 1

4

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

50120583m50120583m50120583m50120583m

50120583m 50120583m 50120583m 50120583m

50120583m50120583m50120583m50120583m

(a)

NC-PBS NC-Lf1 C-PBS C-Lf10

05

1

15

2

25

3

Hist

olog

ical

scor

e

lowastlowast

lowast

(b)

Figure 4 Histological examination and scores of different groups ofmice (a) Histological examination of noncolitis control group noncolitis(Lf1) control colitis group and Lf1-treated group of mice A-B NC-PBS (noncolitis control) showing normal mucosal and epithelial layeralong with intact glands with no sign of inflammation infiltration ulceration and cryptitis C-D NC-Lf1 (noncolitis (Lf1) control) showingnormal histology of noncolitis (Lf1) control similar to noncolitis control EndashH C-PBS (colitis control) showing severity of colitis in colitiscontrol group (1) Inflammation infiltrate (lymphocytes and neutrophils) (2) gland destruction (3) cryptitis (4) loss of entire crypt witherosion of surface epithelium and I-L C-Lf1 (colitis-Lf1 treatment groups) showing normalization of histological score in colitis- (Lf1) treatedgroup with no cryptitis no ulceration andminimal-to-moderate infiltration (b) Histological scoreschanges induced in different mice colonin different mice groups in different treatments NC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control andC-Lf1 colitis-Lf1 treatment groups lowastData are significantly different within the group (119875 lt 0001 Bonferroni post hoc test) The data fordifferent groups were reported as mean plusmn standard deviation

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

2

1

1

2

1

4

3

2

1 1

4

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

50120583m50120583m50120583m50120583m

50120583m 50120583m 50120583m 50120583m

50120583m50120583m50120583m50120583m

(a)

NC-PBS NC-Lf1 C-PBS C-Lf10

05

1

15

2

25

3

Hist

olog

ical

scor

e

lowastlowast

lowast

(b)

Figure 4 Histological examination and scores of different groups ofmice (a) Histological examination of noncolitis control group noncolitis(Lf1) control colitis group and Lf1-treated group of mice A-B NC-PBS (noncolitis control) showing normal mucosal and epithelial layeralong with intact glands with no sign of inflammation infiltration ulceration and cryptitis C-D NC-Lf1 (noncolitis (Lf1) control) showingnormal histology of noncolitis (Lf1) control similar to noncolitis control EndashH C-PBS (colitis control) showing severity of colitis in colitiscontrol group (1) Inflammation infiltrate (lymphocytes and neutrophils) (2) gland destruction (3) cryptitis (4) loss of entire crypt witherosion of surface epithelium and I-L C-Lf1 (colitis-Lf1 treatment groups) showing normalization of histological score in colitis- (Lf1) treatedgroup with no cryptitis no ulceration andminimal-to-moderate infiltration (b) Histological scoreschanges induced in different mice colonin different mice groups in different treatments NC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control andC-Lf1 colitis-Lf1 treatment groups lowastData are significantly different within the group (119875 lt 0001 Bonferroni post hoc test) The data fordifferent groups were reported as mean plusmn standard deviation

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

0 3 6 9 1250

55

60

65

70

75

80

85

Days

NC-Lf1C-Lf1

M100 bp

M500bpS1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Lf1

Log

(cfu

g)

Figure 5 Persistence and RAPD pattern of faecal isolates andstandard culture Lf1 using primer 275 in faecal samples of mouseNC-Lf1 noncolitis (Lf1) control and C-Lf1 colitis-Lf1 treatmentgroups The data for different groups were reported as mean plusmnstandard deviation

