Role And Mechanism Of Gut Flora-bile Acid-FXR/CCL21 Axis In The Pathogenesis Of Colitis

Part 1 Intestinal dysbiosis and bile acid dysmetabolism of colitis in miceObjective:To explore the correlation between the intestinal dysbiosis and bile acid metabolic disorders in mice with acute colitis.Methods:C57BL/6 mice were randomly divided into normal control group and DSS group.The normal control group received no intervention.The DSS group was treated with 2.5%DSS drinking water for 5 days to induce colitis,and sacrificed after 7 days.Fresh stool specimens on day 0 and day 7 of the DSS group were collected for 16S rDNA sequencing and fecal bile acid targeted determination.Analyze the characteristics of intestinal microbiota dysbiosis and bile acid metabolic disorders,and conduct correlation analysis between them.Results:16S rDNA sequencing analysis:Compared with that before modeling,the alpha diversity of the fecal flora of DSS-induced colitis mice was significantly reduced and the diversity index chaol,observed_species,PD_whole_tree,and shannon index reduced significantly(P values were 0.003,0.001,0.041 and 0.043,respectively).PCoA analysis showed that the fecal flora of mice with colitis was significantly different from that before modeling.After DSS modeling,the composition and structure of mouse fecal flora changed significantly.Further LEfSe discriminant analysis showed that Lactobacillus,Bifidobacterium and Staphylococcus were significantly reduced after DSS modeling,and Turicibacter,Lachnospiraceae_NK4A136_group,Bacteroides,Enterococcus,Escherichia-Shigella,Clostridium_sensu_stricto₁ and Pasteurella were significantly increased after DSS modeling.PICRUST2 function prediction and PCA analysis indicated that the function of mouse intestinal flora changed significantly after DSS modeling.KEGG analysis identified 43 significantly changed signal pathways,among which the top 5 pathways are Biosynthesis of ansamycins,Secondary bile acid biosynthesis,D-Glutamine and D-glutamate metabolism,D-Alanine metabolism and Fatty acid biosynthesis pathways.Fecal bile acid targeted metabolomics analysis:PCA analysis showed that the fecal bile acid profile of colitis mice changed significantly compared with that before DSS modeling.The total bile acid content was significantly reduced(P=0.016).Specifically,the unconjugated bile acid,primary bile acid and secondary bile acid were significantly reduced(P values were 0.017,0.008 and 0.043),the ratio of secondary bile acid to the primary bile acid decreases(P=0.032).According to the VIP value(VIP>1)based on OPLS-DA model and the P value of significance test(P<0.05),the differential bile acids before and after DSS modeling were jointly screened.A total of 11 differential bile acids were identified including 12-ketoLCA,TUDCA,TDCA,THDCA,isoDCA,?UDCA,TCDCA,6-ketoLCA,TCA,TLCA and NorDCA.Pearson correlation analysis showed that the abundance of Bifidobacterium and Lactobacillus were significantly positively correlated with the content of most differential bile acids.Conclusion:The structure and function of the fecal flora of mice with DSS-induced colitis changed significantly,mainly manifested as decreased diversity,decreased beneficial bacteria,increased harmful bacteria and conditional pathogenic bacteria,and decreased secondary bile acid synthesis function.The fecal bile acid profile of colitis mice changed significantly,the total bile acid level decreased,and the ratio of secondary to primary bile acid decreased.The abundance of Bifidobacterium and Lactobacillus in the feces of mice with colitis was significantly positively correlated with the content of most differential secondary bile acids.Part 2 Effect of Bifidobacterium infantis on colonic inflammation and intestinal flora-bile acid metabolismObjective:To analyze the effects of Bifidobacterium infantis on colon inflammation,intestinal flora,bile acid metabolism and intestinal transcriptome in mice,and to preliminarily reveal that Bifidobacterium infantis can relieve colon inflammation by regulating the intestinal flora and bile acid metabolism.Methods:C57BL/6 mice were randomly divided into normal control group,DSS group and Bifidobacterium infantis group.The DSS group was treated with saline by gavage for 7 days while modeling,and the Bifidobacterium infantis group was treated with Bifidobacterium infantis by gavage for 7 days while modeling.Evaluate the colonic inflammation of the mice,and collect fresh stool samples on day 0 and day 7 for 16S rDNA sequencing and fecal bile acid targeted determination.The mice were killed and their colon was collected for RNA sequencing.Multi-omics