Effect On Autophagy To Osteoblast Irradiated By X-ray

Purpose: To examine the effects of X-irradiation on the proliferation,apoptosis and mineralization in MC3T3-E1 mouse osteoblasts,and to elucidate its possible molecular mechanism from the perspective of autophagy.Methods: MC3T3-E1 cells were cultured in vitro and irradiated with a medical linear accelerator at a pulse rate of 6MV/min.The groups were divided into the following cells: blank control group,0.25 Gy group,0.5 Gy group,1 Gy group,2 Gy group and 4 Gy group.The MTT colorimetric assay was was applied to screen the concentration of doxycycline(DOX).The intervention time of DOX autophagy was determined by MDC staining.The concentration of 2.5 nM DOX and the intervention time of 48 h were used for the experiment.Monodansylcadaverine(MDC)staining was used to observe the autophagy after irradiated by X-ray for 72 hours.The cell proliferation was detected by colony formation assay.The alizarin red staining was used to observe the number of mineralized nodules after 21 days of osteogenic induction.Alkaline phosphates(ALP)kit was used to detect mineralization ability.Hoechst 33342 staining and flow cytometry were used to detect apoptosis of osteoblasts and Western Blot was used to detect the protein levels of LC3,Atg5,Beclin-1,Caspase-3,Bcl-2,Bax,p62 and Runx2.Results:MDC staining showed that the fluorescence intensity increased with the increasing of irradiation dose.The results of colony formation assay showed that irradiation inhibited MC3T3-E1 cells proliferation in a dose dependent manner(P<0.05).The data of alizarin red staining and ALP detection showed that the number of mineralized nodules and the ALP activity in the irradiated groups were decreased(P<0.05).Increased nucleus condensation and apoptotic bodies in the irradiated groups after 72 hours was observed by Hochest33342 cell stainning.The results of flow cytometry showed that the apoptosis rate increased after irradiation(P<0.05).The data of western blot showed that the relative expression levels of Beclin-1,Atg5,Lc3 II/Lc3 I and Caspase3 except for 0.25 Gy protein increased significantly in a dose dependent manner(P<0.01).The expression level of p62 and Bcl-2/Bax protein was decreased,the difference was statistically significant(P<0.01).The expression level of Runx2 was significantly reduced except for 0.25 Gy(P <0.01).MDC staining results showed that after added 2.5nM DOX,the fluorescence intensity of each irradiation group was basically disappeared at 48 h.The colony formation assay showed that the cell proliferation activity of DOX treated groups increased(P<0.05).Alizarin red staining and ALP detection showed that the number of mineralized nodules and the ALP activity in the DOX treated groups were increased(P<0.05).Increased nucleus condensation and apoptotic bodies in the DOX treated groups after 48 hours was observed by Hochest33342 cell stainning.The results of flow cytometry showed that the apoptosis rate increased in the DOX treated groups(P<0.05).The data of western blot showed that the relative expression level of Beclin-1,Atg5,Lc3 II/Lc3 I and Bcl-2/Bax except for 0.25 Gy protein were significantly decreased(P<0.05).The protein expression level of p62 was decreased at radiation doses of 1 Gy-4 Gy,the difference was statistically significant(P<0.05).The expression level of Runx2 was significantly increased(P<0.05).The protein expression of Caspase3 was increased at radiation doses of 2 Gy and 4 Gy,and the difference was statistically significant(P<0.05).Conclusion : X-ray can induce apoptosis of MC3T3-E1 osteoblasts in vitro.Inhibite autophagy can aggravate osteoblasts apoptosis.The mechanism may be related to the activation of Caspase-3 protein.X-ray can inhibit the mineralization ability of MC3T3-E1 osteoblasts in vitro.autophagy inhibition can reduce the damage of X-ray mineralization ability of osteoblasts.The mechanism may be related to the up-regulation of ALP activity and the osteogenic gene Runx2.