Effect of osteoprotegerin on nitric oxide production and endothelial nitric oxide synthase activity in osteoclasts / 中国组织工程研究

ABSTRACT

BACKGROUND:Osteoprotegerin and nitrogen monoxidum play a key role in the prevention and treatment of osteoporosis. However, the correlation of the two in inhibiting the proliferation and differentiation of osteoclasts remains unclear. OBJECTIVE:To observe the effect of different doses of osteoprotegerin on nitric oxide production and endothelial nitric oxide synthase activity in osteoclasts. METHODS:Tartrate resistant acid phophatase staining was used to test whether the induced cells are osteoclasts. Osteoclasts were divided into six groupsblank control group (no reagent);negative control group (DMEM);four osteoprotegerin groups (10, 25, 50, 75μg/L of osteoprotegerin). Annexinv-FITC kit and flow cytometry were used to test the apoptosis rate of osteoclasts. Real-time PCR was used to detect the expression of osteoclasts marker gene, TRAP mRNA and protein kinase K mRNA. Nitric oxide production and nitric oxide synthase activity were determined using the corresponding kits. Four osteoprotegerin groups were added with L-NAME, a kind of nitric oxide synthase inhibitor, to test the changes of the apoptosis rates of osteoclasts and the changes of the expression of TRAP mRNA and protein kinase K mRNA of osteoclasts. RESULTS AND CONCLUSION:Osteoprotegerin inhibited the differentiation of osteoclasts and induced the apoptosis. Osteoprotegerin concentration had a positive correlation with the apoptosis rate of osteoclasts, and a negative correlation with the numbers of osteoclasts and expression of marker gene TRAP mRNA and protein kinase K mRNA in osteoclasts. Osteoprotegerin boosted the nitric oxide production and endothelial nitric oxide synthase activity, and osteoprotegerin concentration was positively correlated to the nitric oxide production and endothelial nitric oxide synthase activity. After Raw264.7 cells were in vitro cultured, osteoprotegerin and nitric oxide play a synergic role in inhibiting osteoclasts production and promoting the apoptosis. We speculate that there is osteoprotegerin/endothelial nitric oxide synthase/nitric oxide signal pathway.