Yishen Gushu formula exerts osteoprotective effects in OVX-Induced PMOP rats: role of ferroptosis Inhibition and iron metabolism correction

Postmenopausal osteoporosis (PMOP) is a common bone disease among middle-aged and elderly women. Ferroptosis, a form of programmed cell death, plays a crucial role in the regulation of bone metabolism. Yishen Gushu Formula (YSGSF), a traditional Chinese medicine (TCM) compound, has clinical potential for treating PMOP, yet its specific mechanism of action remains unclear. This study aims to investigate the specific mechanism by which YSGSF treats PMOP by inhibiting ferroptosis through mediating the NCOA4/GPX4 pathway. The rat PMOP model was established by OVX and treated with YSGSF for 12 consecutive weeks. HE staining and micro-CT were used to examine bone tissue morphology and structure. Serum levels of E2, PINP, and β-CTX were measured by ELISA. RNA-seq analysis was performed to explore the potential mechanism of YSGSF. Oxidative stress markers (ROS, MDA, SOD, GSH, FE) in rat serum were determined. The regulatory effects of YSGSF on NCOA4, FTH1, GPX4, and SLC7A11 were evaluated by IHC and qRT-PCR. Erastin was used in vitro to induce ferroptosis in ROS17/28 osteoblasts, with cell viability and ferroptosis-related protein levels detected by CCK8 and Western blotting. YSGSF improved bone metabolism in OVX rats, alleviated osteoporotic morphological changes, promoted the overall structural recovery of trabecular bone, and increased bone density. RNA-seq suggested that YSGSF may exert its therapeutic effect on PMOP by inhibiting ferroptosis. Subsequent results indicated that YSGSF improved iron metabolism in bone tissue and suppressed iron-dependent oxidative damage. It reduced serum iron ion accumulation and inhibited the production of ROS and MDA. IHC and qRT-PCR confirmed that YSGSF suppressed the expression of NCOA4, upregulated FTH1, and further increased the expression levels of GPX4 and SLC7A11. In vitro cell experiments demonstrated that YSGSF ameliorates Erastin-induced ferroptosis in osteoblasts, improving the expression of related proteins and cell viability. YSGSF ameliorates OVX-induced PMOP, potentially by inhibiting osteoblast ferroptosis through modulation of the NCOA4/GPX4 pathway.