Research Article Therapeutic Effect of Chung-Pae, an...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 659459 11 pageshttpdxdoiorg1011552013659459

Research ArticleTherapeutic Effect of Chung-Pae an Experimental HerbalFormula on Acute Lung Inflammation Is Associated withSuppression of NF-120581B and Activation of Nrf2

Kyun Ha Kim1 Do-Hyun Kim2 Nara Jeong1 Kwan-Il Kim2 Yong Ho Kim3 Mei Lee2

Jun-Yong Choi45 Hee Jae Jung2 Sung-Ki Jung2 and Myungsoo Joo1

1 Division of Applied Medicine School of Korean Medicine Pusan National University Yangsan 626-870 Republic of Korea2Division of Allergy Immune and Respiratory System Department of Internal Medicine College of Oriental MedicineKyung Hee University Seoul 130-701 Republic of Korea

3 College of Korean Medicine Daegu Haany University Daegu 706-060 Republic of Korea4Department of Korean Medical Science School of Korean Medicine Pusan National University Yangsan 626-870 Republic of Korea5 Department of Internal Medicine Korean Medicine Hospital Pusan National University Yangsan 626-870 Republic of Korea

Correspondence should be addressed to Sung-Ki Jung jskes1gmailcom and Myungsoo Joo mjoopusanackr

Received 2 April 2013 Accepted 24 July 2013

Academic Editor Muhammad Nabeel Ghayur

Copyright copy 2013 Kyun Ha Kim 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

Acute lung injury (ALI) is an inflammatory disease with high mortality but therapeutics against it is unavailable Recently weelaborated a formula named Chung-pae (CP) that comprises four ethnic herbs commonly prescribed against various respiratorydiseases inAsian traditionalmedicine CP is being administered in aerosol to relieve various respiratory symptoms of patients in ourclinic Here we sought to examine whether CP has a therapeutic effect on ALI and to uncover the mechanism behind it Reporterassays show thatCP suppressed the transcriptional activity of proinflammatoryNF-120581B and activated that of anti-inflammatoryNrf2Similarly CP suppressed the expression ofNF-120581Bdependent proinflammatory cytokines and induced that ofNrf2 dependent genesin RAW2647 An aerosol intratracheal administration of CP effectively reduced neutrophilic infiltration and the expression of pro-inflammatory cytokines hallmarks of ALI in the lungs of mice that received a prior intraperitoneal injection of lipopolysaccharideThe intratracheal CP administration concomitantly enhanced the expression of Nrf2 dependent genes in the lung Therefore ourresults evidenced a therapeutic effect of CP on ALI in which differential regulation of the two key inflammatory factors NF-120581Band Nrf2 was involved We propose that CP can be a new therapeutic formula against ALI

1 Introduction

Acute lung injury (ALI) is a severe inflammatory disease withsubstantial morbidity and mortality in human Hallmarks ofALI include neutrophilic infiltration to the lung parenchymaabnormal lung compliance and impaired gas exchange [1ndash3] While systemic inflammation by bacterial infection isthe major cause of ALI severe multiple trauma aspirationpneumonia and complications of mechanical ventilationoften lead to ALI [1] Despite extensive studies and clinicaltrials therapeutic measures against the disease have beenelusive [4]

Lipopolysaccharide (LPS) a cell wall component ofGram-negative bacteria is known as a major activator ofinflammatory response that leads to ALI It binds to TLR4 toactivate NF-120581B which largely accounts for the production ofproinflammatory cytokines including tumor necrosis factor-120572 (TNF-120572) and interleukin (IL)-1 -6 -8 -10 -12 and -15families [5] These cytokines contribute to recruiting neu-trophils to the lungs where neutrophils clear up infectiousagents Since NF-120581B is also found to be chronically activein many other inflammatory diseases such as inflammatorybowel disease arthritis and gastritis [5] therapeutics strate-gies have been focused on attenuating NF-120581B activity and

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Composition of Chung-pae

Scientific name Weight (g)Ephedrae Herba 2Pogostemonis (Agastachis) Herba 2Caryophylli Flos 1Zingiberis Rhizoma Crudus 1Total 6

the production of Proinflammatory cytokines which turnsout to be ineffective [6] On the other hand accumulatingevidence suggests that Nrf2 plays an essential role in protect-ing against various inflammatory diseases including acutepulmonary injury smoke-induced emphysema and asthma[7ndash9] Although Nrf2 is emerging as a potential therapeutictarget for inflammatory diseases [9 10] no effective thera-peutics against ALI is available yet

Several herbal medicinal formulas have been prescribedfor the treatment of various lung diseases in Asian traditionalmedicine [11] Therefore it is possible that these formulas area resource for the development of a new therapeutic measureagainst deadly respiratory diseases Popular herbal formulasagainst respiratory diseases include Ma-Huang Tang that hasbeen used against cough with rigor Junghangshichae-tangagainst cough with phlegm and nausea Gwackhyangjungki-san against cold with nausea and abdominal pain and Kyeji-tang against cold with headache and fever [11] Although eachherbal formula is typically composed of several herbs it con-tains a key herb that accounts for the efficacy of a formula Forinstance Ephedrae Herba is the key ingredient of Ma-HuangTang Caryophylli Flos is the key herb in Junghangshichae-tang and Pogostemonis (Agastachis) Herba and ZingiberisRhizomaCrudus are key herbs inGwackhyangjungki-san andKyeji-tang respectively In the hope of obtaining a herbalmedicine effective on a broad spectrum of respiratory dis-eases we formulated a new herbal medicine named Chung-pae (CP) that is composed of these key herbs contained inthe four formulas CP is being used as a complementarymea-sure in the respiratory clinic at Kyung-HeeOrientalMedicineHospital Seoul Korea to relieve the symptoms of patientswith dyspnea and cough Although it seemed that CP helpsimprove the symptoms experimental evidence supporting itseffect is scarce Here we sought to examine whether CP hasa therapeutic effect on respiratory diseases and if it doeshow CP exerts its effect To this end we used an LPS-inducedALI mouse model Since CP is being administered in aerosolto the patients we delivered CP in aerosol to the lung anddetermined the effect of CP on ALI in mice To obtain aninsight on the underlying mechanism we tested whetherCP affects the activities of Proinflammatory factor NF-120581Bandor anti-inflammatory factor Nrf2 Our findings showthat aerosol delivery of CP to the lung effectively reducedthe hallmarks of ALI which was appeared to be associatedwith suppression of Proinflammatory factor NF-120581B andactivation of anti-inflammatory factor Nrf2

2 Material and Methods

21 Preparation of the Water Extract of Chung-Pae Theherbs composing of Chung-pae (CP) shown in Table 1 werepurchased from Kwang-Myoung-Dang herb store (PusanRepublic of Korea) and identified by Professor J Y Choi(School of Korean Medicine Pusan National UniversityYangsan Republic of Korea) The voucher specimen (num-ber pnukh004) is kept in the herbarium stock room of theSchool of Korean Medicine Pusan National University Acrude decoction ofCPwas obtained by boiling 60 g ofChung-pae in 400mLdistilledwater for 2 hours followed by filtrationthrough 045 120583m filter The resultant decoction was concen-trated to 50mL by a low-pressure evaporator and underwentfreeze-drying processes to yield 6 g of powder Appropriateamount of the powder was dissolved in phosphate bufferedsaline (PBS) prior to experiment

22 Reagents and Antibodies 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide Sulforaphane and E coliLPS (serotype 055 B5) for animal study were from SigmaChemical Co (St Louis MO USA) TLR4-specific E coliLPS was purchased fromAlexis Biochemical (San Diego CAUSA)

