Effects Of Total Flavone Of Abelmoschus Manihot On Alleviating Experimental Colitis In Mice By Modulating "Bile Acid-gut Microbiota" Homeostasis Via Farnesoid X Receptor-Associated Pathways

BackgroundUlcerative colitis(UC)belongs to the category of inflammatory bowel disease(IBD).Western diet has a significant impact on the distribution of gut microbiota and its metabolites.Abnormal changes in the intestinal microenvironment may be the initiating factors for the occurrence and development of UC.Westernization of dietary patterns may be one of the reasons for the increase in the incidence of UC in developing countries.The total flavone of Abelmoschus manihot(TFA),a compound extracted from the flowers of Abelmoschus manihot(L.)Medic,has been widely used for the treatment of Crohn’s disease,chronic glomerulonephritis and other diseases.In our previous study,TFA could relieve the symptoms of DSS-induced colitis in mice and inhibit the inflammation of colon.Therefore we explore the possible mechanism of TFA for UC based on the metabolism of bile acids and 16S rRNA sequencing.Experiment 1PurposeIn previous studies,TFA showed a significant improvement effect on DSS-induced colitis in mice,but its effect on intestinal flora and bile acid metabolism and its potential targets are still unclear,and further research is needed.Through animal experiments,detection of fecal bacteria and targeted bile acid metabolism,the regulation effect of TFA on intestinal flora and bile acids in DSS-induced colitis mice was explored.Further explore its possible targets and provide precise targets for the treatment of UC with traditional Chinese medicine.Methods50 male C57BL/6J mice were randomized into five groups,Normal group,DSS group,TFA(high dose)+DSS group,TFA(low dose)+DSS group,and prednisone acetate(PAT)+ DSS group.The colitis model was established by drinking 2.5%DSS solution for 7 days.TFA and PAT was administered 10 days.After the experiment,the symptoms of colitis in mice were evaluated by weight,disease activity index(DAI),colon length and histological scores.qPCR and ELISA were used to detect IL-1β,TNF-α,IL-6 and other pro-inflammatory factors,and the changes of intestinal barrier function in mice were assessed by immunohistochemistry and PAS-AB staining.The distribution of gut microbiota in mice was detected by 16S rRNA sequencing.The content of the bile acids in feces of mice was detected by High-performance liquid chromatography and mass spectrometry(HPLC-MS).The changes of FXR-SHP signaling pathway in mice were detected by qPCR,Western Blot and immunohistochemistry.ResultDSS-induced colitis in mice showed significant body weight loss and colonic damage.The TFA(high dose)group significantly improved colon shortening and histological damage in mice with colitis,inhibited the expression of pro-inflammatory factors(TNF-α mRNA,IL-1β mRNA,IL-18 mRNA),and relieved the intestinal mucosal barrier damage and increase the expression of MUC2 mRNA,KLF4 mRNA and ZO-1 mRNA.TFA(high-dose)group significantly improved the gut microbiota imbalance in mice with colitis and enriched the beneficial bacteria ansia.KEGG pathway analysis showed that the differential bacteria were closely related to secondary bile acids.The TFA(high dose)group could balance the disorder of bile acids metabolism in feces of mice.Treatment with TFA reversed the abnormally reduced levels of CDCA,UDCA,DCA and TCA in colitis mice.Correlation analysis showed that there was a significant correlation between the altered gut microbiota and major bile acids after the intervention of the TFA.FXR and its downstream targets were detected by qPCR,Western blot and immunohistochemistry.The results showed that TFA could activate intestinal FXR and promote the expression of its downstream target gene SHP.Further in vitro experiments showed that TFA could directly activate FXR to increase the expression of the downstream target gene SHP in vitro,which further focused on FXR as a possible potential target.Experiment 2PurposeIn order to further verify the role of TFA in regulating the homeostasis of "bile acidmicrobiota",we constructed FXR knockout mice and performed DSS-induced colitis modeling on the basis of FXR knockout mice.To investigate whether the treatment of TFA in DSS-induced enteritis mice,the regulation of intestinal flora disturbance and the improvement of bile acid homeostasis are mediated by FXR.MethodsThe FXR knockout mice with C57BL/6J background were used for DSS-induced modeling to further explore effects of TFA on alleviating experimental colitis in mice by modulating bile acid-gut microbiota axis homeostasis via farnesoid X receptor-associated pathways.40 FXR-null mice were randomized into four groups,Normal group,DSS group,TFA(high dose)+DSS group,and TFA(low dose)+DSS group.The colitis model was established by drinking 2.5%DSS solution for 7 days.TFA was administered 10 days.After the experiment,the symptoms of colitis in mice were evaluated by weight,DAI,colon length and histological scores.qPCR and ELISA were used to detect IL-1β,TNF-α,IL-6 and other pro-inflammatory factors,and the changes of intestinal barrier function in mice were assessed by immunohistochemistry and PAS-AB staining.The distribution of gut microbiota in mice was detected by 16S rRNA sequencing.The content of the bile acids in feces of mice was detected by HPLC-MS.ResultThe deletion of FXR greatly reduced the therapeutic effect of TFA on mice with colitis.The treatment of TFA could not improve the weight loss,bloody diarrhea and other symptoms of mice,and could not inhibit the expression of pro-inflammatory factors.The protective effect of TFA on the intestinal barrier was significantly attenuated in FXR-null mice.There was no significant change in the structure and diversity of gut microbiota before and after TFA intervention.The abundance of Akkermansia in FXR-null mice was low,and TFA intervention could not promote the abundance of Akkermansia.The regulation of gut microbiota distribution and the enrichment of probiotics were extremely limited in FXR-null mice treated with TFA.The results of bile acid metabolism showed that TFA intervention could not reverse the bile acid abnormalities caused by DSS,and Venn diagrams showed no difference in metabolites before and after TFA intervention.ConclusionTFA may regulate the homeostasis of bile acid-gut microbiota axis by activating FXR to promote the expression of its downstream target gene SHP and improve the symptoms of mice with colitis.