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:

• non-alcoholic fatty liver disease
• farnesoid X receptor
• gastrointestinal microbiome
• bile acid
• metabolic diseases

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81000968, No. 81101540, No. 81101637, No. 81172273, No. 81272388, No. 81301820, No. 81472673), the Fund of Shanghai Science and Technology Commission (16ZR1406100,15410710100), the Doctoral Fund of Ministry of Education of China (20120071110058), and the National Clinical Key Special Subject of China. The authors would like to thank the members of Professor Xi-Zhong Shen’s laboratory for helpful discussions and critical reading of the manuscript.

Abbreviation list

ACE, abundance-based coverage estimator; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; BSEP, bile salt export pump; CA, cholic acid; CDCA, chenodeoxycholic acid; Ct, cycle threshold; CYP7A1, cholesterol 7α-hydroxylase; CYP8B1, sterol 12α-hydroxylase; DCA, deoxycholic acid; Fgf15, fibroblast growth factor 15; FMT, fecal microbiota transplantation; FXR, farnesoid X receptor; GAPDH, glyceraldehyde phosphate dehydrogenase; GGT, γ-glutamyl transpeptidase; HE, hematoxylin-eosin; HFD, high fat diet; HLD-C, high-density lipoprotein-cholesterol; IL, interleukin; LBP, lipopolysaccharide-binding protein; LCA, lithocholic acid; LDA, linear discriminant analysis; LDL-C, low-density lipoprotein-cholesterol; LEfSe, linear discriminant analysis with effect size; LPS, lipopolysaccharide; MCA, muricholic acid; MRM, multiple reaction monitor; MS, mass spectrometry; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NIH, National Institutes of Health; NMDS, nonmetric multidimensional scaling; PBC, primary biliary cholangitis; PC1, the first principal component; PCoA, principal coordinate analysis; RT-qPCR, reverse transcription quantitative real-time PCR; SCFA, short chain fatty acid; SD, standard deviation; SHP, small heterodimer partner; TB, total bilirubin; TBS, Tris-buffered saline; TCA, tauro-cholic acid; TCDCA, tauro-chenodeoxycholic; TNF-α, tumor necrosis factor-α; TαMCA, tauro-α-muricholic acid; TβMCA, tauro-β-muricholic acid; UDCA, ursodeoxycholic acid; UPLC, ultra-high-performance liquid chromatography; ZO-1, tight junction protein-1; αMCA, α-muricholic acid; βMCA, β-muricholic acid.

Supplementary materials

Table S1 cDNA target genes and primers used for quantitative real-time PCR

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.

Table S2 The differential expressed bacteria among three groups from phylum to genus level

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.

Table S3 Bile acid levels in liver, ileum, cecum, feces and gallbladder

Figure S3 Pie charts of conjugated bile acids and unconjugated bile acids in liver, ileum, cecum, feces and gallbladder.

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.

Disclosure

The authors report no conflicts of interest in this work.