analysis was performed to explore the role of Bifidobacterium infantis in the pathogenesis of colitis.Results:Compared with the DSS group,the disease activity score and colon histological score of the Bifidobacterium infantis group were reduced(P values were 0.009,0.046),and the colon inflammation is significantly relieved.16S rDNA sequencing data analysis:The alpha diversity of the fecal flora of mice in the Bifidobacterium infantis group did not change significantly compared with the DSS group(P values were 0.200,0.343,0.343 and 0.486,respectively).PCoA analysis indicated that there were some differences in the fecal flora between the two groups.The abundance of Bifidobacterium and Lactobacillus in the fecal of the Bifidobacterium infantis group showed an increasing trend compared with the DSS group,and the abundance of Bacteroides,Enterococcus,Escherichia-Shigella,Clostrium_sensu_stricto₁ and Pasterurella had a decreasing trend,but the differences were not statistically significant(P values were 0.886,0.343,0.886 0.886,0.514,0.886 and>0.999,respectively).Fecal bile acid targeted metabolomics analysis:Compared with the DSS group,the total fecal bile acids,unconjugated bile acids,and secondary bile acids of the Bifidobacterium infantis group showed an increasing trend,but the differences were not statistically significant(P value were 0.114,0.114 and 0.200,respectively).Compared with the normal control group,the fecal bile acid THDCA content of the DSS group was lower(P=0.016),while the Bifidobacterium infantis group had a significantly higher THDCA content than the DSS group(P=0.029).Intestinal transcriptomics analysis:RNA-seq and differential expression analysis of Bifidobacterium infantis group and DSS group showed that there were 450 differential expression genes(Differential Expression Genes,DEGs)between the two groups,including 401 up-regulated and 49 down-regulated DEGs.Functional enrichment analysis shows that there are a variety of biological processes related to immune inflammation,such as leukocyte migration,leukocyte chemotaxis and leukocyte cell-cell adhesion,etc.,and KEGG pathways such as cytokine-cytokine receptor interaction,IL-17 signaling pathway and cell adhesion molecules,participating in the relieving process of colon inflammation mediated by Bifidobacterium infantis.GEO database analysis showed that the bile acid receptor FXR was significantly reduced in UC patients.Western blot results showed that the expression level of colonic FXR was significantly lower than that of the normal control group,while the expression level of FXR in the Bifidobacterium infantis group was significantly higher than that of the DSS group.Conclusion:Bifidobacterium infantis can alleviate DSS-induced colonic inflammation in mice.The results of multi-omics analysis showed that Bifidobacterium infantis can regulate the intestinal flora and bile acid metabolism of colitis mice.RNA-seq showed that Bifidobacterium infantis had a significant impact on the immune and inflammation-related signal transduction pathways in colon of colitis mice.The expression of bile acid receptor FXR is down-regulated in the colon tissues of UC patients and colitis mice,while the intervention of Bifidobacterium infantis can up-regulate the colonic expression of FXR in colitis mice.FXR may play an important role in the alleviation of colon inflammation mediated by Bifidobacterium infantis.Part 3 Bifidobacterium infantis participates in the alleviation of colonic inflammation via FXR/CCL21 axis in miceObjective:Analyze the effect of bile acid receptor FXR on intestinal flora and bile acid metabolism of colitis mice,and explore the potential mechanism of the interaction between FXR and its downstream target genes in alleviating colon inflammation.Methods:Wild-type C57BL/6 mice were divided into WT DSS group and WT Bifidobacterium infantis group,FXR knockout mice were divided into KO DSS group and KO Bifidobacterium infantis group.The four groups of mice were given 2.5%DSS drinking water for 5 days to induce colitis.The WT and KO Bifidobacterium infantis groups were treated with Bifidobacterium infantis by gavage for 7 days,and the DSS groups were treated with saline by gavage.Evaluate the colon inflammation and collect the fresh fecal specimens on day 0 and day 7 for 16S rDNA sequencing and fecal bile acid targeted determination.The mice were killed on day 7 and colon tissues were collected for RNA sequencing.Multi-omics analysis was conducted to explore the role of FXR in mediating the protective effect of Bifidobacterium infantis on colon inflammation.Bioinformatics analysis was