Effect Of Dahuang Mudan Decoction On Inflammatory Bowel Disease By Regulating Flagellin/TLR5 Signaling Pathway

Objective:Inflammatory bowel disease is a chronic condition of unknown origin characterized by severe inflammation and mucosal destruction in the intestine,locating in colon and rectum.It's symptom showing abdominal pain,diarrhea,bloody stool,and so on.The course of IBD is fairy long,easy to attack repeatedly and difficult to cure thoroughly,which has a great impact on both human health and spirit.In addition,The odds of IBD developing into cancer is 5 to 10 times higher than normal.At present,the etiology and pathogenesis remains unclear.But it is now widely acknowleged that it result from inappropriate intestine immune response by environment,gut microbiota and infection factors,in genetically predisposed individuals,inducing intestinal tissue damage and persistent inflammation.The significant role of gut microbiota in IBD having being proved by a number of research,espically the pathogen-associated molecular pattern of intestinal flora,such as lipopolysaccharide,peptidoglycan,flagellin,and so on.There is lack of effective treatment,on account of uncertain etiology and complicated condition.Clinical trials showed that Traditional Chinese medicine has a unique efficacy in the treatment of IBD,conventional Chinese medicine as follows,Shengling Baishu Powder,Pulsatilla Decoction,Dahuang Mudan Decoction,and so like.Dahuang Mudan Decoction derives from Synopsis of Golden Chamber,authored by Zhongjing Zhang,with purging heat,dispelling dampness,breaking stasis and eliminating carbuncles,a conventional Chinese medicine of intestinal carbuncle resulting from damp-heat and blood stasis.It has being applied to inflammatory disease,for instance,ulcerative colitis and acute appendicitis,and so on,in modern clinical treatment.However,the mechanism of Dahuang Mudan Decoction remains unclear.It has been said that Dahuang Mudan Decoction and its active constituents is able to regulate the gut microbiota of IBD mice.Flagellin is structure component of the intestinal bacterial flagellum,as well as an important pathogen-associated molecular patterns.It is recognized and bound by TLR5,involving MyD88/IRAK/TRAF6 signaling pathway,activating NF-?B,and resulting in the level of pro inflammatory cytokines gene expression increased,such as IL-8?MIP3,inducing proinflammatory response.Studies have shown that flagellin/TLR5 plays a crucial role in the occurrence and development of IBD.In addition,paeoniflorin is the main ingredient in total glycosides,and accounts for about 90,which is also one of important active ingredient of Dahuang Mudan Decoction So,it is hypothesized that treatment of inflammatory bowel disease with Dahuang Mudan Decoction may be related to reglute flagellin/TLR5/NF-KB signaling pathway.Therefore,the aim of this study is to explore the mechanism of Dahuang Mudan Decoction from flagellin/TLR5/NF-?B signaling pathway.Methods:1.Quality control of Dahuang Mudan DecoctionThe fingerprints of 10 batches of Dahuang Mudan Decoction were detected by high performance liquid chromatography(HPLC),with the standard of trigonelline,amygdalin,paeoniflorin,Paeonol,aloe-emodin,rhein,emodin and chrysophanol being selected as quality control.2.Animal model and administrationC57BL/6 mice were divided into 6 groups according to random group method:control group,model group,mesalazine group,low dose group of Dahuang Mudan Decoction(DL),Dahuang Mudan Decoction middle dose group(DM),Dahuang Mudan Decoction high dose group(DH).Except the contro group intaking distilled water,other groups were treated with 3%dextran sulfate sodium(DSS)solution for 7 days to induce IBD model,and then given distilled water instead for another 7 days.The control group and model group were given distilled water by oral administration,and other groups was given corresponding drug at the dosage of 0.2mL/10g for 14 consecutive days.3.Effect of Dahuang Mudan Decoction on IBD MiceThe IBD mouse model was induced by free drinking of 3%DSS solution for 7 days continuously.The efficacy of Dahuang Mudan Decoction was demonstrated by the following indicators,the percentage of body weight loss,DAI score,peripheral white blood cell count,colon morphological changes,colon length change,and histopatho