Research Of The Mechanism Of Qing-Chang-Hua-Shi Formula For Ulcerative Colitis Treatment Based On The Host-microbiota Metabolism Of Bile Acids

Purpose:Qing-Chang-Hua-Shi formula(QHF),composed with Baitowweng,Baizhi,Diyu,Huangqin,Chaobaishao and Huangqi,has been wildly used in clinical practice for ulcerative colitis(UC)treatment by clearing the damp and promoting the mucosal healing.In previous studies,we found that there were disturbed in metabolism of bile acids in serum and feces of patients with UC.Our clinical trials showed that QHF could introduce and maintain the remission of this disease.Moreover,we also found that QHF could relieve the symptoms of TNBS-induced colitis in rats and inhibit the inflammation of colon.Therefor we study the mechanism of QHF for UC based on the metabolism of bile acids.Methods1.Colitis mouse model was built by administering 2%dextran sodium sulphate(DSS)in drinking water for 7 days followed by 14 days break for a total of three cycles.The changed of body weight,DAI and colon length of mice were recorded,the histology score was evaluated by research and the expression of the proinflammatory cytokines in colon were detected by qRT-PCR.Moreover,the therapeutic effect of each herb in QHF was also evaluated in DSS-induced acute colitis.2.The content of the bile acids(BAs)in the serum,feces and colon of mice was detected by High-performance liquid chromatography and mass spectrometry(HPLC-MS).Using the Peakview and MultiQuant to get the data,the change of the BAs metabolism after QHF treatment was analysis.Furthermore,the intestinal barrier of mice with or without QHF treatment was also evaluated using different methods,such as western blot,alcian blue/periodic acid-Schiff(AB/PAS)staining and immunohistochemistry.And the changed of intestinal immune was also analysis by western blot.3.Microbial DNA was extracted from ileocecal valve contents samples,the V3-V4 hypervariable regions of the bacteria 16S rRNA gene thermocycler PCR system.Operational taxonomic units(OTUs)were clustered with 97%similarity cutoff using UPARSE with a novel’greedy’ algorithm that performs chimera filtering and OTU clustering simultaneously.Based on these,alpha-diversity,beta-diversity and differential species were analysis.The therapeutic effect of QHF was also evaluated on DSS-induced colitis in Pseudo-germ-free(PGF)mice.Whatsmore,the expression of Cyp7a1 in liver and FXR/FGF15 pathway in ileum was detected by qRT-PCR and western blot.Results1.QHF could attenuate DSS-induced colitis,by reliving the weight loss and shortage of colon length.As for the six herbs of QHF,all of them could attenuate DSS-induced acute colitis,in which Baitouweng,Baizhi and Diyu inhibiting inflammation in colon of mice,Huangqi and Baishao promoting the proliferation of crypt stem cells and Baitouweng and Baizhi protecting goblet cells.Briefly,Baitouweng,Baizhi and Diyu showed more therapeutic effect than Huangqi,Baishao and Huangqin.2.QHF could balance the disorder of bile acids metabolism in serum,feces and colon of mice.Compared with normal mice,the concentration of DCA,UDCA and T-DCA was decreased and CA was increased in serum.In feces,multiple unconjugated bile acids,including chenodeoxycholic acid(CDCA)、β-muricholic acid(β-MCA)and ursodeoxycholic acid(UDCA),were increased in colitis mice.We found the conjugation bile acids were decreased,while the unconjugated bile acids were increased in colon of colitis mice.And the upregulated ratio of unconjugated bile acids and conjugated bile acids in colitis mice was reversed after QHF treatment.Moreover,we also found that the NLPR3/Capsase1/IL-1β pathway was actived in colon tissue in DSS group,and was inactived in DSS+QHF group.3.There is different between DSS-induced chronic colitis mice and normal mice in gut microbiota.In briefly,the diversity was lower and the dysbiosis in gut microbiota of colitis mice.In phylum level,the Firmicutes and the Bacteroides were the most.In the gut microbiota of colitis mice,the level of Firmicutes was decreased while the level of Bacteroides was increased.And this dysregulation was reversed in DSS+QHF group.Far more analysis,the gut microbiota,at genus level,taking the deconjugation of bile acids was upregulated in DSS group and was downregulated in DSS+QHF group.This means that QHF could regulate bile acids metabolism through gut microbiota.And QHF also shown the protective effect on PGF mice.As for the vital part of Cyp7a1 on bile acids metabolism,we detected the expression of this enzyme in liver,and found it was upregulated in DSS group.QHF could inhibit the active of Cyp7a1.Moreover,we also found the FXR/FGF15 pathway was inactive in ileum of colitis mice,and QHF could upregulate the expression of FXR and FGF15 to change the metabolism of bile acids.Research ConclusionQing-chang-Hua-shi formula could attenuate DSS-induced colitis in mice by balancing the metabolism of bile acids through regulating the gut microbiota and FXR/FGF15 pathway,then promoted the proliferation of the crypt stem cells,inhibited the apoptosis of the epithelial cells and.maintained the number and function of goblet cells to protect the intestinal barrier,as well as inhibited the NLRP3/Caspase1/IL-1β pathway to regulate the intestinal immune.