Research Article Xianfanghuomingyin, a Chinese...

Research ArticleXianfanghuomingyin a Chinese Compound MedicineModulates the Proliferation and Differentiation ofT Lymphocyte in a Collagen-Induced Arthritis Mouse Model

Bo Nie1 Xue Li1 Yi Wei1 Meng Chen2 Jingwei Zhou3 Lixia Lou1 Bin Dong1

Aiming Wu1 Dongmei Zhang1 Lingqun Zhu1 Jiuli Zhao1 and Limin Chai1

1Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing Dongzhimen HospitalBeijing University of Chinese Medicine Beijing China2School of Preclinical Medicine Beijing University of Chinese Medicine Beijing China3Department of Rheumatology Dongzhimen Hospital Beijing University of Chinese Medicine Beijing China

Correspondence should be addressed to Limin Chai liminchaihotmailcom

Received 5 May 2016 Revised 17 July 2016 Accepted 26 July 2016

Academic Editor Young-Su Yi

Copyright copy 2016 Bo Nie et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In traditional Chinesemedicine (TCM) xianfanghuomingyin (XFHM) is used to treat autoimmune diseases including rheumatoidarthritis (RA) Here we studied the mechanisms underlying its treatment effects especially its anti-inflammatory effects in acollagen-induced arthritis (CIA) mouse model We found that cartilage destruction and pannus formation were alleviated bytreatment with XFHM The abnormal differentiation of Th1 and Th17 cells was downregulated significantly by XFHM and Th2and Treg cells were upregulated Moreover the expression levels of specific cytokines and transcription factors related to Th1 cells(interferon 120574 [IFN120574] T-bet) and Th17 cells (interleukin- [IL-] 17) and the nuclear receptor retinoic acid receptor-related orphanreceptor-gamma (ROR120574) were downregulated Serum IL-4 and GATA-3 which contribute to Th2 cells differentiation increasedsignificantly after XFHMadministrationThese results indicate that XFHMcan restore the balance of T lymphocytes and reestablishthe immunological tolerance to inhibit autoinflammatory disorder of RA Taken together XFHM can be used as a complementaryor alternative traditional medicine to treat RA

1 Introduction

Rheumatoid arthritis (RA) characterized by inflammationof synovial joints is a chronic and systemic autoimmunedisease which leads to a progressive destruction of articularcartilage and periarticular structures [1 2] The pathogenesisof RA is not well understood Various inflammatory cellsincluding T and B cells macrophages dendritic cells andnatural killer cells infiltrate RA joints and induce the pro-liferation of cells of the synovial lining [3 4] These cellsconstitute the synovial lining participate in inflammationcontribute to synovial proliferation pannus formation andcartilage destruction and subchondral bone erosion [5]

The involvement of autoreactive T cells in the pathogen-esis of RA well established T helper 1 (Th1) and Th17 cell

subsets play key roles in the pathogenesis of RA Abnormaldifferentiation of Th1 cells with an increased secretion ofinterferon 120574 (IFN120574) and a disturbed balance of Th1-Th2 cellsis also shown in autoimmune diseases including RA [6 7] Inrecent years the discovery of IL-17-producing Th17 cells haschanged the initial recognition of Th1 cell driven disease ofRA Th17 cells and IL-17 trigger synovial inflammation con-tributing to cartilage and bone destruction [8 9] Collagen-induced arthritis (CIA) is suppressed significantly in IL-17-deficient mice suggesting that Th17 cells play a crucial rolein the pathogenesis of CIA mice [10] Regulatory T (Treg)cells that have lost regulatory control of effector T cells alsopromotes the pathogenesis of RA [7] Loss of the suppressioneffect of Treg cells on effector T cells can lead to loss of controlof the autoimmune inflammation in RA and subsequent

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 6356871 14 pageshttpdxdoiorg10115520166356871

2 Evidence-Based Complementary and Alternative Medicine

pathological damage process Transferring CD4+CD25+ Tregcells into CIA mice can slow the progression of CIA andreduce the production of acute-phase proteins [11]

Xianfanghuomingyin (XFHM) described in ancientherbal formulae (JiaozhuFurenLiangfang which is a Chinesemedical treatise compiled by XueJi in the Song Dynasty ofAncient China) is a typical traditional Chinese medicine(TCM) formula which has been used for over 1000 yearsThe formulae combined plant species and minerals that wereprescribed based on clinical experience Multiple compo-nents of the formula could interact with multiple targets andexert synergistic therapeutic efficacies [12 13] The optimizedformula of XFHM (once named Lijie capsule) used in thisstudy is comprised of 12 different herb Resent evidenceindicates that XFHM can be used to treat osteoarthritis ofthe knee [14] and hip joint synovitis [15] Additionally ourprevious studies have suggested that XFHM may induce theapoptosis in lymphocytes in the peripheral blood (PB) of ratswith adjuvant arthritis [16]

Here we investigated the possible anti-inflammatorymechanisms underlying the regulation of T lymphocyte dif-ferentiation to maintain immunological tolerance followingXFHM treatment in a CIAmousemodel Leflunomide (LEF)a disease-modifying antirheumatic drug (DMARD) for themanagement of RA [17] was used as the positive controlTheaim of the present study was to identify the specific cellulartargets involved in the antiautoimmune effects of XFHM

2 Materials and Methods

21 Drugs The XFHM formula comprises Atractylodeslancea Ligusticum chuanxiong Hort Paeonia veitchii LynchTail of Radix Angelicae sinensis Angelica dahurica Radix Sa-poshnikoviae Boswellia carteri Birdw Commiphora myrrhaAstragalus membranaceus Caulis Lonicerae JaponicaeGentiana macrophylla Pall and Rehmannia glutinosaLibosch at a ratio of 2 2 2 2 3 3 1 1 8 6 3 3Quality control was carried out by infrared fingerprintspectrum (IRFP) techniques The IRFP graph is shownin S1 Fig (see Supplementary Material available online athttpdxdoiorg10115520166356871)

22 High Performance Liquid Chromatography-ElectrosprayIonizationMass Spectrometer (HPLC-ESIMS119899) AnalysisHPLC-ESIMSn analysis was performed on a Shimadzu20LC (Kyoto Japan) coupled to a diode array detector andTripleTOF 4600 + CDS mass spectrometer (AB Sciex MAUSA) The chromatographic separations were carried outon a Agilent Poroshell C18 (21mm times 100mm 27 120583m) Themobile phase consisted of a combination of A (05permil formicacid and 2mM acetic acid) and B (05permil formic acid and2mM acetic acid in acetonitrile methyl alcohol [1 1]) witha linear gradient 0ndashminus10min (5ndashminus20 B) or 10ndashminus22min(20ndashminus95 B) The flow rate was 04mLmin the sampleinjection volume was 5 120583L and the column and sampletemperature were 40∘CThe diode array detector (DAD) wasset at 200 220 250 and 280 nm for real-time monitoringof the peak intensity Mass spectra were simultaneously

acquired using electrospray ionization with positive andnegative ionization (POS and NEG) modes at fragmentationvoltages (40 psi) over the range of119898119911 50ndash1250

23 Mice DBA1J male mice (119899 = 6 per group 7-8 weeksold) were purchased from HFK Bioscience Co Ltd (BeijingChina) Animal care and use were in accordance with institu-tional guidelines and all animal experiments were approvedby the Institutional Animal Care and Use Committee ofthe National Institute of State Scientific and TechnologicalCommission

24 Induction of CIA Mice except mice in the normalgroup were immunized intradermally at the base of the tailwith 150120583g of bovine type II collagen (CII) (Sigma CAUSA) emulsified with an equal volume of complete Freundrsquosadjuvant (CFA) (Sigma CA USA) Mice were boosted byintradermal injection with 150 120583g of CII emulsified withincomplete Freundrsquos adjuvant (IFA) on the 21st day afterimmunization

25 Drug Treatment Drug treatment began after boosterimmunization and lasted for 28 days Mice were randomlydivided into four groups as follows normal group mice fedthe control diet and orally given sterile saline model groupmice fed the same as the normal group LEF group (Batchnumber 130126 Cinkate Corporation Beijing China) micefed the control diet and orally daily given 2mgkg LEF for 28days XFHM group mice fed the control diet and orally given53 gkg XFHM daily for 28 days The mice were sacrificedon the 29th day after treatment Mice were anesthetized byisoflurane anesthesia (2-3 isoflurane with oxygen supply)PB was obtained by removing eyeballs and the left legs andhind paws and spleens were removed

26 Clinical Scores Clinical CIA scores were monitoredevery 7 days after booster immunization Arthritic severityscores were derived as follows [18] The clinical scores forpaws were classified as 0 (normal joints) 1 (swelling in onedigit or joint inflammation) 2 (swelling in two or three digitsor slight paw swelling) 3 (swelling in more than four digitsand moderate swelling in the entire paw) and 4 (severeswelling and deformation of the paw) The sum of the scoresof all four paws of each mouse represented the total clinicalscore

