Asperosaponin VI inhibition of DNMT alleviates GPX4 suppression-mediated osteoblast ferroptosis and diabetic osteoporosis.

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Diabetic osteoporosis (DOP) is an insidious complication of diabetes with limited therapeutic options. DOP is pathologically associated with various types of regulated cell death, but the precise role of ferroptosis in the process remains poorly understood. Asperosaponin VI (AVI), known for its clinical efficacy in treating bone fractures and osteoporosis, may exert its osteoprotective effects through mechanisms involving ferroptosis, however this has not been established. This study aimed to investigate the role of AVI in modulating ferroptosis in a mouse model of DOP and to explore the underlying mechanisms. We assessed OP alterations in femurs of DOP-conditioned mice and primary bone cells. We generated a strain of osteoblast-specific Gpx4-deficient mice. A combination of micro-CT, immunohistochemistry, immunofluorescence, methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), western blotting (WB), and AVI pull-down assays were employed to elucidate the mechanism and therapeutic target of AVI in DOP. Our findings revealed that femurs from DOP-conditioned mice exhibited significant ferroptosis and suppression of the core anti-ferroptosis factor GPX4, mainly due to hypermethylation of the Gpx4 promoter mediated by DNA methyltransferases DNMT1and DNMT3a. Notably, treatment with AVI effectively reversed the hypermethylation, restored GPX4 expression, and reduced ferroptotic pathologies associated with DOP by inhibiting DNMT1/3a. In primary osteoblasts, AVI alleviated GPX4 suppression and reduced ferroptosis in DOP-conditioned primary osteoblasts through a mechanism dependent on DNMT inhibition and GPX4 restoration. Importantly, the anti-ferroptotic and osteoprotective effects of AVI were abolished in osteoblastic Gpx4 haplo-deficient mice (Gpx4Ob-/+) or when GPX4 was pharmacologically inactivated with RSL3. Our study identifies a pivotal epigenetic ferroptotic pathway that contributes significantly to DOP and uncovers a crucial pharmacological property of AVI that is potentially effective in treating patients with DOP and related osteoporotic disorders.