Multi-target protective effects of Agrimonia pilosa Ledeb. against metabolic dysfunction-associated steatohepatitis in mice

Ledeb. (AP), a traditional herbal medicine rich in flavonoids, phenolics, triterpenoids, and glycosides, has been widely used for hepatic injury and metabolic disorders. experiments, UHPLC-HRMS profiling, network pharmacology, and molecular simulation to elucidate the bioactive constituents and mechanisms of AP against metabolic dysfunction-associated steatohepatitis (MASH). Therapeutic efficacy was evaluated using a MASH mouse model, AML12 hepatocytes, and RAW264.7 macrophages. Active constituents were identified by UHPLC-HRMS, and potential targets were predicted via SwissTargetPrediction and GEO databases, followed by PPI network construction, GO/KEGG enrichment analysis, molecular docking, and molecular dynamics simulation. AP markedly reduced body weight, liver index, and serum AST, ALT, TG, TC, and LDL-c levels, and attenuated hepatic steatosis, inflammation, and fibrosis. In AML12 cells, AP suppressed lipogenesis by downregulating SREBP-1c, FASN, and SCD1, while promoting fatty acid β-oxidation through CPT1A upregulation. In RAW264.7 macrophages, AP inhibited LPS-induced expression of TNF-α, IL-1β, and IL-6. A total of 83 active constituents and 25 key targets were identified, with HMGCR and AXL emerging as hub nodes. Agrimol B (AGB) exhibited favorable binding affinity and structural stability toward both targets. Mechanistically, AGB inhibited HMGCR, reduced SREBP-2 nuclear translocation, and enhanced LXRα/β-mediated cholesterol efflux, maintaining hepatic cholesterol homeostasis. These findings demonstrate that AP ameliorates MASH through coordinated regulation of lipid metabolism, inflammatory suppression, and collagen deposition, with AGB representing a promising bioactive candidate warranting further investigation.