The protective effect and mechanism of the FXR agonist obeticholic acid via targeting gut microbiota in non-alcoholic fatty liver disease

Abstract

Background: It is reported that various diseases such as non-alcoholic fatty liver disease (NAFLD) are associated with imbalance of microbiome. And FXR has been well investigated in liver diseases. Purpose: The objective of this study was to identify the role of farnesoid X receptor agonist obeticholic acid via targeting gut microbiota in NAFLD. Patients and methods: Male C57BL/6 mice were fed either a normal-chow diet or a high-fat diet (HFD). Obeticholic acid(30mg/(kg·d)) and/or a combination of antibiotics were administered orally by gavage to mice for 12 weeks. Gut microbiota profiles were established through 16S rRNA amplicon sequencing. The effects of obeticholic acid on liver inflammation, the gut barrier, endotoxemia, gut microbiome and composition of the bile acid were also investigated. Results: Obeticholic acid treatment can significantly improve obesity, circulation metabolism disorders, liver inflammation and fibrosis, and intestinal barrier damage caused by HFD. Removal of normal commensal bacteria can weaken the effect of obeticholic acid. The gut microbial structure was changed, and abundance of Blautia was increased significantly after treated with obeticholic acid. After obeticholic acid treatment, the concentration of taurine-bound bile acid caused by HFD was reduced in the liver. Conclusion: Taken together, these data suggest that obeticholic acid has aprotective effect on NAFLD via changing the components of gut microbiota, specifically increasing the abundance of Blautia.

Keywords: bile acid; farnesoid X receptor; gastrointestinal microbiome; metabolic diseases; non-alcoholic fatty liver disease.

Figure 1

Obesity portraits. (A) Body weight through the 24-week period. (B) Body weight in the 24th week. (C) Energy intake through the 24-week period. (D) Liver index. *P<0.05; ***P<0.001. Abbreviations: NC, control group fed with control diet; HF, group fed with high fat diet; NI, group fed with the control diet and treated with obeticholic acid (30 mg/(kg·d)); FI, group fed with the high fat diet and treated with obeticholic acid (30 mg/(kg·d)); ns, not statistically significant.

Figure S1

Serum liver function, glucose and inflammatory mediators. (A) Serum ALT, (B) AST, (C) ALP, (D) GGT, (E) TB, (F) glucose, (G) LBP, (H) adiponectin and (I) TNF-α. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transpeptidase; TB, total bilirubin; LBP, lipopolysaccharide-binding protein; TNF-α, tumor necrosis factor-alpha.

Figure S2

Serum and liver lipids. (A) Serum triglyceride, (B) total cholesterol, (C) LDL-C and (D) HDL-C. (E) Liver triglycerides, (F) cholesterol, (G) LDL-C and (H) HDL-C. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Abbreviations: TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein-cholesterol; HDL-C, high-density lipoprotein-cholesterol.

Figure 2

Histopathological staining of liver, ileum and colon tissues. Representative liver HE, oil red O and Masson’s trichrome staining, and HE staining of ileum and colon tissues (200× magnification). Abbreviation: HE, hematoxylin-eosin.

Figure 3

Intestinal barrier function. (A) Relative mRNA expression of ZO-1, (B) occludin, (C) TNF-α, (D) IL-1β and (E) IL-10 in ileum tissue. (F) Protein expression of ZO-1 and occludin in ileum tissue. (G) Relative mRNA expression of ZO-1, (H) occludin, (I) TNF-α, (J) IL-1β and (K) IL-10 in colon tissue. (L) Protein expression of ZO-1 and occludin in colon tissue. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Abbreviations: ZO-1, tight junction protein-1; TNF-α, tumor necrosis factor-α; IL, interleukin.

Figure 4

Score plots of multivariate analysis on OTU level and community barplots. (A) PCoA and (B) NMDS on OTU level. (C) Community barplot on the phylum and (D) genus level. Abbreviations: PCoA, principal coordinate analysis; OTU, operational taxonomic unit; NMDS, nonmetric multidimensional scaling; ZO-1, tight junction protein-1; TNF-α, tumor necrosis factor-α; IL, interleukin..

Figure 5

Difference analysis of gut microbiota. (A) One-way ANOVA bar plot of the top 20 genera with the most abundant expression. (B) LDA scores of taxa enriched on the genus level. Only taxa with an LDA significant threshold >3 are shown. (C) Taxonomic cladogram generated by LEfSe analysis from phylum to genus level. *P<0.05, **P<0.01. Abbreviations: ANOVA, analysis of variance; LEfSe, linear discriminant analysis coupled with effect size; LDA, linear discriminant analysis.

Figure S3

Pie charts of conjugated bile acids and unconjugated bile acids in liver, ileum, cecum, feces and gallbladder.

Figure 6

Bile acid levels in liver, ileum, cecum, feces and gallbladder. Abbreviations: CA, cholic acid; LCA, lithocholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; CDCA, chenodeoxycholic acid; MCA, muricholic acid; αMCA, α-muricholic acid; βMCA, β-muricholic acid; HDCA, hyodeoxycholic acid; TUDCA, tauro-ursodeoxycholic acid; TCDCA, tauro-chenodeoxycholic acid; TCA, tauro-cholic acid; TαMCA, tauro-α-muricholic acid; TβMCA, tauro-β-muricholic acid; THDCA, tauro-hyodeoxycholic acid; TLCA, tauro-lithocholic acid.

Figure 7

mRNA expression of bile acid metabolism. mRNA expression of CYP7A1, CYP8B1, SHP and BSEP. *P<0.05; ** P<0.01; ***P<0.001; ns, not statistically significant. Abbreviations: CYP7A1, hepatic cholesterol 7α-hydroxylase; CYP8B1, sterol 12α-hydroxylase; SHP, small heterodimer partner; BSEP, bile salt export pump.

Figure S4

Obesity portraits, serum biochemical indicators and liver lipids in the four groups. (A) Body weight in the 24th week. (B) Liver index. (C) Energy intake in the 24th week. (D) Serum ALT, (E) AST, (F) ALP, (G) GGT, (H) TB, (I) glucose, (J) LBP, (K) adiponectin and (L) TNF-α. (M) Serum triglyceride, (N) total cholesterol, (O) LDL-C and (P) HDL-C. (Q) Liver triglycerides, (R) cholesterol, (S) LDL-C and (T) HDL-C. *P<0.05; **P<0.01; ***P<0.001; ns, not statistically significant. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transpeptidase; TB, total bilirubin; LBP, lipopolysaccharide-binding protein; TNF-α, tumor necrosis factor-alpha; TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein-cholesterol; HDL-C, high-density lipoprotein-cholesterol.

Figure S5

Histopathological staining of liver, ileum and colon tissues in the four groups. Representative liver HE, oil red O staining and Masson’s trichrome staining, and HE staining of ileum and colon tissues (200×magnification). Abbreviations: HE, hematoxylin-eosin; NV, group fed with the control diet and treated with obeticholic acid (30 mg/(kg·d)) and a combination of antibiotics; FV,  group fed with high fat diet and treated with obeticholic acid (30 mg/(kg·d)) and a combination of antibiotics.