(全)Paeoniflorin abrogatesDSS-induced colitis

Paeoni?orin abrogates DSS-induced colitis via a TLR4-dependent pathway Jingjing Zhang,1,2Wei Dou,1,3Eryun Zhang,1,2Aning Sun,1,2Lili Ding,1Xiaohui Wei,1Guixin Chou,4

Sridhar Mani,3and Zhengtao Wang1,2,4

1Shanghai Key Laboratory of Complex Prescription and MOE Key Laboratory for Standardization of Chinese Medicines,

Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai,China;2Department of Pharmacognosy,China Pharmaceutical University,Nanjing,China;3Departments of Medicine and Genetics,Albert Einstein College of Medicine,New York,New York;and4Shanghai R&D Center for Standardization of Traditional Chinese Medicine, Shanghai,China

Submitted4December2012;accepted in?nal form6November2013

Zhang J,Dou W,Zhang E,Sun A,Ding L,Wei X,Chou G, Mani S,Wang Z.Paeoni?orin abrogates DSS-induced colitis via a TLR4-dependent pathway.Am J Physiol Gastrointest Liver Physiol 306:G27–G36,2014.First published November14,2013; doi:10.1152/ajpgi.00465.2012.—Paeonia lacti?ora Pall is one of the most well-known herbs in China,Korea,and Japan for more than 1,200years.Paeoni?orin,the major bioactive component of peony root,has recently been reported to have anticolitic activity.However, the underlying molecular mechanism is unclear.The present study was to explore the possible mechanism of paeoni?orin in attenuating dextran sulfate sodium(DSS)-induced colitis.Pre-and coadministra-tion of paeoni?orin signi?cantly reduced the severity of colitis and resulted in downregulation of several in?ammatory parameters in the colon,including the activity of myeloperoxidase(MPO),the levels of TNF-?and IL-6,and the mRNA expression of proin?ammatory mediators(MCP-1,Cox2,IFN-?,TNF-?,IL-6,and IL-17).The decline in the activation of NF-?B p65,ERK,JNK,and p38MAPK correlated with a decrease in mucosal Toll-like receptor4(TLR4)but not TLR2or TLR5expression.In accordance with the in vivo results, paeoni?orin downregulated TLR4expression,blocked nuclear trans-location of NF-?B p65,and reduced the production of IL-6in LPS-stimulated mouse macrophage RAW264.7cells.Transient trans-fection assay performed in LPS-stimulated human colon cancer HT-29cells indicated that paeoni?orin inhibits NF-?B transcriptional activity in a dose-dependent manner.TLR4knockdown and overex-pression experiments demonstrated a requirement for TLR4in pae-oni?orin-mediated downregulation of in?ammatory cytokines.Thus, for the?rst time,the present study indicates that paeoni?orin abro-gates DSS-induced colitis via decreasing the expression of TLR4and suppressing the activation of NF-?B and MAPK pathways.

DSS-induced colitis;TLR4;NF-?B;MAPK;paeoni?orin

INTESTINAL EPITHELIUM REPRESENTS the?rst defense in preventing pathogenic invasion of commensal bacteria.It provides a critical interface between microorganisms and their hosts(1, 33).To maintain healthy homeostasis of intestine,mammals have evolved a defensive network called innate and adaptive immune defensive systems(9,31).The innate immunity plays a central role in regulating immune responses to intestinal pathogen-derived microorganisms by recognizing the presence of speci?c bacterial antigens through an extensive family of pattern recognition receptors(PRRs),which are situated in intestinal epithelial cells and recognize conserved microbial molecules,called pathogen-associated molecular patterns(1, 31).Toll-like receptor4(TLR4)is a PRR that has been found in Drosophila melanogaster and mammals,and compelling research has shown that lipopolysaccharide(LPS),which is the major component of the outer membrane of gram-negative bacteria,binds to TLR4and triggers signaling cascades medi-ated by myeloid differentiation factor MyD88to activate the transcription factor NF-?B and eventually results in in?amma-tory response(14,27).There is increasing evidence that LPS is not the only ligand for TLR4.Heparan sulfate,the extra-domain-A of?bronectin,hyaluronic acid,and?brinogen,have signal through TLR4(8).TLR4is strongly upregulated in in?ammatory bowel disease(IBD)patients as well as in acute dextran sulfate sodium(DSS)-induced colitis mice,implying that changes in TLR4expression and subsequent alterations in the innate immune response contribute to the pathogenesis of IBD(14,16).

Recent studies indicated that Lactobacillus suntoryeus,a gut commensal,blocks in?ammatory mediators(Cox2,TNF-?, IL-1,and IL-6)through suppression of TLR4-mediated NF-?B activation in mice with TNBS-induced colitis(19).Lubbad et al.(23)have reported that amelioration of TNBS-induce colitis by curcumin is mediated through TLR4-linked NF-?B inhibi-tion.We recently demonstrated that naringenin ameliorates DSS-induced colitis via transecting TLR4/NF-?B signaling cascades(4).In addition,TLR4is indicated to be linked with MAPK pathway in IBD.TLR4monoclonal antibody (TLR4mAb)ameliorates DSS-induced colitis via suppression of p38MAPK pathway(21).TLR4ligand LPS caused signif-icant phosphorylation(activation)of MAPK in intestinal mu-cosal enterochromaf?n cells isolated from Crohn’s colitis pa-tients(18).Thus it is proposed that many therapeutic agents that abrogate intestinal in?ammation might transect with the TLR4signaling pathway(7,34).

Paeonia lacti?ora Pall is an ornamental and medicinal plant.

A decoction of the dried root of peony has been used in the treatment of rheumatoid arthritis in China for more than1,200 years(13,22).Paeoni?orin,a monoterpene glucoside,is the principal bioactive constituent of peony root(40).Paeoni?orin has reportedly exhibited anti-in?ammation,immunoregulation,anti-arthritis,antihepatitis,pain-relieving,neuromuscular blocking, and antihyperglycemia effects(15,32,40,41).Previous studies have shown that paeoni?orin suppresses TNF-?induced chemo-kine production in human dermal microvascular endothelial cells by blocking the NF-?

B and ERK pathway(2).A recent investigation(article in Chinese)indicated that paeoni?orin effectively ameliorates the symptoms of oxazolone-induced colitis,but the underlying mechanism is not well established (44).On the basis of the key role TLR4plays in intestinal in?ammation,we investigated the effects of paeoni?orin on

Address for reprint requests and other correspondence:W.Dou,1200Cailun Rd.,Rm.5301,Shanghai Univ.of TCM,Shanghai201203,China(e-mail: vivi.dou@https://www.360docs.net/doc/1117233239.html,).

