Therapeutic Mechanisms of Ruangan Huayu Formula in Liver Cirrhosis: Network Pharmacology, Molecular Docking and Experimental Validation

BACKGROUND: Liver cirrhosis remains a challenging global health issue for which current treatment options are largely limited to managing acute liver failure, with no definitive curative therapies available. The Ruangan Huayu Formula (RGHY) has shown promising clinical effects in recent years; however, its comprehensive chemical profile and the specific mechanisms underlying its anti-cirrhotic activity remain to be fully elucidated. METHODS: Ultra-high-performance liquid chromatography-Orbitrap-explorative mass spectrometry (UHPLC-OE-MS) was performed to characterize the chemical profile of the RGHY. Network pharmacology and molecular docking were leveraged to identify potential targets and pathways. Antifibrotic efficacy and underlying mechanisms of RGHY were evaluated using a thioacetamide (TAA)-induced liver fibrosis model in male Sprague-Dawley rats, along with transforming growth factor-beta (TGF-β)-stimulated human hepatic stellate cells (LX-2) in vitro. RESULTS: UHPLC-OE-MS study showed various chemical components of RGHY. Network pharmacology analysis revealed 57 intersecting targets, with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway standing out. Molecular docking studies returned strong binding scores for AKT1, SRC, PIK3CA, and STAT3 as core targets key to RGHY compounds. RGHY was shown to significantly alleviate TAA-induced liver injury and fibrosis by down-regulating the PI3K/AKT pathway. In vitro, RGHY-containing serum and its key bioactive compounds (7,3',4'-trihydroxyflavone and sulfuretin) disrupted TGF-β-induced LX-2 cell activation, migration, and fibrogenesis. The PI3K/AKT signaling pathway was confirmed to involve in mediating the antifibrotic effects of RGHY. CONCLUSION: RGHY and its bioactive compounds 7,3',4'-trihydroxyflavone and effectively attenuated liver fibrosis and hepatic stellate cell activation through multicomponent, multitarget actions, primarily by modulating the PI3K/AKT pathway. These findings provide mechanistic evidence supporting its traditional use and its potential as a therapeutic candidate for liver cirrhosis.