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 CCl₄ for 8 weeks or single CCl₄ 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 + CCl₄ and CCl₄-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.

Background: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a prevalent chronic liver disorder with limited pharmacological therapies, highlighting the need for novel multi-target agents from traditional medicinal plants. Artemisia rupestris L. has hepatoprotective effects, but its active constituents and mechanisms against MAFLD remain unexplored. Methods: The chemical profile of an ultrasonically-assisted 70% ethanol extract of Artemisia rupestris L. (ARE) was determined using UPLC-Q-TOF-MS. Network pharmacology, molecular docking, 100-ns molecular dynamics (MD) simulations, and in vitro experiments in oleic acid-induced HepG2 cells were integrated to identify active compounds, core targets, and pathways, and to validate lipid-lowering and hepatoprotective effects. Results: Seventy-eight compounds were identified, mainly flavonoids, phenolic acids, and terpenoids. Network analysis highlighted AKR1B10 and MAPK14 as core targets and isorupestonic acid and rupestonic acid as key components. MD simulations supported the stable binding of isorupestonic acid to AKR1B10 (RMSD ~1.6–3.2 Å), driven by van der Waals and electrostatic interactions. In vitro, ARE dose-dependently reduced intracellular triglycerides, total cholesterol, and LDL-C, decreased lipid droplets, and lowered AST/ALT levels, with a high-dose efficacy comparable to atorvastatin. Conclusions: Through in vitro experiments and network pharmacology analyses, this study preliminarily elucidated the beneficial effects of ARE on lipid deposition in hepatocytes and its potential mechanism, thereby providing an experimental basis and potential avenues for subsequent in vivo studies on pharmacodynamics, pharmacokinetics, and toxicology.