27 Histology The left legs and hind paws of mice wereremoved fixed with 4 paraformaldehyde in phosphate-buffered saline and then decalcified for 10 days with EDTAand embedded in paraffinThe paraffin sections were stainedwith hematoxylin-eosin (HE) The specimens were observedand photographed under a light microscope (DM RAS2Leica Solms Germany) For Safranin O staining sectionswere placed in hematoxylin for 2min and then washed inwater for 5min Sections were then placed in 01 SafraninO solution for a further 2min and washed again in water for30 sec Finally the sections were passed through a series ofindustrial methylated spirit concentrations (70 to 100) for

Evidence-Based Complementary and Alternative Medicine 3

2min at each concentration The sections were then clarifiedin xylene for 2min Sections were then viewed and imagestaken using a light microscope

28 Immunohistochemistry Immunohistochemical stainingfor IL-1 was performed After sections were deparaffinizedrehydrated and washed the sections were then antigen-retrieved by pepsin and incubated with 03 hydrogen per-oxidase for 20min to block endogenous peroxidase activityfollowed by processing with serum for 30min to blocknonspecific ligations The sections were then treated withrabbit anti-IL-1 (1 100 Santa Cruz Biotechnology CA USA)primary antibody overnight at 4∘C and then washed andincubated with reagents from an immunohistochemical kit(Zhongshan Biotechnology Ltd Beijing China) in com-pliance with the manufacturerrsquos instructions and visualizedby 33-diaminobenzidine tetrahydrochloride (DAB) Finallythe sections were counterstained with hematoxylin Sectionswere then viewed and images taken using a light microscope

29 Enzyme-Linked Immunosorbent Assay (ELISA) Twenty-four hours after the last administration 08mL of PBwas col-lected from eachmouse by eyeball extirpation Blood sampleswere incubated at 25∘C for 60min and sera were isolated bycentrifuging at 3000 rpm at 4∘C for 10min Supernatants werecollected and stored at minus20∘C for protein quantification Theconcentrations of IFN120574 IL-4 and IL-17 were determined byquantitative sandwich ELISA using ELISA kits (eBioscienceSan Diego CA USA) According to the general protocol100 120583L of sample was incubated with 100 120583L of biotin labeledantibody for 60min at 37∘C followed by three washes Onehundredmicroliters of horseradish peroxidase (HRP) labeledavid in fluid was added and incubated for 30min at 37∘Cfollowed by five washesThen 90120583L of substrate solution wasadded and incubated for 25min at 37∘C Finally 50 120583L of stopsolution was added the optical density (OD) value was readwith a Multimode Reader at a wavelength of 450 nm

210 Fluorescence Activated Cell Sorter (FACS) AnalysisSpleens were removed diced and expressed through a 40 120583mNylon mesh All isolated spleen cells were made into single-cell suspensions To quantify the percentage CD3 and CD4positive cells cells were washed and stained with anti-mouse-CD3-PE and anti-mouse-CD4-PE-Cyanine5 anti-bodies (eBioscience) To determine whether the percent-age of Th1 Th2 or Th17 cells cells of all splenocytesand PB were stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ngmL) and ionomycin (Ion) (1 120583gmL)(Sigma CA USA) for 5 h in the presence of GolgiPlug(BD Bioscience NJ USA) according to the manufacturerrsquosprotocol Cells were then washed and stained with anti-mouse-CD4-PE-Cyanine5 antibody (eBioscience) FollowingCD4 staining cells were blocked fixed and permeabilizedusing a FixationPermeabilization kit according to manu-facturersrsquo instructions (BD Bioscience NJ USA) and thenfurther stained with anti-mouse-IFN120574-PE anti-mouse-IL-4-PE-Cyanine7 or anti-mouserat-IL-17A-FITC antibodies Todetermine the percentage of Treg cells cells were washed and

stainedwith anti-mouse-CD4-PE-Cyanine5 and anti-mouse-CD25-PE antibodies Following CD4 and CD25 staining(eBioscience) cells were blocked fixed and permeabilizedusing a FixationPermeabilization kit according to manu-facturersrsquo instructions (BD Bioscience) and stained withanti-mouserat-FOXP3-FITC antibody (eBioscience) Flowcytometry was performed by a FACS Calibur cytometer andanalyzed using CellQuest software (Beckman Coulter CAUSA)

211 Western Blot Analysis The spleens of CIA mice werehomogenized in 1mL of a lysis buffer (Sigma CA USA)The extracts were clarified by spinning at 10000timesg at 4∘Cfor 15min and then diluted with lysis buffer to achieve aconcentration of approximately 2mgmL protein Proteinsamples were separated by 10 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and trans-ferred onto nitrocellulose membranes (Amersham Phar-macia Biotech Uppsala Sweden) The membranes wereincubated with primary antibodies including rabbit anti-ROR120574 polyclonal antibody (Abcam Cambridge MA USA)rabbit anti-mouse-T-bet and anti-mouse-GATA-3 mono-clonal antibodies (Santa Cruz CA USA) and then incubatedwith HRP-conjugated secondary antibody (Santa Cruz) Allimmunoreactive proteins were visualized using SuperSignalsWest Pico Chemiluminescent Substrate (Thermo ScientificRockford IL USA) Densitometry plots of the proteinexpression levels were normalized to GAPDH and expressedrelative to the levels in the normal group

212 Statistical Analysis All data are presented as themean plusmnstandard deviation (SD) Statistical analyses were performedusing SPSS130 (SPSS Inc Chicago IL USA) One-wayanalysis of variance (ANOVA) followed by the Tukey-Kramertest for multiple comparisons was used to compare withthe treatment groups A 119875 value of lt 005 was consideredstatistically significant

3 Results

31 Characteristics of Pure Compounds in XFHM In recentyears liquid chromatography-mass spectrometry (LC-MS)has become an essential for the analysis of herbal con-stituents It is a powerful analytical tool that has been used toidentify compounds in TCM [19ndash21] In this study multistageMS and high resolutionMS were performed in both negativeand positive ion modes to gain complete information onchemical constitutions of XFHM The negative base peakMS spectrum and positive base peak MS spectrum aredisplayed in Figure 1 Twenty-one constituentswere identifiedby the precise mass and relative ion abundance of the targetpeaks The identified compounds and biological activitiesof individual constituents are shown in Table 1 Multiplereports have indicated that these components have biologicalactivities including anti-inflammatory or immune-regulatoryfunctions inhibitory effects on inflammatory proliferation orangiogenesis and increased production of anti-inflammatory


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Figure 1High performance liquid chromatography-electrospray ionizationmass spectrometer ion chromatograms for xianfanghuomingyin(a) Negative base peak mass spectrometry (MS) spectrum (b) positive base peak MS spectrum

Table 1 Chemical components identified in xianfanghuomingyin by high performance liquid chromatography-electrospray ionizationmass

Number Fragments (119898119911) Formula Identification Biological activities1 1520 1320 C

8H8O4

Vanillic acid Anti-inflammatory amp immune-regulatory2 3211 1691 C

16H24O10

Loganic acid Anti-inflammatory amp immune-regulatory3 1350 C

9H8O4

Caffeic acid Inhibition on inflammatory proliferation4 1730 1350 C

16H18O9

Chlorogenic acid Inhibition on inflammatory proliferation5 1951 1211 C

16H20O9

Gentiopicroside Anti-inflammatory amp immune-regulatory6 1791 1511 C

23H28O11

Paeoniflorin Anti-inflammatory amp immune-regulatory7 1791 1490 C

17H26O10

Loganin Anti-inflammatory amp immune-regulatory8 770 1051 C

10H10O3

Methyl 4-hydroxycinnamate Uncertain in autoimmune disease9 3831 4731 C

26H28O14

Apiin Anti-inflammatory amp immune-regulatory10 3071 2611 C

22H28O11

Prim-O-glucosylcimifugin Anti-inflammatory amp immune-regulatory11 4911 2710 C

30H32O15

Galloylpaeoniflorin Uncertain in autoimmune disease12 2591 2351 C

16H18O6

Cimifugin Uncertain in autoimmune disease13 4612 1610 C

29H36O15

Acteoside Anti-inflammatory amp immune-regulatory14 26707 25204 C

22H22O9

Ononin Uncertainty in autoimmune disease15 3011 1671 C

23H26O10

Lactiflorin Uncertainty in autoimmune disease16 2201 2251 2680 2110 C

16H12O5

Wogonin Inhibition of inflammatory cell proliferation17 2701 2851 C

22H20O11

Wogonoside Inhibition for angiogenesis18 1510 1191 C

15H12O5

Naringenin Immunostimulatory for dendritic cells19 1971 2261 2520 2230 C

16H12O4

Formononetin Increased IL-4 production20 2541 2101 C

16H14O4

Isoimperatorin Anti-inflammatory amp immune-regulatory21 62140 65141 C

41H68O14

Astragaloside IV Anti-inflammatory amp immune-regulatory

cytokines These results provide further evidence to supportthe therapeutic effects of XFHM in RA treatment