23 Animals Male C57BL6 mice inbred in a specificpathogen-free (SPF) facility were purchased from SamtacoBio Korea Ltd (Osan Korea) Animals were housed incertified standard laboratory cages and fed with food andwater ad libitum prior to experiment

24 ALIMouseModel and Intrapulmonary Delivery of Chung-Pae All experimental procedures followed the NIH of KoreaGuidelines for the Care and Use of Laboratory Animals andall the experiments were approved by the Institutional Ani-mal Care and Use Committee of Pusan National University(protocol number PNU-2010-00028) Mice anesthetized byZoletil (Virbac Carros cedex France) received a single doseof 10mgkg LPS or sterile saline via intraperitoneal (ip)route At 2 h after ip LPS either PBS or CP (5 and 20mgkgbody weight) in 25 120583L of PBS was loaded in MicroSprayerAerosolizer-Model IA-1C (Penn-Century Wyndmoor PAUSA) and delivered in aerosol to the lung via intratracheal(it) under visual guidance At 24 h after LPS treatmentmice were euthanized by CO

2gas The trachea was exposed

through midline incision and cannulated with a sterile 24-gauge intravascular catheter Bilateral bronchoalveolar lavage(BAL) was performed by two consecutive instillations of10mL of PBS Total cell numbers in BAL fluid were countedwith hemocytometer and then centrifuged by a cytospin andstained for the differentiation of macrophages lymphocytesor neutrophils by Hemacolor (Merck Darmstadt Germany)Three hundred cells in total were counted and one hundredof the cells in each microscopic field were scored The meannumber of cells per field was reported For the analysis oflung tissue mice were perfused with saline and the wholelung was inflated with fixatives After paraffin embedding5 120583m sections were cut and placed on charged slides and

Evidence-Based Complementary and Alternative Medicine 3

stained with hematoxylin and eosin (HampE) staining methodThree separate HampE-stained sections were evaluated in 200xmicroscopic magnifications per mouse

25 Cell Culture RAW 2647 cells (American Type CultureCollection Rockville MD USA) were cultured in DulbeccorsquosModified Eaglersquos Medium (DMEM) containing L-glutamine(200mgL) (Invitrogen Carlsbad CA USA) supplementedwith 10 (vv) heat-inactivated fetal bovine serum (FBS) and100UmL penicillin and 100 120583gmL streptomycin (Invitro-gen Carlsbad CA USA) and maintained in a humidifiedincubator at 37∘C and 5 CO

2prior to experiment

26 Microculture Tetrazolium (MTT) Assay MTT assay wasperformed to evaluate the cytotoxicity of CP RAW2647 cells(10times 104 cellswell) were treatedwithCP for 16 hwhereMTTsolution was added After 4 h incubation in cell culture incu-bator formazan crystals formed in viable cells were dissolvedwith DMSO and the optical density (OD) of formazan wasmeasured at 540 nm with a microplate reader Cell viabilitywas calculated as a percentage against the untreated Allexperiments were performed three times independently

27 Reporter Constructs Reporter Cell Line and LuciferaseAssay To estimate Nrf2 and NF-120581B transcriptional activ-ity we used reporter cell lines stably harboring an NQO-1luciferase reporter and NF-120581Bluciferase reporter con-structs [6 7] Luciferase activity was measured by a luciferaseassay kit (Promega Madison WI USA) per the manufac-turersquos instruction and normalized by the amount of totalproteins of the cell extract

28 Isolation of Total RNA from Cells and RT-PCR TotalRNAwas isolated from right lung homogenateswith TRIZOLreagent (GeneAll Korea) according to the manufacturerrsquosinstructions Two micrograms of total RNA were reverse-transcribed by M-MLV reverse transcriptase (Promega)Target mRNA was quantified by using end-point dilutionPCR including three serial 1 to 5 dilutions (1 1 1 5 1 25and 1 125) of RT products for PCR amplification The levelof GAPDH (glyceraldehyde-3-phosphate dehydrogenase)cDNA from each sample was used to normalize the samplesfor differences in PCR efficiency For PCR amplificationTaqPCRx DNA polymerase (Invitrogen) and the manufac-turerrsquos protocol were used Resultant cDNA was amplifiedby PCR with a set of specific primers The forward andthe reverse primers for NQO-1 were 51015840-GCAGTGCTTTCC-ATCACCAC-31015840 and 51015840-TGGAGTGTGCCCAATGCTAT-31015840 the primers for HO-1 were 51015840-TGAAGGAGGCCACCA-AGGAGG-31015840 and 51015840-AGAGGTCACCCAGGTAGCGGG-31015840the primers for GCLC were 51015840-CACTGCCAGAACACA-GACCC-31015840 and 51015840-ATGGTCTGGCTGAGAAGCCT-31015840 theprimers for IL-1120573 were 51015840-GTGTCTTTCCCGTGGACCTT-31015840 and 51015840-TCGTTGCTTGGTTCTCCTTG-31015840 the primersfor TNF-120572 were 51015840-CTACTCCTCAGAGCCCCCAG-31015840 and51015840-AGGCAACCTGACCACTCTCC-31015840 and the primersfor GAPDH were 51015840-GGAGCCAAAAGGGTCATCAT-31015840and 51015840-GTGATGGCATGGACTGTGGT-31015840 The reaction

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conditions were as follows an initial denaturation at 95∘Cfor 5min followed by 28 cycles of denaturation for 30 sec at95∘C annealing for 30 sec at 58∘C and extension for 40 secat 72∘C with a final extension for 7min at 72∘C Ampliconswere separated in 15 agarose gels GAPDH was used asinternal controls to evaluate relative expressions of TNF-120572IL-1120573 GCLC HO-1 and NQO1 Relative expression of eachgene over GAPDHwas determined by densitometric analysissoftware ImageJ (Wayne Rasband Research Services BranchNational Institute of Mental Health Bethesda MD USA)Reactions were separated in 12 agarose gels in 1 times TBEbuffer at 100V for 30min stained with SYBR safe DNA gelstain (Invitrogen) and visualized under LED light

29 Statistical Analysis To compare the results amonggroups one-way analysis of variance (ANOVA) tests withTukeyrsquos post hoc test was used (with the assistance of InStatGraphpad Software Inc San Diego CA) (119875 values lt 005 areconsidered significant) All experiments were performed atleast three times independently

3 Results

31 The Water Extract of Chung-Pae Suppresses the Tran-scriptional Activity of NF-120581B and the Expression of Proin-flammatory Cytokines in RAW 2647 Cells For the study weprepared and used the water extract of CP First we testedwhether CP has any cellular toxicity RAW 2647 cells amurine macrophage-like cell line were treated with variousamounts of CP from 1 120583gmL to 50120583gmL At 16 h aftertreatments MTT assay was performed As shown in Figure 1CP showed a slight cytotoxicity within the range of 20120583gmLbut a significant cytotoxicity at 50120583gmL However MTTassay at 12 h after treatments showed no cytotoxicity withinthe range of 20120583gmL while 50120583gmL of CP did a significantcytotoxicity (data not shown) Therefore we chose to use


the amounts ranged from 5 120583gmL to 20120583gmL of CP in thisstudy

Next we tested the possibility that CP exerts its effectby regulating NF-120581B activity given that NF-120581B regulatesexpressions of Proinflammatory cytokines and chemokinesincluding TNF-120572 IL-1 6 8 10 12 15 and MIP-1120572 [5] Todetermine whether CP affects the transcriptional activity ofNF-120581B we took a RAW 2647 cell line that stably harborsan NF-120581B-luciferase reporter construct [12] and treated itwith different amounts of CP for 16 h and subsequentlytreated with LPS (01 120583gmL) At 8 h after LPS treatmenttotal cell lysate was prepared for luciferase assay As shownin Figure 2(a) 10 120583gmL or 20120583gmL of CP significantlyreduced luciferase activity driven by activated NF-120581B sug-gesting that CP suppresses the transcriptional activity ofNF-120581B To determine whether decrease of NF-120581B activityresults in reduced expression of NF-120581B dependent geneswe performed similar experiments with RAW 2647 cellsand analyzed the expression of Proinflammatory cytokinesgoverned by NF-120581B As shown in Figure 2(b) expressions ofIL-1120573 and TNF-120572 were similarly decreased by CP treatmentThese results indicate that CP suppresses NF-120581B activitycontributing to suppression of inflammatory gene expression

