Role Of Intestinal Flora And Th17/Treg Cells In Mechanism Of Rhubarb Peony Decoction Ameliorates Inflammatory Bowel Disease

Objective:Inflammatory bowel disease,included ulcerative colitis(UC)and crohn's disease(CD),had the characteristics of chronic persistent,recurrent,non-specific and inflammatory.The incidence of IBD increased yearly in Asia with unknown etiology,and the possible mechanisms of pathogenesis may be related to genetic factors,environment,intestinal flora and immune.Due to the diversity and complexity of pathogenesis of IBD,and limitations of Western medicine,the effective treatment for IBD was insufficient.Thanks to the therapeutic advantage of traditional Chinese medicine(TCM),which were multi-target and multi-channel,let the research of TCM treatment for IBD attract more attention in recent years.Rhubarb peony decoction(RPD),recorded in Synopsis of Golden Chamber,was used for intestinal inflammation.RPD was widely used for IBD in moderm clinical application without clear mechanism.Immune dysfunction and intesitinal dysbacteriosis were accompanied with IBD,as decreasing short chain fatty acids(SCFAs)and T cells differentiation imbalance.The differentiation imbalance between Th17 and Treg participated in the pathogenesis of IBD directly.Th17 and Treg were two subsets of CD4+T cells,with contrary immune functions,Th17 had the pro-inflaimmatory effect as secreting IL-17,IL-21 and IL-22,Treg was immunosuppressive as secreting IL-10 and TGF-?.Usually,there is a balance between Th17 and Treg,which helpe to avoid the invasion of foreign pathogens,and also helpe to maintain the steady-state of the body.Furthermore,studies have shown that,the intestinal flora and its metabolites affected the immune function,and regulated the differentiation of T cells into Th17 and Treg directly.Our previous studies have found that,RPD played the therapeutic role with increasing the number and proportion of peripheral Treg in IBD mice.But it is still unclear that whether the therapeutic mechanism of RPD for IBD related to regulation of intestinal flora and SCFAs,and thereby,affected the differentiation of Th17/Treg.Therefore,we investigated whether RPD suppresses gut inflammation in an in vivo inflammatory model of IBD induced by dextran sulfate sodium(DSS)in mice,and observe the effect of RPD on intestinal flora,SCFAs as well as the balance of Th17/Treg,point to define its potential mechanism of action for IBD.Methods:1.Quality control of RPDHPLC was used to detect emodin,aloe emodin,rhein,rhubarb,physcion,paeoniflorin,paeonol,amygdalin and trigonelline in RPD,and to determine the fingerprint of RPD.2.UC model establishment,grouping and administrationMice drank 2.5%DSS for 5 days to induce acute IBD model.Control group,DSS group,Mesalazine group,and three RPD groups with different dosages(0.25mg/mL,0.5mg/mL,and 1mg/mL)were set.The mice in Mesalazine and RPD groups were administrated at the same time as establishing IBD model for 14 days.The mice in control and DSS groups were administrated with equal volume of sterile water.3.Effect of RPD on in DSS-induced miceThe body weight and traits of stool were recorded daily for disease activity index(DAI).The colon length,spleen index,the number and proportion of granulocyte within peripheral blood,colonic HE staining,MPO activity and colonic IL-1? content were detected.4.Regulation of RPD on intestinal flora in in DSS-induced micePlate counting and 16S rDNA sequencing were used to detect the number and abundance of intestinal flora,as GC-MS was used to detect the content of SCFAs.5.Effect of RPD on Thl7/Treg and cytokines in DSS-induced miceFlow cytometry was used to detect Thl7 and Treg in both spleen and mesenteric lymph nodes(MLN).WB and IHC were used to detect the expression and distribution of IL-17A and Foxp3 in colon.In the meanwhile,flow cytometry and ELISA were used to detect the content of IL-6,IL-17A;IL-21,IL-22,TNF-a,IFN-?,IL-10 and TGF-? in colon.Results:1.Quality control of RPDRPD contain emodin,aloe emodin,rhein,rhubarb,physcion,paeoniflorin,paeonol,amygdalin and trigonelline when detected by HPLC,as well as the fingerprint of RPD.2.Effect of RPD on in DSS-induced miceDSS-induced IBD in mice was simple and easy to copy.Appropriate concentration and modeling time should be chose according to mice species and gender.C57BL/6 mice drank2.5%DSS for 5 days were used in this study for IBD modeling.Decreased body weight,loose and bloody stool,increased DAI and spleen index,shorter colon with edema and ulcers were obvious in DSS-induced mice.The colon was damaged as the results of HE staining have shown.To be more specific,colonic mucosa damaged,intestinal glands and goblet cells reduced,inflammatory cell infiltrated into submucosa,colonic histopathological scores,the number and proportion of granulocyte in peripheral blood,colonic MPO activity as well as content of IL-1? in serum and colon raised significantly(P<0.01,P<0.001).Mesalazine