Silymarin ameliorates diet-induced gallstone formation by regulating gut microbiota-derived GCDCA to suppress ferroptosis-ROS-NFκB signaling pathway

Gallstones (GS), a globally prevalent gastrointestinal disorder in adults, arise from multifactorial pathogenesis with dietary patterns serving as a key contributor. This study aimed to elucidate the therapeutic efficacy of silymarin in a diet-induced gallstone murine model established via lithogenic diet (LD) induction. LD-fed mice exhibited marked hepatic oxidative stress, inflammatory activation, and ferroptosis, accompanied by elevated serum glycochenodeoxycholic acid (GCDCA) levels. Our findings demonstrate that GCDCA triggers dose-dependent ferroptosis in murine hepatocytes, a process reversible by the ferroptosis inhibitor ferrostatin-1 (Fer-1). Mechanistically, GCDCA promotes hepatocyte ferroptosis through SQSTM1-KEAP1-NRF2 axis-mediated suppression of SLC7A11, while simultaneously activating NFκB signaling via ROS generation to inhibit FXR activity. Oral silymarin administration in gallstone-bearing mice exhibited dose-dependent therapeutic effects, with higher doses demonstrating optimal efficacy in: (1) reducing gallstone formation, (2) ameliorating hepatic histopathological damage and lipid accumulation, (3) suppressing hepatocyte ferroptosis, (4) decreasing hepatic inflammatory cytokines, and (5) normalizing serum ALT/AST levels. Further analysis revealed silymarin's ability to restore gut microbiota homeostasis, reduce serum GCDCA concentrations, and inhibit both ferroptosis and NFκB signaling through microbiota-dependent mechanisms. Collectively, silymarin exerts anti-gallstone effects by stabilizing gut microbiota to reduce serum GCDCA, inhibit hepatocyte ferroptosis and ROS-NFκB activation, and restore FXR expression and transcriptional activity. This study provides novel mechanistic insights and proposes microbiota-targeted therapeutic strategies for gallstone management.