The toxicity of XFHM in mice was studied in a parallelexperiment The effects on liver and kidney functions areshown in S2 Fig There were no significant differencesbetween mice in the normal group and mice in the XFHMgroup

32 XFHM Suppresses Histopathological Changes and IL-1120573Expression in Metatarsophalangeal Joints in CIA Mice Afterbooster immunization the clinical CIA scores were deter-minedweekly As shown in Figure 2(a) on day 14 the swellingscore associated with arthritis in the XFHM group was 467plusmn103 compared to 683 plusmn 075 for the normal group TheXFHM and LEF treatments significantly reduced the arthritisseverity scores on days 0ndash28 after booster immunization

Histopathological lesions in the metatarsophalangeal jointsof mice were studied after HE staining (Figure 2(b)) andsynovial hyperplasia pannus formation and destruction ofarticular cartilage were detected in the metatarsophalangealjoints of CIA mice Histopathological lesions were alleviatedby treatment with LEF and XFHM Moderate proliferationof synovial cells was observed in XFHM-treated group Cellmorphology was restored to normal Pannus formation wasless and cartilage surfaces were smoother than those of themodel group Bone destruction was prevented by XFHMtreatment

A decrease in Safranin O staining of the growth platesin CIA mice was evident prior to morphological changesin the chondrocytes [22] There were also differences inSafranin O staining of the growth plates observed amongthe groups (Figure 2(c)) The intensity of Safranin O staining


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Figure 2 Clinical scores of CIA hematoxylin-eosin (HE) staining Safranin O staining and immunohistochemistry for IL-1120573 inmetatarsophalangeal joints (a) The clinical CIA scores monitored every 7 days after the booster immunization Data are presented as meansplusmn SD lowastlowast119875 lt 001 compared to the normal group 119875 lt 005 119875 lt 001 compared to themodel group (b) HE staining inmetatarsophalangealjoints uarr indicates synovium 998771 indicates articular cartilage and lowast indicates pannus (c) Safranin O staining of the growth plate The arrowsindicate Safranin O staining (d) Immunohistochemistry for IL-1120573 The arrows indicate the location of IL-1120573 Original magnification 200x(A) normal group (B) model group (C) group treated with LEF and (D) group treated with XFHM The scale bar corresponds to 60120583mthroughout

in CIA mice decreased significantly relative to the normalmice After being treated with LEF and XFHM the intensityof staining increased Immunohistochemical detection andlocalization showed that IL-1120573was expressed in the synoviumand chondrocytes As showed in Figure 2(d) significantdifferences were observed between normal and CIA miceAbnormal expression of IL-1120573 decreased significantly withLEF and XFHM treatments

33 XFHM Inhibits the Proliferation and Differentiation ofT Lymphocytes in CIA Mice FACS analysis showed that

the ratios of CD3+CD4+ T lymphocytes in PB (Figures3(a) and 3(b)) and spleen (Figures 3(c) and 3(d)) increasedsignificantly in CIA mice (119875 lt 001 or 119875 lt 005) Thesepercentages in the treatment groups were lower than thoseof the model group T lymphocytes counts in spleen of theXFHM group were decreased significantly relative to those ofthe model group (119875 lt 005)

34 XFHM Decreases the Differentiation of Th1 Cells inSpleen of CIA Mice To study the differentiation of Th1 cellslymphocytes from the spleens of CIA mice were stained with


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(A) (B)

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Figure 3 Xianfanghuomingyin attenuated the proliferation of CD3+CD4+ T cells in peripheral blood and spleen (a) and (b)The percentageof CD3+CD4+ T cells in the peripheral bloods of mice after 4 weeks of treatment (c) and (d) The percentage of CD3+CD4+ T cells in spleenof mice after 4 weeks of treatment Results are presented in the bar charts Data are presented as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 comparedto the normal group 119875 lt 005 119875 lt 001 compared to the model group

CD4 antibody and intracellularly with IFN120574 and then aFACS analysis was conducted As shown in Figure 4 thepercentage of Th1 cells in the model group was increasedsignificantly relative to those of the normal group (119875 lt001) However the levels ofTh1 cells in the treatment groupsdecreased significantly relative to those of the model group(119875 lt 001) Because T-box transcription factor (T-bet)regulates the differentiation of Th1 cells and production ofTh1 cytokines particularly IFN120574 [23] we tested the levels ofIFN120574 in sera by ELISA as well as the expression of T-bet inwhole spleen tissue lysates by western blot analysisThe IFN120574levels in sera and the production of T-bet in spleens of the

model group were increased significantly relative to those ofthe normal group (119875 lt 001 or 119875 lt 005) but increasesin IFN120574 and T-bet were suppressed with XFHM treatment(119875 lt 001)

35 XFHM Increases the Differentiation of Th2 Cells in Spleenof CIA Mice Whole spleen cells were surface stained byCD4 antibody and intracellularly stained by IL-4 antibodyto determine the percentage of Th2 cells In addition weexamined the transcriptional regulator GATA-3 that plays arole in Th2 differentiation and cell expansion [24] We alsoassayed the IL-4 levels in sera by ELISA and production of

Evidence-Based Complementary and Alternative Medicine 7CD

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Figure 4 XFHM suppressed the differentiation of CD4+IFN120574+Th1 cells and the production of IFN120574 and T-bet (a) and (b) The percentageof CD4+IFN120574+Th1 cells in spleens isolated from mice treated for 4 weeks (c) The IFN120574 levels in sera of mice detected by ELISA The resultsare presented in the bar chart (d) T-bet was detected in whole spleen tissue lysates by western blot analysis The results are presented in thebar chart GAPDH was used as an internal control Data are presented as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 compared to the normal group119875 lt 005 119875 lt 001 compared to the model group

GATA-3 in spleens by western blot analysis As shown inFigure 5 the percentage of CD4+IL-4+ Th2 cells in spleensand the levels of IL-4 in sera of the model group were lessthan those of the normal group (119875 lt 001 or 119875 lt 005) Thepercentage of Th2 cells and protein expression of GATA-3 inspleens of the XFHM group were higher than those of themodel groups (119875 lt 005)

36 XFHM Downregulates the Differentiation of Th17 Cellsin Spleen of CIA Mice Th17 cell which exclusively producesIL-17 plays a key pathogenic role in autoimmune diseases

Signal transducer and activator of transcription 3 (STAT3)and RAR-related orphan receptor 120574t (ROR120574t) are nuclearreceptors required to generate IL-17-producing CD4+ Th17cells [25 26] We stained spleen cells with CD4 and IL-17antibodies for FACS analysis andmeasured the levels of IL-17in mouse sera and the protein expression levels of STAT3 andROR120574 in spleens to explore the biological effects of XFHMon the regulation of Th17 cells differentiations in spleens ofCIA mice Our data showed that the level of Th17 cells inspleens of the model group was markedly higher than thatof the normal group (119875 lt 001) and IL-17 in sera was also


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Figure 5 XFHM enhanced CD4+IL-4+ Th2 cells differentiation and the production of IL-4 and GATA-3 (a) and (b) The percentage ofCD4+IL-4+Th2 cells in spleen isolated from mice treated for 4 weeks (c) The level of IL-4 in sera of mice detected by ELISA The results arepresented in the bar chart (d) GATA-3 was detected in whole spleen tissue lysates by western blot analysis GAPDH was used as an internalcontrol Data are presented as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 compared to the normal group 119875 lt 005 119875 lt 001 compared to themodel group

increased (Figures 6(a) 6(b) and 6(c)) The percentage ofTh17 cells and the production of STAT3 and ROR120574 in thetreatment groups decreased significantly relative to those ofthe model group and the effects of XFHM were superior tothose of LEF (119875 lt 001) (Figure 6(d))The high levels of IL-17displayed a downwards trend in the XFHM and LEF groups(Figure 6(c))

37 XFHM Induces the Differentiation of Treg Cells in PB andSpleen of CIA Mice Treg cells in PB and spleen were stained

by CD4 and CD25 antibodies and intracellularly strained byFOXP3 antibody and then evaluated by FACS As shown inFigure 7 the percentage of Treg cells in PB was lower inthe model group than in the normal group (119875 lt 001) andthe numbers of Treg cells in the XFHM and LEF groupswere higher than those of the model group Interestingly thepercentage of Treg cells in the XFHM group was significantlyhigher than that of model group (119875 lt 005) These resultsindicate that XFHMcan upregulate differentiation of the Tregsubset in CIA mice


