Effect Of The PKC Pathway On The Osteoblast Apoptosis Induced By Dexamethasone: A Preliminary Report

ObjectiveDetecting the effect of osteoblast apoptosis induced by dexamethasoneby PKC pathway, inducing osteoblast apoptosis by dexamethasone andtreating with the agonist and inhibitor of PKC.MethodsOsteoblasts were isolated by using the enzyme alternately to digest fromneonatal SD rat’s skull and cultured, the third generation cells were taken toidentify by observing cell morphology, Alizarin Red staining calcium nodule anddetecting alkaline phosphatase. The experiment was divided into A, B, C and Dgroups. A was blank group，B was model group，C was phorbol group，D wasstaurosporine group. MTT assay measures cell activity, flow cytomerymeasures apoptosis and the ELISA measures the change of PKC in cellmembrane and cytoplasm. Statistical method: T-test、single-factor analysis ofvariance, significance level P <0.05.ResultsThe cells were isolated by using the enzymes(Invitrogen I and) alternatelyand cultured successfully, Identified as osteoblasts by observing cellmorphology, Alizarin Red staining calcium nodule and detecting alkalinephosphatase. The rate of model group’s osteoblasts apoptosis (18.632±3.763)is significantly increased than the control group’s apoptosis rate (5.071±1.692)， P <0.05，the model is set up. The OD values measured by MTT assay of blank group, model group, phorbol group, star staurosporine were：0.693±0.061,0.504±0.035,0.655±0.017,0.671±0.030respectively withsignificant difference between the groups. Flow cytometry measures apoptosis,the osteoblasts apoptosis rate of blank group, model group, phorbol group andstaurosporine group were5.071±1.692、18.632±3.763、25.655±2.480、8.334±3.336respectively with significant difference between the groups. Thecontent of PKC in cytoplasm and membrane were detected by the Elisa：change largest at10min，almost restore at its normal size at30min and at onehour change but the opposite to its normal size.ConclusionThe agonists(Phorbol) of PKC can significant increase the rate ofosteoblast apoptosis induced by dexamethasone, the inhibitors(staurosporine)of PKC can significant reduce the rate. Dexamethasone can activate PKC in thecytoplasm and transfer it to the cell membrane. PKC pathway is one of themechanism of osteoblast apoptosis, induced by dexamethasone.