Am J Physiol Gastrointest Liver Physiol306:G27–G36,2014. First published November14,2013;doi:10.1152/ajpgi.00465.2012.

TLR4expression and evaluated NF-?B and MAPK pathways in DSS-induced colitis.

MATERIALS AND METHODS

Animals.Healthy8-wk-old female C57BL/6mice(20?2g)were obtained from Shanghai Laboratory Animal Center,Shanghai,China and housed under a speci?c pathogen-free facility at room tempera-ture(25°C)with alternating12:12-h light-dark cycles.Standard mouse chow pellets and water were supplied ad libitum.This study was approved by the Animal Ethics Committee of Shanghai Univer-sity of Traditional Chinese Medicine(SHUTCM).All mice were housed under a speci?c pathogen-free facility at SHUTCM and kept under the same laboratory conditions of temperature(25?2°C)and lighting(12-h light-dark cycle)and were given free access to standard laboratory chow and tap water.

Induction of colitis and administration of paeoni?orin.The exper-iment lasted for10days.Colitis was induced by giving4%(wt/vol) DSS(molecular mass36,000–50,000Da,MP Biomedicals,Solon, OH)in drinking water for7days ad libitum.Eight-week-old female mice were randomly distributed into four groups(n?10per group): Group1,vehicle controls were administered100?l of0.5%(wt/vol) methylcellulose by oral gavage once per day;Group2,paeoni?orin (dissolved in0.5%methylcellulose)at a dose of50mg/kg of body wt via oral gavage once per day;Group3,100?l of0.5%(wt/vol) methylcellulose by oral gavage once per day and4%(wt/vol)DSS in drinking water from day3to day10;Group4,paeoni?orin by oral gavage(50mg/kg)3days prior to or concurrently with DSS treatment and continued to the end of DSS treatment.Total gavage volume was identical for each group.At4h after receiving the last gavage,the mice were killed and the colon was removed.Paeoni?orin(cat.no. 12-1001,HPLC?98%)was obtained from Shanghai R&D Center for Standardization of Traditional Chinese Medicine,Shanghai,China, and the dosage selection(50mg/kg)was based on previous in vivo studies and was con?rmed to result in effective alleviation of acute lung injury and arthritis in mouse disease models(40,43).

Colitis evaluation.Mice were monitored daily for body weight, diarrhea,and bloody stool.Bloody diarrhea event was assessed as described before(3,4).Following measurement of the colon length, the distal colon was taken and?xed in10%(wt/vol)buffered formalin for H&E staining.Histological damage was assessed as a combined score of in?ammatory cell in?ltration(score0–3)and mucosal dam-age(score0–3)by a method previously described(5,24,36).For in?ammatory cell in?ltration in the colon mucosa,rare in?ammatory cells(mononuclear in?ltrates)in the lamina propria were counted as 0;increased numbers of in?ammatory cells,including neutrophils in the lamina propria,were counted as1;con?uence of in?ammatory cells,extending into the submucosa,were scored as2;and a score of 3was given for transmural extension of the in?ammatory cell in?l-tration.For epithelial damage,absence of mucosal damage was counted as0,discrete focal lymphoepithelial lesions were counted as 1,mucosal erosion/ulceration was counted as2,and a score of3was given for extensive mucosal damage and extension through deeper structures of the bowel wall.The two subscores were added and the combined histological score ranged from0(no changes)to6(exten-sive cell in?ltration and tissue damage).

Immunoblotting analysis.The mouse macrophage RAW264.7cells were procured and cultured according to the guidelines of American Type Culture Collection(ATCC,Rockville,MD).When cells reached 70%con?uence,paeoni?orin(50?M)was added and incubated for2 h.Cells were overnight stimulated with LPS(2?g/ml,Escherichia coli055:B5,L6529–1mg,Sigma-Aldrich,St.Louis,MO)and then subjected to cell lysis.Colon tissues were disrupted by homogeniza-tion on ice and centrifuged at4°C(12,000g,15min)and the supernatants were collected.Equal amounts of protein(40?g/lane) were separated on10%SDS-PAGE and transferred to nitrocellulose membrane.Membranes were blocked in5%(wt/vol)skim milk and incubated with antibodies against mouse TLR4(sc-293072,1:1,000,

Santa Cruz Biotechnology,CA),phospho-p65(no.3,033,1:1,000,Cell

Signaling Technology,Beverly,MA),I?B?(no.4,814,1:1,000,Cell

Signaling),phospho-ERK1/2(no.4377,1:1,000,Cell Signaling),

ERK1/2(no.4348,1:1,000),phospho-JNK(no.9255,1:2,000,Cell

Signaling),JNK(no.9252,1:1,000,Cell Signaling),phospho-p38(no.

9215,1:1,000,Cell Signaling),p38(no.9212,1:1,000,Cell Signal-

ing),and?-actin(no.4970,1:2,000,Cell Signaling).Blots were then

washed three times with PBS containing0.1%(wt/vol)Tween-20

(PBST)and incubated with horseradish peroxidase-conjugated sec-

ondary antibodies(Santa Cruz).Blots were again washed with PBST

and then developed by enhanced chemiluminescence detection re-

gents(GE Healthcare,Pittsburgh,PA).The protein bands were

quanti?ed by the average ratios of integral optic density following

normalization to the housekeeping gene?-actin expression.

RNA analysis.When RAW264.7cells reached70%con?uence, cells were treated with or without paeoni?orin(50?M)for2h prior

to LPS(2?g/ml,Sigma-Aldrich)treatment for an additional12h.

RNA was extracted from cultured cells or colon tissues by use of

TRIzol reagent(Life Technologies,Carlsbad,CA).Quantitative real-

time PCR(qPCR)was performed by using cDNA generated from3?g total RNA with the SuperScript II Reverse Transcriptase kit(Life Technologies).The primer sequences used in PCR ampli?cation are as

