Low-dose valproic acid ameliorates osteoporosis by restoring redox homeostasis and suppressing ferritinophagy-dependent ferroptosis via EZH2/H3K27me3 signaling

Valproic acid (VPA), a histone deacetylase inhibitor, exhibits dose-dependent effects on bone homeostasis. This study investigates whether low-dose VPA protects against ovariectomy (OVX)-induced osteoporosis by targeting oxidative stress and ferroptosis. OVX rats received low- (100 mg/kg/d) or high-dose (300 mg/kg/d) VPA for 12 weeks. Bone microstructure was analyzed by micro-CT. Systemic redox status was evaluated by measuring MDA, SOD, GSH, and Fe 2+ . Ferroptosis markers (GPX4, ACSL4, FTH1, NCOA4) were examined. In MC3T3-E1 cells pretreated with VPA (0.5–3 mM), erastin was used to induce ferroptosis. The EZH2/H3K27me3 pathway and osteoclastogenesis were further assessed. OVX induced bone loss, oxidative stress (elevated MDA/Fe 2+ , decreased SOD/GSH), and ferroptosis activation (increased ACSL4/NCOA4, decreased GPX4/FTH1). Low-dose VPA reversed these changes, improved bone density and microarchitecture, and reduced bone resorption. High-dose VPA showed no protective effects. In vitro, 1 mM VPA attenuated erastin-induced lipid peroxidation, mitochondrial damage, and ferroptosis. Mechanistically, VPA activated EZH2/H3K27me3 signaling, enhancing H3K27me3 enrichment at the NCOA4 promoter to suppress ferritinophagy and ferroptosis. VPA also inhibited RANKL-induced osteoclast differentiation. Low-dose VPA ameliorates osteoporosis by restoring redox homeostasis, epigenetically inhibiting NCOA4-mediated ferritinophagy via EZH2/H3K27me3 activation, and suppressing osteoclastogenesis. These findings identify low-dose VPA as a multifaceted anti-osteoporotic agent and highlight the EZH2/H3K27me3/NCOA4 axis as a pivotal regulatory pathway in bone redox biology. • Low-dose VPA (100 mg/kg/d) rescues bone mass in ovariectomized rats by reversing oxidative stress and ferroptosis. • VPA activates EZH2/H3K27me3 to silence NCOA4 , blocking ferritinophagy in osteoblasts ( P < 0.0001). • Dose-dependent duality : Low-dose VPA protects bone, high-dose exacerbates osteoblast damage. • VPA restores redox balance, inhibits ferroptosis, and suppresses osteoclastogenesis ( P < 0.001). • First evidence that low-dose VPA targets the EZH2-ferritinophagy-ferroptosis axis for osteoporosis.