Reductase and Catalase) in Colitis Mouse Model by RT-qPCR Specificity of RT-qPCR products generated from theamplification of reference gene ldquoACTBrdquo and the target genesldquoNrf 2rdquo ldquoSOD2rdquo ldquoTrxR-1rdquo and ldquoCATrdquo using specific primerswas checked on agarose gel Specific single product ofthe desired amplicons that is 102 bp (ldquoNrf 2rdquo) and 112 bp(ldquoACTBrdquo) (Figure 2(S) in Supplementary Material) resultedin a single product of specificmelting temperatures of 8310∘Cand 8320∘C for ldquoNrf 2rdquo and ldquoACTBrdquo respectively by meltcurve analysis (Figure 2(S)) However no Cq values inamplification curve for ldquoNrf 2rdquo gene were obtained in thecolitis-induced mice as well as Lf1 fed-colitis-induced micegroupsThemice fed with probiotic strain alone in noncolitisgroup also did not show any significant level (119875 lt 0231)of expression of ldquoNrf 2rdquo which turned out to be only 1756relative to control group (Figure 7) Similarly no significantlevel of expression of the target gene could be demonstratedin both untreated colitis-induced mice and Lf1-treated colitismice since no amplification of the target gene was recordedin both of the mice groups

With respect to antioxidant enzymes systems spe-cific single products of the desired amplicons that is87 bp (ldquoSOD2rdquo) 65 bp (ldquoTrxR-1rdquo) and 64 bp (ldquoCATrdquo) wereobtained All the curves showed high linearity (Pearsoncorrelation coefficient R2 gt 0978) The slopes of ldquoSOD2rdquoldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo curves were minus3351 minus3363minus3349 and minus3360 respectively which indicated corre-sponding high real-time PCR efficiencies of 1988 198 1989

NC-PBS NC-Lf1 C-PBS C-Lf10

10

20

30

40

50

60

70

80

90

100

MD

A (120583

Mg

tiss

ue)

lowast

lowastlowast

lowastlowast

Figure 6 Comparative evaluation of MDA levels recorded in thecolonic tissue of experimental mice group for different treatmentsNC-PBS noncolitis control NC-Lf1 noncolitis (Lf1) control C-PBS colitis control and C-Lf1 colitis-Lf1 treatment groups lowastDataare significantly different within the group (119875 lt 0001 Bonferronipost hoc test)The data for different groups were reported asmean plusmnstandard deviation

minus120

minus100

minus80

minus60

minus40

minus20

0

20

NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1NC-Lf1 C-PBS C-Lf1

Relat

ive e

xpre

ssio

n (c

hang

e in

fold

)

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

Nrf2 SOD2 TrxR CAT

Figure 7 Relative expression of Nrf 2 SOD2 TrxR and CATin colitis mouse model NC-Lf1 noncolitis (Lf1) control C-PBScolitis control and C-Lf1 colitis-Lf1 treatment groups Data arerepresented as mean plusmn SD number of RT-qPCR experiments (119899) =3 Data showing asterisk mark (lowast) without line shows significantdifference compared with noncolitis control (Bonferroni post hoctest) lowast119875 lt 0001

and 1984 in respect of the targeted genes (Figure 3(a)) Onmelt curve analysis a single product of specific melting tem-peratures of 8259 8353 8262 and 8316∘C was recorded forldquoSOD2rdquo ldquoTrxR-1rdquo ldquoCATrdquo and ldquoACTBrdquo respectively (Figure3(b)) The relative expression of ldquoSOD2rdquo in different micetreatment groups has been recorded in Figure 7 The expres-sion of ldquoSOD2rdquo was significantly (119875 lt 0000) downregulatedin colitis-induced mice at the level of ndash10000-fold relative toPBS control group However when the colitis-induced micegroup was fed with Lf1 the downregulation was considerablyreduced to minus37037-fold indicating that there was actually asubstantial increase (approximately 63) in the expression

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

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Enzyme Research

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International Journal of

Microbiology

BioMed Research International 9

of ldquoSOD2rdquo gene under these conditions On the other handthe mice group fed with Lf1 alone showed a significant level(119875 lt 0000) of upregulation by as much as 6446-fold inldquoSOD2rdquo expression

