Rougan Granules ameliorates liver fibrosis through autophagy regulation: Insights from HPLC-Q-TOF-MS/MS, network pharmacology, and experimental validation

Network pharmacological analysis of RGG was performed based on HPLC-Q-TOF-MS/MS. • RGG alleviates liver fibrosis and suppresses hepatic stellate cell activation. • RGG exerts anti-fibrotic effects through autophagy mediated by the PTEN/AKT/mTOR signaling pathway. To clarify the bioactive constituents and anti-fibrotic mechanisms of Rougan Granules (RGG), this study utilized HPLC-Q-TOF-MS/MS to identify its chemical profile, followed by network pharmacology and molecular docking to explore potential targets. In vivo, RGG was administered at different doses to mice with carbon tetrachloride (CCl 4 )-induced liver fibrosis. The attenuation of liver injury by RGG was evaluated via serum biochemical profiling, histological staining, and western blot analysis. In vitro, platelet-derived growth factor-BB (PDGF-BB) was used to activate LX-2 cells, and the mechanisms of RGG in suppressing hepatic stellate cell (HSC) activation were validated via immunofluorescence and western blot. Furthermore, autophagic flux was monitored using laser scanning confocal microscopy in LX-2 cells transfected with mRFP1-EGFP-LC3B lentivirus. A total of 120 chemical constituents were identified, with network pharmacology analysis indicating that quercetin, genistein, and kaempferol might serve as the primary bioactive components of RGG. Functional enrichment analysis revealed that common targets were significantly enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Molecular docking confirmed that these key components exhibited stable binding affinities for phosphatase and tensin homolog (PTEN) and AKT1. Both in vivo and in vitro experiments validated that RGG upregulates PTEN expression to inhibit the AKT/mechanistic target of rapamycin (mTOR) signaling pathway. Furthermore, RGG was shown to exert anti-fibrotic effects by inducing HSC autophagy through the modulation of this pathway. These findings reveal that RGG attenuates liver fibrosis via PTEN/AKT/mTOR-mediated autophagy, supporting its therapeutic potential.