Umbilical Cord Mesenchymal Stem Cells Alleviate Osteoblast Differentiation Disorder Induced by Oxidative Stress by Inhibiting Ferroptosis Through the miR-218-5p/CBX3 Axis

Objective The pathogenesis of osteoporosis is closely linked to osteoblast impairment caused by oxidative stress. While umbilical cord mesenchymal stem cells (UC-MSCs) are widely recognized as a promising regenerative therapy, the specific pathways underlying their protective effects on oxidatively stressed osteoblasts are not fully understood. This study aimed to determine whether UC-MSCs exerted protective effects on osteoblasts by targeting ferroptosis and to characterize the mediating function of the miR-218-5p/CBX3 signaling axis in this regulatory mechanism. Methods We established an in vitro oxidative stress injury model by treating the mouse osteoblast cell line (MC3T3-E1) with H₂O₂. We co-cultured these cells with UC-MSCs. Cell viability and injury were evaluated using CCK-8 and LDH release assays. ROS, Fe²⁺, MDA, and glutathione (GSH) levels were assessed with probes and biochemical kits to evaluate ferroptosis characteristics. Expression of ferroptosis defense proteins and osteogenic markers was analyzed using RT-qPCR and Western blot analysis. To validate the regulatory relationship between miR-218-5p and CBX3, dual-luciferase reporter assays were employed. Functional rescue experiments were carried out using a specific ferroptosis inducer (RSL3), miRNA inhibitors, overexpression constructs, and siRNA-mediated gene silencing. Results UC-MSCs protected MC3T3-E1 cells from H₂O₂ injury by improving viability, reducing LDH release, and inhibiting ferroptosis (ROS, iron overload, lipid peroxidation). They upregulated glutathione peroxidase 4 (GPX4), SLC7A11, and FTH, restoring ALP activity and osteogenic marker expression. Importantly, the introduction of RSL3 entirely abrogated these UC-MSC-mediated osteoprotective effects, confirming the causal involvement of ferroptosis. Mechanism studies showed that H₂O₂ downregulated the expression of miR-218-5p, which directly targets the 3'UTR of CBX3 and inhibited its protein expression; UC-MSCs could significantly upregulate and restore miR-218-5p levels. Mechanism studies have shown that miR-218-5p effectively replicates the anti-ferroptosis and bone protection effects of UC-MSCs. On the contrary, the use of specific inhibitors to suppress miR-218-5p significantly weakened the protective benefits of UC-MSCs against ferroptosis and osteogenic dysfunction, and the overexpression of CBX3 also reversed the protective effect mediated by UC-MSCs. Conclusion UC-MSCs inhibit oxidative stress-induced osteoblast ferroptosis and promote osteogenesis via the miR-218-5p/CBX3 axis, providing new insights into MSC therapy for osteoporosis.