100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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

(d)Figure 6 XFHM inhibited CD4+IL-17+Th17 cells differentiation and the production of IL-17 STAT3 and ROR120574 (a) and (b)The percentageof CD4+IL-17+Th17 cells in spleen isolated frommice treated for 4 weeks (c)The levels of IL-17 in sera of mice detected by ELISAThe resultsare presented in the bar chart (d) STAT3 and ROR120574 were detected in whole spleen tissue lysates by western blot analysis GAPDH was usedas an internal control Data were presented as means plusmn SD lowastlowast119875 lt 001 compared to the normal group 119875 lt 001 compared to the modelgroup


FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)

Figure 7 XFHM increased CD4+CD25+FOXP3+ Tregs differentiation (a) The percentages of CD4+ T cells in peripheral bloods (PB) andspleens of mice Lymphocytes were gated in total cells ((a1) PB (a2) spleen) (a1) and (a2) are the percentages of CD4+ T cells in PB andspleens (b1) and (b2) are the percentages of CD4+CD25+FOXP3+ Treg cells in PB and spleens of mice after treatment for 4 weeks (c) Theresults are presented in the bar chart Data are presented as means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 compared to the normal group 119875 lt 005119875 lt 001 compared to the model group


4 Discussion

TCM formulae consisting of plants and minerals are oftenprescribed based on clinical experience Several formulaehave been verified as valid complementary or alternativetherapies for the treatment of various diseases [27 28] Inthis study we focused on the Chinese traditional therapyXFHM and its mechanisms of action in improving patho-physiologic characteristics of RA in CIA mice Negative andpositive base peak spectra from XFHM were measured toobtain information on its chemical constituents As shown inFigure 1 and Table 1 21 constituents were identified byHPLC-ESIMSn analysis The experimental evidences indicate thatthese constituents including wanillic acid [29] loganic acid[30] paeoniflorin [31] loganin [32] apiin [33] gentiopicro-side [34] astragaloside IV [35] acteoside [36] prime-O-glucosylcimifugin [37] and isoimperatorin [38] had anti-inflammatory and immune-regulatory effects that inhib-ited the activation of NF-120581B or other signaling pathwaysChlorogenic acid [39] caffeic acid [40] and wogonin [41]had inhibitory effects on proliferation of synoviocytes anddamage to articular chondrocytes In addition wogonosideinhibits LPS-induced angiogenesis both in vitro and in vivo[42] Naringenin can manipulate the immunostimulatoryproperties of dendritic cells [43] and formononetin enhancesIL-4 production in T cells [44] Therefore we suggest thatthe functions of the Chinese herbs in XFHM contribute toits actions on multiple targets and its synergistic therapeuticeffects against RA

RA is an autoimmune rheumatic inflammatory diseaseT cells have been shown to participate in the pathogenesisof RA For many years RA has been considered as Th1-dependent disease Autoreactive T cells may recognize anumber of autoantigens induce Th1 cell differentiation anddisturb the balance of Th1 and Th2 cells triggering theautoimmune response that contributes to the pathogenesisof RA [45] This concept has changed since the discoveryof Th17 cells Th17 cells characterized by production of thehighly inflammatory cytokine IL-17 mediate joint pathologyThese cells which also produce INF120574 and other inflammatorycytokines initiate and prolong synovitis in multiple jointsand facilitate pannus development which eventually leadto cartilage and bone destruction [46 47] In additiondeficiencies in the number and function of Treg cells andresistance of effector T cells to Treg cell mediated suppressionare involved in the development of synovial inflammation [7]CIAmice are a recognized animalmodel for RA researchTheproliferation and differentiation of T cells in CIA mice werereflected in pathological changes similar to RA [48]

In the pathogenesis of RA antigen-presenting cell pro-vides immune cues that instruct the differentiation of naıveCD4+ precursor cells to optimally counteract infectiousthreat [49] Separate lineages of effectorTh-cell differentiatedfrom naıve CD4+ precursor cells participate in immunologi-cal response TheTh1 cell lineage is characterized by produc-tion of IFN120574 whereas the Th2 cell lineage is characterizedby production of IL4 IL5 and IL13 [50] Differentiation ofTh1 and Th2 effector cells is governed by the cytokines IL-12and IL-4 as well as by signaling through the Notch receptor

[51] Binding of the initial autoparacrine produced IFN120574to its receptor activates STAT1 and then strongly promotesthe expression of the Tbx21 gene which encodes T-bet T-bet enhances the transcriptional competence and increasesproduction of INF120574 Furthermore T-bet increases expres-sion of the IL-12 receptor 1205732 chain and further enhancesIFN120574 production T-bet also prevents Th2 differentiationby inhibiting expression of the GATA-3 gene [52] For thedifferentiation of Th2 cells IL-4 receptor signaling stronglypromotes expression of the IL-4 and GATA-3 genes GATA-3 encompasses the IL-4 IL-5 and IL-13 genes and promotesproduction of these cytokines Finally IL-4 enhances Th2-cell differentiation in a feed-forward loop GATA-3 can alsoinhibit the expression of the IL-12 receptor1205732 chain to restrictTh1 differentiation [53] Expression of the Tbx21 gene canincrease expression of T-bet leading to transactivation ofthe IFN120574 gene by binding to NF-120581B p50 or inhibiting ofIL-4 receptor signaling [54] Many reports have suggestedthat IL-17 and Th17 cells play important roles in inductionand propagation of autoimmunity in animal models aswell as human autoimmune disease including RA [55]Th17 cells that are specific for self-antigens initiate inflam-mation and cause severe autoimmunity The activation ofnaıve T cells induced by proinflammatory cytokines initiatesdifferentiation of Th17 cells leading to the expression oftranscription factor ROR-120574t and production of IL-17 [56]Our results show that XFHM can inhibit the proliferationand activation of CD3+CD4+ T cells and conversely promotethe conversion of T cells into CD4+CD25+FOXP3+ Treg cellsHowever XFHM treatment does not regulate the productionof granulocyte-macrophage colony-stimulating factor (GM-CSF) which is a critical cytokine for the differentiation ofTreg cells (S3 Fig) Furthermore XFHM can also suppressthe differentiations of Th1 and Th17 cells and increase thedifferentiation of Th2 cells According to these results wesuggest that XFHM maintains immunological tolerance byregulating the differentiation of T cell subsets to restore thebalance between T lymphocytes To illustrate the regulatorymechanisms of XFHM the production of defining proin-flammatory cytokines and transcription factors related toTh cells was evaluated Interestingly XFHM decreased thelevels of IFN120574 T-bet (Th1) IL-17 STAT3 and ROR120574 (Th17)but increased the production of IL-4 and GATA-3 (Th2) toregulate the differentiations of Th subsets

LEF is a low molecular weight isoxazole derivative usedin RA therapies It has functions for anti-inflammatory andimmunosuppressive function [57] The active metabolite ofLEF can prevent the interaction of T cells with antigen-presenting cells that forms an immunologic synapse regulatethe proliferation and differentiation of Th cells to main-tain the immunological tolerance and relieve RA immune-disorders [58] However a recent study suggests that LEFhas myelosuppressive and hepatotoxic potential based on arat model of RA [59] Adverse drug reactions to LEF suchas pruritus loss of appetite weakness dizziness diarrheaand erythema also occur frequently in clinical follow-upsurvey XFHM consists of 12 types of medicinal herbs Theprescription is determined based on clinical symptoms andthe compatibility of herbs Multiple components can act on


various targets and have synergistic therapeutic effects Fur-ther use of XFHM as an alternative therapy for RA can allowpatients to avoid myelosuppressive hepatotoxic and otherpotential adverse reactions of modern pharmacotherapiesincluding LEF treatment In addition our study also indicatesthat the therapeutic effects of XFHM are equal to or exceedthose of LEF

In conclusion our data suggest that XFHM canmodulatethe differentiations of Th1 Th2 and Th17 cells and promotethe differentiation of Treg cells restoring the normal bal-ance of Ths cells and maintaining immunological toleranceinterfere with immune cell invasion of synovial membranesand prevent cartilage and bone destruction in joints of RAjoints XFHM therefore should be used as a complementaryor alternative traditional medicine in the treatment of RA

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

BoNie Xue Li YiWeiMeng Chen and Jingwei Zhou contri-buted equally to this work

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (81173228)

References

[1] K Andreas C Lubke T Haupl et al ldquoKey regulatorymoleculesof cartilage destruction in rheumatoid arthritis an in vitrostudyrdquo Arthritis Research ampTherapy vol 10 no 1 p R9 2008

[2] U Muller-Ladner T Pap R E Gay M Neidhart and S GayldquoMechanisms of disease themolecular and cellular basis of jointdestruction in rheumatoid arthritisrdquo Nature Clinical PracticeRheumatology vol 1 no 2 pp 102ndash110 2005

