Protective Role of Alpha-Lipoic Acid Against Methotrexate-Induced Osteotoxicity: Mechanisms of Oxidative Stress Regulation and MAPK Pathway Inhibition

Background/Objectives: Osteotoxicity is a severe complication of Methotrexate (MTX) chemotherapy, characterized by oxidative stress and disrupted bone remodeling. The primary objective of this study was to investigate the cytoprotective mechanisms of the antioxidant Alpha-Lipoic Acid (ALA) against MTX-induced osteotoxicity, specifically focusing on its modulation of oxidative stress, apoptosis, and Mitogen-Activated Protein Kinase (MAPK) signaling pathways. Methods: Murine osteocyte-like MLO-Y4 cells were cultured and exposed to a fixed dose of MTX (10−5 M), either alone or concurrently with ALA (50 μmol/L) for 48 h. Biochemical profiling was performed using specific enzyme-linked immunosorbent assays (ELISA) and colorimetric kits to evaluate pro- and anti-apoptotic proteins (Caspase-3, Bax, Bcl-2, Wee1, GRP78, GADD153, AIF), active MAPK components (p-JNK, p-ERK), and standard oxidative stress parameters (TAS, TOS, SOD, GPx). Results: MTX treatment induced significant cellular stress, evidenced by elevated Caspase-3, Bax, p-JNK, and p-ERK levels, alongside a critical reduction in Bcl-2 expression. MTX also markedly increased TOS while depleting TAS, SOD, and GPx levels. Conversely, co-treatment with ALA significantly mitigated these cytotoxic responses. ALA restored the Bax/Bcl-2 balance, effectively downregulated both p-JNK and p-ERK activation, and substantially reinforced the cellular antioxidant defense system by enhancing TAS, SOD, and GPx activities, although recovery to baseline control levels was partial. Conclusions: ALA exerts robust in vitro cytoprotective effects against MTX-induced osteotoxicity in MLO-Y4 cells by counteracting oxidative stress and inhibiting aberrant apoptotic and MAPK signaling. These findings establish a mechanistic baseline, underscoring the need for subsequent in vivo dose–response studies to validate ALA’s therapeutic potential in chemotherapy management.