performed to predict FXR target genes,and preliminarily verifies the effect of FXR on candidate target genes in an inflammatory cell model induced by rhTNF-a.Then,CUT&Tag and qPCR was conducted to verify the targeted binding between FXR and CCL21.Results:The weight,disease activity score and colonic histological score of the KO Bifidobacterium infantis group had no significant changes compared with the KO DSS group(P values were 0.185,0.421 and 0.857,respectively),and the colon inflammation was not relieved.16S rDNA sequencing data analysis:On day 0,compared with WT groups,the alpha diversity of the fecal flora of KO groups was significally reduced;PCoA analysis showed that there was a significant difference in the composition of the fecal flora between the two groups;LEfSe analysis found that compared with WT groups,Ruminococcaceae_UCG₀14 was significantly enriched in KO groups,while Bifidobacterium and Turicibacter were significantly reduced.On day 7,compared with the KO DSS group,the alpha diversity of fecal flora of the KO Bifidobacterium infantis group did not change significantly;PCoA analysis showed that there was a slight distinction of the fecal flora between the two groups.LEfSe analysis found that,for KO DSS group,the abundance of Turicibacter,Escherichia-Shigella,Romboutsia,Enterococcus,and Akkermansia increased,while the abundance of Jeotgalicoccus and Lachnoclostridium decreased,among which the abundance of Turicibacter,Escherichia-Shigella,Romboutsia and Akkermansia showed a decreasing trend after administration of Bifidobacterium infantis,but the difference was not statistically significant.Fecal bile acid targeted metabolomics analysis:On day 0,compared with WT groups,the ratio of secondary bile acid to primary bile acid in FXR KO mice obviously decreased(P=0.005);On day 7,for KO DSS group,the levels of fecal conjugated bile acid and primary bile acid increased significantly(P values were 0.049 and 0.007,respectively),the ratio of unconjugated to conjugated bile acid and the ratio of secondary to primary bile acid decreased significantly(P values were 0.028 and 0.014,respectively).For KO Bifidobacterium infantis group,the levels of fecal conjugated bile acid and primary bile acid showed a decreasing trend,and the ratio of unconjugated to conjugated bile acid showed an increasing trend,but their differences were not statistically significant(P value were 0.057,0.629 and 0.229,respectively),and the ratio of secondary to primary bile acid showed no increasing trend.On day 0,the fecal bile acid differential analysis of KO groups and WT groups identified five differential bile acids,including(3MCA,dehydroLCA,isoLCA,alloLCA and LCA.On day 7,there was no significant difference in fecal bile acid between the KO Bifidobacterium infantis group and KO DSS group.Intestinal transcriptomics analysis:There are 471 DEGs between the KO DSS group and WT DSS group including 242 up-regulated DEGs and 229 down-regulated DEGs.Functional enrichment analysis showed that there were 19 significantly differential KEGG pathways between the two groups,including several immuno-inflammation-related signaling pathways such as cell adhesion molecules,cytokine-cytokine receptor interaction,and leukocyte transendothelial migration.Bioinformatics analysis screened out 7 FXR candidate target genes,including CCL21,TIE1,CDC25B,MMRN2,SLCLA3,PLIN1 and SLC1A1.Preliminary verification of CCL21 on HT29 cell level:An inflammatory cell model was established by rhTNF-?(100ng/ml)stimulation.qRT-PCR showed that CCL21 was significantly increased when incubated with rhTNF-a for 24h(P=0.0001).Treatment with FXR agonist GW4064 can significantly reduce the mRNA level of CCL21 in inflammatory cell model(P<0.0001).CUT&Tag-qPCR results showed that the level of CCL21 in experimental group was significantly higher than that of the IgG negative control group.Conclusion:After FXR knockout,the alleviating effect of Bifidobacterium infantis on colitis in mice is weakened.FXR knockout has a significant effect on intestinal flora and bile acid metabolism of mice.After FXR knockout,the effect of Bifidobacterium infantis on intestinal flora and bile acid metabolism of colitis mice is weakened.Bioinformatics analysis suggests that FXR may alleviate colonic inflammation by targeting CCL21 expression.Results at the cellular level confirm that activating FXR can inhibit the expression of CCL21 in inflammatory cell models.Further,the targeted binding between FXR and CCL21 was verified by CUT&Tag.Part 4 Preliminary investigation of the transformation mechanisms from ulcerative colitis to carcinogenesisObjective:To