The expression of ldquothioredoxinrdquo was also found to besignificantly (119875 lt 0000) downregulated in colitis-inducedmice at the level of minus4292-fold with respect to noncolitisPBS control mice However when the colitis-induced micewere fed with Lf1 there was no significant (119875 lt 0428)relative change in expression profile with respect to controlgroup although the expression level decreased from minus4292-fold in the colitis group to minus1328-fold in colitis-Lf1 miceThese results indicate that downregulation of the expressionwas not that drastic when Lf1 was used in the colitis-inducedmice group in comparison to the untreated colitis group Onthe other hand the noncolitis mice group fed with Lf1 aloneresulted in a significant level (119875 lt 0031) of upregulation ofldquothioredoxinrdquo gene expression to the extent of 2656-foldThemice group fed with Lf1 did not show any significant level(119875 lt 0493) of relative changes (minus129) in expression profilewith respect to control group However expression of ldquoCATrdquogene could not be demonstrated in other treatment groupssince amplification could not be recorded in the colitis-induced mice as well as Lf1 fed-colitis-induced mice groups

4 Discussion

Low-grade inflammations accompanied by acute phaseoxidative stress to the affected tissues in the gut are thehallmark of IBDs Hence to neutralize the damaging effectsof oxidative stress antioxidant supplements are popularlyconsumed with the belief that loading of antioxidant capacitythrough external sources could improve the ability to down-sizerestrict potential oxidative damage caused by reactiveoxygen species (ROS) However recent advances made inunderstanding redox homeostasis maintained via the ldquoNrf 2rdquosignalling pathway may challenge the concept of artificiallysupplying the body with antioxidants In this situation thefeedback nature of the redox systemmust be considered fullyas too much ingestion of antioxidants may actually diminishthe bodyrsquos endogenous defensive antioxidative capability withserious health implications Hence the better and saferoption to deal with the severe oxidative stress induced dam-age in the gut would be by exploring antioxidative potentialsof probiotic bacteria since excessive antioxidative responsecoming from the probiotic strains may be downregulatedby the stabilizing systems operating in the whole organismsthereby suggesting the feasibility of the probiotic therapy tomanage IBD from a broader perspective The major focusof this study centred on ldquoNrf 2rdquo signalling pathway and itsmodulation by probiotic strains under both in vitro and invivo conditions Another important issue that figured promi-nently in the study was to find whether the antioxidativeefficacy of Lf1 driven by ldquoNrf 2rdquo signalling pathway that wassuccessfully demonstrated in HT-29 cells could be replicatedin colitis mice model or not This was considered importantsince the rationale for selecting Lf1 for demonstrating itsefficacy in in vivo studies in colitis mice model in this

investigation was based on its in vitro ldquoNrf 2rdquo modulatoryactivity and also by virtue of showing high antioxidativeactivity as reported previously [13]

The outcome of this study indicated that L fermentumLf1 was quite effective in reducing the severity of colitis inmice by bringing about significant changes in DAI scorebody weight gain or loss colon length faecal character andfaecal bleeding (bloody stool) besides histological damageseverity scores A drastic weight loss in mice was recordedthat became highly significant starting from the eighth dayto the thirteenth day (119875 lt 005) due to severe experimentalcolitis However significant differencewas recorded inweightgain in colitis control versus colitis-Lf1 treatment groupsOur observations in this regard are in accordance withearlier reports statingweight gain in probiotics-treated group[38 39] The probiotic-treated group of mice had longercolon relative to colitis control as well as noncolitis controlgroups [39] The shortening of the colon under severe colitisconditions might be attributed to thickening of the colonepithelial wall that could bring about contraction of thecolon length DAI scores in Lf1 treatment group decreasedgradually and reached as low as zero on the 13th day insome mice Significant differences (119875 lt 005) in the DAIscores of treatment and colitis control groups demonstratedthe efficacy of Lf1 against colitis which is consistent with thesimilar observations in DAI results recorded by several otherinvestigators [38ndash40] Significant differences in histologicalscores amongst the colitis control and treatment groupswere also recorded which implies that Lf1 was effective inpreventing histological damage of treatedmice to a significantlevel (119875 lt 005) which is perfectly in line with those ofearlier reports on improvement in histological damage incolitis mice fed with probiotics [38ndash40]

