Abstract

Background: Liver fibrosis is the consequence of chronic liver diseases that can progress to cirrhosis and liver failure. However, the role and mechanism of gut leakiness in liver fibrosis are poorly understood, and there is no FDA-approved drug to treat liver fibrosis. In this study, we aimed to investigate the causal role of protein acetylation and gut dysbiosis in promoting gut leakiness, endotoxemia, and liver fibrosis and the protection by melatonin (MT), a safe agent recognized by FDA, through the gut-liver axis.

Methods: Young male Sprague-Dawley rats were divided into 12 groups (4 groups x 3 timepoints): Control (vehicle), MT (10 mg/kg/day, oral), TAA (200 mg/kg/dose twice a week, i.p.), and MT+TAA for 1, 2, or 4 weeks (n=6-8/group). Histological analyses were determined by Picrosirius red, H&E, and TUNEL staining. Liver injury was evaluated by serum transaminases (ALT, AST) and markers for apoptosis and fibrosis. Gut dysbiosis was measured via 16S metagenomic sequencing and metabolomic analyses of cecum samples. Gut leakiness was assessed by serum endotoxin (LPS), while immunoblots and immunoprecipitation were performed to detect protein alterations. For additional mechanistic studies, similar experiments were performed in wild-type (WT) and gut- or liver-specific Sirt1-KO mice using TAA (200 mg/kg/dose twice a week, i.p.) for 5 weeks (n=6-8/group).

Results: TAA caused hyperacetylation (Ac-Lys) of gut and liver proteins via selective suppression of Sirtuin 1 (SIRT1) deacetylase along with gut dysbiosis, leading to gut leakiness, endotoxemia, and liver fibrosis, all of which were improved by MT. Gut tight and adherent junction proteins (TJ/AJs) were acetylated and degraded via ubiquitin-dependent proteolysis. Decreased TJ/AJs with increased intestinal deformation, enterocyte apoptosis, and serum endotoxin were observed as early as 1 week, while liver fibrosis and injury markers, hepatocyte apoptosis, and serum ALT and AST were markedly elevated 2 and 4 weeks after TAA exposure. TAA increased the abundance of pathogenic bacteria (Proteobacteria, Ruminococcaceae, etc.) but decreased the levels of short-chain fatty acids (Butyric, Propionic acids, etc.). Interestingly, TAA decreased only SIRT1 activity and protein levels, but not other six SIRT isoforms, and caused hyperacetylation of liver proteins related to inflammation (Ac-NFκB) and apoptosis (Ac-FOXO1). Pretreatment with MT ameliorated all these changes at 1, 2, and 4 weeks. TAA-exposed gut- or liver-specific Sirt1-KO mice also showed markedly decreased gut TJ/AJs with greater enterocyte apoptosis, endotoxemia, liver fibrosis, and hyperacetylation of gut and liver proteins than those of WT mice.

Conclusion: This study showed the novel mechanisms of the decreased SIRT1-mediated protein hyperacetylation along with gut dysbiosis in promoting gut leakiness and liver fibrosis and the protective role of melatonin through the gut-liver axis.