Effect Of Osteoprotegerin On Nitric Oxide Production And Endothelial Nitric Oxide Synthase Activity In Osteoclasts

Objective:Raw264.7cells were cultured and induced by RANKL into osteoclasts in vitro. Adding different doses of osteoprotegrin(OPG) to observe the nitric oxide(NO) expression and endothelial nitric oxide synthase (eNOS) activity.Method:Experiments are carried in four steps.Firstly,Raw264.7cells are cultured and induced by RANKL(50ng/ml),tartrate resistant acid phophatase staining was uesd to tesify the induced cells are osteoclasts.Secondly,osteoclasts are devided into six groups, group A is blank control group;group B is negative control, add DMEM.; group C、 D. E^F was given10,25,50,75ng/ml of OPG respectively. Propyl iodide organism insert DNA quantitative staining method was used to test the apoptosis rats of osteoclasts. Real time PCR was used to test the expression of marker gene TRAP mRNA and Protein kinase K mRNA of osteoclasts. Thirdly, NO detection kit and nitric oxide synthase(eNOS) activity kit were used to test NO production and eNOS activity. Fourthly, group C、D、E、F were added with L-NAME, a kind of nitric oxide synthase inhibitor, to test the changes of apotosis rats of osteoclasts and the changes of the expression of marker gene TRAP mRNA and Protein kinase K mRNA of osteoclast.Results: (1)With the increase of concentration of OPG, the number of osteoclasts decreased and apoptosis rate increased gradually, there is a significant difference compared with control groups.(p<0.05).(2) With the increase of concentration of OPG, the production of NO increased, while the activity of eNOS increased firstly then decreased, the difference is significant compared with control groups.(3) With the increase of concentration of OPG, expressions of specific marker enzymes TRAP mRNA and protein kinase K mRNA decreased. However, after the joining of eNOS inhibitor, expression of two specific enzymes increased than before.Conclusions:(1)Under the condition of Raw264.7cells cultured in vitro, OPG can inhibit the formations of osteclasts.(2)Within a certain concentration range,OPG can boost the NO production and eNOS activity, when reach certain concentration the activity of eNOS decreased,the difference is significant compare with blank control group and negative control group.(3)Under the condition of Raw264.7cells cultured in vitro, in the experiment design of OPG concentration range,OPG has synergistic effect with NO in inhibiting osteoclasts formation and boosting its apoptosis rat.