The significant differences in the MDA content in thecolitis control versus the colitis-Lf1 treatment group as shownin our study suggest that Lf1 exhibited a strong antioxidativeproperty in colon as well and prevented lipid peroxidationThese findings are in concordance with that of Ito et alwho demonstrated the highest inhibitory activity against lipidperoxidation in liposomes by Streptococcus thermophilus YIT2001 [41] Our findings on the antioxidative efficacy of Lf1 interms of lipid peroxidation inhibition in colitis-Lf1 mice canalso be supported by a similar study conducted in Sprague-Dawley rats previously [42] wherein different probiotic andprebiotic based treatments alone or in combination priorto colitis induction with DSS were also shown to evoke asignificant decrease in the levels of lipid peroxidation in pro-biotic group Similarly two additional studies carried out bySingh et al [43] and Kaushal and Kansal [44] demonstratedantioxidative efficacy of low-fat probiotic dahi prepared withregular dahi cultures plus L acidophilus and L casei as wellas B bifidum by significantly reducing the levels of TBARSin colon carcinogenesis in ratsmice in comparison to DMHcontrol rats [43] These observations further substantiate thefindings of our study and reaffirmed the antioxidative efficacyof Lf1 strain against colitis

41 Relative Expression Studies Since the selection of Lf1 wasbased on its ability to induce ldquoNrf 2rdquo expression in HT-29

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

cells the expression of the same was also investigated underin vivo conditions using colitis model This was consideredimportant because Nrf 2ARE signalling pathway plays acrucial role in vascular homeostasis and the defence ofendothelial and gut epithelial cells against oxidative stressencountered during inflammatory process However surpris-ingly Lf1 could not induce ldquoNrf 2rdquo expression in the micemodel This was quite unexpected and inconsistent with theresponse recorded in HT-29 cells Although the exact causeof differential ldquoNrf 2rdquo expression under in vitro and in vivoconditions cannot be explained precisely due to lack of anysupportive data it might be attributed to some inhibitoryfactor present in the complex mice gut milieu that couldinterfere with the processing and nuclear translocation offunctional ldquoNrf 2rdquo Hence it would be interesting to identifysuch unknown factors that eventually may provide a clue toexplain this hypothesis Our findings in this regard howeverare at variance with results of previous research groupswho demonstrated increased level of ldquoNrf 2rdquo expression inrat model with liver fibrosis fed with blueberry after 21days of treatment [45 46] Another possible reason forthe failure of ldquoNrf 2rdquo expression in colitis mice with Lf1could be ascribed to differences in the treatment periodsin the two studies and also due to different mechanismsusing different animal models followed in the two studiesFurthermore it is to be clarified that the results from themiceexperiment were recorded only up to 13 days which mightbe inadequate to demonstrate the effect unlike the 21 daysused by Wang et al [45 46] Moreover strain specificity ofthe probiotic cultures could also partly account for variationin the results thereby necessitating further standardizationof the experimental design to optimize the ldquoNrf 2rdquo expressionconditions in the animalmodels Hence in order to showcasethe exact cause of this contradiction in results under in vitroand in vivo conditions more robust in-depth studies areneeded to unravel the intricacy of this process by optimizingthe experimental conditions and proper design of the animalstudies This is important from regulatory perspectives sinceEU has planned new restrictions and regulations for animalmodels

The antioxidative potentials of Lf1 were found to bereinforced with a powerful arsenal of several enzymatic andnonenzymatic factors such as antioxidative enzymes andother important molecules which could play a protective roleduring the oxidative stress induced conditions confronted inthe gut Our findings on ldquoSOD2rdquo expression in the mousemodel evoked with Lf1 are well supported by the outcomeof some previous studies [47ndash49] wherein an upregulationof ldquoSOD2rdquo was also recorded with their respective probioticfood preparations Their results showed that probiotic dahicould increase the activity of superoxide dismutase in RBCsand colorectal tissues of rats after treatment with probioticdahi However our results on upregulation (6-446-fold) ofldquoSOD2rdquo in noncolitic mice when fed with Lf1 for a periodof 12 days are at variance with those earlier reports on theeffect of daily consumption of probiotic and conventionalyoghurt on oxidant and antioxidant parameters in plasmaof young healthy women [50 51] Both studies reportedthat the probiotic and the conventional groups did not have

any significant difference in ldquoSODrdquo levels before and afterprobiotic intervention (119875 gt 005) This could be attributedto the use of a different probiotic strain and also the hostspecificity used in the two studies