logical alterations of colon tissue.4.Detection of flagellin antibody in serumThe serum of the mouse was collected and the ELISA kit was used to detect the content of flagellin antibody in serum.5.Effects of Dahuang Mudan Decoction on TLR5 and NF-?B p65 in colonic tissueThe expression of TLR5 in colonic tissue was detected by immunohistochemistry and Western blot,and NF-?B p65 in colonic tissue were detected by Western blot.6.Effects of Dahuang Mudan Decoction on TLR5/NF-?B Signaling PathwayHT-29 cells was stimulated by flagellin,and cytokines in cell supernatant was measure by CBA kit.The level of TLR5 mRNA and p65 mRNA was detected by RT-PCR.Then,the relative expression of TLR5,MyD88,I?B and NF-?B p65 was detected by Western blot.Result:1.Quality control of Dahuang Mudan DecoctionThe fingerprints of rhubarb peony soup were detected by high performance liquid chromatography,with the standard of fenugreekin,amygdalin,paeoniflorin,paeonol,aloe emodin,rhein,emodin and chrysophanol as control.The precision,stability and repeatability of high performance liquid phase method were measued to ensure the feasibility,stability and reliability of the experiment.In addition,the detection reslut of 10 batches of rhubarb peony soup fingerprints,at the same chromatographic conditions,showing that the similarity of each batch fingerprint was above 0.90 excepting one sample,which indicated that the experimental compound was stable and laid the foundation for further experiments.2.Effect of Dahuang Mudan Decoction on IBD MiceCompared with the control group,the weight of the model group decreased,and the difference was statistically significant starting from the seventh day(P<0.01);The DAI score increased(P<0.001);The total number of white blood cells,granulocyte,blood platelet and red blood cell distribution width increased(P<0.01,P<0.001);The swelling,brittle,broken and shorten colon(P<0.001);The colon tissue structure is not complete under the microscope,showing that intestinal epithelium and intestinal gland disorder or even fall off,ulcers significantly,with infiltrating a large number of lymphocyte in lamina propria and submucosal,accompanied by vasodilation.Compared with the model group,the weight of the Dahuang Mudan Decoction group with 3 dose was gradually to normal(P<0.05,P<0.01,P<0.001);The DAI score decreased(P<0.001);The total number of white blood cells,granulocyte,blood platelet and red blood cell distribution width decreased(P<0.05,P<0.01,P<0.001);Colonic tissue morphology and the length of colon gradually to normal(P<0.05);The colon tissue structure is relatively complete under the microscope,showing that intestinal epithelium and intestinal gland neatly arranged,ulcers reduced,with infiltrating a less number of lymphocyte in lamina propria and submucosal.In conclusion,it shows that Dahuang Mudan Decoction had a therapeutic effect on IBD mice.3.Effects of Dahuang Mudan Tang on Flagellin/TLR5/NF-?B Signaling Pathway in IBD miceThe flagellin antibody in the serum was increased,comparing with control group(P<0.001),and decreased in the Dahuang Mudan Decoction 3 groups(P<0.05,P<0.01).Compared with the control group,the expression of TLR5 and NF-?B p65 in the colon tissue increased(P<0.001),and Dahuang Mudan Decoction down-regulate the expression of TLR5 and NF-?B p65(P<0.05,P<0.01,P<0.001)4.Effects of Dahuang Mudan Tang on TLR5/NF-?B Signaling Pathway in HT-29 CellsCompared with the control group,flagellin promoted the secretion of IL-8,IL-6,IL-1?,and TNF(P<0.01,P<0.001),but Dahuang Mudan Decoction reduced the secretion of those cytokine(P<0.05,P<0.01,P<0.001).At the same time,the expression of TLR5 mRNA and p65 mRNA in HT-29 induced by flagellin increased(P<0.05,P<0.01),and the Dahuang Mudan Decoction inhibited TLR5 mRNA and p65 mRNA overexpression.In addition,the expression of TLR5,MyD88,and NF-?B p65 increased,but the expression of I?B reduced,showing that flagellin is able to activate TLR5/NF-?B Signaling Pathway and Dahuang Mudan Decoction regulate it to cure IBD.Conclusion:1.The level of anti-flagellin antibody in serum and expression of TLR5 in colon of IBD mice increased2.The Dahuang Mudan Decoction has a therapeutic effect on IBD mice,and its therapeutic mechanism is related to its regulation of flagellin/TLR5/NF-?B signaling pathway.