Dexamethasone Induced Apoptosis Of Rat Osteoblast Via The Mitochondria-mediated Pathway

Objective To investigate the effect of dexamethasone(Dex)induced apoptosis of osteoblasts in vitro and to explore the molecular mechanism of dexamethasone in the apoptosis of osteoblasts.Methods The primary osteoblasts were isolated and cultured in vitro from SD rats.The morphology of osteoblasts were observed under invert microscope.The osteoblasts were identified by alizarin red staining and alkaline phosphatase(ALP)staining.Different concentrations of dexamethasone(0 mol/L,10-8 mol/L,10-6 mol/L,10-4mol/L)were used in the primary osteoblasts of SD rats for 24 hours.The nucleus morphology was observed by DAPI staining.The apoptosis rate of osteoblasts was detected by flow cytometry.The morphology of nuclei and mitochondria was observed by transmission electron microscopy,and the mitochondrial transmembrane potential was detected by JC-1 fluorescence probe.Western blot examined the expression of Bax in each group.Analysis of experimental data by One-Way ANOVA.Results1.After 10-8 mol/L,10-6 mol/L,and 10-4 mol/L dexamethasone intervention for 24 hours,DAPI staining showed that the osteoblasts of 10-8mol/L dexamethasone group were rarely apoptotic,and the apoptosis of osteoblasts in 10-6 mol/L and 10-4 mol/L dexamethasone group was obvious,the higher the concentration of dexamethasone,the more obvious the apoptotic phenomena,such as nuclear condensation and nuclear cracking.2.After different concentrations of dexamethasone intervention on osteoblasts for 24 hours,the apoptotic rate was detected by flow cytometry.Compared with the blank control group,the apoptotic rate of osteoblasts in 10’6 mol/L and 10-4 mol/L dexamethasone groups increased significantly(P<0.05).However,the apoptosis rate of 10-8 mol/L dexamethasone group was not significantly changed(P>0.05).Compared with 10-8 mol/L dexamethasone group,the apoptotic rate of osteoblasts in 10-6 mol/L and 10-4 mol/L dexamethasone group was significantly higher(P<0.05).Compared with 10-6 mol/L dexamethasone group,the apoptotic rate of osteoblasts in 10-4 mol/L dexamethasone group was significantly higher(P<0.05).These results suggest that dexamethasone can induce osteoblast apoptosis in a concentration dependent manner.3.After different concentrations of dexamethasone intervention on osteoblasts for 24 hours,the transmission electron microscopy showed that the nuclei of osteoblasts were regular and the structures of mitochondrion in the cytoplasm were intact in the blank control group.And the nuclei of osteoblasts were more regular and the structures of mitochondrion were more complete in 10-8 mol/L dexamethasone group.In 10-6 mol/L dexamethasone group,the nuclei of osteoblasts appeared nuclear pyknosis and the number of mitochondria in the cytoplasm was significantly reduced.However,in 10-6mol/L dexamethasone group,the nuclei of osteoblasts pyknoses were obvious,the size of mitochondrion was different,the swelling and vacuolar changes of mitochondria increased.These results suggest that the apoptosis of osteoblasts induced by dexamethasone may be related to the structural changes of mitochondria.These results suggest that dexamethasone can induce nuclear pyknosis and mitoehondrial decrease in rat osteoblasts,leading to apoptosis.With the inerease of dexamethasone concentration,the effect of dexamethasone on osteoblasts apoptosis is more obvious.4.After different concentrations of dexamethasone intervention on osteoblasts for 24 hours,and JC-1 detected mitochondrial membrane potential of osteoblasts.Compared with the blank control group,the proportion of cells with decreased mitochondrial membrane potential in 10-6 mol/L and 10-4 mol/L dexamethasone group was significantly increased(P.<0.05),however,there was no significant change in the proportion of cells with decreased mitochondrial membrane potential in 10-8 mol/L dexamethasone group(P>0.05).Compared with the 10-8 mol/L dexamethasone group,the proportion of cells with decreased mitochondrial membrane potential was significantly increased in the 10-6 mol/L and 10-4 mol/L dexamethasone groups(P<0.05).Compared with the 10-6 mol/L dexamethasone group,the proportion of cells with decreased mitochondrial membrane potential increased in the 10-4 mol/L dexamethasone group(P<0.05).It was suggested that dexamethasone could induce a dose-dependent increase in the proportion of cells with decreased mitochondrial membrane potential in rat osteoblasts.5.After different concentrations of dexamethasone intervention on osteoblasts for 24 hours,and Western blotting results showed that the expression of Bax in osteoblasts of 10-8 mol/L dexamethasone was not significantly increased(P>0.05),while the expression of Bax in osteoblasts of 10-6 mol/L and 10-4 mol/L dexamethasone was significantly increased(P<0.05).Compared with 10-8 mol/L dexamethasone group,the expression of Bax protein in osteoblasts of 10-6 mol/L and 10-4 mol/L dexamethasone group increased significantly(P<0.05).Compared with 10-6 mol/L dexamethasone group,the expression of Bax protein in osteoblasts of 10-4 mol/L dexamethasone group increased significantly(P<0.05).It is suggested that dexamethasone ean induce osteoblast apoptosis by increasing Bax protein expression in a concentration dependent manner.Conclusion 10-6mol/L and 10-4 mol/L dexamethasone can induce apoptosis of rat osteoblasts,and its mechanism may be related to mitochondrial pathway.