follows:5=-CTGAAGCTGTTGCGTTACAT-3=/5=-ACTACGTCTGA-

CTCCGAGGG-3=for mTLR2,5=-TTCAGAGCCGTTGGTGTATC-

3=/5=-CCCATTCCAGGTAGGTGTTT-3=for mTLR4,5=-GAATCA-

GAATCGCCTTTCGT-3=/5=-ACCAGGTCGTTAAATATCCC-3=for

mTLR5,5=-AGTTGACCCGTAAATCTGA-3=/5=-TGAAAGGGAATAC-

CATAACA-3=for mMCP-1,5=-GAAGTCTTTGGTCTGGTGCCT-3=/5=-

GCTCCTGCTTGAGTATGTCG-3=for mCox2,5=-AGCAACAACATA-

AGCGTCAT-3=/5=-CCTCAAACTTGGCAATACTC-3=for mIFN-?,

5=-CGTGGAACTGGCAGAAGAGG-3=/5=-AGACAGAAGAGCGTGG-

TGGC-3=for mTNF-?,5=-ACCACGGCCTTCCCTACTTC-3=/5=-

CATTTCCACGATTTCCCAGA-3=for mIL-6,5=-TCAGACT-

ACCTCAACCGTTCC-3=/5=-ATGTGGTGGTCCAGCTTTCC-3=for

mIL-17,5=-CAGCCTTCCTTCTTGGGTAT-3=/5=-TGGCATAGAG-

GTCTTTACGG-3=for?-actin.PCR reactions were carried out by using

SYBR Premix ExTaq Mix(Takara Bio,Shiga,Japan)in an ABI Prism

7900HT Sequence Detection System(Life Technologies).Thermal cycler

parameters were as follows:1cycle of95°C for30s,40cycles of denatur-

ation(95°C,5s),and combined annealing/extension(60°C,30s).Gene

expression changes were calculated by the comparative Ct method and the

values were normalized to endogenous reference?-actin.

Determination of TNF-?and IL-6levels.Colon segments were

homogenized in ice-cold PBS.The homogenates were centrifuged at

3,000g for10min and the supernatants were assayed to determine

levels of TNF-?and IL-6.RAW264.7macrophages were pretreated

with paeoni?orin(50?M)for2h and incubated with LPS(2?g/ml,

Sigma-Aldrich)for12h,the supernatants were harvested and the level

of IL-6was determined.The level of each cytokine was evaluated

using ELISA kits according to the manufacturer’s protocols(R&D

Systems,Minneapolis,MN)and the results were expressed in pico-

grams per milligram of protein for tissue samples and picograms per

milliliter for cell mediums.

Determination of MPO activity.Tissue myeloperoxidase(MPO) activity,which is linearly related to neutrophil in?ltration in in?amed tissue,was assayed to monitor the degree of in?ammation.MPO activity in colon tissues was measured as previously described(5)and the values were expressed as units per milligram of protein.

NF-?B nuclear translocation immuno?uorescence.RAW264.7

cells were seeded in eight-chamber slides(BD Biosciences,Bedford,

MA)at a density of5?104cells per well.Cells were allowed to

adhere at37°C overnight and paeoni?orin(50?M)was added for2

h.Then cells were overnight stimulated with LPS(2?g/ml,Sigma-

Aldrich)and?xed with4%(wt/vol)paraformaldehyde solution at

20°C for10min.After washing in PBS,cells were permeabilized with

0.3%(wt/vol)Triton X-100in PBS at room temperature for20min.

G28PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

After incubation in blocking buffer of0.1%(wt/vol)Triton X-100, 1%(wt/vol)BSA,and3%(wt/vol)donkey serum,cells were then incubated with rabbit NF-?B p65antibody(no.8242,1:50,Cell Signaling)overnight at4°C.After washing in PBS,cells were further incubated with Alexa Fluor488-conjugated donkey anti-rabbit IgG (A-21,206,1:500,Life Technologies)at room temperature for45min. To stain the nuclei,we added1?g/ml of DAPI(Life Technologies) in PBS at room temperature.Fluorescence photographs were obtained by use of an Olympus CKX41?uorescence microscope.

NF-?B luciferase reporter assay.Human colorectal carcinoma HT-29cells were purchased from ATCC(Manassas,VA),and1?106 HT-29cells were electroporated with pGL4.32[luc2P/NF-?B-RE/ Hygro]reporter vector(Promega,WI)by using the Lonza Nucleo-fector II instrument(Amaxa Biosystems,Germany)according to the manufacturer’s protocol.The pGL4.32reporter is a NF-?B reporter vector containing NF-?B response elements and?re?y luciferase gene.Cells were then seeded into48-well plate following transfection. After overnight incubation,cells were incubated with or without paeoni?orin(25,50,and100?M)for2h followed by an additional incubation with LPS(2?g/ml,Sigma-Aldrich)for12h.At the end of the experiments,cells were washed once with PBS,then lysed in0.1 ml1?Passive Lysis Buffer(Promega).Cell-free lysates were ob-tained by centrifugation at10,000g for2min at4°C.The effects of paeoni?orin and LPS on the activation of NF-?B promoter in trans-fected cells were determined by luciferase activity.Luciferase activity in cell lysate was quanti?ed using a luciferase assay system(Promega) and a Glomax20/20Luminometer(Promega).The results were presented as relative light units and data were expressed as fold induction of control cells.

Gene silencing and overexpression.We electroporated1?106 RAW264.7cells with TLR4siRNA(m)(sc-40261,Santa Cruz Biotechnology)targeting the mouse TLR4mRNA using Lonza Nucleofector II instrument(Amaxa Biosystems,Germany)according to the manufacturer’s protocol.Control siRNA(sc-16240,Santa Cruz Biotechnology),a nontargeting siRNA,was used as a negative con-trol,and1?106HT-29cells were electroporated with human TLR4 expression plasmid pcDNA3-TLR4-YFP(Addgene).Cells were then seeded into six-well plate following transfection.Cells were incubated with or without paeoni?orin(50?M)and were stimulated with LPS (2?g/ml)for12h.At the end of the incubation,cells were rinsed, scraped,and used in qRT-PCR or Western blot analyses.

Statistical analysis.The data were analyzed with use of a SPSS 16.0statistical package.Multiple comparisons were performed by one-way analysis of variance followed by LSD t-test.A value of P?0.05was considered statistically signi?cant,and all results are pre-sented as means?SD.

