Abstract
AIMS: This study aimed to investigate the therapeutic mechanism of red yeast rice extract (RYRE) in high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) through untargeted metabolomics analysis and experimental validation. METHODS: An untargeted metabolomics analysis based on UHPLC-QTOF/MS was performed to identify differential metabolites in liver tissues. A NAFLD model was established in hamsters by HFD feeding. Forty hamsters were randomly allocated into four groups (n = 10 per group): control (CON), model (MOD), red yeast rice extract (Xuezhikang, XZK), and simvastatin (SVT). Serum levels of lipids (TG, CHO, HDL-C, LDL-C), liver function parameters (ALT, AST, ALB, ALP, γ-GT, TBiL, DBiL, TBA), and inflammatory cytokines (TNF-α, IL-4, IL-1β, TGF-β) were measured by biochemical assays and ELISA. Liver tissues were subjected to Oil Red O, hematoxylin-eosin (HE), and Masson staining for histopathological evaluation. Additionally, qPCR and immunohistochemistry were employed to investigate the underlying signaling pathways. RESULTS: XZK significantly reduced serum levels of DBiL, TBiL, TBA, CHO, TG, and LDL-C, while increasing HDL-C in HFD-fed hamsters. Both XZK and SVT markedly decreased pro-inflammatory cytokines. Untargeted metabolomics identified 135 differential metabolites between the MOD and XZK groups (83 down-regulated, 52 up-regulated), which were primarily involved in carbon metabolism, lipid metabolism, and hormone metabolism. Mechanistically, XZK attenuated the JNK/AP-1/TNF-α signaling pathway, as evidenced by reduced mRNA expression of cFos, cJUN, JAK1/2/3, and TNF-α, along with decreased protein levels of p-JNK, cFos, cJUN, and TNF-α. Notably, the p-cJun/total cJun ratio showed a distinct regulatory pattern, suggesting complex modulation of AP-1 subunit phosphorylation. CONCLUSION: XZK effectively ameliorates hepatic steatosis, dyslipidemia, and inflammation in HFD-induced NAFLD hamsters. The therapeutic effects are mediated through restoration of metabolic homeostasis and suppression of the JNK/AP-1/TNF-α signaling pathway.