32 CP Activates the Transcriptional Activity of Nrf2 andInduces Expression of Nrf2-Dependent Genes in RAW 2647Cells Accumulating evidence suggest that Nrf2 is a mas-ter anti-inflammatory factor that prevents from acute lunginflammation [8 13 14] Therefore we tested the possibilitythat CP affects Nrf2 activity contributing to the effect ofCP We used an Nrf2-luciferase reporter cell line derivedfrom RAW 2647 cells [15] and treated it with increasingamounts of CP (1 5 10 20120583gmL) At 16 h after treatmenttotal cell lysate was prepared for luciferase assay As shown inFigure 3(a) similar to treatment with sulforaphane (5120583M) awell-documentedNrf2 activator [16] CP treatment increasedthe luciferase activity in a dose dependentmanner suggestingthat CP activates the transcriptional activity ofNrf2 To deter-mine whether activation of Nrf2 results in the expression ofNrf2 dependent genes we performed similar experiments todetermine whether CP treatment induces the expression ofNrf2 dependent genes RAW 2647 cells were treated withCP as described above and total RNA of the treated cellswas extracted for semiquantitative RT-PCRanalyses ofNQO-1 HO-1 and GCLC (Figure 3(b)) prototypical Nrf2 targetgenes [17 18] As shown in Figure 3(b) CP treatment inducedthe expression of Nrf2 dependent genes Combined with CPsuppressing NF-120581B these results suggest that CP exert ananti-inflammatory function by both suppressing NF-120581B andactivating Nrf2

33 Intratracheal Delivery of CP Ameliorates NeutrophilicInfiltration to the Lung in an LPS-Induced ALI Mouse ModelSince our results show that CP activated Nrf2 a key tran-scription factor that has been known to protect from acutelung injury (ALI) [14] we testedwhether CPhas a therapeuticeffect on ALI To this end we set up an LPS-inducedALI mouse model Mice received an ip LPS (10mgkg

body weight) for the induction of lung inflammation At 2 hafter LPS injection various amounts of CPwere administeredto the lungs of LPS-treated mice Given that daily dose of CPto patients is 5mgkg body weight and that our results indi-cated that CPwas effective in activatingNrf2 from 5 120583gmL to20120583gmL (Figure 3) we tested two different doses 5mgkgequivalent to the dose for patients and 20mgkg of CP SinceCP is being prescribed in a form of inhalant CP was loadedin a microsprayer and delivered in aerosol to the lung viatrachea At 24 h after LPS treatment mice were euthanizedand the lungs were harvested for the analysis of the effectof CP on neutrophilic infiltration to the lung As shown inFigure 4(a) HampE staining of lung sections shows that whilecontrols received sham or CP (20mgkg) (119899 = 5group) onlymaintained alveolar structure intact (top two panels) LPS-injected mice developed a characteristic lung structure dueto inflammation (1st panel in the bottom) However it CPadministration either 5mgkg or 20mgkg ameliorated theinflammatory lung structure (bottom 2 panels from left)

To determine whether CP regulates neutrophilic infil-tration we performed bronchoalveolar lavage (BAL) andcounted the infiltrates in BAL fluid As shown in Figures4(b) and 4(c) while LPS administration increased the cel-lular infiltration to the lung in which neutrophils werepredominant (3rd columns from the left) both doses of CPsignificantly reduced the number of neutrophils in the lung(4th and 5th columns) Together these results show thatCP posttreatment relieved neutrophilic lung inflammationinduced by LPS suggesting that CP has a therapeutic effecton ALI

34 Intratracheal Delivery of CP Reduces the Expressionof Proinflammatory Cytokines and Activates That of Nrf2-Dependent Genes in the Lung Since our results withmacrophage implicatedCP in suppressingNF-120581B activity andactivating Nrf2 we tested whether the therapeutic effect ofCP on lung inflammation in ALI mice is associated withsuppressed NF-120581B and activated Nrf2 We treated mice asdescribed in Figure 4 and harvested the lungs of mice (119899 =5group) Total RNA in the lung was extracted quantitatedand analyzed by semiquantitative RT-PCR As shown inFigure 5(a) expressions of Proinflammatory cytokine genessuch as TNF-120572 and IL-1120573 were decreased by it CP admin-istration Meanwhile expressions of Nrf2 dependent genessuch as NQO-1 HO-1 and GCLC were enhanced by itCP administration (Figure 5(b)) Both suppression of theexpression of Proinflammatory cytokines and enhancementof Nrf2-dependent gene expression became apparent whenmice received a higher dose of CP In any event these resultssuggest that the therapeutic effect of CP is associated withsuppression of NF-120581B and activation of Nrf2

4 Discussion

In this study we sought to obtain experimental evidencethat CP has a therapeutic effect on ALI and to unveilunderlying mechanisms for the effect Since CP comprisesfour herbs that have been reported to have anti-inflammatory


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Figure 2 Chung-pae suppresses NF-120581B transcriptional activity and NF-120581B dependent gene expression (a) The transcriptional activity NF-120581B was measured in an NF-120581B reporter cell line derived from RAW 2647 cells The cell line was pretreated with indicated amounts of CP for16 h and then subsequently with TLR4 specific LPS (01 120583gmL) for 8 h Luciferase activity was normalized by the amount of total proteins incell lysate Treatment with 5120583gmL of CP was not statistically significant lowast119875 was less than 005 compared to the LPS-treated Data representthe mean plusmn SEM of three independent experiments (b) RAW 2647 cells were treated with CP and LPS as in (a) Total RNA was extractedand analyzed by semiquantitative RT-PCR for TNF-120572 and IL-1120573 The intensity of each PCR band was measured by densitometric analysis(ImageJ) and the relative expression of each gene was calculated over GAPDH lowast119875 was less than 005 compared to the LPS treated Data arepresented as the mean plusmn SEM of 3 separate experiments

activities in vitro we hypothesized that the effect of CP onrespiratory symptoms is related to the anti-inflammatoryactivities exerted by its constituents Our results show thatCP suppressed neutrophilic infiltration to the lung and the

production of Proinflammatory cytokines hallmarks of ALIin an LPS-induced ALI mouse model which was associatedwith suppression of Proinflammatory transcription factorNF-120581B and activation of anti-inflammatory factor Nrf2


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Figure 3 Chung-pae activates Nrf2 transcriptional activity and induces Nrf2 dependent genes (a) An Nrf2 reporter cell line derived fromRAW2647 cells was treated with SFN (5mM) or the indicated amounts of CP for 16 hours Luciferase activity was normalized by the amountof total proteins Data are shown in the mean plusmn SEM of three independent measurements lowast119875 lowastlowast119875 and lowastlowastlowast119875 were less than 005 001 and0001 respectively compared to untreated control (b) RAW 2647 cells were treated with CP similar to (a) Total RNA was analyzed bysemiquantitative RT-PCR for NQO-1 HO-1 and GCLC The intensity of each PCR band was measured by densitometric analysis (ImageJ)and relative expression of each gene was calculated over GAPDH lowast119875 and lowastlowast119875 were less than 005 compared to the untreated Data arepresented as the mean plusmn SEM of 3 separate experiments

