Redox, inflammatory, and ferroptosis pathway modulation by methotrexate and dexamethasone in primary human RPE cells...

Oxidative stress, inflammation, and ferroptosis drive retinal disease. Methotrexate (MTX) and dexamethasone (DEX) are clinically used to counter these insults, but their effects on retinal pigment epithelium (RPE) homeostasis remain unclear. This study investigates how MTX, DEX, and their combination affect cell viability, redox balance, inflammatory signaling, and ferroptosis-related gene expression in human RPE cells under basal and oxidative stress conditions.

Human RPE cells were exposed to 100 µM H₂O₂ for 24 h and treated with MTX (100 µg/ml), DEX (50 µg/ml), or their combination. Cell viability was assessed using MTT assay. Total oxidant status (TOS), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were measured from cell lysates. IL-6, IL-8, IL-10, and TGF-β levels were quantified by ELISA, and GPX4, ACSL4, and SLC7A11 mRNA expression was measured by qPCR.

Under basal conditions, treatments did not affect viability. DEX and combination increased TOS, with MDA elevated by combination only. CAT activity increased with DEX. MTX reduced IL-8, DEX reduced IL-6, and combination reduced IL-6, IL-8, and TGF-β. GPX4, ACSL4, and SLC7A11 decreased with all treatments. Under oxidative stress, viability declined. DEX restored CAT activity; IL-6 and IL-8 were reduced by both drugs, while combination additionally reduced TGF-β and IL-10. GPX4 increased with MTX and combination, ACSL4 with combination, and SLC7A11 with MTX.

MTX and DEX exert context-dependent effects in RPE cells. Basally, they induce adaptive preconditioning; under oxidative stress, combination therapy triggers a coordinated protective response. These findings support strategies integrating redox modulation, immune regulation, and ferroptosis control to preserve RPE function.