Abstract

Background:

Increased intestinal permeability is one of the multiple hits in the pathogenesis of non-alcoholic steatohepatitis (NASH). Bile salt hydrolase (BSH) is a gut bacterial enzyme that hydrolyzes conjugated bile acids (BAs) into unconjugated BAs. Our previous study reported that inhibition of BSH with a gut-restricted small molecule inhibitor, AAA-10, increased conjugated BAs and prevented the development of intestinal permeability and liver steatosis in an early onset, diet-induced rat steatosis model. This study sought to evaluate whether AAA-10 could treat pathogenic intestinal permeability and liver damage in models of early NASH and NASH with fibrosis.

Methods:

8-week-old Wistar rats were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for either 3 or 6 weeks to develop early NASH or NASH with fibrosis, respectively. Rats were randomly assigned into two groups and gavaged twice daily with either 10 mg/kg of AAA-10 or control vehicle for 1 or 2 weeks. Molecular and histologic assessments on ileum and liver tissue were performed. RNA sequencing was performed to assess alteration of the global transcriptomic landscape of ileum and liver. In vitro human hepatic stellate cells (HSCs) and ex vivo human precision-cut liver slices (PCLS) were used to assess the effect of treatment with conjugated and/or unconjugated BAs.

Results: Increased BSH activity and gut permeability were observed with liver fibrosis. After AAA-10 treatment, liver chemistry indices including ALT and AST improved. In both early NASH and NASH with fibrosis models, BSH inhibition reduced gut permeability by decreasing translocation of FITC-dextran and ileal expression of occludin, a tight junction protein upregulated and translocated to the membrane to compensate for disrupted tight junctions in NASH. BSH inhibition was associated with lowered liver collagen deposition, improved fibrosis score and reduced expression of pro-fibrogenic genes. Consistently, RNAseq analysis showed that the global transcriptomic signatures of intestinal function including absorption ability were restored after BSH inhibition, suggesting reduced intestinal permeability. Liver inflammatory and fibrogenic signatures were inhibited after AAA-10 treatment. BSH inhibition also enhanced 113 good prognosis genes and reduced 73 poor prognosis genes in the liver previously shown to be associated with progressive liver disease and HCC. Conjugated BA treatment attenuated TGF-β induced fibrogenic genes in in vitro human primary HSCs and TWNT4 cells and in ex vivo human PCLS; these changes were reversed by unconjugated BA treatment.

Conclusion:

BSH inhibition with AAA-10 treatment reduced gut permeability and alleviated liver fibrosis in rats with early NASH and NASH with fibrosis. These data suggest that inhibitors of gut bacterial BSHs could be developed as treatments for NASH and other diseases characterized by pathogenic intestinal permeability.