alleviated the weight loss especially at day 9 and day 11(P<0.05,P<0.01),and reduced DAI especially at day 8 and day 10(P<0.05,P<0.001),compared to that of DSS group.The colonic histopathology score,spleen index,the number and proportion of granulocyte within peripheral blood colon,colonic MPO activity and content of IL-1? in colon,of Mesalazine were decreased significantly compared with DSS group(P<0.05,P<0.01).But Mesalazine was not effective on promoting colon structure.All three dosages of RPD were significantly protective for colon structure,as decreased ulcers,spleen index,MPO activity,IL-1? and the number and proportion of granulocyte compared with DSS group(P<0.01,P<0.001).3.Effect of RPD on structure and abundance of intestinal flora in DSS-induced mice3.1 Results of plate counting assayThe results of plate counting shown that,bifidobacterium decreased(P<0.001).Lactobacillaceae and Bacteroides fragilis increased without significant difference,as Escherichia coli increased significantly of DSS-induced mice(P<0.05).All three dosages of RPD increased Bifidobacterium,decreased Lactobacillaceae,Bacteroides fragilis and Escherichia coli compared with DSS group(P<0.05,P<0.01).3.2 Results of 16S rDNA sequencing assayIntestinal flora diversity and abundance of mice with IBD decreased,as the results of 16S rDNA sequencing have shown.The relative abundance of Actinobacteria and Firmicutes decreased,and the relative abundance of Bacteroidetes,Proteobacteria and Verrucomicrobia increased significantly at phylum level.Bifidobacteerium,Butyricicoccus and Desulfovibrio decreased,as Bacteroides,Butyricimonas,Escherichia and Akkermansia increased significantly at genus level.The abundance and diversity of intestinal flora was not changed by Mesalazine obviously.The relative abundance of Actinobacteria and Firmicutes were significantly increased by RPD.as Proteobacteria and Bacteroidetes decreased.The relative abundance of Butyricicoccus and Desulfovibrio were increased,Bacteroides and Butyricimonas were decreased significantly,as the diversity of intestinal flora was increased by RPD.Therefore,RPD was positive for ameliorating intestinal microecology.3.3 Results of GC-MS assayAcetic acid,propionic acid and butyric acid of mice with IBD decreased,as compared with control(P<0.01,P<0.05 and P<0.01).Mesalazine increased SCFAs without significant difference,as compared with UC mice.All three dosages of RPD increased SCFAs,0.5g/mL and 1g/mL RPD increased butyric acid significantly as compared with DSS group(P<0.01,P<0.05 or P<0.001).4.Effect of RPD on Th17/Treg and cytokines in DSS-induced mice4.1 Regulation of RPD on Thl7/TregMLN Th17 of UC mice increased,Treg decreased(P<0.001).Mesalazine and RPD,especially 0.5mg/mL RPD and lmg/mL RPD,decreased Th17,as increasing Treg(P<0.01,P<0.001).Spleen Thl7 and Treg of UC mice increased(P<0.01,P<0.001).The proportion of Th17 increased,and Treg decreased with Mesalazine administration.RPD,especially 0.5g/mL RPD and lg/mL RPD,restored proportion of Th17 and Treg significantly(P<0.05,P<0.001).The expression of IL-17A and Foxp3 in lamina propria of colon increased significantly in IBD mice induced by DSS.Msalazine,0.5g/mL RPD and 1g/mL RPD then significantly decreased the expression of IL-17A and Foxp3 in lamina propria of colon.4.2 Regulation of RPD on cytokinesCytokines in colon of IBD mice,such as IFN-?,IL-6,TNF-a,IL-17A,IL-22 and IL-10,were significantly higher than control(P<0.05 or P<0.001).IL-21 was increased without significant difference as compared with control.And TGF-? decreased significantly(P<0.001).Mesalazine decreased IFN-?,TNF-?,IL-17A and IL-10(P<0.01 or P<0.001),while decreased IL-21 and IL-22 without significant difference compared with DSS group.TGF-?was increased without significant difference compared with DSS group.RPD was effective for regulation of inflammatory cytokines.0.25g/mL RPD decreased IFN-?,IL-6,IL-17A,IL-22 and IL-10 significantly(P<0.01 or P<0.001).0.5g/mL and lg/mL RPD decreased not only cytokines as 0.25g/mL RPD did,but also TNF-a as well(P<0.01 or P<0.001).IL-21 was decreased by all three dosages of RPD without significant differece compared with DSS group.TGF-? was increased by all three dosages of RPD,but was only increased by 1g/mL RPD significantly(P<0.01).Conclusion:1.The intestinal inflammation and ulceration of IBD mice induced by DSS was obvious.Intestinal flora was changed,as the diversity of intestinal flora decreased,probiotics decreased and opportunistic pathogen increased.So did decreased SCFAs' secretion,Th17/Treg imbalance,and inflammatory cytokines disorder.2.RPD was effective for IBD mice induced by DSS,regulated the diversity and quantity of intestinal flora,and restored proportion of Th17/Treg and cytokines secretion.The therapeutic mechanism may be related to the regulation of intestinal dysbacteriosis,recovery of intestinal immune function and the balance between Th17/Treg.