A Pathophysiological Hepatocyte Senescence Model Mimicking Lipid Overload and Oxidative Stress via dietary OA/PA and H ₂ O ₂ Co-Treatment

Alleviating hepatocyte senescence offers potential for treating chronic liver diseases. However, current models of hepatocyte senescence fail to recapitulate the multifactorial pathology of aged livers, primarily relying on oxidative stress induction that produces oversimplified phenotypes. To address this issue, integrative transcriptomic and fatty acid determination of aged murine liver tissues revealed profound dysregulation of fatty acid metabolism, characterized by pathological accumulation of diet-related fatty acids oleic acid (OA) and palmitic acid (PA). We developed a physiomimetic senescence induction paradigm combining OA/PA with hydrogen peroxide (H₂O₂) exposure across four human and murine hepatic cell systems. This dual-hit approach generated more prominent senescence hallmarks, including senescence-associated β-galactosidase activity, lipid droplet formation, redox imbalance, and cell cycle arrest. Transcriptomic analysis revealed enhanced aging hallmark genes regulation in THLE-2 cells cotreated with OA/PA and H₂O₂ than in those treated with H₂O₂ alone. Crucially, the anti-aging drug rapamycin reversed senescence phenotypes and lipid accumulation exclusively in the dual-induction model, validating its pathophysiological relevance. Our work establishes aberrant lipid metabolism as an essential accelerator of hepatocyte senescence. A fatty acid-rich environment mimicking high-lipid dietary conditions combined with H₂O₂ exposure induces hepatocytes resembling aged livers, and this updated hepatocyte senescence model provides an advanced tool for screening dietary interventions or functional foods targeting age-related liver disorders.