Correlation Between Inflammatory Response And Intestinal Flora And Bile Acid Metabolism In UC Patients With Two TCM Syndromes

BackgoundGut microbiota and metabolites are considered to be involved in the pathogenesis of inflammatory bowel disease(IBD).Basic experiments have shown that imbalanced intestinal flora and altered bile acid profiles affect ulcerative colitis(ulcerative colitis UC)intestinal inflammation recently.It was found that bile acid receptors Takeda G-protein-coupled receptor 5(TGR5)and Vitamin D receptor(VDR)participated in intestinal inflammatory responses via regulating NF-?B signaling.However,the differences in the fecal bile acid profiles and the expression of bile acid receptors in UC patients with different TCM syndromes and the relationship between bile acids,gut microbiota and inflammation in patients with UC are still unclear.We hypothesized that altered profiles of fecal bile acids might be correlated with gut microbiota,and inflammatory responses in UC patients with different TCM syndromes,and gut microbiota bile acid metabolites might play a role in the pathogenesis of UC.ObjectivesThe aim of this study was to investigate the changes in fecal bile acids and analyze the relationship between bile acids,gut microbiota and inflammation in UC patients with different TCM syndromes.MethodsActive UC patients including large intestine damp-heat syndrome,spleen-deficiency dampness-obstruction syndrome and age,sex,and body mass index-matched healthy controls(HCs)who met the established inclusion criteria from April 2019 to January 2020 in the China-Japan Friendship Hospital Hospital were recruited.Fresh stool samples,serum samples,and intestinal mucosa samples were collected from the subjects.The study used 16S rDNA sequencing technology to detect the differences in intestinal flora between UC patients and HCs.Fecal bile acids were measured by targeted metabolomic approaches.Mucosal TGR5 and VDR expression was analyzed using immunohistochemistry,and serum inflammatory cytokine levels were measured with ELISA.Correlations between these parameters were analyzed.Results1.Demographics,clinical characteristics and psychological states of study subjects? Thirty-two UC patients(21 cases of UC large intestine damp-heat syndrome and 11 cases of UC spleen-deficiency dampness-obstruction syndrome)and twenty-three HCs were enrolled in this study.There were no significant differences in age,gender,and BMI among the three groups.? There was no significant difference in the duration of disease,Mayo score of disease activity,scope of lesions,severity of disease,and clinical type between the UC large intestine damp-heat syndrome group and the spleen-deficiency dampness-obstruction syndrome group.Quality of life score of patients with damp-heat syndrome of large intestine was significantly lower than that of patients with spleen-deficiency dampness-obstruction syndrome.38.10%of UC large intestine damp-heat syndrome group and 36.36%of patients with spleen-deficiency damp-heat syndrome had different degrees of anxiety symptoms.The HADs depression score of UC showed a tendency to increase in patients with spleen-deficiency dampness-obstruction syndrome but failed to reach a significant level.2.Diversity of intestinal flora and screening of different key gut microbiota of each group of subjects? The community alpha diversity index Chao1,Shannon,and Simpson of UC large intestine damp-heat syndrome group and spleen-deficiency dampness-obstruction syndrome group were significantly lower than those of the control group(P<0.01),and the flora diversity index of UC large intestine damp-heat syndrome group was lower than that of the spleen-deficiency dampness-obstruction syndrome group but failed to reach a significant level.? Firmicutes,Ruminococcaceae,Clostridium XlVb,Faecalibacterium and Roseburia were significantly decreased in patients with UC(P<0.01).However,Proteobacteria,Enterobacteriaceae,Escherichia/Shigella,Enterococcus were significantly enriched in the UC group(P<0.01).? Compared with spleen-deficiency dampness-obstruction syndrome group,the abundance of Escherichia/Shigella,Enterobacteriaceae,and Erysipelotrichaceae in the UC large intestine damp-heat syndrome group was significantly higher(P<0.01),and Faecalibacterium,Ruminococcus,Parvimonas Fusicatenibacter significantly decreased in the UC large intestine damp-heat syndrome group(P<0.05).3.Analysis of the difference in fecal bile acids of each group of subjects and the correlations between fecal bile acids,intestinal microbes and disease activity Mayo