清肠消澼饮调控FXR信号通路修复肠上皮屏障治疗溃疡性结肠炎小鼠的作用机制OA

Objective To investigate the mechanism by which Qingchang Xiaopi Yin(QXY)repairs the intestinal epithelial barrier and treats ulcerative colitis(UC)in mice through modulating the farnesoid X receptor(FXR)signaling pathway.Methods Forty C57BL/6 mice were randomly assigned to four groups:normal group,model group,QXY group,and mesalazine group,with 10 mice in each group.Twenty FXR knockout(FXR-/-)C57BL/6 mice were randomly divided into three groups:an FXR-/-normal group(n=6),an FXR-/-model group(n=7),and an FXR-/-QXY group(n=7).The UC model was induced by administering 3%dextran sulfate sodium(DSS)in drinking water ad libitum for 7 consecutive days.Concurrently,mice in the QXY group and FXR-/-QXY group received daily rectal enemas of QXY solution(14.71 g·kg-1),and the mesalazine group received daily rectal enemas of mesalazine suspension(1 g·kg-1).All groups were treated at a volume of 10 mL·kg-1 for 10 consecutive days.Differentially expressed genes(DEGs)were identified by transcriptomic analysis of colon tissue and subjected to GO functional enrichment and KEGG pathway enrichment analyses.FXR protein expression was detected by immunohistochemistry.The mRNA expression levels of FXR,IBABP,ASBT,TNF-α,IL-1β,IL-6,ZO-1,Occludin,and Claudin1 were measured by qRT-PCR.Mucin expression was assessed by Alcian blue-nuclear fast red staining,and tight junction protein expression was detected by immunofluorescence.Histopathological changes were observed by HE staining.The gut microbiota composition was analyzed by 16S rDNA sequencing,and the levels of bile acid metabolites in cecal contents were measured by targeted metabolomics.Results(1)A total of 95 DEGs(70 upregulated and 25 downregulated)were identified between the model group and the QXY group.These DEGs were mainly enriched in cholesterol metabolism,primary bile acid biosynthesis,focal adhesion,and bile secretion pathways,with a close association between the key gene targets FXR(Nr1h4)and CyP7a1.The biological processes involved were predominantly related to immune-inflammatory responses and bile acid metabolism.(2)Compared with the normal group,the model group showed significantly decreased protein expression of FXR and mRNA expression of FXR and IBABP(P＜0.05,P＜0.01),significantly increased mRNA expression of TNF-α,IL-1β,and IL-6(P＜0.01),significantly reduced mucin expression(P＜0.01),significantly downregulated protein expression of tight junction proteins ZO-1 and Occludin(P＜0.01),and significantly decreased mRNA expression of ZO-1,Occludin,and Claudin1(P＜0.01)in colon tissue.Compared with the model group,the QXY group exhibited significantly upregulated protein expression of FXR and mRNA expression of FXR,IBABP,and ASBT(P＜0.05,P＜0.01),significantly downregulated mRNA expression of TNF-α,IL-1β,and IL-6(P＜0.01),significantly increased mucin expression(P＜0.01),significantly enhanced protein expression of ZO-1 and Occludin(P＜0.05,P＜0.01),and significantly elevated mRNA expression of ZO-1,Occludin,and Claudin1(P＜0.01)in colon tissue.(3)In the FXR-/-mouse experiments,compared with the FXR-/-normal group,the FXR-/-model group demonstrated a significant decrease in body weight(P＜0.01),significant shortening of colon length(P＜0.01),significant increases in the disease activity index(DAI)and histopathological scores(P＜0.01),markedly downregulated FXR protein expression(P＜0.01),significantly reduced expression of mucin and tight junction proteins ZO-1 and Occludin(P＜0.01),significantly decreased alpha diversity indices(Sobs,Shannon,Simpson,Chao1,Ace,and Pielou)of the gut microbiota(P＜0.05,P＜0.01),a clear separation in the principal coordinate analysis plot,significantly decreased relative abundances of Lactobacillus,Mammaliicoccus,Limosilactobacillus,Adlercreutzia and Blautia(P＜0.01),significantly increased relative abundance of Bacteroides(P＜0.01),an increasing trend in the ratio of primary to secondary bile acids and in the levels of CA and CDCA in the cecal contents(P＞0.05),a significantly increased β-UDCA level(P＜0.05),and significantly decreased levels of LCA,UCA,7-ketoLCA,and DCA(P＜0.01).Compared with the FXR-/-model group,none of these indices in the FXR-/-QXY group were significantly altered(P＞0.05),indicating that FXR knockout may abolish the therapeutic effect of QXY on DSS-induced UC in mice.Conclusion QXY exerts a protective effect against DSS-induced ulcerative colitis in mice by restoring gut microbiota homeostasis,modulating bile acid metabolism,enhancing intestinal epithelial barrier integrity,and suppressing inflammatory responses.This mechanism is intimately associated with the activation of the FXR signaling pathway.