Empagliflozin shields brain from methotrexate toxicity: Nrf2/HO-1/GPX4 activation prevents ferroptosis and chemo-brain in rats.

BACKGROUND: Methotrexate (MTX) can induce "chemo-brain," but mechanisms remain incompletely defined. Ferroptosis has been implicated in MTX toxicity. We investigated whether Empagliflozin (EMPA) protects against MTX-induced neurotoxicity by modulating ferroptosis-related pathways. METHODS: Male Sprague-Dawley rats (n = 32) received Control, EMPA (10 mg/kg/day oral, 14 days), MTX (20 mg/kg, i.p., day 7), or MTX + EMPA. Behavior (open field, Y-maze, rotarod) was tested on days 9-10. On day 15, hippocampi were analyzed for oxidative stress (MDA, GSH, SOD, CAT, GPx), ferroptosis markers (tissue iron, 4-hydroxynonenal [4-HNE]), cytokines, apoptosis (Bax/Bcl-2, caspase-3/-9), and mRNA levels of Nrf2, HO-1, GPX4, PTGS2 and ACSL4. Nrf2, HO-1, GPX4 and histology were assessed by immunohistochemistry. RESULTS: MTX caused cognitive and motor deficits, hippocampal damage, increased tissue iron (45.05 ± 4.89-76.37 ± 6.84 µg/g) and 4-HNE (119.10 ± 15.47-248.02 ± 23.92 pmol/mg), elevated MDA, depleted antioxidants, and downregulated Nrf2/HO-1/GPX4. PTGS2 and ACSL4 were strongly upregulated, with parallel cytokine surges and apoptotic activation. EMPA alone was neutral. MTX + EMPA significantly improved behavior, partially restored Nrf2/HO-1/GPX4, reduced PTGS2/ACSL4 expression, and attenuated iron overload, 4-HNE/MDA, inflammation and apoptosis, while preserving hippocampal architecture. CONCLUSIONS: EMPA mitigates MTX-induced neurotoxicity by re-engaging Nrf2/HO-1/GPX4, limiting iron-dependent lipid peroxidation (tissue iron,4-HNE) and dampening PTGS2/ACSL4-linked ferroptosis alongside inflammatory and apoptotic cascades. These findings support EMPA as a potential adjunct to reduce MTX-related cognitive impairment and warrant evaluation in chronic, clinically relevant regimens.