score in all subjects? The concentrations of fecal secondary bile acids such as lithocholic acid(LCA),deoxycholic acid(DCA),glycodeoxycholic acid(GDCA),glycolithocholic acid(GLCA),taurolithocholic acid(TLCA)in UC patients were significantly lower than those in HCs(P<0.05);The concentrations of primary bile acids such as taurocholic acid(TCA),cholic acid(CA),taurochenodeoxycholic acid(TCDCA),and glycochenodeoxycholic acid(GCDCA)in UC patients were significantly higher than those in HCs(P<0.05).? The total concentration of fecal secondary bile acids of the UC large intestine damp-heat syndrome group and the spleen-deficiency dampness-obstruction syndrome group was significantly lower than that of the HCs group(P<0.01),and the UC large intestine damp-heat syndrome group was significantly lower than that of the spleen-deficiency dampness-obstruction group(P=0.048);Compared with the HCs group,the PBA/SBA of the UC large intestine damp-heat syndrome group and the spleen-deficiency dampness-obstruction syndrome group was significantly higher than that of the HCs group(P<0.05),and the UC large intestine damp-heat syndrome group was significantly higher than that of the UC spleen-deficiency dampness-obstruction group(P=0.019).The ratio of conjugated bile acids to unconjugated bile acids in UC large intestine damp-heat syndrome group and spleen-deficiency dampness-obstruction group was significantly higher than that of HCs group(P<0.01),but there was no significant difference between large intestine damp-heat syndrome and spleen-deficiency dampness-obstruction group? Clostridium XlVb,Ruminococcus,Faecalibacterium,Roseburia were positively correlated with fecal DCA and LCA(P<0.01),and negatively related to fecal CA,CDCA,TCA(P<0.01).Enterococcus,Veillonella,Klebsiella,Streptococcus were negatively correlated with fecal DCA and LCA(P<0.01),and positively related to fecal CA and CDCA,TCA(P<0.01)?The Mayo score of UC disease activity was negatively correlated with fecal LCA(r=-0.507,P=0.0036)and DCA(r=-0.474,P=0.0071),and positively correlated with fecal TCA(r=0.403,P=0.0245)and PBAs/SBAs(r=0.683,P=0.0001)4.The expression of bile acid receptor TGR5,VDR and NF-?B p65 in the intestinal mucosa and serum pro-inflammatory cytokine levels of subjects in each group?The expression levels of TGR5 and NF-?B p65 in the intestinal mucosa of the UC large intestine damp-heat syndrome group and the spleen-deficiency dampness-obstruction syndrome group were significantly higher than those of the healthy control group(P<0.01),and the expression level of NF-?B p65 was significantly increased in UC large intestine damp-heat syndrome group compared with spleen-deficiency dampness-obstruction syndrome group(P=0.022);the expression level of VDR in the control group was significantly higher than that in the UC large intestine damp-heat syndrome group(P=0.014).?The levels of serum pro-inflammatory cytokines TNF-?,IL-6,IL-1?,IL-1? and IL-2 in UC large intestine damp-heat syndrome group and spleen-deficiency dampness-obstruction syndrome group were significantly higher than those of HCs(P<0.01),The levels of TNF-?and IL-6 was significantly increased in UC large intestine damp-heat syndrome group compared with spleen-deficiency dampness-obstruction syndrome group(P<0.05).? The levels of serum pro-inflammatory cytokines IL-2,IL-1?,IL-1?,TNF-?,IL-6 were negatively correlated with fecal DCA and LCA(P<0.01),and the levels of IL-1?,TNF-? were positively correlated with fecal TCA(P<0.05).ConclusionsDysregulation of gut microbiota and altered constitution of fecal bile acid metabolites may participate in the pathogenesis of UC via regulating inflammatory responses by the bile acid receptors TGR5 and VDR.1.The constitution intestinal flora and bile acid profiles of patients with UC large intestine damp-heat syndrome and spleen deficiency damp-block syndrome are different,and dysregulation of gut microbiota related to bile acid metabolism affects the conversion of fecal primary bile acid to secondary bile acid,which may lead to altered constitution of fecal bile acid metabolites in patients with different syndromes of UC.2.The fecal bile acid profiles of UC patients is closely related to serum inflammatory cytokines.Altered fecal bile acid composition may participate in the inflammatory response by regulating the bile acid receptor TGR5/VDR/NF-?B signaling pathway.3.Dysregulation of gut microbiota,altered constitution of fecal bile acids and abnormal expression of bile acid receptors may be involved in the degree of inflammation of UC and TCM syndrome types.