RESULTS

Paeoni?orin pre-and coadministration ameliorates DSS-induced colitis.The protective ef?cacy of paeoni?orin on colitis was assessed by body weight change,bloody diarrhea event,colon length,and histological analysis.There was no weight loss observed in mice administered vehicle or paeoni-?orin alone(groups1and2,respectively).The body weight of mice in group3dramatically decreased from day3onward following DSS treatment.Administration of paeoni?orin effec-tively suppressed body weight loss(Fig.1A).Diarrhea symp-toms appeared on or shortly after day3.At days3to7,all the mice on the DSS-only treatment(group3)experienced both diarrhea and bloody diarrhea,whereas none of the mice receiv-ing vehicle or paeoni?orin alone(groups1and2,respectively) exhibited diarrhea at any point during the studies.By day10, all the group3mice were euthanized because of critical weight loss.In contrast,the mice receiving both DSS and paeoni?orin (group4),exhibited less diarrhea and bloody diarrhea than did the group3mice(Fig.1B).Colon shortening is a surrogate marker of colonic in?ammation(28).After7days of treatment with DSS in drinking water,there was a signi?cant shortening (P?0.05)of the colon length of mice in group3compared with the mice receiving both DSS and paeoni?orin(group4). This indicates that the oral administration of paeoni?orin signi?cantly ameliorated the symptom of colon shortening (Fig.1,C and D).In addition,mice treated with vehicle or paeoni?orin alone exhibited intact crypt-villus structures and epithelial layer.DSS administration resulted in a paucity of intact crypt-villus structures and large in?ammatory exudates across the thickness of the bowel wall.By contrast,paeoni?o-rin administration to DSS-exposed mice resulted in signi?cant protection of the colon crypt structures and less severe histo-logical in?ammation(Fig.1,E and F).This result strongly suggested that the inhibition of in?ammatory in?ltration was a mechanism for the protective effects of paeoni?orin in DSS-induced colitis.Next,to determine whether paeoni?orin could also ameliorate the severity on DSS-induced colitis,coadmin-istration experiments were performed.Administering paeoni-?orin at the same time as DSS treatment would replicate the clinical ease of administration that would occur in conjunction with in?ammatory relapses in IBD.The results for the body weight loss,diarrhea event rate,colon shortening,and colon histology(Fig.1,A–F)indicated that paeoni?orin coadminis-tration with DSS can also decrease the severity of DSS-induced colitis even though the attenuated effect is less pronounced than using the preadministration schedule.

Paeoni?orin decreases TLR4mRNA and protein expression. Considering TLR4is an upstream signaling protein involved in NF-?B activation and the pathogenesis of IBD,we hypothe-sized that the protective effect of paeoni?orin is mediated by interference with TLR4.To determine the effect of paeoni?orin on TLR4expression,qPCR and immunoblotting analyses were carried out.The results demonstrated that mRNA(Fig.2A)and protein(Fig.2B)expression of TLR4were signi?cantly in-duced in colon mucosa after7-day DSS treatment compared with normal control group.The relative increase in mRNA and protein expression of TLR4after DSS treatment was signi?-cantly downregulated in mice pretreated with paeoni?orin.The in vitro data performed in mouse macrophage RAW264.7cells con?rmed with the results(Fig.2,C and D).The results indicated that paeoni?orin might decrease the severity of DSS-induced colitis via targeting TLR4pathway.Indeed,to deter-mine the effects of paeoni?orin treatment on alternative TLRs known to be dysregulated in IBD,namely,TLR2and TLR5 mRNA expression abundance(6,30,37),qPCR was per-formed.Interestingly,TLR2mRNA expression was signi?-cantly upregulated,whereas TLR5mRNA expression re-mained unchanged in DSS-induced colitis.In addition,neither control nor in?amed colons exhibit signi?cant difference in the expression of TLR2and TLR5after paeoni?orin preadminis-tration(Fig.2E).Collectively,the data strongly indicated that TLR4but not TLR2or TLR5appears to account for the anti-in?ammatory effect of paeoni?orin.

Paeoni?orin treatment blocks NF-?B activation in vivo and in vitro.NF-?B is a central regulator of immune response and NF-?B activation is indicated to be an important step in the development of human IBD(27).On the basis of this rationale, we hypothesized that the anti-in?ammatory effect of paeoni-

G29

PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

G30PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

Fig.1.Preadministration(pre-)and coadministration(co-)of paeoni?orin(Paeo)attenuated clinical symptoms in dextran sulfate sodium(DSS)-induced colitis mice.A:body weight changes.Data plotted as percentage of basal body weight.B:occurrence of bloody diarrhea.Data plotted as percentage of total mice that had bloody diarrhea on different days(point of time)of DSS treatment.C:macroscopic view of the colon.D:colon length.E:representative H&E-stained colon sections(magni?cation?200).F:histology score.Values were expressed as means?SD(n?10).*P?0.05,**P?0.01,***P?0.001vs.DSS-treated group.

?orin in response to DSS-induced colitis,correlated with the blockade of NF-?B activation.A signi?cant increase(P?0.001)in the protein expression of phospho-p65was observed in colon mucosa of DSS-induced model group(Fig.3A). Furthermore,DSS-induced I?B?phosphorylation/degradation and phosphorylation of NF-?B p65were dramatically inhibited by coadministration DSS plus paeoni?orin(P?0.001).In accordance with the in vivo data,paeoni?orin inhibited NF-?B p65nuclear translocation as indicated in LPS-stimulated mouse macrophage RAW264.7cells(Fig.3B).On the other hand,induction of NF-?B-mediated luciferase activity in hu-man colorectal carcinoma HT-29cells by LPS was inhibited by paeoni?orin treatment(25,50,and100?M)in a dose-depen-dent manner(Fig.3C).These results indicated that paeoni?orin signi?cantly blocks the NF-?B signaling pathway in DSS-induced colitis by suppressing I?B?degradation,blocking NF-?B p65nuclear translocation,and inhibiting NF-?B-medi-ated transcriptional activity.

Paeoni?orin inhibits mRNA expression of proin?ammatory mediators and cytokines.To further determine the impact of paeoni?orin on NF-?B signaling cascade,we investigated the expression levels of representative downstream signaling genes involved in NF-?B activation.qPCR analyses of several NF-?B target proin?ammatory mediator genes were carried out.The results showed that mRNA expression of MCP-1,Cox2,IFN-?, TNF-?,IL-6,and IL-17was remarkably induced in in?amed colons of mice exposed to DSS(group3).In contrast,the increase in in?ammatory mediators and cytokines following DSS treat-ment was signi?cantly decreased(P?0.001)in mice receiving paeoni?orin administration(Fig.4,A–F).The results indicated

Fig.2.Effects of paeoni?orin on the expres-

sion of TLR2,TLR4,and TLR5.The mRNA

(A)and protein(B)expression of TLR4in

colon samples isolated from mice(n?6per

group),and the mRNA(C)and protein(D)

expression of TLR4in RAW264.7cells(n?

3per treatment).Quanti?cation of the TLR4

protein expression was performed by densi-

tometric analysis of the blots.E:the mRNA

expression of TLR2and TLR5in colon

samples isolated from mice(n?6per

group).Data were expressed as means?SD

of3independent experiments.***P?0.001

vs.DSS or LPS-treated group;ns,no signif-

icance.

G31

PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

that paeoni?orin ameliorates DSS-induced colitis which correlates with the suppression of NF-?B signaling.