CP is a composite formula ofEphedraeHerba CaryophylliFlos Pogostemonis (Agastachis) Herba and Zingiberis Rhi-zoma Crudus In Asian traditional medicine these herbshave been mainly prescribed for respiratory diseases exceptCaryophylli Flos For instance Ephedrae Herba has beenknown to be effective in reducing wheezing asthma andedema Pogostemonis (Agastachis) Herba is mainly used forcold and nausea in summer and Zingiberis Rhizoma Crudus

is mainly for common cold nausea and cough with phlegm[19] On the other hand Caryophylli Flos has been tradition-ally used for abdominal disorders including vomiting hiccuppain diarrhea and lack of appetite [19] However recentstudies have shown that these herbs have multiple effectsFor instance Ephedrae Herba has antiallergic antiasthmaticanticoagulant bronchodilator smooth muscle relaxant andvasoconstrictor activities [20ndash22] Pogostemonis (Agastachis)


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Figure 4 Aerosol intratracheal Chung-pae administration suppresses acute neutrophilic lung inflammation in LPS-induced ALI animalmodel (a) HampE stained lung sections of C57BL6 mice C57BL6 mice received sham (top panels) or an ip injection of LPS (bottom panels)At 2 h after the treatments mice received 20mgkg (top 2nd panel and bottom 3rd panel) or 5mgkg (bottom 2nd panel) of CP in aerosolvia trachea At 24 h after LPS administration the lungs of mice were analyzed by histological examination Data are representatives of at leastfive different areas of a lung (200x magnifications) Total cells (b) and neutrophils and macrophages (c) in BAL fluid were scored lowast119875 was lessthan 005 compared to the mice treated with LPS only Data are presented as the mean plusmn SEM of 5 mice per group

Herba shows antiemetic antiviral (influenza) antitumorand smooth muscle relaxant activities [20 23] ZingiberisRhizoma Crudus does analgesic antibacterial antiemeticantimutagenic antiulcer hepatoprotective and smoothmus-cle relaxant activities [20 24ndash26] and Caryophylli Flosdoes analgesic anticoagulant antiulcer and smooth muscle

relaxant activities [20 27 28] Nevertheless the fours herbshave shown to have anti-inflammatory activity in common

Although it is not fully understood how CP exerts itsanti-inflammatory activity it appears that CP targets NF-120581B NF-120581B is a protein complex that promotes inflam-mation by expressing Proinflammatory cytokines and is


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Figure 5 Aerosol intratracheal Chung-pae administration suppresses the expression of Proinflammatory cytokines and enhances theexpression ofNrf2-dependent genes in the lungs ofmiceMice (119899 = 5group) received 5mgkg or 20mgkg of CP 2 h after an ip LPS injectionAt 24 h after LPS injection the lungs of variously treatedmice were harvested for semiquantitative RT-PCR analysis of Proinflammatory genes(a) The intensity of each PCR band was measured by densitometric analysis (ImageJ) and relative expression of each gene was calculatedover GAPDH lowast119875 was less than 005 compared to the mice treated with LPS only (b) Similarly the expression of Nrf2 dependent genes inthe lung was analyzed Mice treated with 20mgkg of CP increased the expression of Nrf2 dependent genes Expressions of these genes wereenhanced by CP lowast119875 was less than 005 compared to untreated control and lowastlowast119875 was less than 005 compared to the LPS treated


found ubiquitously in most cells including lung parenchymalcells [29] Aberrant NF-120581B activity has been known tobe associated with many inflammatory diseases includinginflammatory bowel disease [30] arthritis [31] sepsis [32]gastritis [33] asthma [34] COPD [34] and atherosclerosis[5] In addition the link between NF-120581B and inflamma-tion in septic ALI has well been documented [35] Thusregulation of NF-120581B activity has been regarded as a rea-sonable therapeutic target for these inflammatory diseasesTwo constituents of CP Ephedrae Herba and ZingiberisRhizoma Crudus are known to suppress NF-120581B activityin RAW 2647 macrophages stimulated with LPS [36 37]and the other two constituents Pogostemonis (Agastachis)Herba and Caryophylli Flos suppress cytokine productionin LPS-stimulated murine macrophages [38 39] Althoughprecise mechanisms by which the herbs suppress NF-120581Bremain unknown it is presumable that CP composed ofthe four herbs suppresses NF-120581B Indeed our results showthat CP suppressed NF-120581B activity and the production ofProinflammatory cytokines driven by NF-120581B in RAW 2647cells and in the lung

Nrf2 has been found abundantly in tissues and organs thatbear a high level of oxidative stress such as lungs liver brainGI tract kidney spleen heart and muscles [40 41] Recentstudies have shown that Nrf2 is critical to fend the cytotoxiceffects of oxidative stress [42] and plays an important rolein regulating lung inflammation [40 41] These studiessuggest that Nrf2 is an emerging therapeutic target againstinflammatory diseases Our results show that CP activatedNrf2 activating the expression of Nrf2 dependent genes inRAW 2647 cells and in the lung These results indicate thatCP activates Nrf2 and its regulatory genes contributing toanti-inflammatory function of CP

Given our results that CP activated Nrf2 it is conceivablethat Nrf2 activated by CP suppresses NF-120581B contributingto the anti-inflammatory effect of CP Supportive to thisnotion it has been reported that Nrf2 can directly suppressthe functions of NF-120581B and AP-1 resulting in reducedexpression of Proinflammatory cytokines elicited by LPS[43 44] However sulforaphane a potent activator of Nrf2suppresses the expression of Proinflammatory cytokines bypreventing oligomerization of TLR4 triggered by LPS [45]suggesting that Nrf2 may not directly suppress NF-120581B Inaddition neither overexpression of Nrf2 nor activation ofNrf2 by kaurenoic acid suppresses the expression of repre-sentative Proinflammatory genes including IL-1120573 and TNF-120572in RAW 2647 cells [46] Although strongly activating Nrf2the fruit hull of Gleditsia sinensis does not affect the functionof NF-120581B in RAW 2647 cells [15] Although these resultscannot exclude the possibility that CP suppresses NF-120581B viaNrf2 the impact of Nrf2 on NF-120581B activity remains to beelucidated Nevertheless our results suggest that CP has apotent anti-inflammatory activity by both suppressingNF-120581Band activating Nrf2

As described above there are a plethora of reportssuggesting that the four herbs composed of CP haveanti-inflammatory functions However these studies mostlyreported a preventive rather than a therapeutic effect of theherbs because in most cases the herbs were treated prior

to the onset of inflammatory response In this study unlikethose studies we attempted to address whether CP has atherapeutic effect on inflammatory lung disease In additionsince CP is in use as an inhalant in clinic we would like toretain a clinical relevancy by delivering CP in aerosol to thelungs of mice To this end we first injected LPS to the miceto induce ALI and thereby lung inflammation and 2 h laterdelivered CP in aerosol directly to the lung using a micro-sprayer According to our assessment we routinely deliver CPto more than 80 of the lung (data not shown) Our resultsshow that CP was highly effective in reducing neutrophilicinfiltration to the lung incurred by ALI suggesting that CPis a fast-acting therapeutics against acute lung inflammationsuch as ALI In addition CP posttreatment to ALI micesuppressed the expression of Proinflammatory cytokinesin the lung accompanied by increased expression of Nrf2dependent genes which was consistent with our results withRAW 2647 cells Therefore our results suggest that thetherapeutic effect of CP on ALI is at least in part attributed tosuppression of NF-120581B activity and activation of Nrf2 activitywhich may be served as an underlying mechanism for thetherapeutic effect of CP on ALI