[3] I B McInnes and G Schett ldquoThe pathogenesis of rheumatoidarthritisrdquoTheNew England Journal of Medicine vol 365 no 23pp 2205ndash2219 2011

[4] S You S-A Yoo S Choi et al ldquoIdentification of key regulatorsfor the migration and invasion of rheumatoid synoviocytesthrough a systems approachrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no1 pp 550ndash555 2014

[5] B Bartok and G S Firestein ldquoFibroblast-like synoviocytes keyeffector cells in rheumatoid arthritisrdquo Immunological Reviewsvol 233 no 1 pp 233ndash255 2010

[6] T Kobezda S Ghassemi-Nejad K Mikecz T T Glant andZ Szekanecz ldquoOf mice and men how animal models advanceour understanding of T-cell function in RArdquo Nature ReviewsRheumatology vol 10 no 3 pp 160ndash170 2014

[7] E J Wehrens B J Prakken and F Van Wijk ldquoT cells outof control-impaired immune regulation in the inflamed jointrdquoNature Reviews Rheumatology vol 9 no 1 pp 34ndash42 2013

[8] A Laurence and J J OrsquoShea ldquoTH-17 differentiation ofmice andmenrdquo Nature Immunology vol 8 no 9 pp 903ndash905 2007

[9] W B van den Berg and PMiossec ldquoIL-17 as a future therapeutictarget for rheumatoid arthritisrdquo Nature Reviews Rheumatologyvol 5 no 10 pp 549ndash553 2009

[10] S Nakae A Nambu K Sudo and Y Iwakura ldquoSuppressionof immune induction of collagen-induced arthritis in IL-17-deficient micerdquo The Journal of Immunology vol 171 no 11 pp6173ndash6177 2003

[11] M E Morgan R Flierman L M van Duivenvoorde et alldquoEffective treatment of collagen-induced arthritis by adoptivetransfer of CD25+ regulatory T cellsrdquo Arthritis amp Rheumatismvol 52 no 7 pp 2212ndash2221 2005

[12] T Liu H Cao Y Ji et al ldquoInteraction of dendritic cells andT lymphocytes for the therapeutic effect of Dangguiliuhuangdecoction to autoimmune diabetesrdquo Scientific Reports vol 5Article ID 13982 2015

[13] N-H Yim A Kim Y P Jung T Kim C J Ma and J Y MaldquoFermented So-Cheong-Ryong-Tang (FCY) induces apoptosisvia the activation of caspases and the regulation of MAPKsignaling pathways in cancer cellsrdquo BMC Complementary andAlternative Medicine vol 15 no 1 article 336 2015

[14] J-F Zeng M-Z Li M Li B-Q Sun W-H Zhou andF Yang ldquoXianfang huoming decoction jiawei iontophoresisanalysis combined with clinical of arthroscopic debridementfor osteoarthritis of kneeirdquo Chinese Journal of ExperimentalTraditional Medical Formulae vol 20 no 11 pp 199ndash202 2014

[15] D X Gao Xuan Z Yu and Z Wanliang ldquoClinical observationon the treatment of hip joint synovitis in children with internaland external application of Xianfang Huoming Yinrdquo Rheuma-tism and Arthritis vol 4 no 4 p 3 2015

[16] C L Tang Nong Z Lijun L Guobiao and H Xiaoqi ldquoInflu-ences of Lijie Capsules on expressions of Fas and FasL genes inlymphocytes in rats with adjuvant arthritisrdquo Journal of BeijingUniversity of Traditional Chinese Medicine vol 29 no 7 p 42016

[17] G Murdaca F Spano and F Puppo ldquoUse of leflunomide plusTNF-120572 inhibitors in rheumatoid arthritisrdquo Expert Opinion onDrug Safety vol 12 no 6 pp 801ndash804 2013

[18] H-B Hsiao C-C Hsieh J-B Wu H Lin and W-C LinldquoKinsenoside inhibits the inflammatory mediator release in atype-II collagen induced arthritis mouse model by regulatingthe T cells responsesrdquo BMC Complementary and AlternativeMedicine vol 16 no 1 article 80 2016

[19] Q Chen S Xiao Z Li N Ai and X Fan ldquoChemical andmetabolic profiling of Si-Ni decoction analogous formulae byhigh performance liquid chromatography-mass spectrometryrdquoScientific Reports vol 5 Article ID 11638 2015

[20] S Wang P Chen Y Xu X Li and X Fan ldquoCharacterizationof the chemical constituents in Da-Huang-Gan-Cao-Tang byliquid chromatography coupled with quadrupole time-of-flighttandemmass spectrometry and liquid chromatography coupledwith ion trap mass spectrometryrdquo Journal of Separation Sciencevol 37 no 14 pp 1748ndash1761 2014

[21] Y Yan C-Z Chai D-WWang X-Y Yue D-N Zhu and B-YYu ldquoHPLC-DAD-Q-TOF-MSMS analysis andHPLC quantita-tion of chemical constituents in traditional Chinese medicinalformula Ge-Gen Decoctionrdquo Journal of Pharmaceutical andBiomedical Analysis vol 80 pp 192ndash202 2013

[22] K Takahi J Hashimoto K Hayashida et al ldquoEarly closure ofgrowth plate causes poor growth of long bones in collagen-induced arthritis ratsrdquo Journal of Musculoskeletal NeuronalInteractions vol 2 no 4 pp 344ndash351 2002


[23] C A Singer ldquoT-bet is induced by interferon-120574 to mediate che-mokine secretion and migration in human airway smoothmuscle cellsrdquo American Journal of PhysiologymdashLung Cellularand Molecular Physiology vol 300 no 4 pp L633ndashL641 2011

[24] J Zhu H Yamane J Cote-Sierra L Guo and W E PaulldquoGATA-3 promotes Th2 responses through three differentmechanisms induction of Th2 cytokine production selectivegrowth of Th2 cells and inhibition of Th1 cell-specific factorsrdquoCell Research vol 16 no 1 pp 3ndash10 2006

[25] Q Ruan V Kameswaran Y Zhang et al ldquoThe Th17 immuneresponse is controlled by the RelndashROR120574ndashROR120574T transcrip-tional axisrdquoThe Journal of Experimental Medicine vol 208 no11 pp 2321ndash2333 2011

[26] T J Harris J F Grosso H R Yen et al ldquoCutting edge an invivo requirement for STAT3 signaling inTH17 development andTH17-dependent autoimmunityrdquo The Journal of Immunologyvol 179 no 7 pp 4313ndash4317 2007

[27] T W Corson and C M Crews ldquoMolecular understandingandmodern application of traditional medicines triumphs andtrialsrdquo Cell vol 130 no 5 pp 769ndash774 2007

[28] T Xue and R Roy ldquoStudying traditional Chinese medicinerdquoScience vol 300 no 5620 pp 740ndash741 2003

[29] W-S Choi P-G Shin J-H Lee and G-D Kim ldquoThe regula-tory effect of veratric acid on NO production in LPS-stimulatedRAW2647 macrophage cellsrdquo Cellular Immunology vol 280no 2 pp 164ndash170 2012

[30] S Wei H Chi H Kodama and G Chen ldquoAnti-inflammatoryeffect of three iridoids in human neutrophilsrdquo Natural ProductResearch vol 27 no 10 pp 911ndash915 2013

[31] J Ni D Yang L Song and C Li ldquoProtective effects of paeoni-florin on alveolar bone resorption and soft-tissue breakdownin experimental periodontitisrdquo Journal of Periodontal Researchvol 51 no 2 pp 257ndash264 2016

[32] N Yamabe J S Noh C H Park et al ldquoEvaluation of loganiniridoid glycoside from Corni Fructus on hepatic and renalglucolipotoxicity and inflammation in type 2 diabetic dbdbmicerdquo European Journal of Pharmacology vol 648 no 1ndash3 pp179ndash187 2010

[33] T Mencherini A Cau G Bianco R Della Loggia R PAquino and G Autore ldquoAn extract of Apium graveolens vardulce leaves structure of the major constituent apiin andits anti-inflammatory propertiesrdquo Journal of Pharmacy andPharmacology vol 59 no 6 pp 891ndash897 2007

[34] L Zhao J Ye G-T Wu X-J Peng P-F Xia and Y Ren ldquoGen-tiopicroside prevents interleukin-1 beta induced inflammationresponse in rat articular chondrocyterdquo Journal of Ethnopharma-cology vol 172 pp 100ndash107 2015

[35] W-J Zhang and B Frei ldquoAstragaloside IV inhibits NF-120581Bactivation and inflammatory gene expression in LPS-treatedmicerdquo Mediators of Inflammation vol 2015 Article ID 27431411 pages 2015

