Unraveling berberine’s multi-target mechanism in combating early-stage masld: a network pharmacology and in vitro study

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) poses a global health burden with limited therapies. Berberine (BBR) shows promise against MASLD, but its molecular mechanisms remain unclear. Network pharmacology predicted BBR-MASLD intersecting targets, followed by protein-protein interaction (PPI) network construction, GO/KEGG enrichment, and molecular docking. In vitro validation used FFA-induced HepG2 steatosis cells with Oil Red O staining, triglyceride assay, RT-qPCR, and Western blot for p-AKT/AKT. From 147 intersecting targets, six functional core targets were identified: AKT1, IL6, TP53, TNF, IL1B, and BCL2. Enrichment analyses implicated insulin resistance, lipid metabolism, and inflammation. Molecular docking confirmed strong BBR–core protein binding. In vitro, BBR dose-dependently reduced lipid accumulation and triglyceride levels, downregulated lipogenic (SREBP-1c) and pro-inflammatory (IL-6, IL-1β, TNF-α) genes, and restored insulin signaling (AKT1) and apoptosis-related (TP53) gene expression.BBR increased the p-AKT (Ser473)/total AKT ratio as shown by Western blot. BBR attenuates lipid accumulation and partially reverses steatosis-related transcriptional changes in an FFA-induced HepG2 model, including restoration of AKT1 signaling at both mRNA and protein phosphorylation levels. These findings suggest that BBR exerts protective effects againstearly hepatocellular steatosist hrough coordinated regulation of lipid metabolism, inflammation, and stress-related targets. This study provides a network-based rationale for further in vivo and mechanistic investigation in MASLD, and identifies potential pharmacodynamic biomarkers for future clinical trials.