explore the early abnormal changes of gene expression and biological processes in the process of UC carcinogenesis.To analyze the characteristics of intestinal dysbiosis in the mouse model of colitis-associated colorectal cancer induced by AOM/DSS,and to preliminarily explore the effect of FXR on intestinal flora and the role of this effect in the transformation from inflammation to cancer in mice.Methods:Through GEO database mining and WGCNA,the early abnormal changes of gene expression and biological processes in the process of UC carcinogenesis are explored.As for animal experiments,wild-type C57BL/6 mice were divided into WT control group and WT AOM/DSS group,FXR knockout mice were used as KO AOM/DSS group.The WT control group was given no intervention,and the WT and KO AOM/DSS groups were administered AOM(12.5mg/kg)intraperitoneally and three cycles of 2%DSS drinking water orally to induce colitis-associated colorectal cancer.Fresh stool samples before and after the experiment were collected for metagenomic sequencing.The mice were sacrificed after 12 weeks and the formation of tumors was evaluated.Results:A total of 13 controls,18 UC and 6 CAD in GSE47908 were enrolled in WGCNA.Six functional modules were identified based on 4929 DEGs between CAD and control group(log2 FC cut-off is 0.3).Green and blue modules were selected because of their consistent correlation with UC and CAD,and the highest correlation coefficient with the progress of UC-associated carcinogenesis.Functional enrichment analysis revealed that genes of these two modules were significantly enriched in biological processes,including mitochondrial dysfunction,cell-cell junction,and immune responses.Thirteen hub genes(SLC25A3,ACO2,AIFM1,ATP5A1,DLD,TFE3,UQCRC1,ADIPOR2,SLC35D1,TOR1AIP1,PRR5L,ATOX1,and DTX3)were identified.As for animal experiments,the weight loss of the KO AOM/DSS group was more significant than that of the WT AOM/DSS group.The total number of colon tumors and both of the number of tumors with a long diameter of 1-2mm and>2mm were higher than that of the WT AOM/DSS group.Metagenomic sequencing analysis of fecal flora of FXR knockout mice and wild type mice:Before modeling,FXR knockout mice had a significant decrease in the gene number of flora compared with wild mice(P=0.002).NMDS analysis showed that the composition of the flora was significantly changed in FXR knockout mice,and LEfSe analysis identified 21 differential bacterial genera and 67 differential bacterial species.Then,128 KEGG pathways with significant differences between FXR knocked mice and wild type mice were selected by differential analysis of functional abundance.Metagenomic sequencing analysis of the fecal flora of wild mice before and after AOM/DSS modeling:The number of fecal flora genes of wild mice after modeling was significantly lower than that before modeling(P=0.004).NMDS analysis showed that the composition of intestinal flora changed obviously after modeling,and LEfSe analysis screened out 23 differential genera and 64 differential species.Functional abundance differential analysis identified 126 significantly differential KEGG pathways.Take the intersection of the above two LEfSe differential analysis,then we found 13 common differential genera and 36 common differential species.Similarly,we also take the intersection of the above two functional abundance differential analysis and then identified 71 common KEGG pathways.The 13 bacterial genera include Mucispirillum,Prevotella,Unclassified_Lachnospiraceae,Roseburia,Oscillibacter,Clostridioides,Bilophila,Unclassified_Firmicutes,Butyrivibrio,Eubacterium,Ruminococcus,Acetivibrio,and Flavonifractor,the abundance of all of which was significantly changed after modeling in wild-type mice,and was also significantly changed in FXR knocked out mice compared with wild type mice before modeling.Conclusion:GEO database mining and WGCNA reveals that the abnormal changes of mitochondrial function,cell-cell junction,and immune responses may play crucial roles in the process of UC carcinogenesis.And 13 hub genes were identified as the potential key genes,including SLC25A3?ACO2?AIFM1?ATP5A1?DLD?TFE3?UQCRC1?ADIPOR2?SLC35D1?TORI AIP1?PRR5L?ATOX1,and DTX3.FXR knockout promotes the transformation from colitis to cancer.Both FXR knockout and AOM/DSS-induced colitis-associated colorectal cancer can reduce the number of genes in fecal flora of mice,and significantly change the structure and function of the flora.Metagenomic sequencing analysis revealed that 13 bacterial genera and 36 bacterial species were simultaneously affected by FXR knockout and AOM/DSS modeling.