[36] W Jing M Chunhua and W Shumin ldquoEffects of acteoside onlipopolysaccharide-induced inflammation in acute lung injuryvia regulation ofNF-120581Bpathway in vivo and in vitrordquoToxicologyand Applied Pharmacology vol 285 no 2 pp 128ndash135 2015

[37] N Chen Q Wu G Chi et al ldquoPrime-O-glucosylcimifuginattenuates lipopolysaccharide-induced acute lung injury inmicerdquo International Immunopharmacology vol 16 no 2 pp139ndash147 2013

[38] L Moon Y M Ha H J Jang et al ldquoIsoimperatorin cimiside eand 23-O-acetylshengmanol-3-xyloside from Cimicifugae Rhi-zome inhibit TNF-120572-induced VCAM-1 expression in human

endothelial cells involvement of PPAR-120574 upregulation andPI3K ERK12 and PKC signal pathwaysrdquo Journal of Ethnophar-macology vol 133 no 2 pp 336ndash344 2011

[39] L Lou Y Liu J Zhou et al ldquoChlorogenic acid and luteolin syn-ergistically inhibit the proliferation of interleukin-1120573-inducedfibroblast-like synoviocytes through regulating the activationof NF-120581B and JAKSTAT-signaling pathwaysrdquo Immunopharma-cology and Immunotoxicology vol 37 no 6 pp 499ndash507 2015

[40] M M Mia and R A Bank ldquoThe pro-fibrotic properties oftransforming growth factor on human fibroblasts are counter-acted by caffeic acid by inhibiting myofibroblast formation andcollagen synthesisrdquo Cell and Tissue Research vol 363 no 3 pp775ndash789 2016

[41] J S Park H J Lee D Y Lee et al ldquoChondroprotective effects ofwogonin in experimental models of osteoarthritis in vitro andin vivordquo Biomolecules and Therapeutics vol 23 no 5 pp 442ndash448 2015

[42] Y Chen N Lu Y Ling et al ldquoWogonoside inhibits lipopoly-saccharide-induced angiogenesis in vitro and in vivo via toll-like receptor 4 signal transductionrdquo Toxicology vol 259 no 1-2pp 10ndash17 2009

[43] Y-R Li D-Y Chen C-L Chu et al ldquoNaringenin inhibitsdendritic cellmaturation and has therapeutic effects in amurinemodel of collagen-induced arthritisrdquo Journal of NutritionalBiochemistry vol 26 no 12 pp 1467ndash1478 2016

[44] J Park S H Kim D Cho and T S Kim ldquoFormononetin aphyto-oestrogen and its metabolites up-regulate interleukin-4production in activated T cells via increased AP-1 DNA bindingactivityrdquo Immunology vol 116 no 1 pp 71ndash81 2005

[45] M De Carli M M DrsquoElios G Zancuoghi S Romagnani andG Del Prete ldquoReview human Th1 and Th2 cells functionalproperties regulation of development and role in autoimmu-nityrdquo Autoimmunity vol 18 no 4 pp 301ndash308 1994

[46] O Snir J Backlund J Bostrom et al ldquoMultifunctional Tcell reactivity with native and glycosylated type II collagen inrheumatoid arthritisrdquo Arthritis and Rheumatism vol 64 no 8pp 2482ndash2488 2012

[47] A Von Delwig J Locke J H Robinson and W-F NgldquoResponse ofTh17 cells to a citrullinated arthritogenic aggrecanpeptide in patients with rheumatoid arthritisrdquo Arthritis andRheumatism vol 62 no 1 pp 143ndash149 2010

[48] X Yuan B Tong Y Dou X Wu Z Wei and Y DaildquoTetrandrine ameliorates collagen-induced arthritis in mice byrestoring the balance between Th17 and Treg cells via the arylhydrocarbon receptorrdquo Biochemical Pharmacology vol 101 pp87ndash99 2016

[49] M L Kapsenberg ldquoDendritic-cell control of pathogen-drivenT-cell polarizationrdquo Nature Reviews Immunology vol 3 no 12pp 984ndash993 2003

[50] AKAbbas KMMurphy andA Sher ldquoFunctional diversity ofhelper T lymphocytesrdquo Nature vol 383 no 6603 pp 787ndash7931996

[51] D Amsen C G Spilianakis and R A Flavell ldquoHow are TH1and TH2 effector cells maderdquo Current Opinion in Immunologyvol 21 no 2 pp 153ndash160 2009

[52] I M Djuretic D Levanon V Negreanu et al ldquoTranscriptionfactors T-bet and Runx3 cooperate to activate Ifng and silenceIl4 in T helper type 1 cellsrdquoNature Immunology vol 8 no 2 pp145ndash153 2007

[53] K M Ansel I Djuretic B Tanasa and A Rao ldquoRegulation ofTh2 differentiation and Il4 locus accessibilityrdquoAnnual Review ofImmunology vol 24 pp 607ndash656 2006


[54] D Amsen J M Blander G R Lee K Tanigaki T Honjo andR A Flavell ldquoInstruction of distinct CD4 T helper cell fates bydifferent notch ligands on antigen-presenting cellsrdquoCell vol 117no 4 pp 515ndash526 2004

[55] H H Hofstetter S M Ibrahim D Koczan et al ldquoTherapeuticefficacy of IL-17 neutralization in murine experimental autoim-mune encephalomyelitisrdquo Cellular Immunology vol 237 no 2pp 123ndash130 2005

[56] E Bettelli T Korn and V K Kuchroo ldquoTh17 the thirdmemberof the effector T cell trilogyrdquo Current Opinion in Immunologyvol 19 no 6 pp 652ndash657 2007

[57] F C Breedveld and J-M Dayer ldquoLeflunomide mode ofaction in the treatment of rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 59 no 11 pp 841ndash849 2000

[58] M Zeyda M Poglitsch R Geyeregger et al ldquoDisruption ofthe interaction of T cells with antigen-presenting cells by theactive leflunomide metabolite teriflunomide involvement ofimpaired integrin activation and immunologic synapse forma-tionrdquo Arthritis and Rheumatism vol 52 no 9 pp 2730ndash27392005

[59] S E Bilasy S S Essawy M F Mandour E A I Ali andS A Zaitone ldquoMyelosuppressive and hepatotoxic potential ofleflunomide and methotrexate combination in a rat model ofrheumatoid arthritisrdquoPharmacological Reports vol 67 no 1 pp102ndash114 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


pathological damage process Transferring CD4+CD25+ Tregcells into CIA mice can slow the progression of CIA andreduce the production of acute-phase proteins [11]

Xianfanghuomingyin (XFHM) described in ancientherbal formulae (JiaozhuFurenLiangfang which is a Chinesemedical treatise compiled by XueJi in the Song Dynasty ofAncient China) is a typical traditional Chinese medicine(TCM) formula which has been used for over 1000 yearsThe formulae combined plant species and minerals that wereprescribed based on clinical experience Multiple compo-nents of the formula could interact with multiple targets andexert synergistic therapeutic efficacies [12 13] The optimizedformula of XFHM (once named Lijie capsule) used in thisstudy is comprised of 12 different herb Resent evidenceindicates that XFHM can be used to treat osteoarthritis ofthe knee [14] and hip joint synovitis [15] Additionally ourprevious studies have suggested that XFHM may induce theapoptosis in lymphocytes in the peripheral blood (PB) of ratswith adjuvant arthritis [16]

Here we investigated the possible anti-inflammatorymechanisms underlying the regulation of T lymphocyte dif-ferentiation to maintain immunological tolerance followingXFHM treatment in a CIAmousemodel Leflunomide (LEF)a disease-modifying antirheumatic drug (DMARD) for themanagement of RA [17] was used as the positive controlTheaim of the present study was to identify the specific cellulartargets involved in the antiautoimmune effects of XFHM

2 Materials and Methods

21 Drugs The XFHM formula comprises Atractylodeslancea Ligusticum chuanxiong Hort Paeonia veitchii LynchTail of Radix Angelicae sinensis Angelica dahurica Radix Sa-poshnikoviae Boswellia carteri Birdw Commiphora myrrhaAstragalus membranaceus Caulis Lonicerae JaponicaeGentiana macrophylla Pall and Rehmannia glutinosaLibosch at a ratio of 2 2 2 2 3 3 1 1 8 6 3 3Quality control was carried out by infrared fingerprintspectrum (IRFP) techniques The IRFP graph is shownin S1 Fig (see Supplementary Material available online athttpdxdoiorg10115520166356871)