Our results with regard to upregulation (2656) ofldquoTrxR-1rdquo in noncolitic mice when fed with Lf1 for a periodof 13 days and nonsignificant effect of probiotic feeding incolitis mice group cannot be substantiated due to lack ofany published reports on these lines Nevertheless there arefew studies reporting the positive effects of upregulationof thioredoxin reductase in the alleviation of cardiac dys-function and protection against oxidative damage broughtabout by chemoprotective agents such as resveratrol [52]Almost a similar trend was recorded with regard to catalasein our study as Lf1 did not evoke any significant effecton expression of catalase and in fact there was not evenbasal level expression recorded in the colitic mice groupOur findings in this regard are almost similar to those ofprevious published report on the clinical efficacy of probioticpreparations in healthy womenwhich reported that themeanactivities of catalase decreased significantly (119875 lt 0001) inthe probiotic group after consuming yoghurt daily for fourweeks [50] Nonetheless some previous studies show thatfeeding of probiotic dahi increased the activities of catalasein different tissues in their respective studies [47 48] Besidesthis there was an interesting study carried out by Chamariet al that reported that catalase levels in plasma of younghealthy women in a randomized clinical trial in probioticgroup increased significantly after probiotic intervention incomparison with baseline (119875 lt 0001) and the mean changesin catalase level were significantly different in probiotic andconventional groups (119875 lt 0001) [51]

5 Conclusion

From the outcome of this study it can be concluded thatour indigenous Lactobacillus fermentum strain Lf1 is ade-quately equipped with the multifactorial antioxidative andanti-inflammatory defense arsenal not only to protect itsown survival but also to confer protection to the host cellsagainst the hostile oxidative stress confronted in the mice gutduring colitis However it remains to be seen whether theantioxidative efficacy of this strain is mediated by activationof ldquoNrf 2rdquo stress response pathway or through other routes inestablished animal models Nevertheless it has the potentialto be explored as prospective functional therapeutics tomanage IBDs by establishing its clinical efficacy in well-designed double-blind placebo-controlled human trials

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the support provided byDirector National Dairy Research Institute (Karnal India)

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 11

and Indian Council of Agricultural Research (India) tocarry out the study The authors are also indebted to theMinistry of Food Processing Industries (MoFPI India) forfinancial support during the execution of the project Thefinancial support received from the National Dairy ResearchInstitute (NDRI India) in terms of providing fellowshipto the first author of the paper to carry out her MasterrsquosProgramme is greatly appreciatedThe authors also gratefullyacknowledge the help rendered by Mr Mohinder SinghAttendant Small Animal House NDRI Sunita Grover andVirender Kumar Batish conceived and designed the experi-ments Ritu Chauhan Aparna Sudhakaran VasanthakumariHarsh Panwar Rashmi H Mallapa and Raj Kumar Duaryperformed the experiments RajKumarDuary SunitaGroverand Virender Kumar Batish analyzed and interpreted thedata Sunita Grover and Virender Kumar Batish contributedreagentsmaterialsanalysis tools Raj Kumar Duary SunitaGrover and Virender Kumar Batish prepared the paper

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] N Kavian W Marut A Servettaz et al ldquoReactive oxygenspeciesmdashmediated killing of activated fibroblasts by arsenictrioxide ameliorates fibrosis in a murine model of systemicsclerosisrdquo Arthritis amp Rheumatism vol 64 no 10 pp 3430ndash3440 2012

[3] H Zhu andY R Li ldquoOxidative stress and redox signalingmech-anisms of inflammatory bowel disease updated experimentaland clinical evidencerdquo Experimental Biology and Medicine vol237 no 5 pp 474ndash480 2012

[4] H-R Kim A Lee E-J Choi et al ldquoAttenuation of experimentalcolitis in glutathione peroxidase 1 and catalase double knockoutmice through enhancing regulatory T cell functionrdquo PLoS ONEvol 9 no 4 Article ID e95332 2014

