Study on the apoptotic effect of dexamethasone on osteoblast and its mechanism / 中华风湿病学杂志

ABSTRACT

Objective To investigate the effect of Dexamethasone (Dex) on the proliferation and apoptosis of osteoblasts in vitro and to explore its underlying mechanism.Methods Osteoblasts were acquired by primary culturing from new born SD rats.The inverted microscope was used to observe the cellular appearance.The cells were identified by alkaline phosphatase staining and alizarin red staining.The third generation osteoblasts were divided into four groups.Cells were incubated with different concentrations (0,10-8 mol/L,10-7 mol/L,10-6 mol/L) of dexamethasone for 12 hours,24 and 48 hours.Cell Counting Kit-8 was performed to evaluate the inhibitory effect on cell proliferation.The apoptosis rate was analyzed by flow cytometry with Annexin Ⅴ-FITC/PI double staining.The fluorescence microscopy was used to test the nuclear alteration and the expression of caspase-3.Western blot assay was applied to detect the expression of Bcl-2,Bad,caspase-3 and phosphorylated Akt.One-Way analysis of variance was used to determine the difference between groups.LSD-t was used to compare the difference between any two groups.Results Com-pared with the control group,dexamethasone at dose of 10-8 mol/L,10-7 mol/L and 10-6 mol/L inhibited the proliferation of osteoblasts,most evidently in 48 hours (0.980±0.028 vs 1.143±0.017,t=5.454,P＜0.05;0.798±0.057 vs 1.143±0.017,t=1 1.555,P＜0.05;0.728±0.031 vs 1.143±0.017,t=13.908,P＜0.05).Dexamethasone at dose of 10-7 mol/L and 10-6 mol/L induced apoptosis of osteoblasts at 48 hours,showing significant difference compared with control group [(9.8± 2.6)％ vs (4.1±0.8)％,t=3.508,P＜0.05;(12.4±2.6)％ vs (4.1±0.8)％,t=5.140,P＜0.05].However,10-8 mol/L of dexamethasone had no apparent effect in inducing apoptosis of osteoblasts [(4.9±1.2)％ vs (4.1±0.8)％,t=0.470,P ＞0.05].The immunofluorescene staining result showed that the expression of caspase-3 protein was significantly increased in 10-7 mol/L and 10-6 mol/L dex group (t=4.320,8.475,P＜0.05).The Western blotting results showed that dexamethasoneat the concentration of 10-7 mol/L,10-6 mol/L could significantly increase the expression of Bad and caspase-3 and down-regulate the expression of Bcl-2 and p-Akt.The expression of Bcl-2 was markedly reduced by 53.8％,78.4％ (t=4.019,5.988;P＜0.05),The expression of p-Akt decreased by 37％,49.6％ (t=2.067,3.491;P＜0.05),the expression of Bad protein increased by 276.9％ and 334.8％ respectively (t=7.342,8.872;P＜0.05),the expression of caspase-3 protein were increased by 138.0％ and 193.9％ (t=5.510,7.750;P＜0.05).Conclusion Dexamethasone is capable of inhibiting the proliferation of osteoblast,as well as augmenting the apoptosis.The mechanism of this process is probably related to reduction of the level of Bcl-2 expressionand up-regulation the expression of Bad,caspase-3 with the effects of inhibiting the PI3K/Akt signaling pathway.