5 Conclusion

Here we provide experimental evidence that CP had atherapeutic effect on ALI by using mice which was mediatedby differential regulation of the activities of NF-120581B and Nrf2Our findings suggest that CP can be a new therapeuticformula against ALI In addition our study suggests thepossibility that inhalation of ethnic herbal medicine is anadministration route for the treatment of acutely developinginflammatory lung diseases such as ALI

Conflict of Interests

The authors do not have a commercial or other associationthat might have a conflict of interests

Authorsrsquo Contribution

Kyun Ha Kim and Do-Hyun Kim contributed equally to thispaper

Acknowledgment

This research was supported by Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) funded by the Ministry of Education Science andTechnology (2012R1A1A2044346 to Myungsoo Joo)

References

[1] R S Irwin and J M Rippe Irwin and Rippersquos Intensive CareMedicine Lippincott Williams amp Wilkins Philadelphia PaUSA 2003

[2] Y G Zhu J M Qu J Zhang H N Jiang and J F XuldquoNovel interventional approaches for ALIARDS cell-based


gene therapyrdquo Mediators of Inflammation vol 2011 Article ID560194 7 pages 2011

[3] J Devaney M Contreras and J G Laffey ldquoClinical reviewgene-based therapies for ALIARDS where are we nowrdquoCritical Care vol 15 no 3 p 224 2011

[4] L B Tolle and T J Standiford ldquoDanger-associated molecularpatterns (DAMPs) in acute lung injuryrdquo The Journal of Pathol-ogy vol 229 no 2 pp 145ndash156 2013

[5] C Monaco E Andreakos S Kiriakidis et al ldquoCanonical path-way of nuclear factor 120581B activation selectively regulates proin-flammatory and prothrombotic responses in human atheroscle-rosisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 15 pp 5634ndash5639 2004

[6] A P Wheeler and G R Bernard ldquoTreating patients with severesepsisrdquoTheNew England Journal of Medicine vol 340 no 3 pp207ndash214 1999

[7] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004

[8] T Rangasamy J Guo W A Mitzner et al ldquoDisruption ofNrf2 enhances susceptibility to severe airway inflammation andasthma in micerdquo Journal of Experimental Medicine vol 202 no1 pp 47ndash59 2005

[9] T E Sussan T Rangasamy D J Blake et al ldquoTargeting Nrf2with the triterpenoid CDDO-imidazolide attenuates cigarettesmoke-induced emphysema and cardiac dysfunction in micerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 1 pp 250ndash255 2009

[10] A Boutten D Goven E Artaud-Macari J Boczkowski andM Bonay ldquoNRF2 targeting a promising therapeutic strategyin chronic obstructive pulmonary diseaserdquo Trends in MolecularMedicine vol 17 no 7 pp 363ndash371 2011

[11] J K Chen and T T Chen Chinese Herbal Formulas andApplications Pharmacological Effects amp Clinical Research Art ofMedicine Press City of Industry Calif USA 2009

[12] J H Lyu K H Kim H W Kim et al ldquoDangkwisoo-san anherbal medicinal formula ameliorates acute lung inflammationvia activation of Nrf2 and suppression of NF-120581Brdquo Journal ofEthnopharmacology vol 140 no 1 pp 107ndash116 2012

[13] R K Thimmulappa H Lee T Rangasamy et al ldquoNrf2 is acritical regulator of the innate immune response and survivalduring experimental sepsisrdquo Journal of Clinical Investigationvol 116 no 4 pp 984ndash995 2006

[14] K Chan and Y W Kan ldquoNrf2 is essential for protection againstacute pulmonary injury in micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 96 no22 pp 12731ndash12736 1999

[15] J Y Choi M J Kwun K H Kim et al ldquoProtective effectof the fruit hull of Gleditsia sinensis on LPS-induced acutelung injury is associated with Nrf2 activationrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID974713 11 pages 2012

[16] J A Johnson D A Johnson A D Kraft et al ldquoThe Nrf2-ARE pathway an indicator and modulator of oxidative stressin neurodegenerationrdquo Annals of the New York Academy ofSciences vol 1147 pp 61ndash69 2008

[17] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy of

Sciences of the United States of America vol 93 no 25 pp14960ndash14965 1996

[18] W A Solis T P Dalton M Z Dieter et al ldquoGlutamate-cysteine ligase modifier subunit mouse Gclm gene structureand regulation by agents that cause oxidative stressrdquo Biochemi-cal Pharmacology vol 63 no 9 pp 1739ndash1754 2002

[19] D Bensky S Clavey and E Stoger Chinese Herbal MedicineMateria Medica Eastland Press Seattle Wash USA 2004

[20] J K Chen T T Chen and L Crampton Chinese MedicalHerbology and Pharmacology Art of Medicine Press City ofIndustry Calif USA 2004

[21] Y G Liu and J B Luo ldquoEffects of among compositions of HerbaEphedrae decoction on genic xpression of 5-lipoxygenase acti-vating protein IL-4 and leukotriene C 4 in asthmatic micerdquoZhongguo Zhongyao Zazhi vol 32 no 3 pp 246ndash249 2007

[22] H Q Zhai S F Zhang M C Gao et al ldquoEffects of herbaEphedra sinicae and fructus schisandrae Chinensis on pathol-ogy of rats with bleomycin A5-induced idiopathic pulmonaryfibrosisrdquo Zhong Xi Yi Jie He Xue Bao vol 9 no 5 pp 553ndash5572011

[23] Y C Li S Z Peng H M Chen et al ldquoOral administration ofpatchouli alcohol isolated from Pogostemonis Herba augmentsprotection against influenza viral infection in micerdquo Interna-tional Immunopharmacology vol 12 no 1 pp 294ndash301 2012

[24] F Aimbire S C Penna M Rodrigues K C Rodrigues R AB Lopes-Martins and J A A Sertie ldquoEffect of hydroalcoholicextract of Zingiber officinalis rhizomes on LPS-induced ratairway hyperreactivity and lung inflammationrdquo ProstaglandinsLeukotrienes and Essential Fatty Acids vol 77 no 3-4 pp 129ndash138 2007

[25] P L Kuo Y LHsuM SHuangM J Tsai andYCKo ldquoGingersuppresses phthalate ester-induced airway remodelingrdquo Journalof Agricultural and FoodChemistry vol 59 no 7 pp 3429ndash34382011

[26] M N Ghayur A H Gilani and L J Janssen ldquoGinger atten-uates acetylcholine-induced contraction and Ca2+ signallingin murine airway smooth muscle cellsrdquo Canadian Journal ofPhysiology and Pharmacology vol 86 no 5 pp 264ndash271 2008

[27] C B Magalhaes D R Riva L J Depaula et al ldquoIn vivo anti-inflammatory action of eugenol on lipopolysaccharide-inducedlung injuryrdquo Journal of Applied Physiology vol 108 no 4 pp845ndash851 2010

[28] A Saini S Sharma and S Chhibber ldquoInduction of resistanceto respiratory tract infection with Klebsiella pneumoniae inmice fed on a diet supplemented with tulsi (Ocimum sanctum)and clove (Syzgium aromaticum) oilsrdquo Journal of MicrobiologyImmunology and Infection vol 42 no 2 pp 107ndash113 2009

[29] T D Gilmore ldquoIntroduction to NF-120581B players pathwaysperspectivesrdquo Oncogene vol 25 no 51 pp 6680ndash6684 2006

[30] I Atreya R Atreya andM FNeurath ldquoNF-120581B in inflammatorybowel diseaserdquo Journal of Internal Medicine vol 263 no 6 pp591ndash596 2008

[31] S S Makarov ldquoNF-ΚB in rheumatoid arthritis a pivotalregulator of inflammation hyperplasia and tissue destructionrdquoArthritis Research vol 3 no 4 pp 200ndash206 2001