[5] A W Girotti ldquoMechanisms of lipid peroxidationrdquo Journal ofFree Radicals in Biology and Medicine vol 1 no 2 pp 87ndash951985

[6] D Bonnefont-Rousselot J P Bastard M C Jaudon and JDelattre ldquoConsequences of the diabetic status on the oxi-dantantioxidant balancerdquo Diabetes amp Metabolism vol 26 no3 pp 163ndash176 2000

[7] T Nguyen C S Yang and C B Pickett ldquoThe pathways andmolecular mechanisms regulating Nrf2 activation in responseto chemical stressrdquo Free Radical Biology and Medicine vol 37no 4 pp 433ndash441 2004

[8] D Gao Q Li Z Liu et al ldquoAntidiabetic potential of Rhodiolasachalinensis root extract in streptozotocin-induced diabeticratsrdquo Methods and Findings in Experimental and Clinical Phar-macology vol 31 no 6 pp 375ndash381 2009

[9] P Matafome T Louro L Rodrigues et al ldquoMetformin andatorvastatin combination further protect the liver in type 2diabetes with hyperlipidaemiardquo DiabetesMetabolism Researchand Reviews vol 27 no 1 pp 54ndash62 2011

[10] B Lallemant A Evrard C Combescure et al ldquoReference geneselection for head and neck squamous cell carcinoma geneexpression studiesrdquo BMC Molecular Biology vol 10 article 782009

[11] T Kullisaar M Zilmer M Mikelsaar et al ldquoTwo antioxidativelactobacilli strains as promising probioticsrdquo International Jour-nal of Food Microbiology vol 72 no 3 pp 215ndash224 2002

[12] T Kullisaar E Songisepp M Mikelsaar K Zilmer TVihalemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[13] A A Achuthan R K Duary A Madathil et al ldquoAntioxidativepotential of lactobacilli isolated from the gut of Indian peoplerdquoMolecular Biology Reports vol 39 no 8 pp 7887ndash7897 2012

[14] K Truusalu R Mikelsaar P Naaber et al ldquoEradication ofSalmonella typhimurium infection in amurinemodel of typhoidfever with the combination of probiotic Lactobacillus fermen-tumME-3 and ofloxacinrdquo BMCMicrobiology vol 8 article 1322008

[15] K Truusalu T Kullisaar P Hutt et al ldquoImmunological antiox-idative and morphological response in combined treatmentof ofloxacin and Lactobacillus fermentum ME-3 probiotic inSalmonella typhimurium murine modelrdquo APMIS vol 118 no11 pp 864ndash872 2010

[16] P Gionchetti F Rizzello A Venturi et al ldquoOral bacteriotherapyas maintenance treatment in patients with chronic pouchitisa double-blind placebo-controlled trialrdquo Gastroenterology vol119 no 2 pp 305ndash309 2000

[17] R Bibiloni R N Fedorak G W Tannock et al ldquoVSL3probiotic-mixture induces remission in patients with activeulcerative colitisrdquo American Journal of Gastroenterology vol100 no 7 pp 1539ndash1546 2005

[18] M A Zocco L Z dal Verme F Cremonini et al ldquoEfficacy ofLactobacillusGG inmaintaining remission of ulcerative colitisrdquoAlimentary Pharmacology amp Therapeutics vol 23 no 11 pp1567ndash1574 2006

[19] E Miele F Pascarella E Giannetti L Quaglietta R N Baldas-sano and A Staiano ldquoEffect of a probiotic preparation (VSL3)on induction and maintenance of remission in children withulcerative colitisrdquo American Journal of Gastroenterology vol104 no 2 pp 437ndash443 2009

[20] A Tursi G Brandimarte A Papa et al ldquoTreatment of relapsingmild-to-moderate ulcerative colitis with the probiotic VSL3 asadjunctive to a standard pharmaceutical treatment a double-blind randomized placebo-controlled studyrdquo The AmericanJournal of Gastroenterology vol 105 no 10 pp 2218ndash2227 2010

