Natural small molecule hinokitone mitigates NASH fibrosis by targeting regulation of FXR-mediated hepatocyte apoptosis

Hinokitone exerts an inhibitory effect on NASH fibrosis. Mechanistically, Hinokitone attenuates hepatocyte apoptosis by targeting the upregulation of hepatocyte FXR, which in turn indirectly inhibits HSC activation. • Natural small molecule HO inhibits liver fibrosis in a mouse NASH model. • Hepatocyte FXR mediates the effect of HO anti-liver fibrosis. • HO inhibits hepatocyte lipid deposition and hepatic inflammation by binding to and upregulating the protein level of FXR proteins in hepatocytes, thereby exerting an anti-apoptotic effect on hepatocytes. • HO indirectly preventing hepatic stellate cell activation through inhibiting hepatocyte apoptosis and thus inhibits liver fibrosis. Liver fibrosis is the common fate of NASH and poses a major health threat with very limited pharmacological treatments. This study aims to investigate the preventive effect of hinokitone (HO), an isolated compound from Agathis dammara , on NASH fibrosis and its underlying mechanism. To investigate the effect of HO on NASH fibrosis, C57BL/6 mice were either fed a high-fat diet (HFD) in conjunction with intraperitoneal injection of CCl 4 for 8 weeks or single CCl 4 for 14 days to establish mouse liver fibrosis model, and HO was administered by gavage simultaneously. To elucidate the underlying mechanisms, HepG2 cells were stimulated by palmitic acid (PA) or tumor necrosis factor α plus actinomycin-D (Act-D + TNFα) to induce hepatocyte apoptosis model. Furthermore, hepatocyte Farnesoid-X-receptor (FXR) specifically knocked out mice were established by the albumin-Cre-loxP recombination enzyme system to ascertain the role of FXR in the anti-NASH fibrosis effects of HO. The results showed that HO presented dose-dependent anti-liver fibrosis efficacy in NASH mice induced by HFD + CCl 4 and CCl 4 -induced mouse liver fibrosis. Cellularly, HO significantly inhibited PA-induced lipotoxic apoptosis and Act-D + TNFα-induced exogenous apoptosis in hepatocytes, which in turn prevented HSC activation. Mechanistically, bioinformatics analysis and surface plasmon resonance assay had identified hepatocyte FXR as a target of HO. Specifically, HO directly bound to FXR and upregulated its protein level by inhibiting proteasomal degradation. In turn, HO attenuated hepatocyte lipid deposition through upregulating the FXR’s downstream target genes SHP and CES1 , and reduced cleaved-CASP8 level, thereby inhibiting hepatocyte apoptosis. Furthermore, HO lost its function in the inhibition of hepatocyte apoptosis and liver fibrosis when knockout hepatocyte FXR. In conclusion, HO has an inhibitory effect on NASH fibrosis. This effect is mediated by targeting upregulation of hepatocyte FXR, which in turn attenuates hepatocyte apoptosis and thus indirectly inhibits the activation of HSCs.