[32] E Abraham ldquoNuclear factor-120581B and its role in sepsis-associatedorgan failurerdquo Journal of InfectiousDiseases vol 187 supplement2 pp S364ndashS369 2003

[33] N Moorchung A N Srivastava A K Sharma B R AchyutandBMittal ldquoNuclear factor 120581-B and histopathology of chronicgastritisrdquo Indian Journal of PathologyampMicrobiology vol 53 no3 pp 418ndash421 2010


[34] M R Edwards N W Bartlett D Clarke M Birrell M Belvisiand S L Johnston ldquoTargeting the NF-120581B pathway in asthmaand chronic obstructive pulmonary diseaserdquo Pharmacology andTherapeutics vol 121 no 1 pp 1ndash13 2009

[35] M D Schwartz E E Moore F A Moore et al ldquoNuclear factor-120581B is activated in alveolarmacrophages frompatients with acuterespiratory distress syndromerdquo Critical Care Medicine vol 24no 8 pp 1285ndash1292 1996

[36] I S Kim Y J Park S J Yoon and H B Lee ldquoEphedrannin Aand B from roots of Ephedra sinica inhibit lipopolysaccharide-induced inflammatorymediators by suppressing nuclear factor-120581B activation in RAW 2647 macrophagesrdquo InternationalImmunopharmacology vol 10 no 12 pp 1616ndash1625 2010

[37] H Y Lee S H Park M Lee et al ldquo1-Dehydro-[10]-gingerdionefrom ginger inhibits IKK120573 activity for NF-120581B activation andsuppressesNF-120581B-regulated expression of inflammatory genesrdquoThe British Journal of Pharmacology vol 167 no 1 pp 128ndash1402012

[38] Y F Xian Y C Li S P Ip Z X Lin X P Lai and Z R SuldquoAnti-inflammatory effect of patchouli alcohol isolated fromPogostemonis Herba in LPS-stimulated RAW2647 macro-phagesrdquo Experimental and Therapeutic Medicine vol 2 no 3pp 545ndash550 2011

[39] T F Bachiega J P B de Sousa J K Bastos and JM Sforcin ldquoClove and eugenol in noncytotoxic concentra-tions exert immunomodulatoryanti-inflammatory action oncytokine production by murine macrophagesrdquo Journal of Phar-macy and Pharmacology vol 64 no 4 pp 610ndash616 2012

[40] J M Lee J Li D A Johnson et al ldquoNrf2 a multi-organprotectorrdquo FASEB Journal vol 19 no 9 pp 1061ndash1066 2005

[41] H Y Cho S P Reddy and S R Kleeberger ldquoNrf2 defends thelung from oxidative stressrdquo Antioxidants and Redox Signalingvol 8 no 1-2 pp 76ndash87 2006

[42] R Gold L Kappos D L Arnold et al ldquoPlacebo-controlledphase 3 study of oral BG-12 for relapsingmultiple sclerosisrdquoTheNew England Journal of Medicine vol 367 no 12 pp 1098ndash11072012

[43] W Li T O Khor C Xu et al ldquoActivation of Nrf2-antioxidantsignaling attenuates NF120581B-inflammatory response and elicitsapoptosisrdquo Biochemical Pharmacology vol 76 no 11 pp 1485ndash1489 2008

[44] G H Liu J Qu and X Shen ldquoNF-120581Bp65 antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitatingrecruitment ofHDAC3 toMafKrdquoBiochimica et Biophysica Actavol 1783 no 5 pp 713ndash727 2008

[45] H S Youn Y S Kim Z Y Park et al ldquoSulforaphane suppressesoligomerization of TLR4 in a thiol-dependent mannerrdquo Journalof Immunology vol 184 no 1 pp 411ndash419 2010

[46] J H Lyu G S Lee K H Kim et al ldquoEnt-kaur-16-en-19-oicacid isolated from the roots of Aralia continentalis inducesactivation of Nrf2rdquo Journal of Ethnopharmacology vol 137 no3 pp 1442ndash1449 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Composition of Chung-pae

Scientific name Weight (g)Ephedrae Herba 2Pogostemonis (Agastachis) Herba 2Caryophylli Flos 1Zingiberis Rhizoma Crudus 1Total 6

the production of Proinflammatory cytokines which turnsout to be ineffective [6] On the other hand accumulatingevidence suggests that Nrf2 plays an essential role in protect-ing against various inflammatory diseases including acutepulmonary injury smoke-induced emphysema and asthma[7ndash9] Although Nrf2 is emerging as a potential therapeutictarget for inflammatory diseases [9 10] no effective thera-peutics against ALI is available yet

Several herbal medicinal formulas have been prescribedfor the treatment of various lung diseases in Asian traditionalmedicine [11] Therefore it is possible that these formulas area resource for the development of a new therapeutic measureagainst deadly respiratory diseases Popular herbal formulasagainst respiratory diseases include Ma-Huang Tang that hasbeen used against cough with rigor Junghangshichae-tangagainst cough with phlegm and nausea Gwackhyangjungki-san against cold with nausea and abdominal pain and Kyeji-tang against cold with headache and fever [11] Although eachherbal formula is typically composed of several herbs it con-tains a key herb that accounts for the efficacy of a formula Forinstance Ephedrae Herba is the key ingredient of Ma-HuangTang Caryophylli Flos is the key herb in Junghangshichae-tang and Pogostemonis (Agastachis) Herba and ZingiberisRhizomaCrudus are key herbs inGwackhyangjungki-san andKyeji-tang respectively In the hope of obtaining a herbalmedicine effective on a broad spectrum of respiratory dis-eases we formulated a new herbal medicine named Chung-pae (CP) that is composed of these key herbs contained inthe four formulas CP is being used as a complementarymea-sure in the respiratory clinic at Kyung-HeeOrientalMedicineHospital Seoul Korea to relieve the symptoms of patientswith dyspnea and cough Although it seemed that CP helpsimprove the symptoms experimental evidence supporting itseffect is scarce Here we sought to examine whether CP hasa therapeutic effect on respiratory diseases and if it doeshow CP exerts its effect To this end we used an LPS-inducedALI mouse model Since CP is being administered in aerosolto the patients we delivered CP in aerosol to the lung anddetermined the effect of CP on ALI in mice To obtain aninsight on the underlying mechanism we tested whetherCP affects the activities of Proinflammatory factor NF-120581Bandor anti-inflammatory factor Nrf2 Our findings showthat aerosol delivery of CP to the lung effectively reducedthe hallmarks of ALI which was appeared to be associatedwith suppression of Proinflammatory factor NF-120581B andactivation of anti-inflammatory factor Nrf2

2 Material and Methods

21 Preparation of the Water Extract of Chung-Pae Theherbs composing of Chung-pae (CP) shown in Table 1 werepurchased from Kwang-Myoung-Dang herb store (PusanRepublic of Korea) and identified by Professor J Y Choi(School of Korean Medicine Pusan National UniversityYangsan Republic of Korea) The voucher specimen (num-ber pnukh004) is kept in the herbarium stock room of theSchool of Korean Medicine Pusan National University Acrude decoction ofCPwas obtained by boiling 60 g ofChung-pae in 400mLdistilledwater for 2 hours followed by filtrationthrough 045 120583m filter The resultant decoction was concen-trated to 50mL by a low-pressure evaporator and underwentfreeze-drying processes to yield 6 g of powder Appropriateamount of the powder was dissolved in phosphate bufferedsaline (PBS) prior to experiment

22 Reagents and Antibodies 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide Sulforaphane and E coliLPS (serotype 055 B5) for animal study were from SigmaChemical Co (St Louis MO USA) TLR4-specific E coliLPS was purchased fromAlexis Biochemical (San Diego CAUSA)