[21] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[22] C Dai C Zheng F Meng Z Zhou L Sang and M JiangldquoVSL3 probiotics exerts the anti-inflammatory activity viaPI3kAkt and NF-120581B pathway in rat model of DSS-inducedcolitisrdquo Molecular and Cellular Biochemistry vol 374 no 1-2pp 1ndash11 2013

[23] R Toumi K Abdelouhab H Rafa et al ldquoBeneficial role of theprobiotic mixture ultrabiotique on maintaining the integrity ofintestinalmucosal barrier inDSS-induced experimental colitisrdquoImmunopharmacology and Immunotoxicology vol 35 no 3 pp403ndash409 2013

[24] E Miyauchi T Ogita and J Miyamoto ldquoBifidobacteriumlongum alleviates dextran sulfate sodium-induced colitis bysuppressing IL-17A response involvement of intestinal epithe-lial costimulatory moleculesrdquo PLoS ONE vol 8 Article IDe79735 2013

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 BioMed Research International

[25] H Yoon Y S Yoon M S Kim M J Chung and D Y YumldquoA probiotic preparation duolac-gold ameliorates dextran sul-phate sodium-induced mouse colitis by downregulating theexpression of IL-6rdquo Toxicology Research vol 30 no 1 pp 27ndash32 2014

[26] R Crittenden S Karppinen S Ojanen et al ldquoIn vitro fer-mentation of cereal dietary fibre carbohydrates by probioticand intestinal bacteriardquo Journal of the Science of Food andAgriculture vol 82 no 8 pp 781ndash789 2002

[27] J K Kaushik A Kumar R K Duary A K Mohanty S Groverand V K Batish ldquoFunctional and probiotic attributes of anindigenous isolate of Lactobacillus plantarumrdquo PLoS ONE vol4 no 12 Article ID e8099 2009

[28] M E Sanders and T R Klaenhammer ldquoInvited review thescientific basis of Lactobacillus acidophilusNCFM functionalityas a probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash331 2001

[29] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[30] T Ringel-Kulka O S Palsson D Maier et al ldquoProbioticbacteria Lactobacillus acidophilus NCFM and Bifidobacteriumlactis Bi-07 versus placebo for the symptoms of bloating inpatients with functional bowel disorders a double-blind studyrdquoJournal of Clinical Gastroenterology vol 45 no 6 pp 518ndash5252011

[31] R K Duary V K Batish and S Grover ldquoImmunomodulatoryactivity of two potential probiotic strains in LPS-stimulatedHT-29 cellsrdquo Genes and Nutrition vol 9 no 3 p 398 2014

[32] V A Sudhakaran H Panwar R Chauhan et al ldquoModulationof anti-inflammatory response in lipopolysaccharide stimulatedhuman THP-1 cell line and mouse model at gene expressionlevel with indigenous putative probiotic lactobacillirdquo Genes ampNutrition vol 8 no 6 pp 637ndash648 2013

[33] T Asakawa and S Matsushita ldquoColoring conditions of thiobar-bituric acid test for detecting lipid hydroperoxidesrdquo Lipids vol15 no 3 pp 137ndash140 1980

[34] H S Cooper S N S Murthy R S Shah and D J SedergranldquoClinicopathologic study of dextran sulfate sodium experimen-tal murine colitisrdquo Laboratory Investigation vol 69 no 2 pp238ndash250 1993

[35] B G Meerveld and K R Tyler ldquoCPI-1189 protects againstdextran sulfate sodium-induced colitis in micerdquo AmericanJournal of Pharmacology and Toxicology vol 1 no 3 pp 54ndash592006

[36] A Pospiech and B Neumann ldquoA versatile quick-prep ofgenomic DNA from Gram-positive bacteriardquo Trends in Genet-ics vol 11 no 6 pp 217ndash218 1995

[37] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[38] M V Herıas J F J G Koninkx J G Vos J H J Huis Inrsquot Veldand J E van Dijk ldquoProbiotic effects of Lactobacillus casei onDSS-induced ulcerative colitis in micerdquo International Journal ofFood Microbiology vol 103 no 2 pp 143ndash155 2005

