Curculigoside attenuates osteoporosis through regulating DNMT1 mediated osteoblast activity

This work aims to study the function of curculigoside in osteoporosis and explore whether DNMT1 is closely involved in osteoblast activity. After OB-6 osteoblasts were treated with hydrogen peroxide (H₂O₂), a curculigoside treatment group was set up and a series of biological tests including MTT, flow cytometry, western blotting, ROS fluorescence intensity, mitochondrial membrane potential, and ELISA experiments were performed to verify the effect of curculigoside on the activity of osteoblasts. Then, alkaline phosphatase (ALP) activity, alizarin red staining, PCR, and western blotting assays were performed to detect the effects of curculigoside on osteoblast function. By constructing DNMT1 knockdown and overexpression OB-6 cell lines, the effect of DNMT1 on osteoblast function was verified. In addition, the expression level of Nrf2 in each group was detected to speculate the mechanism of DNMT1 in osteoporosis. The cell activity and level of bcl-2 and SOD were significantly increased; the cell apoptosis, ROS fluorescence intensity, mitochondrial membrane potential, MDA and level of caspase-3, Bax, and CAT was reduced in curculigoside treatment group compared with H₂O₂-induced OB-6 osteoblasts. Meanwhile, the ALP activity, number and area of bone mineralized nodules, and gene and protein expression of OSX and OPG were significantly elevated in curculigoside group. Moreover, DNMT1 knockdown had a similar promotion effect on osteoblast function as curculigoside, and DNMT1 overexpression could reverse the promotion effect of curculigoside on osteoblast function. Further mechanistic studies speculated that DNMT1 might play a role in osteoporosis by affecting Nrf2 methylation. Curculigoside enhances osteoblast activity through DNMT1 controls of Nrf2 methylation.