Gramine limits alcohol-associated liver injury by suppressing NOX1-linked ferroptotic responses and mitochondrial dysfunction

Alcohol-associated liver injury involves oxidative stress, mitochondrial dysfunction, and ferroptosis-related damage. NADPH oxidase 1 (NOX1), together with its regulatory GTPase Ras-related C3 botulinum toxin substrate 1 (RAC1), forms a regulatory module that governs redox signaling. Yet small molecules that engage NOX1 and suppress these coupled injury programs remain insufficiently defined. Here, we identified gramine as a candidate NOX1-engaging compound and delineated its protective mechanism in ethanol-associated hepatocellular injury. Alcohol challenge induced a hepatic NOX1/RAC1 response, recapitulated in ethanol-injured hepatocytes. Silencing Nox1 or Rac1 blunted ethanol-driven injury phenotypes, limiting injury-associated enzyme release, reactive oxygen species accumulation, mitochondrial membrane potential disturbance, mitochondrial network disruption, and DNA fragmentation while partially preserving glutathione peroxidase 4 (GPX4). Molecular docking predicted a gramine-NOX1 interaction, a cellular thermal shift assay supported target engagement, and cycloheximide chase analysis showed gramine accelerated NOX1 protein turnover. In ethanol-exposed hepatocytes, gramine conferred concentration-associated protection, improving cell viability, and reducing injury-associated enzyme release, lipid droplet signals, and DNA fragmentation. Post-ethanol gramine treatment reduced injury-associated enzyme release. NOX1 overexpression undermined gramine-mediated protection and reinstated ferrous iron accumulation, glutathione redox imbalance, reactive oxygen species production, mitochondrial dysfunction, and damage-associated signaling. Erastin weakened gramine-mediated protection, implicating inhibition of ferroptotic responses in the protective mechanism. In alcohol-gavaged mice, gramine reduced hepatic NOX1 abundance, serum injury markers, Fe²⁺ levels, lipid accumulation, and lipid peroxidation-associated changes, while preserving GPX4 abundance and limiting histopathological injury. These findings support gramine as a NOX1-engaging small molecule that limits alcohol-associated liver injury by suppressing NOX1-linked ferroptotic responses and mitochondrial dysfunction.