Abstract: Objective To explore the protective mechanism of melatonin on the oxidative stress damage of osteoblasts induced by hydrogen peroxide. Methods Primary osteoblasts were extracted by tissue explants adherent method from ten newborn SD rats within 24 hours and purified by differential adhesion method. Cells were identified by alkaline phosphatase and Alizarin Red S staining when passaged to the second generation. The identified primary osteoblasts were treated with different concentrations of H2O2 for different time and CCK8 assay was used to detect cell proliferation. Choose an appropriate concentration and time to establish an oxidative stress damage model and intervene with melatonin and EX527. The experiment was divided into control group, H2O2 group, melatonin group and EX527 group. Alkaline phosphatase and Alizarin Red S staining were performed on the four groups, the content of reactive oxygen species, malondialdehyde and superoxide dismutase were detected, the apoptosis rate was detected by flow cytometry and the western blotting method was used to detect the expression of Bax, Bcl2, BMP2, RUNX2, SIRT1 and p66SHC. Results The primary osteoblasts were successfully extracted after identification. After treated with 400 μmol/L H2O2 for 4 hours, the cell viability decreased to 52 % which was suitable for establishing oxidative stress damage model, alkaline phosphatase activity and mineralization ability decreased, ROS content, MDA content increased and SOD activity decreased, the apoptosis rate increased accompanied by SIRT1, BMP2, RUNX2, Bcl2 expression decreased and p66SHC, Bax expression increased. Melatonin pretreatment alleviated the oxidative damage of H2O2 on osteoblasts and partially restored the activity and mineralization ability of osteoblasts. The SIRT1 inhibitor EX527 can reverse the above effects of melatonin. Conclusion Melatonin can inhibit the H2O2-induced oxidative damage of osteoblasts and promote osteogenesis via SIRT1/p66SHC pathway |