ABSTRACT: Objective: This study investigated the therapeutic mechanism by which Xuezhikang (XZK) alleviates non-alcoholic fatty liver disease (NAFLD) in mice, with a specific focus on the interplay between the gut microbiota, metabolic alterations, and hepatic ferroptosis. Methods: An NAFLD mouse model was induced via a high-fat diet (HFD) and subsequently treated with XZK for 10 weeks. Gut microbiota composition in cecal contents was profiled via 16S rRNA gene sequencing, while untargeted metabolomics was conducted using UPLC-LTQ-Orbitrap-MS. Additionally, an integrated analysis was performed to evaluate correlations between differential metabolites and specific gut microbial taxa. Hepatic ferroptosis was assessed by measuring the expression of key proteins using immunohistochemistry and Western blotting. Results: XZK treatment significantly ameliorated serum lipid profiles, liver function, and systemic inflammation in NAFLD mice. Regarding the microbiome, XZK reversed gut dysbiosis by restoring microbial community structure, decreasing the abundance of Proteobacteria and opportunistic pathogens (Lawsonia, unclassified Enterobacteriaceae, Enterococcus, etc), while enriching beneficial taxa (Bacteroides, Adlercreutzia, unclassified S24-7, etc). Metabolomic analysis identified 73 differential metabolites (54 upregulated and 19 downregulated), and pathway enrichment confirmed the critical role of ferroptosis in NAFLD pathogenesis. Correlation analysis demonstrated significant associations between differential genera, differential metabolites, and NAFLD-related indicators including ALT, AST, TC, TG, MDA, 4-HNE, Fe, and IL-6. Notably, XZK attenuated hepatic oxidative stress and inhibited the ferroptosis pathway by regulating the expression of glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2). Conclusion: XZK ameliorates NAFLD by restoring gut microbiota homeostasis, correcting metabolic dysregulation, and inhibiting hepatic ferroptosis, thereby validating the gut microbiota-metabolite-ferroptosis axis as a key therapeutic target.
