[Construction of an osteoblast injury model and study on the protective effects of biosynthetic vitamin D_3].

OBJECTIVE

To construct a D-galactose(D-Gal)-induced osteoblast injury model mimicking senile osteoporosis and investigate the protective effect of biosynthetic vitamin D_3.

METHODS

Using Human osteoblast cell line hFOB1.19, cell viability was detected by Cell Counting Kit-8(CCK-8), and apoptosis was assessed by Calcein AM/propidium iodide(PI) double staining. The optimal concentration and duration of D-Gal for modeling were determined. Cells were divided into control, D-Gal model, and intervention groups treated with D-Gal plus biosynthetic vitamin D_3 at concentrations of 10~(-10), 10~(-9), 10~(-8), 10~(-7) and 10~(-6) mol/L. Multiple assays were performed: CCK-8 for viability, colorimetry for alkaline phosphatase(ALP) activity, ELISA for osteocalcin(OC) content, β-galactosidase staining for cellular senescence, and DCFH-DA fluorescent probe for reactive oxygen species(ROS) levels. Statistical analysis was conducted using one-way ANOVA followed by Dunnett's test.

RESULTS

A stable osteoblast injury model was successfully established using 60 mg/mL D-Gal treatment for 24 hours. Compared with the control group, the model group exhibited significantly reduced cell viability((51.11±7.36)% vs. (108.90±16.01)%, P<0.01), ALP activity((14.63±0.80)μmol/L vs. (394.40±1.39) μmol/L, P<0.01), and OC content((837.40±4.34) pg/mL vs. (2642.00±58.92) pg/mL, P<0.01), along with increased ROS levels and the proportion of senescent cells. After intervention with biosynthetic vitamin D_3, the 10~(-8) mol/L and 10~(-7) mol/L groups showed significant improvement. The 10~(-7) mol/L group demonstrated the most pronounced effects, markedly enhancing cell viability((75.71±9.04)% vs. (51.11±7.36)%, P<0.05) and ALP activity((253.60±4.16) μmol/L vs. (14.63±0.80) μmol/L, P<0.01), while also reducing ROS levels and the proportion of senescent cells.

CONCLUSION

A stable in vitro model of senile osteoporosis-related osteoblast injury was successfully constructed. Biosynthetic vitamin D_3 can significantly ameliorated D-Gal-induced impairments of osteoblast proliferation and differentiation, as well as alleviated cellular senescence and oxidative stress.