22 High Performance Liquid Chromatography-ElectrosprayIonizationMass Spectrometer (HPLC-ESIMS119899) AnalysisHPLC-ESIMSn analysis was performed on a Shimadzu20LC (Kyoto Japan) coupled to a diode array detector andTripleTOF 4600 + CDS mass spectrometer (AB Sciex MAUSA) The chromatographic separations were carried outon a Agilent Poroshell C18 (21mm times 100mm 27 120583m) Themobile phase consisted of a combination of A (05permil formicacid and 2mM acetic acid) and B (05permil formic acid and2mM acetic acid in acetonitrile methyl alcohol [1 1]) witha linear gradient 0ndashminus10min (5ndashminus20 B) or 10ndashminus22min(20ndashminus95 B) The flow rate was 04mLmin the sampleinjection volume was 5 120583L and the column and sampletemperature were 40∘CThe diode array detector (DAD) wasset at 200 220 250 and 280 nm for real-time monitoringof the peak intensity Mass spectra were simultaneously

acquired using electrospray ionization with positive andnegative ionization (POS and NEG) modes at fragmentationvoltages (40 psi) over the range of119898119911 50ndash1250

23 Mice DBA1J male mice (119899 = 6 per group 7-8 weeksold) were purchased from HFK Bioscience Co Ltd (BeijingChina) Animal care and use were in accordance with institu-tional guidelines and all animal experiments were approvedby the Institutional Animal Care and Use Committee ofthe National Institute of State Scientific and TechnologicalCommission

24 Induction of CIA Mice except mice in the normalgroup were immunized intradermally at the base of the tailwith 150120583g of bovine type II collagen (CII) (Sigma CAUSA) emulsified with an equal volume of complete Freundrsquosadjuvant (CFA) (Sigma CA USA) Mice were boosted byintradermal injection with 150 120583g of CII emulsified withincomplete Freundrsquos adjuvant (IFA) on the 21st day afterimmunization

25 Drug Treatment Drug treatment began after boosterimmunization and lasted for 28 days Mice were randomlydivided into four groups as follows normal group mice fedthe control diet and orally given sterile saline model groupmice fed the same as the normal group LEF group (Batchnumber 130126 Cinkate Corporation Beijing China) micefed the control diet and orally daily given 2mgkg LEF for 28days XFHM group mice fed the control diet and orally given53 gkg XFHM daily for 28 days The mice were sacrificedon the 29th day after treatment Mice were anesthetized byisoflurane anesthesia (2-3 isoflurane with oxygen supply)PB was obtained by removing eyeballs and the left legs andhind paws and spleens were removed

26 Clinical Scores Clinical CIA scores were monitoredevery 7 days after booster immunization Arthritic severityscores were derived as follows [18] The clinical scores forpaws were classified as 0 (normal joints) 1 (swelling in onedigit or joint inflammation) 2 (swelling in two or three digitsor slight paw swelling) 3 (swelling in more than four digitsand moderate swelling in the entire paw) and 4 (severeswelling and deformation of the paw) The sum of the scoresof all four paws of each mouse represented the total clinicalscore

27 Histology The left legs and hind paws of mice wereremoved fixed with 4 paraformaldehyde in phosphate-buffered saline and then decalcified for 10 days with EDTAand embedded in paraffinThe paraffin sections were stainedwith hematoxylin-eosin (HE) The specimens were observedand photographed under a light microscope (DM RAS2Leica Solms Germany) For Safranin O staining sectionswere placed in hematoxylin for 2min and then washed inwater for 5min Sections were then placed in 01 SafraninO solution for a further 2min and washed again in water for30 sec Finally the sections were passed through a series ofindustrial methylated spirit concentrations (70 to 100) for









3 Results



Inte

nsity

1

2

349

11

13

16

18

19

2021


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0e01e52e53e54e55e56e57e58e5

(a)

Inte

nsity


2

5

67 8

10

12

1416

1517

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

00e010e520e530e540e550e560e570e580e590e510e6

(b)




8H8O4


16H24O10


9H8O4


16H18O9


16H20O9


23H28O11


17H26O10


10H10O3


26H28O14


22H28O11


30H32O15


16H18O6


29H36O15


22H22O9


23H26O10


16H12O5


22H20O11


15H12O5


16H12O4


16H14O4


41H68O14








NormalModel

LEFXFHM

0

2

4

6

8

10

12

14

16

Clin

ical

scor

e

7 14 21 280(Days)

lowastlowast

lowastlowast

lowastlowast

(a)

(A) (B)

(C) (D)

lowast

lowast

lowast

(b)

(A) (B)

(C) (D)

(c)

(A) (B)

(C) (D)

(d)







100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100 101 102 103

CD4

CD4

CD4

CD4

CD3 CD3 CD3 CD3


03

12

571 120

169478

342

550

02

122

326

118482

398307 02 20 156

747 78

763 77

14118

88731

16318

94707

18223

0

10

20

30

40

50

0

5

10

15

20

25

Perc

enta

ges o

fCD

3+

CD4+

cells

()

Perc

enta

ges o

fCD

3+

CD4+

cells

()


lowastlowast

lowast

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

(A) (B)

(C) (D)

(A) (B)

(C) (D)(a)

(b)

(c)

(d)






4CD

4

347

583

11

26

41

37

585

336 37

33

607

333

517 43

49190

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

IFN120574 IFN120574

(A) (B)

(C) (D)

(a)


Perc

enta

ges o

fCD

4+

IFN120574+

cells

()

0

2

4

6lowastlowast

(b)

0

50

100

150

200

Fluo

resc

ence

inte

nsity


lowastIFN120574

(c)

T-bet

GAPDH


05

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

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

lowastlowast

(d)






100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

247

733

12

08

08

13

784

194

06752

10232

729

245

08

18

CD4

CD4

IL-4 IL-4

L1 L2

L3 L4

L1 L2

L3 L4

L1 L2

L3 L4

l1 l2

l3 l4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of


CD4+

IL-4

+ce

lls (

)

00

05

10

15

20

lowast

(b)

IL-4


100

200

300

Fluo

resc

ence

inte

nsity

lowastlowast

(c)

GATA-3

GAPDH


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

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(d)






100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results



Inte

nsity

1

2

349

11

13

16

18

19

2021


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0e01e52e53e54e55e56e57e58e5

(a)

Inte

nsity


2

5

67 8

10

12

1416

1517

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

00e010e520e530e540e550e560e570e580e590e510e6

(b)




8H8O4


16H24O10


9H8O4


16H18O9


16H20O9


23H28O11


17H26O10


10H10O3


26H28O14


22H28O11


30H32O15


16H18O6


29H36O15


22H22O9


23H26O10


16H12O5


22H20O11


15H12O5


16H12O4


16H14O4


41H68O14








NormalModel

LEFXFHM

0

2

4

6

8

10

12

14

16

Clin

ical

scor

e

7 14 21 280(Days)

lowastlowast

lowastlowast

lowastlowast

(a)

(A) (B)

(C) (D)

lowast

lowast

lowast

(b)

(A) (B)

(C) (D)

(c)

(A) (B)

(C) (D)

(d)







100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100 101 102 103

CD4

CD4

CD4

CD4

CD3 CD3 CD3 CD3


03

12

571 120

169478

342

550

02

122

326

118482

398307 02 20 156

747 78

763 77

14118

88731

16318

94707

18223

0

10

20

30

40

50

0

5

10

15

20

25

Perc

enta

ges o

fCD

3+

CD4+

cells

()

Perc

enta

ges o

fCD

3+

CD4+

cells

()


lowastlowast

lowast

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

(A) (B)

(C) (D)

(A) (B)

(C) (D)(a)

(b)

(c)

(d)






4CD

4

347

583

11

26

41

37

585

336 37

33

607

333

517 43

49190

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

IFN120574 IFN120574

(A) (B)

(C) (D)

(a)


Perc

enta

ges o

fCD

4+

IFN120574+

cells

()

0

2

4

6lowastlowast

(b)

0

50

100

150

200

Fluo

resc

ence

inte

nsity


lowastIFN120574

(c)

T-bet

GAPDH


05

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

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

lowastlowast

(d)






100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

247

733

12

08

08

13

784

194

06752

10232

729

245

08

18

CD4

CD4

IL-4 IL-4

L1 L2

L3 L4

L1 L2

L3 L4

L1 L2

L3 L4

l1 l2

l3 l4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of


CD4+

IL-4

+ce

lls (

)

00

05

10

15

20

lowast

(b)

IL-4


100

200

300

Fluo

resc

ence

inte

nsity

lowastlowast

(c)

GATA-3

GAPDH


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

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(d)






100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Inte

nsity

1

2

349

11

13

16

18

19

2021


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0e01e52e53e54e55e56e57e58e5

(a)

Inte

nsity


2

5

67 8

10

12

1416

1517

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

00e010e520e530e540e550e560e570e580e590e510e6

(b)