Paeoni?orin reduces the production of TNF-?and IL-6. After7days of DSS administration,the levels of colonic TNF-?and IL-6increased markedly(Table1).Paeoni?orin administra-tion signi?cantly prevented the production of TNF-?and IL-6 in the in?amed colon.Consistent with the in vivo results, IL-6secretion from RAW264.7macrophages was enhanced by LPS treatment,but the enhancement was signi?cantly suppressed by paeoni?orin administration(Fig.4G).The data indicated that paeoni?orin inhibits the production of proin?ammatory cytokines and thereby suppresses in?am-matory responses.

Paeoni?orin decreases the activity of MPO in the in?amed colon.MPO activity,a marker for neutrophil in?ltration into the in?amed tissue,was low in the colonic tissues of control mice(group1and group2)and markedly increased in mice with DSS-induced colitis(Table1).These results,along with the histological con?rmation,demonstrate increased neutrophil in?ltration in DSS-induced colitis mice.The increased MPO activity in mice with DSS-induced colitis was signi?cantly reduced after administration of paeoni?o-rin.This?nding suggested that paeoni?orin exerts anti-in?ammatory effect by reducing neutrophil in?ltration in the colonic mucosa.

LPS-mediated cytokine expression is blunted by paeoni?orin and directly dependent on TLR4.We have demonstrated that paeoni?orin decreases the expression of TNF-?and IL-6(Fig. 4,D and E).To explore the mechanistic involvement of TLR4 in the regulation of cytokines expression by paeoni?orin,TLR4 gene expression was silenced by TLR4siRNA transfection.As illustrated in Fig.5A(top),treatment of RAW264.7cells with TLR4siRNA signi?cantly decreased the expression of TLR4. Exposure of the control siRNA transfected RAW264.7cells to

Fig.3.Paeoni?orin inhibited NF-?B activity

in vivo and in vitro.A:total protein from

colon samples(n?6per group)was ex-

tracted and Western blot was performed.

Quanti?cation of the phospho-p65and I?B?

protein expression was performed by densito-

metric analysis of the blots.Data were ex-

pressed as means?SD of3independent

experiments.B:RAW264.7cells were treated

as described in MATERIALS AND METHODS.

NF-?B p65localization was visualized un-

der a?uorescence microscope(magni?ca-

tion?200).C:HT-29cells were transiently

transfected with pGL4.32[luc2P/NF-?B-RE/

Hygro].Cells were incubated with vehicle or

paeoni?orin(25,50,and100?M)for2h

followed by an additional incubation with

LPS(2?g/ml)for12h.Cells were lysed and

the lysates were analyzed by use of a lu-

ciferase assay system.NF-?B promoter-

driven luciferase activity was expressed as

fold values of control cells(designated as1).

Data are expressed as means?SD of qua-

druplicate of2independent experiments.

###P?0.001vs.vehicle-treated group;

**P?0.01,***P?0.001vs.DSS-treated

group or LPS-treated group.

G32PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

LPS caused a signi?cant increase in the mRNA expression of TNF-?and IL-6(data not shown),and these effects were pre-vented by that cotreatment with paeoni?orin(Fig.5A,bot-tom).Interestingly,the regulatory effect of paeoni?orin was lost in cells lacking the expression of TLR4,indicating a key role for TLR4in mediating the paeoni?orin effect(Fig.5B). To complement the study described above,we overex-pressed TLR4in colon cancer HT-29cells.A robust in-crease in the mRNA expressions of TNF-?and IL-6was observed in LPS-stimulated HT-29cells transfected with TLR4expression vector pcDNA3-TLR4-YFP;this effect was attenuated by cotreatment with paeoni?orin(Fig.5B).Taken together,these results indicated that the effect of paeoni?orin in the downregulation of cytokines is dependent on TLR4expression.

Paeoni?orin suppresses the MAPK pathway signaling.To investigate whether MAPK implicates in the regulatory effects of paeoni?orin on DSS-induced in?ammation,we assessed the activation of signaling molecules including ERK1/2,JNK,and p38MAPKs.As shown in Fig.5C,DSS induced strong phosphorylation(activation)of ERK1/2,JNK,and p38in the in?amed colon.Paeoni?orin treatment markedly inhibited the DSS-induced increase of phosphorylation of ERK1/2,JNK, and p38.The results suggested that MAPK signaling suppres-

Fig.4.Effect of paeoni?orin on the expression of

in?ammatory mediators.A–F:mRNA expres-

sion of MCP-1,Cox2,IFN-?,TNF-?,IL-6,

and IL-17was assessed by quantitative real-

time PCR in colon samples isolated from

mice(n?6per group).Expression was

normalized to?-actin.G:the levels of IL-6

were determined by ELISA.Values are ex-

pressed as means?SD of triplicates of2

independent experiments.*P?0.05,

***P?0.001vs.DSS-treated group or LPS-

treated group.

G33

PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

sion may also contribute to the anti-in?ammatory effect of paeoni?orin.

DISCUSSION

IBD,consisting of Crohn’s disease(CD)and ulcerative colitis(UC),is a chronic in?ammatory disease of the gastro-intestinal tract.The combined prevalence of CD and UC is ?300–400cases/100,000individuals in Western countries (17).Therapeutic strategies currently available for the manage-ment of IBD generally include administration of5-aminosalicy-lates or sulfasalazine,antibiotics,glucocorticoids,immunosup-pressive agents(6-mercaptopurine,azathioprine,methotrexate, cyclosporine,tacrolimus)and biologics such as anti-TNF agents (in?iximab,adalimumab,certolizumab).However,despite their ef?cacy,some patients are unresponsive to these therapies and often suffer from numerous side effects that preclude the contin-uation of the treatment(17).Consequently,alternative medicine is becoming an increasingly attractive approach for the treatment of various IBD among patients unresponsive to or unwilling to take standard medicines(25).Among these alternative approaches is the use of herbal medicines;a substantial body of evidence demonstrates that various herbal ingredients or naturally occurring compounds present signi?cant anticolitic effects(20,39).How-ever,limited scienti?c evidence regarding the mechanistic under-standing of their actions has prevented their prevalent use(20).

Table1.Effects of paeoni?orin on MPO activity and the

levels of TNF-?and IL-6in DSS-induced colitis mice

Group TNF-?,pg/mg pr IL-6,pg/mg pr MPO,U/mg pr

Vehicle20.8?1.914.8?3.7 4.3?0.5

Paeoni?orin29.9?2.815.5?1.1 2.7?0.3

DSS212.7?9.2*103.6?6.9*19.6?1.4*

DSS?paeoni?orin53.8?3.4?42.3?3.7?13.7?1.6?

