| Objective To investigate the effect of Dexamethasone（Dex）on the proliferation,apoptosis and osteogenesis of osteoblast in vitro and to explore its underlying mechanism.Methods Osteoblasts were acquired by primary culturing from new born SD rats.Cells were divided into different groups as follows:（1）Group with different concentrations of dexamethasone:control group（0 mol/L Dex）,10-8mol/L Dex group、10-7mol/L Dex group and 10-6mol/L Dex group,incubated for 24h;（2）To further verify the effect of dexamethasone on proliferation and function of osteoblasts,cells were divided into control group、740Y-P group、Dex group、740Y-P+Dex group,incubated for 48h.Cell Counting Kit-8 assay was performed to evaluate the cell proliferation.Annexin V-FITC/PI double staining was performed to analyze the apoptosis rate.Alkaline Phosphatase staining was used to detect the activity of ALP in osteoblast.The fluorescence microscopy was applied to detect the expression of ALP and caspase-3.Western blot assay further examined the expression of ALP,OCN,caspase-3,caspase-9,p-PI3K and p-Akt in each group.One-Way analysis of variance was used to analyze the experimental data.Results1.Compared with the control group,dexamethasone at dose of 10-8mol/L,10-7mol/L and 10-6mol/L inhibited the proliferation of osteoblasts,most evidently in 48 hours.Compared with the 10-8mol/L group,the osteoblasts proliferation of 10-7mol/L and 10-6mol/L Dex group were both.Compared to the10-7mol/L group,the proliferation of 10-6mol/L Dex group were markedly reduced.2.Dexamethasone at concentration of 10-7mol/L and 10-6mol/L induced apoptosis of osteoblasts at 48 hours,showing significant difference compared to the control group（P<0.05）.However,10-8mol/L of Dex had no apparent effect in inducing apoptosis of osteoblasts（P>0.05）3.The activity of Alkaline phosphatase in all timegroups was significantly lower than control group（P<0.05）.Compared to the 24h group,the ALP activity of osteoblast in 48h group were markedly decreased.4.The immunofluorescene staining result showed that the expression of caspase-3 protein was significantly increased andALP protein expression was markedly reduced in 24h and 48h group（P<0.05）.5.The Western blotting results showed that dexamethasone at the concentration of 10-7mol/L、10-6mol/L could significantly reduce the expression of ALP,OCN,p-PI3K and p-Akt and up regulate the expression of caspase-3and caspase-9（P<0.05）.6.Compared with the control group,10μmol/L740Y-P could enhance the proliferation of osteoblast and up-regulate the expression of p-Akt（P<0.05）.There is no significant change of ALP activity and apoptosis rate was observed in 740Y-P treated cells.Compared to the Dex group,the proliferation rate and ALP activity of 740Y-P+Dex group were markedly increased（P<0.05）,and the expression of OCN,ALPand p-Akt protein increased significantly.Compared with the Dex group,the apoptosis rate and the expressions of caspase-3 and caspase-9 were markedly reduced（P<0.05）.Conclusion 10-6mol/L dexamethasone can inhibit the proliferation of osteoblasts,augments the apoptosis and supress the expressions of OCN,ALP and p-Akt protein and its mechanism may be related to the down-regulation of PI3K/Akt phosphorylation... |
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