8H8O4


16H24O10


9H8O4


16H18O9


16H20O9


23H28O11


17H26O10


10H10O3


26H28O14


22H28O11


30H32O15


16H18O6


29H36O15


22H22O9


23H26O10


16H12O5


22H20O11


15H12O5


16H12O4


16H14O4


41H68O14








NormalModel

LEFXFHM

0

2

4

6

8

10

12

14

16

Clin

ical

scor

e

7 14 21 280(Days)

lowastlowast

lowastlowast

lowastlowast

(a)

(A) (B)

(C) (D)

lowast

lowast

lowast

(b)

(A) (B)

(C) (D)

(c)

(A) (B)

(C) (D)

(d)







100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100 101 102 103

CD4

CD4

CD4

CD4

CD3 CD3 CD3 CD3


03

12

571 120

169478

342

550

02

122

326

118482

398307 02 20 156

747 78

763 77

14118

88731

16318

94707

18223

0

10

20

30

40

50

0

5

10

15

20

25

Perc

enta

ges o

fCD

3+

CD4+

cells

()

Perc

enta

ges o

fCD

3+

CD4+

cells

()


lowastlowast

lowast

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

(A) (B)

(C) (D)

(A) (B)

(C) (D)(a)

(b)

(c)

(d)






4CD

4

347

583

11

26

41

37

585

336 37

33

607

333

517 43

49190

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

IFN120574 IFN120574

(A) (B)

(C) (D)

(a)


Perc

enta

ges o

fCD

4+

IFN120574+

cells

()

0

2

4

6lowastlowast

(b)

0

50

100

150

200

Fluo

resc

ence

inte

nsity


lowastIFN120574

(c)

T-bet

GAPDH


05

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

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

lowastlowast

(d)






100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

247

733

12

08

08

13

784

194

06752

10232

729

245

08

18

CD4

CD4

IL-4 IL-4

L1 L2

L3 L4

L1 L2

L3 L4

L1 L2

L3 L4

l1 l2

l3 l4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of


CD4+

IL-4

+ce

lls (

)

00

05

10

15

20

lowast

(b)

IL-4


100

200

300

Fluo

resc

ence

inte

nsity

lowastlowast

(c)

GATA-3

GAPDH


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

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(d)






100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NormalModel

LEFXFHM

0

2

4

6

8

10

12

14

16

Clin

ical

scor

e

7 14 21 280(Days)

lowastlowast

lowastlowast

lowastlowast

(a)

(A) (B)

(C) (D)

lowast

lowast

lowast

(b)

(A) (B)

(C) (D)

(c)

(A) (B)

(C) (D)

(d)







100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100 101 102 103

CD4

CD4

CD4

CD4

CD3 CD3 CD3 CD3


03

12

571 120

169478

342

550

02

122

326

118482

398307 02 20 156

747 78

763 77

14118

88731

16318

94707

18223

0

10

20

30

40

50

0

5

10

15

20

25

Perc

enta

ges o

fCD

3+

CD4+

cells

()

Perc

enta

ges o

fCD

3+

CD4+

cells

()


lowastlowast

lowast

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

(A) (B)

(C) (D)

(A) (B)

(C) (D)(a)

(b)

(c)

(d)






4CD

4

347

583

11

26

41

37

585

336 37

33

607

333

517 43

49190

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

IFN120574 IFN120574

(A) (B)

(C) (D)

(a)


Perc

enta

ges o

fCD

4+

IFN120574+

cells

()

0

2

4

6lowastlowast

(b)

0

50

100

150

200

Fluo

resc

ence

inte

nsity


lowastIFN120574

(c)

T-bet

GAPDH


05

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

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

lowastlowast

(d)






100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

247

733

12

08

08

13

784

194

06752

10232

729

245

08

18

CD4

CD4

IL-4 IL-4

L1 L2

L3 L4

L1 L2

L3 L4

L1 L2

L3 L4

l1 l2

l3 l4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of


CD4+

IL-4

+ce

lls (

)

00

05

10

15

20

lowast

(b)

IL-4


100

200

300

Fluo

resc

ence

inte

nsity

lowastlowast

(c)

GATA-3

GAPDH


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

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(d)






100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100 101 102 103

CD4

CD4

CD4

CD4

CD3 CD3 CD3 CD3


03

12

571 120

169478

342

550

02

122

326

118482

398307 02 20 156

747 78

763 77

14118

88731

16318

94707

18223

0

10

20

30

40

50

0

5

10

15

20

25

Perc

enta

ges o

fCD

3+

CD4+

cells

()

Perc

enta

ges o

fCD

3+

CD4+

cells

()


lowastlowast

lowast

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

(A) (B)

(C) (D)

(A) (B)

(C) (D)(a)

(b)

(c)

(d)






4CD

4

347

583

11

26

41

37

585

336 37

33

607

333

517 43

49190

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

IFN120574 IFN120574

(A) (B)

(C) (D)

(a)


Perc

enta

ges o

fCD

4+

IFN120574+

cells

()

0

2

4

6lowastlowast

(b)

0

50

100

150

200

Fluo

resc

ence

inte

nsity


lowastIFN120574

(c)

T-bet

GAPDH


05

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

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

lowastlowast

(d)






100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

247

733

12

08

08

13

784

194

06752

10232

729

245

08

18

CD4

CD4

IL-4 IL-4

L1 L2

L3 L4

L1 L2

L3 L4

L1 L2

L3 L4

l1 l2

l3 l4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of


CD4+

IL-4

+ce

lls (

)

00

05

10

15

20

lowast

(b)

IL-4


100

200

300

Fluo

resc

ence

inte

nsity

lowastlowast

(c)

GATA-3

GAPDH


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

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(d)






100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4CD

4

347

583

11

26

41

37

585

336 37

33

607

333

517 43

49190

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

l1 l2

l3 l4

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

IFN120574 IFN120574

(A) (B)

(C) (D)

(a)


Perc

enta

ges o

fCD

4+

IFN120574+

cells

()

0

2

4

6lowastlowast

(b)

0

50

100

150

200

Fluo

resc

ence

inte

nsity


lowastIFN120574

(c)

T-bet

GAPDH


05

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

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

lowastlowast

(d)






100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

247

733

12

08

08

13

784

194

06752

10232

729

245

08

18

CD4

CD4

IL-4 IL-4

L1 L2

L3 L4

L1 L2

L3 L4

L1 L2

L3 L4

l1 l2

l3 l4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of


CD4+

IL-4

+ce

lls (

)

00

05

10

15

20

lowast

(b)

IL-4


100

200

300

Fluo

resc

ence

inte

nsity

lowastlowast

(c)

GATA-3

GAPDH


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

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(d)






100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

247

733

12

08

08

13

784

194

06752

10232

729

245

08

18

CD4

CD4

IL-4 IL-4

L1 L2

L3 L4

L1 L2

L3 L4

L1 L2

L3 L4

l1 l2

l3 l4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of


CD4+

IL-4

+ce

lls (

)

00

05

10

15

20

lowast

(b)

IL-4


100

200

300

Fluo

resc

ence

inte

nsity

lowastlowast

(c)

GATA-3

GAPDH


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

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(d)






100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103 100 101 102 103

100 101 102 103100 101 102 103

CD4

100

101

102

103

CD4

IL-17 IL-17

210

13

0908

30 24217

08

38

761

750730

232

753

24 193G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

G1 G2

G3 G4

(A) (B)

(C) (D)

(a)Pe

rcen

tage

s of

CD4+

IL-17+

cells

()


lowastlowast

0

2

4

6

(b)

IL-17


lowastlowast

0

50

100

150

Fluo

resc

ence

inte

nsity

(c)

ROR120574

ROR120574

STAT3

GAPDH

STAT3



lowastlowast

lowastlowast

00

03

06

09

12

Rela

tive e

xpre

ssio

n

00

05

10

15

20

Rela

tive e

xpre

ssio

n

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



FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















FS li

n

SS lin CD4

FS li

n

SS lin CD4

A 650 H 308 H 160A 728

100 101 102 103 100 101 102 10310230 0 1023

35 39

(a1) (a2)

(a)

CD25

CD25

FOXP3

CD25

CD25

FOXP3

98

00

04

03

893

103

862

130

02

06

07896

0989

02897

201

14

25

79

19

91

27

254

636

716736

703

193

7049

188

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

E1 E2

E3 E4

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100 101 102 103

FOXP3100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103

FOXP3100 101 102 103

100 101 102 103100 101 102 103

(b1) (b2)

(A) (B) (A) (B)

(C) (D) (C) (D)

(b)

Perc

enta

ges o

f


00

02

04

06

08

10

0

2

4

6

8

10

lowastlowast

lowast


CD4+CD25+FOXP3+

cells

()

Perc

enta

ges o

fCD4+CD25+FOXP3+

cells

()

(c1) (c2)

(c)



4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4 Discussion









Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Competing Interests




Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Xianfanghuomingyin, a Chinese...

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