Colon segments from mice(n?6per group)were excised and homoge-

nized.The supernatants were assayed for the determination of the activity of

MPO and the levels of TNF-?and IL-6.Values are expressed as means?SD

of triplicates of2independent experiments.*P?0.001vs.vehicle-treated

group,?P?0.01,?P?0.001vs.dextran sulfate sodium(DSS)-treated group.

Fig.5.Role of TLR4in paeoni?orin-medi-

ated downregulation of cytokines and effect

of paeoni?orin on the suppression of MAPK

pathway.A:RAW264.7cells were electro-

porated with TLR4siRNA(m)or control

siRNA.Cells were treated with or without

paeoni?orin(50?M)and were stimulated

with LPS(2?g/ml)for12h.Cells were

harvested and analyzed by Western blot

(top)or qRT-PCR(bottom).**P?0.01vs.

vehicle-treated control siRNA-transfected

cells.B:HT-29cells were electroporated

with human TLR4expression vector.Cells

were treated as described above and ana-

lyzed by Western blot(top)or qRT-PCR

(bottom).*P?0.05vs.LPS-treated untrans-

fected cells;###P?0.001vs.LPS-treated

transfected cells.C:expression of the phos-

pho-ERK,phospho-JNK,and phospho-p-38

in colon tissues was analyzed by Western

blot(n?4).Quanti?cation of the protein

expression was performed by densitometric

analysis of the blots.**P?0.01,***P?

0.001vs.DSS-treated group.

G34PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

Oral administration of DSS in mice induces colitis resem-bling human UC.This model corresponds well to the clinical signs of UC in human and can serve as a reliable model for the studies of this disease(26).In the present study,we demon-strated that pre-and coadministration of paeoni?orin signi?-cantly ameliorated the severity of DSS-induced colitis in mice. We have reported that baicalein,a natural?avonoid with anti-in?ammatory activity,ameliorated DSS-induced colitis via targeting Cdx2/PXR axle and inhibiting NF-?B signaling genes(3).Since NF-?B activation is an important step in the progress of human IBD,we hypothesized that the anticolitic effect of paeoni?orin might be related to NF-?B inhibition. The results con?rmed that paeoni?orin ameliorates DSS-in-duced colitis by inhibiting NF-?B activation.Consistent with the in vivo inhibition of I?B?degradation and phosphorylation of NF-?B p65,in vitro evaluation of NF-?B activity suggested a direct role of paeoni?orin in the inhibition of NF-?B signal-ing:LPS treatment of mouse macrophage RAW264.7cells resulted in nuclear translocation of NF-?B and could be re-versed by paeoni?orin treatment;LPS treatment of transiently transfected human colon cancer HT-29cells revealed paeoni-?orin inhibited NF-?B luciferase reporter activity in a dose-dependent manner.Actually,we found that the administration of paeoni?orin not only inhibited NF-?B activity but also decreased the activity of MPO,reduced the production of in?ammatory cytokines(TNF-?and IL-6),downregulated in-?ammatory mediators(MCP-1,Cox2,IFN-?,TNF-?,IL-6, and IL-17),and limited the in?ammatory(histological)re-sponse.Together,these molecular changes resulted in signi?-cant amelioration of DSS-induced colitis.

Although the pathogenesis of IBD is not fully understood,it is generally accepted that the in?ammation is a result of an aberrant immune response to antigens of the host gut micro-biota in genetically susceptible individuals(12).The intestinal lumen contains a vast array of different substances that may interact with the host,such as dietary factors,microbial com-ponents,and environmental pollutants(19).Many of these xenobiotics interact with NF-?B via activation of Toll-like receptors such as TLR4(27).Recently,TLR4-mediated NF-?B activation in the intestine was proved to play a causative role in the disruption of mucosal homeostasis(7).TLR4activation in fact leads to intestinal injury and generally contributes to the pathogenesis of IBD(10).TLR4knockout mice have long been used for the study of TLR4signaling in the pathogenesis of IBD;however,the underlying effects are still elusive.It has been reported that TLR4knockout mice exposed to DSS developed signi?cantly less in?ammation and deletion of TLR4attenuated the colitis in TLR5knockout mice(11,35). Recent work by Fukata et al.(10)demonstrated that the absence of TLR4lowered the susceptibility to colitis-associ-ated colorectal cancer.In contrast,lacking TLR4expression seems to be also detrimental.TLR4-de?cient mice were re-ported to have worse colitis after DSS than wild-type counter-parts but intriguingly were protected from colitis-associated cancer(29,42).The molecular basis for this discrepancy is unclear,whereas some complementary mechanisms might be activated in the absence of TLR4.In our study,TLR4was upregulated in the in?amed colons as well as in LPS-stimulated RAW264.7cells;however,the upregulation of TLR4was signi?cantly reversed by paeoni?orin treatment.To explore the mechanistic involvement of TLR4in the action of paeoni?orin,colon cancer HT-29cells were transfected with human TLR4 expression vector.We found that overexpression of TLR4in HT-29cells potentiated LPS-induced upregulation of TNF-?and IL-6,whereas this effect was attenuated by cotreated with paeoni?orin.In addition,we observed that exposure to the TLR4ligand LPS caused a signi?cant increase in the mRNA expression of TNF-?and IL-6in RAW264.7cells transfected with the control siRNA,and these effects were effectively prevented by that cotreatment with paeoni?orin,whereas si-lencing the expression of TLR4almost completely abrogated the ability of paeoni?orin to counteract the effects of LPS. Thus our results support the hypothesis that paeoni?orin ap-pears to decrease the proin?ammatory cytokines through a TLR4-dependent manner.

Several previous studies have demonstrated that paeoni?orin regulated the key molecules involved in in?ammation.For exam-ple,paeoni?orin suppresses the in?ammatory mediators IL-1, TNF-?,and PGE2in type II collagen induced arthritic rats(40). Zhou et al.(43)have showed that paeoni?orin protects against LPS-induced acute lung injury through downregulation of the activation of p38,JNK,and NF-?B pathways.Chen et al.(2) showed that paeoni?orin suppresses TNF-?induced chemokine production in human dermal microvascular endothelial cells, which is relevant to NF-?B and ERK pathways suppression.Wu et al.(38)suggested that paeoni?orin suppresses NF-?B activa-tion through modulation of I?B?and enhancement of5-?uorou-racil-induced apoptosis in human gastric carcinoma cells.Here we reported that paeoni?orin treatment also signi?cantly inhibited the DSS-induced activation of ERK1/2,JNK,and p38MAPKs. Taken together,these in vitro and in vivo results provide the?rst evidence that the bene?cial effects of paeoni?orin in DSS-induced colitis might be related to the downregulation of TLR4expression and the blockade of the activation of NF-?B and MAPK path-ways.These results may give insight into the further evaluation of paeoni?orin as a therapeutic agent or potential adjunct to IBD therapy.