23 Animals Male C57BL6 mice inbred in a specificpathogen-free (SPF) facility were purchased from SamtacoBio Korea Ltd (Osan Korea) Animals were housed incertified standard laboratory cages and fed with food andwater ad libitum prior to experiment

24 ALIMouseModel and Intrapulmonary Delivery of Chung-Pae All experimental procedures followed the NIH of KoreaGuidelines for the Care and Use of Laboratory Animals andall the experiments were approved by the Institutional Ani-mal Care and Use Committee of Pusan National University(protocol number PNU-2010-00028) Mice anesthetized byZoletil (Virbac Carros cedex France) received a single doseof 10mgkg LPS or sterile saline via intraperitoneal (ip)route At 2 h after ip LPS either PBS or CP (5 and 20mgkgbody weight) in 25 120583L of PBS was loaded in MicroSprayerAerosolizer-Model IA-1C (Penn-Century Wyndmoor PAUSA) and delivered in aerosol to the lung via intratracheal(it) under visual guidance At 24 h after LPS treatmentmice were euthanized by CO

2gas The trachea was exposed

through midline incision and cannulated with a sterile 24-gauge intravascular catheter Bilateral bronchoalveolar lavage(BAL) was performed by two consecutive instillations of10mL of PBS Total cell numbers in BAL fluid were countedwith hemocytometer and then centrifuged by a cytospin andstained for the differentiation of macrophages lymphocytesor neutrophils by Hemacolor (Merck Darmstadt Germany)Three hundred cells in total were counted and one hundredof the cells in each microscopic field were scored The meannumber of cells per field was reported For the analysis oflung tissue mice were perfused with saline and the wholelung was inflated with fixatives After paraffin embedding5 120583m sections were cut and placed on charged slides and








120

100

80

60

40

20

0

minus 1 5 10 20 50CP (120583gmL)

lowast lowast

lowastlowast




3 Results










4 Discussion



0

2

4

6

8

10

5 1020 20

Relat

ive l

ucife

rase

activ

ity

(fold

indu

ctio

n)


minus minus

+ + + +

lowastlowast

CP (120583gmL)

(a)

5 1020 20

minus minus

minus minus

+ + + +

5 1020 20

minus minus

minus minus

+ + + +

TNF-120572

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRN

A

IL-1120573

GA

PDH

mRN

A

lowast lowast

5 1020 20

minus minus

minus minus

+ + + +

15

10

05

00

15

10

05

00

CP (120583gmL)

CP (120583gmL)LPS (01 120583gmL)

LPS (01 120583gmL)

CP (120583gmL)LPS (01 120583gmL)

(b)





5

4

3

2

1


Relat

ive l

ucife

rase

activ

ity

(fo

ld in

duct

ion) lowast

lowast

lowastlowast

lowastlowastlowast

0

(a)

minus 5 10 20 SFN

minus 5 10 20 SFN


CP (120583gmL)

lowast

lowast

lowast


08

06

04

0402

02

08

06

04

02

000000

NQO1

NQO1 HO-1

HO-1

GCLC

GCLC

GAPDH

NQ

O1

GA

PDF

mRN

A

HO

-1G

APD

F m

RNA

GCL

CG

APD

F m

RNA

01

03

(b)







50120583m50120583m50120583m

50120583m 50120583m

(a)

Tota

l cel

l (times10

4)

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

60

40

20

0

lowast

lowast

(b)

Cel

l num

ber (times10

4)


LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

50

40

30

20

10

0

lowast

lowast

(c)






TNF-120572

TNF-120572

IL-1120573

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRA

N

IL- 1120573

GA

PDH

mRN

A

10

08

06

04

02

00

01

05

00

lowast lowast

15


minus minus

10 10 10

20 5 20


minus minus

10 10 10

20 5 20


LPS (mgkg)


minus minus

10 10 10

20 5 20

(a)


minus minus

10 10 10

20 5 20

LPS (mgkg)


minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)

10

08

06

04

02

00

15

01

05

00

06

02

04

00

lowastlowast

lowastlowast

lowastlowast

GCLC

GCLC

GAPDH

NQ

O1

GA

PDH

mRN

A

HO

-1G

APD

H m

RNA

GCL

CG

APD

H m

RNA

HO-1

HO-1

NQO1

NQO1

(b)








5 Conclusion






Acknowledgment


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























120

100

80

60

40

20

0

minus 1 5 10 20 50CP (120583gmL)

lowast lowast

lowastlowast




3 Results










4 Discussion



0

2

4

6

8

10

5 1020 20

Relat

ive l

ucife

rase

activ

ity

(fold

indu

ctio

n)


minus minus

+ + + +

lowastlowast

CP (120583gmL)

(a)

5 1020 20

minus minus

minus minus

+ + + +

5 1020 20

minus minus

minus minus

+ + + +

TNF-120572

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRN

A

IL-1120573

GA

PDH

mRN

A

lowast lowast

5 1020 20

minus minus

minus minus

+ + + +

15

10

05

00

15

10

05

00

CP (120583gmL)

CP (120583gmL)LPS (01 120583gmL)

LPS (01 120583gmL)

CP (120583gmL)LPS (01 120583gmL)

(b)





5

4

3

2

1


Relat

ive l

ucife

rase

activ

ity

(fo

ld in

duct

ion) lowast

lowast

lowastlowast

lowastlowastlowast

0

(a)

minus 5 10 20 SFN

minus 5 10 20 SFN


CP (120583gmL)

lowast

lowast

lowast


08

06

04

0402

02

08

06

04

02

000000

NQO1

NQO1 HO-1

HO-1

GCLC

GCLC

GAPDH

NQ

O1

GA

PDF

mRN

A

HO

-1G

APD

F m

RNA

GCL

CG

APD

F m

RNA

01

03

(b)







50120583m50120583m50120583m

50120583m 50120583m

(a)

Tota

l cel

l (times10

4)

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

60

40

20

0

lowast

lowast

(b)

Cel

l num

ber (times10

4)


LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

50

40

30

20

10

0

lowast

lowast

(c)






TNF-120572

TNF-120572

IL-1120573

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRA

N

IL- 1120573

GA

PDH

mRN

A

10

08

06

04

02

00

01

05

00

lowast lowast

15


minus minus

10 10 10

20 5 20


minus minus

10 10 10

20 5 20


LPS (mgkg)


minus minus

10 10 10

20 5 20

(a)


minus minus

10 10 10

20 5 20

LPS (mgkg)


minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)

10

08

06

04

02

00

15

01

05

00

06

02

04

00

lowastlowast

lowastlowast

lowastlowast

GCLC

GCLC

GAPDH

NQ

O1

GA

PDH

mRN

A

HO

-1G

APD

H m

RNA

GCL

CG

APD

H m

RNA

HO-1

HO-1

NQO1

NQO1

(b)








5 Conclusion






Acknowledgment


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























4 Discussion



0

2

4

6

8

10

5 1020 20

Relat

ive l

ucife

rase

activ

ity

(fold

indu

ctio

n)


minus minus

+ + + +

lowastlowast

CP (120583gmL)

(a)

5 1020 20

minus minus

minus minus

+ + + +

5 1020 20

minus minus

minus minus

+ + + +

TNF-120572

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRN

A

IL-1120573

GA

PDH

mRN

A

lowast lowast

5 1020 20

minus minus

minus minus

+ + + +

15

10

05

00

15

10

05

00

CP (120583gmL)

CP (120583gmL)LPS (01 120583gmL)

LPS (01 120583gmL)

CP (120583gmL)LPS (01 120583gmL)

(b)





