Berbamine attenuates acetaminophen-induced liver injury by engaging GCLC and enhancing ferroptosis-regulatory antioxidant pathways

Objective Acetaminophen (APAP)-induced liver injury (ALI), the major cause of acute drug-induced hepatotoxicity, lacks effective therapies. Berbamine (BBM), a bisbenzylisoquinoline alkaloid derived from the genus Berberis , exhibits diverse pharmacological properties but its efficacy against ALI and involvement in ferroptosis remains unexplored. This study aims to investigate the hepatoprotective effects and mechanisms of BBM against ALI and ferroptosis. Methods The in vivo model of ALI and the in vitro ferroptosis models were established using APAP and RSL3, respectively. Models received BBM or Ferrostatin-1 (Fer-1) treatment. The direct target of BBM was identified through molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assays. Results In vivo, BBM pretreatment dose-dependently alleviated APAP-induced hepatic damage, inflammation, and ferroptosis markers, including lipid peroxidation, GSH depletion, and PTGS2 upregulation, while upregulating GPX4. Meanwhile, the hepatoprotective effects of BBM against ALI matched Fer-1, confirming ferroptosis as an ALI driver. In vitro, BBM inhibited RSL3-induced ferroptosis, reducing ROS, lipid peroxidation, and mitochondrial dysfunction. These effects were mediated by NRF2/GCLC/GPX4 axis activation and FSP1 upregulation. Crucially, BBM directly bound GCLC confirmed by Molecular docking, CETSA, and DARTS. Conclusion BBM protects against ALI by mitigating ferroptosis, oxidative stress, and inflammation, in part through direct modulation of GCLC and coordinated activation of the NRF2/GCLC/GPX4 axis together with FSP1. These findings provide pharmacological evidence supporting the potential of BBM as a promising therapeutic candidate for ALI.