GRANTS

This work was supported by the National Natural Science Foundation of China(81273572,U1032604),the Natural Science Foundation of Shanghai (12ZR1431400),Innovation Program of Shanghai Municipal Education Com-mission(13YZ043),National Institutes of Health Grant RO1CA127231(S. Mani),Damon Runyon Foundation Clinical Investigator Award(CI1502)(S. Mani),and the Albert Einstein College of Medicine.

DISCLOSURES

No con?icts of interest,?nancial or otherwise,are declared by the author(s).

AUTHOR CONTRIBUTIONS

J.Z.,E.Z.,A.S.,and L.D.performed experiments;J.Z.,E.Z.,and G.C. analyzed data;J.Z.,W.D.,and A.S.interpreted results of experiments;W.D. and S.M.conception and design of research;W.D.and S.M.drafted manu-script;W.D.,X.W.,G.C.,S.M.,and Z.W.edited and revised manuscript;W.D., S.M.,and Z.W.approved?nal version of manuscript.

REFERENCES

1.Broz P,Ohlson MB,Monack DM.Innate immune response to Salmonella

typhimurium,a model enteric pathogen.Gut Microbes3:62–70,2012.

2.Chen T,Guo ZP,Jiao XY,Jia RZ,Zhang YH,Li JY,Huang XL,Liu

HJ.Peoni?orin suppresses tumor necrosis factor-?induced chemokine production in human dermal microvascular endothelial cells by blocking nuclear factor-?B and ERK pathway.Arch Dermatol Res303:351–360, 2011.

G35

PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4

3.Dou W,Mukherjee S,Li H,Venkatesh M,Wang H,Kortagere S,

Peleg A,Chilimuri SS,Wang ZT,Feng Y,Fearon ER,Mani S.

Alleviation of gut in?ammation by Cdx2/Pxr pathway in a mouse model of chemical colitis.PLoS One7:e36075,2012.

4.Dou W,Zhang J,Sun A,Zhang E,Ding L,Mukherjee S,Wei X,Chou

G,Wang ZT,Mani S.Protective effect of naringenin against experimen-tal colitis via suppression of Toll-like receptor4/NF-?B signalling.Br J Nutr Mar18:1–10,2013.

5.Dou W,Zhang J,Zhang E,Sun A,Ding L,Chou G,Wang Z,Mani S.

Chrysin ameliorates chemically induced colitis in the mouse through modulation of a PXR/NF-?B signaling pathway.J Pharmacol Exp Ther 345:473–482,2013.

6.Erridge C,Duncan SH,Bereswill S,Heimesaat MM.The induction of

colitis and ileitis in mice is associated with marked increases in intestinal concentrations of stimulants of TLRs2,4,and5.PLoS One5:e9125,2010.

7.Fiorotto R,Scirpo R,Trauner M,Fabris L,Hoque R,Spirli C,

Strazzabosco M.Loss of CFTR affects biliary epithelium innate immu-nity and causes TLR4-NF-?B-mediated in?ammatory response in mice.

Gastroenterology141:1498–1508,2011.

8.Fort MM,Mozaffarian A,St?ver AG,Correia JdaS,Johnson DA,

Crane RT,Ulevitch RJ,Persing DH,Bielefeldt-Ohmann H,Probst P, Jeffery E,Fling SP,Hershberg RM.A synthetic TLR4antagonist has anti-in?ammatory effects in two murine models of in?ammatory bowel disease.J Immunol174:6416–6423,2005.

9.Fries W,Comunale S.Ulcerative colitis:pathogenesis.Curr Drug

Targets12:1373–1382,2011.

10.Fukata M,Chen A,Vamadevan AS,Cohen J,Breglio K,

Krishnareddy S,Hsu D,Xu R,Harpaz N,Dannenberg AJ,Subbara-maiah K,Cooper HS,Itzkowitz SH,Abreu MT.Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors.Gastro-enterology133:1869–1881,2007.

11.Grabig A,Paclik D,Guzy C,Dankof A,Baumgart DC,Erckenbrecht

J,Raupach B,Sonnenborn U,Eckert J,Schumann RR,Wiedenmann B,Dignass AU,Sturm A.Escherichia coli strain Nissle1917ameliorates experimental colitis via toll-like receptor2-and toll-like receptor4-de-pendent pathways.Infect Immun74:4075–4082,2006.

12.Guarner F.Prebiotics in in?ammatory bowel diseases.Br J Nutr98,

Suppl1:S85–S89,2007.

13.He DY,Dai SM.Anti-in?ammatory and immunomodulatory effects of

paeonia lacti?ora pall.,a traditional chinese herbal medicine.Front Phar-macol2:10,2011.

14.Hou YC,Chu CC,Ko TL,Yeh CL,Yeh SL.Effects of alanyl-glutamine

dipeptide on the expression of colon-in?ammatory mediators during the recovery phase of colitis induced by dextran sulfate sodium.Eur J Nutr 52:1089–1098,2013.

15.Hsu FL,Lai CW,Cheng JT.Antihyperglycemic effects of paeoni?orin

and8-debenzoylpaeoni?orin,glucosides from the root of Paeonia lacti-?ora.Planta Med63:323–325,1997.

16.Im JP,Ye BD,Kim JM,Jung HC,Song IS,Kim JS.Rectal adminis-

tration of lipopolysaccharide and ovalbumin ameliorates acute murine colitis.Dig Dis Sci56:2292–2298,2011.

17.Jobin C.Probiotics and ileitis:could augmentation of TNF/NF?B activity

be the answer?Gut Microbes1:196–199,2010.

18.Kidd M,Gustafsson BI,Drozdov I,Modlin IM.IL1beta-and LPS-

induced serotonin secretion is increased in EC cells derived from Crohn’s disease.Neurogastroenterol Motil21:439–450,2009.

19.Lee JH,Lee B,Lee HS,Bae EA,Lee H,Ahn YT,Lim KS,Huh CS,

Kim https://www.360docs.net/doc/1117233239.html,ctobacillus suntoryeus inhibits pro-in?ammatory cytokine expression and TLR-4-linked NF-kappaB activation in experimental coli-tis.Int J Colorectal Dis24:231–237,2009.

