Urolithin C reveals anti-NAFLD potential via AMPK-ferroptosis axis and modulating gut microbiota

The pharmacology of urolithin C (UroC) on non-alcoholic fatty liver disease (NAFLD) is largely undetermined. We sought to investigate the potential for NAFLD improvement by administration of UroC and the underlying mechanisms. We verified the therapeutic effect of UroC on choline-deficient amino acid-defined high fat diet (CDAHFD) induced NAFLD mice via evaluating NAFLD activity score (NAS), AST, ALT, hepatic phosphorylated AMPK, and 4-hydroxynonenal. Oleic acid-induced AML12 cell was appraised by oil red staining and western blotting to explore the effect and mechanism of UroC in vitro. Transcriptional regulation of UroC was explored by liver RNA sequencing, gut microbiota composition was explored by 16SrRNA sequencing, and colorectal tight junctional proteins were detected by western blotting and immunohistochemistry. The detrimental effects of CDAHFD included the increased liver index, AST, ALT, hepatic 4-hydroxynonenal, impaired intestinal mucosal barrier, and most importantly, pathological damage in liver. Oral administration of UroC largely protected against these harmful alterations. Remarkably, both RNA sequencing and western blotting results indicated an activation in hepatic AMPK signaling pathway which was thought to inhibit ferroptosis response to UroC in vivo, while no change were found in AMPK-ferroptosis axis response to UroC in oleic acid-induced AML12 cells, hinted an indispensable linkage between UroC and hepatic AMPK, presumably the gut-liver axis. Furthermore, UroC could neither alleviate lipid deposition nor inhibit ferroptosis in vitro. The 16SrRNA showed UroC partially counteracted the dysbiosis induced by CDAHFD. Specifically, UroC reversed the elevated proportion of Firmicutes/Bacteroidota and enhanced the level of Parabacteroides goldsteinii and Lactobacillus vaginalis, which played a beneficial role in metabolic disorders. Oral administration of Urolithin C protected against the detrimental impact of CDAHFD via regulating AMPK-ferroptosis axis, maintaining intestinal mucosal barrier and counteracting gut dysbiosis.