Integrated proteomic and metabolomic analysis reveals the mechanism of astragalus complanatus flavonoids against liver fibrosis in rats

Abstract

Astragali Complanati Semen (ACS) is a traditional Chinese medicinal herb conventionally applied to tonify the liver and kidney. As the primary bioactive ingredients of ACS, Astragalus complanatus flavonoids (ACF) exert hepatoprotective and anti-fibrotic effects. This study aimed to elucidate the antifibrotic mechanisms of ACF through integrated proteomic and metabolomic analyses combined with experimental validation. A CCl 4 -induced rat model of LF and TGF-β1-activated hepatic stellate cell (HSC) model were established to evaluate the antifibrotic effects of ACF. Integrated proteomic and metabolomic analyses were performed to identify differentially expressed proteins (DEPs), differentially expressed metabolites (DMs), and associated signaling pathways. Key molecular targets were validated using quantitative real-time PCR and Western blotting, while pharmacological inhibitors were employed to investigate the involvement of the FAK/AKT signaling pathway in ACF-mediated inhibition of HSC activation. Compared with the LF model group, ACF treatment significantly ameliorated liver function indicators, relieved histopathological injury and collagen deposition, decreased serum fibrosis markers, downregulated the protein levels of Col I, Col IV and α-SMA, and reduced oxidative stress and inflammatory response. Proteomic screening uncovered 401 differentially expressed proteins (DEPs), which were primarily enriched in extracellular matrix (ECM)–receptor interaction, focal adhesion and PI3K/AKT signaling pathways. Protein–protein interaction analysis further pinpointed FN1, CD44, ITGAV, FAK and AKT1 as core target proteins. In vitro experiments demonstrated that ACF blocked TGF-β1-triggered HSC activation, lowered the expression of α-SMA and ITGAV, and inhibited the phosphorylation of FAK and AKT. Pharmacological blockade of either FAK with PF-562271 or AKT with MK2206 strengthened the anti fibrogenic activity of ACF, verifying that the FAK/ AKT axis mediates the anti-HSC activation effect of ACF. Metabolomic analysis identified 108 differentially expressed metabolites (DMs), mainly enriched in glutathione metabolism, primary bile acid biosynthesis and carbohydrate metabolism. Integrated multi-omics analysis confirmed close correlations between DEPs and DMs within ECM–receptor interaction, PI3K/AKT signaling and primary bile acid biosynthesis pathways. Specifically, glycochenodeoxycholic acid, dulcitol and chenodeoxycholate showed strong correlations with coretarget proteins. ACF achieves comprehensive anti-LF effects via suppressing FAK/AKT1 signaling to restrain HSC activation, while coordinately modulating pivotal metabolic pathways.