20.Li R,Alex P,Ye M,Zhang T,Liu L,Li X.An old herbal medicine with

a potentially new therapeutic application in in?ammatory bowel disease.

Int J Clin Exp Med4:309–319,2011.

21.Liu Y,Zhang Z,Wang L,Li J,Dong L,Yue W,Chen J,Sun X,Zhong

L,Sun D.TLR4monoclonal antibody blockade suppresses dextran-sulfate-sodium-induced colitis in mice.J Gastroenterol Hepatol25:209–214,2010.

22.Liu ZQ,Zhou H,Liu L,Jiang ZH,Wong YF,Xie Y,Cai X,Xu HX,

Chan K.In?uence of co-administrated sinomenine on pharmacokinetic fate of paeoni?orin in unrestrained conscious rats.J Ethnopharmacol99: 61–67,2005.

23.Lubbad A,Oriowo MA,Khan I.Curcumin attenuates in?ammation

through inhibition of TLR-4receptor in experimental colitis.Mol Cell Biochem322:127–135,2009.24.Okayasu I,Hatakeyama S,Yamada M,Ohkusa T,Inagaki Y,Nakaya

R.A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.Gastroenterology98:694–702,1990.

25.Recio MC,Andujar I,Rios JL.Anti-in?ammatory agents from plants:

progress and potential.Curr Med Chem19:2088–2103,2012.

26.Shah YM,Ma X,Morimura K,Kim I,Gonzalez FJ.Pregnane X

receptor activation ameliorates DSS-induced in?ammatory bowel disease via inhibition of NF-?B target gene expression.Am J Physiol Gastrointest Liver Physiol292:G1114–G1122,2007.

27.Siddique I,Khan I.Mechanism of regulation of Na-H exchanger in

in?ammatory bowel disease:role of TLR-4signaling mechanism.Dig Dis Sci56:1656–1662,2011.

28.Siegmund B,Lehr HA,Fantuzzi G,Dinarello CA.IL-1beta-converting

enzyme(caspase-1)in intestinal in?ammation.Proc Natl Acad Sci USA 98:13249–13254,2001.

29.Sussman DA,Santaolalla R,Strobel S,Dheer R,Abreu MT.Cancer in

in?ammatory bowel disease:lessons from animal models.Curr Opin Gastroenterol28:327–333,2012.

30.Tanaka K.Expression of Toll-like receptors in the intestinal mucosa of

patients with in?ammatory bowel disease.Expert Rev Gastroenterol Hepatol2:193–196,2008.

31.Teng X,Xu LF,Zhou P,Sun HW,Sun M.Effects of trefoil peptide3

on expression of TNF-alpha,TLR4,and NF-kappaB in trinitrobenzene sulphonic acid induced colitis mice.In?ammation32:120–129,2009.

32.Tsuboi H,Hossain K,Akhand AA,Takeda K,Du J,Rifa’i M,Dai Y,

Hayakawa A,Suzuki H,Nakashima I.Paeoni?orin induces apoptosis of lymphocytes through a redox-linked mechanism.J Cell Biochem93: 162–172,2004.

33.Uematsu S,Fujimoto K.The innate immune system in the intestine.

Microbiol Immunol54:645–657,2011.

34.Ungaro R,Fukata M,Hsu D,Hernandez Y,Breglio K,Chen A,Xu R,

Sotolongo J,Espana C,Zaias J,Elson G,Mayer L,Kosco-Vilbois M, Abreu MT.A novel Toll-like receptor4antagonist antibody ameliorates in?ammation but impairs mucosal healing in murine colitis.Am J Physiol Gastrointest Liver Physiol296:G1167–G1179,2009.

35.Vijay-Kumar M,Sanders CJ,Taylor RT,Kumar A,Aitken JD,

Sitaraman SV,Neish AS,Uematsu S,Akira S,Williams IR,Gewirtz AT.Deletion of TLR5results in spontaneous colitis in mice.J Clin Invest 117:3909–3921,2007.

36.Wallace BD,Wang H,Lane KT,Scott JE,Orans J,Koo JS,Venkatesh

M,Jobin C,Yeh LA,Mani S,Redinbo MR.Alleviating cancer drug toxicity by inhibiting a bacterial enzyme.Science330:831–835,2010.

37.Wei SC,Rosenberg IM,Cao Z,Huett AS,Xavier RJ,Podolsky DK.

Tribbles2(Trib2)is a novel regulator of toll-like receptor5signaling.

In?amm Bowel Dis18:877–888,2012.

38.Wu H,Li W,Wang T,Shu Y,Liu P.Paeoni?orin suppress NF-kappaB

activation through modulation of I kappaB alpha and enhances5-?uorou-racil-induced apoptosis in human gastric carcinoma cells.Biomed Phar-macother62:659–666,2008.

39.Yan F,Wang L,Shi Y,Cao H,Liu L,Washington MK,Chaturvedi R,

Israel DA,Cao H,Wang B,Peek RM Jr,Wilson KT,Polk DB.

Berberine promotes recovery of colitis and inhibits in?ammatory re-sponses in colonic macrophages and epithelial cells in DSS-treated mice.

Am J Physiol Gastrointest Liver Physiol302:G504–G514,2012.

40.Zhang LL,Wei W,Wang NP,Wang QT,Chen JY,Chen Y,Wu H,Hu

XY.Paeoni?orin suppresses in?ammatory mediator production and reg-ulates G protein-coupled signaling in?broblast-like synoviocytes of col-lagen induced arthritic rats.In?amm Res57:388–395,2008.

41.Zhang W,Dai SM.Mechanisms involved in the therapeutic effects of

Paeonia lacti?ora Pallas in rheumatoid arthritis.Int Immunopharmacol14: 27–31,2012.

42.Zheng L,Riehl TE,Stenson WF.Regulation of colonic epithelial repair

in mice by Toll-like receptors and hyaluronic acid.Gastroenterology137: 2041–2051,2009.

43.Zhou H,Bian D,Jiao X,Wei Z,Zhang H,Xia Y,He Y,Dai Y.

Paeoni?orin protects against lipopolysaccharide-induced acute lung injury in mice by alleviating in?ammatory cell in?ltration and microvascular permeability.In?amm Res60:981–990,2011.

44.Zhou Y,Wang H,Li YS,Tao YW,Zhang JY,Zhang ZQ.Paeoni?orin

increases beta-defensin expression and attenuates lesion in the colonic mucosa from mice with oxazolone-induced colitis.Yao Xue Xue Bao45: 37–42,2010.(In Chinese).

G36PAEONIFLORIN ABROGATES DSS-INDUCED COLITIS VIA TLR4