Tempol Attenuates Methotrexate-Induced Osteotoxicity via Antioxidant Mechanisms: Impairment of Protection by GPX4...

Osteotoxicity is a well-recognized adverse effect of Methotrexate (MTX) therapy, primarily driven by oxidative stress and impaired bone remodeling. This study aimed to investigate the protective effects of Tempol, a membrane-permeable nitroxide antioxidant, against MTX-induced osteotoxicity, and to assess how these effects are influenced by ML210, a glutathione peroxidase 4 (GPX4) inhibitor.

Murine osteocyte-like MLO-Y4 cells were treated with MTX alone, Tempol alone, or a combination of MTX with Tempol and ML210. Apoptotic markers (caspase-3, Bax, Bcl-2), MAPK signaling proteins (p-JNK, p-ERK), and oxidative stress parameters (TAS, TOS, SOD, GPx) were measured via ELISA to evaluate the redox and apoptotic responses.

MTX significantly induced apoptosis, as evidenced by increased caspase-3 activity and Bax expression, along with decreased Bcl-2 levels. MTX also activated the MAPK pathway by upregulating p-JNK and p-ERK. Furthermore, MTX decreased TAS, SOD, and GPx levels, while increasing TOS. Tempol treatment successfully reversed these effects, restoring apoptotic balance, inhibiting MAPK activation, and enhancing antioxidant capacity. However, co-treatment with ML210 markedly attenuated Tempol's protective effects, resulting in sustained oxidative stress, elevated apoptotic markers, and persistent MAPK pathway activation. This suggests that Tempol's cytoprotective actions are dependent on functional GPX4 activity.

Tempol exhibits strong potential as an adjunctive antioxidant therapy to counteract MTX-induced osteotoxicity. Nevertheless, its efficacy is significantly influenced by the status of the endogenous antioxidant enzyme GPX4. These findings underscore the need for further investigation into Tempol's mechanism of action in redox-dependent pathways and its suitability in clinical settings, especially where GPX4 function may be compromised.