5

4

3

2

1


Relat

ive l

ucife

rase

activ

ity

(fo

ld in

duct

ion) lowast

lowast

lowastlowast

lowastlowastlowast

0

(a)

minus 5 10 20 SFN

minus 5 10 20 SFN


CP (120583gmL)

lowast

lowast

lowast


08

06

04

0402

02

08

06

04

02

000000

NQO1

NQO1 HO-1

HO-1

GCLC

GCLC

GAPDH

NQ

O1

GA

PDF

mRN

A

HO

-1G

APD

F m

RNA

GCL

CG

APD

F m

RNA

01

03

(b)







50120583m50120583m50120583m

50120583m 50120583m

(a)

Tota

l cel

l (times10

4)

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

60

40

20

0

lowast

lowast

(b)

Cel

l num

ber (times10

4)


LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

50

40

30

20

10

0

lowast

lowast

(c)






TNF-120572

TNF-120572

IL-1120573

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRA

N

IL- 1120573

GA

PDH

mRN

A

10

08

06

04

02

00

01

05

00

lowast lowast

15


minus minus

10 10 10

20 5 20


minus minus

10 10 10

20 5 20


LPS (mgkg)


minus minus

10 10 10

20 5 20

(a)


minus minus

10 10 10

20 5 20

LPS (mgkg)


minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)

10

08

06

04

02

00

15

01

05

00

06

02

04

00

lowastlowast

lowastlowast

lowastlowast

GCLC

GCLC

GAPDH

NQ

O1

GA

PDH

mRN

A

HO

-1G

APD

H m

RNA

GCL

CG

APD

H m

RNA

HO-1

HO-1

NQO1

NQO1

(b)








5 Conclusion






Acknowledgment


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

5 1020 20

Relat

ive l

ucife

rase

activ

ity

(fold

indu

ctio

n)


minus minus

+ + + +

lowastlowast

CP (120583gmL)

(a)

5 1020 20

minus minus

minus minus

+ + + +

5 1020 20

minus minus

minus minus

+ + + +

TNF-120572

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRN

A

IL-1120573

GA

PDH

mRN

A

lowast lowast

5 1020 20

minus minus

minus minus

+ + + +

15

10

05

00

15

10

05

00

CP (120583gmL)

CP (120583gmL)LPS (01 120583gmL)

LPS (01 120583gmL)

CP (120583gmL)LPS (01 120583gmL)

(b)





5

4

3

2

1


Relat

ive l

ucife

rase

activ

ity

(fo

ld in

duct

ion) lowast

lowast

lowastlowast

lowastlowastlowast

0

(a)

minus 5 10 20 SFN

minus 5 10 20 SFN


CP (120583gmL)

lowast

lowast

lowast


08

06

04

0402

02

08

06

04

02

000000

NQO1

NQO1 HO-1

HO-1

GCLC

GCLC

GAPDH

NQ

O1

GA

PDF

mRN

A

HO

-1G

APD

F m

RNA

GCL

CG

APD

F m

RNA

01

03

(b)







50120583m50120583m50120583m

50120583m 50120583m

(a)

Tota

l cel

l (times10

4)

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

60

40

20

0

lowast

lowast

(b)

Cel

l num

ber (times10

4)


LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

50

40

30

20

10

0

lowast

lowast

(c)






TNF-120572

TNF-120572

IL-1120573

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRA

N

IL- 1120573

GA

PDH

mRN

A

10

08

06

04

02

00

01

05

00

lowast lowast

15


minus minus

10 10 10

20 5 20


minus minus

10 10 10

20 5 20


LPS (mgkg)


minus minus

10 10 10

20 5 20

(a)


minus minus

10 10 10

20 5 20

LPS (mgkg)


minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)

10

08

06

04

02

00

15

01

05

00

06

02

04

00

lowastlowast

lowastlowast

lowastlowast

GCLC

GCLC

GAPDH

NQ

O1

GA

PDH

mRN

A

HO

-1G

APD

H m

RNA

GCL

CG

APD

H m

RNA

HO-1

HO-1

NQO1

NQO1

(b)








5 Conclusion






Acknowledgment


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5

4

3

2

1


Relat

ive l

ucife

rase

activ

ity

(fo

ld in

duct

ion) lowast

lowast

lowastlowast

lowastlowastlowast

0

(a)

minus 5 10 20 SFN

minus 5 10 20 SFN


CP (120583gmL)

lowast

lowast

lowast


08

06

04

0402

02

08

06

04

02

000000

NQO1

NQO1 HO-1

HO-1

GCLC

GCLC

GAPDH

NQ

O1

GA

PDF

mRN

A

HO

-1G

APD

F m

RNA

GCL

CG

APD

F m

RNA

01

03

(b)







50120583m50120583m50120583m

50120583m 50120583m

(a)

Tota

l cel

l (times10

4)

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

60

40

20

0

lowast

lowast

(b)

Cel

l num

ber (times10

4)


LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

50

40

30

20

10

0

lowast

lowast

(c)






TNF-120572

TNF-120572

IL-1120573

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRA

N

IL- 1120573

GA

PDH

mRN

A

10

08

06

04

02

00

01

05

00

lowast lowast

15


minus minus

10 10 10

20 5 20


minus minus

10 10 10

20 5 20


LPS (mgkg)


minus minus

10 10 10

20 5 20

(a)


minus minus

10 10 10

20 5 20

LPS (mgkg)


minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)

10

08

06

04

02

00

15

01

05

00

06

02

04

00

lowastlowast

lowastlowast

lowastlowast

GCLC

GCLC

GAPDH

NQ

O1

GA

PDH

mRN

A

HO

-1G

APD

H m

RNA

GCL

CG

APD

H m

RNA

HO-1

HO-1

NQO1

NQO1

(b)








5 Conclusion






Acknowledgment


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















50120583m50120583m50120583m

50120583m 50120583m

(a)

Tota

l cel

l (times10

4)

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

60

40

20

0

lowast

lowast

(b)

Cel

l num

ber (times10

4)


LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

50

40

30

20

10

0

lowast

lowast

(c)






TNF-120572

TNF-120572

IL-1120573

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRA

N

IL- 1120573

GA

PDH

mRN

A

10

08

06

04

02

00

01

05

00

lowast lowast

15


minus minus

10 10 10

20 5 20


minus minus

10 10 10

20 5 20


LPS (mgkg)


minus minus

10 10 10

20 5 20

(a)


minus minus

10 10 10

20 5 20

LPS (mgkg)


minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)

10

08

06

04

02

00

15

01

05

00

06

02

04

00

lowastlowast

lowastlowast

lowastlowast

GCLC

GCLC

GAPDH

NQ

O1

GA

PDH

mRN

A

HO

-1G

APD

H m

RNA

GCL

CG

APD

H m

RNA

HO-1

HO-1

NQO1

NQO1

(b)








5 Conclusion






Acknowledgment


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TNF-120572

TNF-120572

IL-1120573

IL-1120573

GAPDH

TNF-120572

GA

PDH

mRA

N

IL- 1120573

GA

PDH

mRN

A

10

08

06

04

02

00

01

05

00

lowast lowast

15


minus minus

10 10 10

20 5 20


minus minus

10 10 10

20 5 20


LPS (mgkg)


minus minus

10 10 10

20 5 20

(a)


minus minus

10 10 10

20 5 20

LPS (mgkg)


minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)CP (mgkg)

minus minus

minus minus

10 10 10

20 5 20

LPS (mgkg)

10

08

06

04

02

00

15

01

05

00

06

02

04

00

lowastlowast

lowastlowast

lowastlowast

GCLC

GCLC

GAPDH

NQ

O1

GA

PDH

mRN

A

HO

-1G

APD

H m

RNA

GCL

CG

APD

H m

RNA

HO-1

HO-1

NQO1

NQO1

(b)








5 Conclusion






Acknowledgment


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusion






Acknowledgment


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 Therapeutic Effect of Chung-Pae, an...

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