Total flavonoids of Rhizoma drynariae influence ferroptosis in osteoblasts via miR-205-5p/GPX4 axis

Purpose: This research investigated the biological effects of total flavonoids of Rhizoma Drynariae (TFRD) on osteoporosis in mice. Methods: Mice were subjected to bilateral ovariectomy (OVX) to generate the osteoporosis model used, which was then intragastrically administered TFRD daily at a dose of 75 mg/kg. Bone loss was examined histologically using H & E staining. Moreover, erastin-treated primary osteoblasts were used to further analyze the effect of TFRD on ferroptosis. Reactive oxygen species (ROS), ferrous iron level, and CCK-8 method were employed to determine the protective influence of TFRD against ferroptosis in erastin-treated osteoblasts. The relationship between microRNA-205-5p (miR-205-5p) and glutathione peroxidase 4 (GPX4) was determined using luciferase assay. Results: TFRD treatment inhibited OVX-induced bone mass loss, downregulated ferrous ion content, and increased the level of GPX4 in vivo (p < 0.01). Besides, TFRD treatment inhibited erastin-induced increases in ROS level and ferrous iron level, and promoted cell viability in vitro (p < 0.01). Moreover, TFRD increased GPX4 protein expression level (p < 0.01). Results from gain and loss experiments showed that GPX4 increased cell viability, and reduced ferrous ion and ROS levels in erastin-induced osteoblasts (p < 0.01, p < 0.001, respectively). Furthermore, miR-205-5p directly targeted GPX4 and negatively regulated GPX4 expression (p < 0.01, p < 0.001, respectively). Upregulation of GPX4 reversed the effect of miR-205-5p overexpression on cell viability, and reduced ferrous iron level and ROS levels in osteoblasts significantly. Conclusion: TFRD may serve as an inhibitor of osteoblast ferroptosis during osteoporosis formation by down-regulating the expression of miR-205-5p which directly targets GPX4 in mouse osteoblasts. These findings indicate the therapeutic potential and underlying mechanism of action of TFRD.