[39] L Chen X Wang Y Cui et al ldquoTherapeutic effects of fourstrains of probiotics on experimental colitis in micerdquo WorldJournal of Gastroenterology vol 15 no 3 pp 321ndash327 2009

[40] L R Fitzpatrick J Small R A Hoerr E F Bostwick LMaines and W A Koltun ldquoIn vitro and in vivo effects of the

probiotic Escherichia coli strain M-17 immunomodulation andattenuation of murine colitisrdquo British Journal of Nutrition vol100 no 3 pp 530ndash541 2008

[41] M Ito K Ohishi Y Yoshida W Yokoi and H Sawada ldquoAnti-oxidative effects of lactic acid bacteria on the colonic mucosaof iron-overloaded micerdquo Journal of Agricultural and FoodChemistry vol 51 no 15 pp 4456ndash4460 2003

[42] N Osman D Adawi G Molin S Ahrne A Berggren andB Jeppsson ldquoBifidobacterium infantis strains with and withouta combination of Oligofructose and Inulin (OFI) attenuateinflammation in DSS-induced colitis in ratsrdquo BMC Gastroen-terology vol 6 article 31 2006

[43] N K Singh A Kumar and P R Sinha ldquoChemopreventive effectof probiotic dahi (curd) containing Lactobacillus acidophilusand Lactobacillus casei on 1 2 dimethyhydrazine induced coloncarcinogenesis in ratsrdquo International Journal of Probiotics andPrebiotics vol 2 no 4 pp 195ndash202 2007

[44] D Kaushal and V K Kansal ldquoProbiotic Dahi containing Lacto-bacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkersof ageing in micerdquoMolecular Biology Reports vol 39 no 2 pp1791ndash1799 2012

[45] A N Wang X W Yi H F Yu B Dong and S Y Qiao ldquoFreeradical scavenging activity of Lactobacillus fermentum in vitroand its antioxidative effect on growing-finishing pigsrdquo Journalof Applied Microbiology vol 107 no 4 pp 1140ndash1148 2009

[46] Y Wang M Cheng B Zhang M Mu and J Wu ldquoEffects ofblueberry on hepatic fbrosis and transcription factor Nrf2 inratsrdquoWorld Journal of Gastroenterology vol 16 no 21 pp 2657ndash2663 2010

[47] H Yadav S Jain and P R Sinha ldquoOral administration of dahicontaining probiotic Lactobacillus acidophilus and Lactobacilluscasei delayed the progression of streptozotocin-induced dia-betes in ratsrdquo Journal of Dairy Research vol 75 no 2 pp 189ndash195 2008

[48] S Rajpal and V K Kansal ldquoProbiotic Dahi containing Lac-tobacillus acidophilus and Bifidobacterium bifidum stimulatesantioxidant enzyme pathways in ratsrdquo Milchwissenschaft vol64 no 3 pp 287ndash290 2009

[49] A DrsquoSouza L Fordjour A Ahmad et al ldquoEffects of probioticsprebiotics and synbiotics on messenger RNA expression ofcaveolin-1 NOS and genes regulating oxidative stress in theterminal ileumof formula-fed neonatal ratsrdquoPediatric Researchvol 67 no 5 pp 526ndash531 2010

[50] E Fabian and I Elmadfa ldquoThe effect of daily consumptionof probiotic and conventional yoghurt on oxidant and anti-oxidant parameters in plasma of young healthy womenrdquo Inter-national Journal for Vitamin and Nutrition Research vol 77 no2 pp 79ndash88 2007

[51] M Chamari A Djazayery M Jalali et al ldquoThe effect of dailyconsumption of probiotic and conventional yogurt on someoxidative stress factors in plasma of young healthy womenrdquoARYA Atherosclerosis Journal vol 4 no 4 pp 175ndash179 2008

[52] M Thirunavukkarasu S V Penumathsa S Koneru et alldquoResveratrol alleviates cardiac dysfunction in streptozotocin-induced diabetes role of nitric oxide thioredoxin and hemeoxygenaserdquo Free Radical Biology andMedicine vol 43 no 5 pp720ndash729 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology