Abstract

Background Metabolic dysfunction-associated steatohepatitis (MASH) presents a growing global health challenge with limited therapeutic options. Rhein, an active anthraquinone derived from the traditional medicine rhubarb ( Rheum palmatum L. ), has demonstrated potential in alleviating metabolic liver disorders, yet its precise mechanism of action against MASH remains unclear. Methods An integrated strategy combining network pharmacology, molecular docking, and experimental validation was employed. A “Rhein-MASH” target network was constructed to identify core targets. The in vivo therapeutic effects and dose dependency of Rhein were assessed in a choline-deficient, high-fat diet (CDAHFD)-induced mouse MASH model. In vitro mechanisms were investigated in PA/OA-treated AML12 hepatocytes using functional assays, pharmacological inhibitors, and siRNA-mediated gene knockdown. Key signaling molecules and gene expression were analyzed via Western blot, qPCR, and immunohistochemistry. Results Rhein treatment improved hepatic steatosis, inflammation, and liver injury in MASH mice in a dose-dependent manner without altering systemic lipid profiles. It selectively upregulated the expression of key genes involved in fatty acid β-oxidation ( Acadl , Cpt1a ) while suppressing pro-inflammatory cytokines ( Tnf-α , Il-1β ). Network pharmacology and molecular docking identified EGFR as a core target, with Rhein demonstrating potent binding affinity (docking score: −7.9 kcal/mol). In vitro experiments revealed that Rhein acts through EGFR to induces its autophosphorylation, and sequentially activates the downstream PI3K/AKT signaling pathway. This signaling module coordinately enhances fatty acid oxidation–related gene expression and reduces inflammatory gene expression partly through PPARα, thereby effectively ameliorating lipid accumulation and inflammatory phenotypes in hepatocytes. Conclusion This study elucidates that Rhein ameliorates MASH by activating the EGFR/PI3