Jiangcaoxiang alleviates hepatic fibrosis through the Nrf2/GPX4 signaling pathway: Elucidated via integrated network pharmacology, multi-omics profiling, and experimental validation

This study aimed to systematically elucidate the therapeutic effects and underlying molecular mechanisms of Jiangcaoxiang (JCX) formula against Hepatic fibrosis (HF) by integrating multi-omics, network pharmacology and both in vivo and in vitro experimental validation. Potential bioactive compounds and their targets were predicted via network pharmacology and molecular docking. Key target genes were screened through transcriptomics, and untargeted metabolomics uncovered differential hepatic metabolites and metabolic pathways. In vivo, histopathological alterations in liver tissue were evaluated by hematoxylin–eosin (H&E) and Masson staining. Serum biochemical indices were quantified with assay kits, while ELISA measured serum levels of type III collagen, type IV collagen, IL-1β, and TNF-α. In vitro, Cell viability was assessed by CCK-8 assay. Intracellular ROS accumulation was detected by DCFH-DA fluorescence staining, and MDA levels were measured to evaluate lipid peroxidation. Finally, Western blotting and RT-qPCR were employed to assess the Nrf2/GPX4 signaling pathways. JCX improved liver function, reduced fibrotic deposition, restored antioxidant enzyme activity, and attenuated inflammatory responses in rats with HF. Bioinformatics analysis identified Nrf2, GPX4, NOX4, IL-17A, and HIF-1α as key targets of JCX. Multi-omics profiling showed that JCX significantly regulated glutathione metabolism, upregulated Nrf2 and GPX4 protein expression, and downregulated IL-17A and HIF-1α protein expression. In BRL-3A cells, JCX treatment reduced intracellular ROS levels in H₂O₂-treated cells and activated the Nrf2/GPX4 signaling pathway. The Nrf2 inhibitor ML385 markedly reversed the therapeutic effects of JCX both in vivo and in vitro. This study demonstrates the therapeutic potential of JCX for HF and elucidates its multi-target pharmacological mechanism, thereby providing a foundation for further investigation and clinical application. • JCX activates the Nrf2/GPX4 signaling pathway to alleviate hepatic fibrosis. • JCX inhibits iron overload-related oxidative damage in hepatocytes. • JCX suppresses IL-17A and HIF-1α to mitigate hepatic inflammatory injury.