Background: Methotrexate (MTX) is widely used as a chemotherapeutic and immunosuppressive agent, but is frequently associated with hepatotoxicity. Ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, is increasingly recognized as a major contributor to drug-induced liver injury. Peroxisome proliferator-activated receptor α (PPARα) serves as a critical regulator of lipid metabolism and antioxidant defense. However, its role in MTX-induced hepatocyte ferroptosis remains unclear. This study aimed to determine whether PPARα modulates MTX-induced liver injury by regulating oxidative stress and ferroptosis in vivo and in vitro.

Methods: A mouse model of MTX-induced liver injury was established, and hepatic histopathology, serum biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST)), inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin-6 (IL-6)), oxidative stress markers (4-hydroxynonenal (4-HNE), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH)), and ferroptosis-related proteins (glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), acyl-CoA synthetase long-chain family member 4 (ACSL4), arachidonate 15-lipoxygenase (ALOX15), transferrin receptor 1 (TFR1)) were assessed. PPAR-related signaling molecules (PPARα, PPARγ, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α)) were analyzed using Western blotting. In vitro, AML-12 hepatocytes were exposed to MTX with or without the PPARα agonist GW7647 to evaluate lipid peroxidation, antioxidant capacity, and ferroptosis markers.

Results: MTX treatment induced significant liver injury, characterized by hepatocellular swelling, vacuolization, elevated ALT/AST (p < 0.05), increased proinflammatory cytokines (p < 0.05), enhanced lipid peroxidation (p < 0.05), and reduced antioxidant capacity (p < 0.05). Ferroptosis-associated markers were significantly altered (decreased GPX4; increased ACSL4, ALOX15, and TFR1; p < 0.05). MTX also disrupted PPAR signaling (reduced PPARα and PGC-1α; increased PPARγ; p < 0.05). Activation of PPARα by GW7647 restored PGC-1α expression, enhanced antioxidant defenses (GPX4, SLC7A11, GSH, SOD; p < 0.05), suppressed ferroptosis-related lipid-remodeling enzymes (p < 0.05), and reduced lipid peroxidation (p < 0.05), thereby alleviating MTX-induced hepatocellular damage in vivo and in vitro.

Conclusion: PPARα activation mitigates MTX-induced liver injury by reprogramming lipid metabolism and enhancing antioxidant defenses, ultimately suppressing ferroptosis. These findings highlight the therapeutic potential of targeting the PPARα-ferroptosis pathway as an innovative strategy to counteract MTX-associated hepatotoxicity.