Experimental Study Of ATG5 Regulating Osteoblast Apoptosis Through Endoplasmic Reticulum Stress In Steroid-induced Osteonecrosis Of The Femoral Head

Objective:Programmed osteocyte death may be one of the pathogenesis of steroid-induced avascular necrosis of the femoral head(SANFH).The current research results show that autophagy-related gene 5(ATG5)may be involved in the regulation of osteoblast apoptosis through the activation of unfolded protein response(UPR)pathway under endoplasmic reticulum stress(ERS),but the exact regulatory mechanism is not clear.The purpose of this study is to explore the role of ERS in the pathogenesis of SANFH by studying the mechanism of ATG5 involved in regulating osteoblast apoptosis,and to provide a theoretical basis for the development of new therapeutic targets for SANFH.Method:Firstly,rat bone marrow mesenchymal stem cells(BMSCs)were extracted and cultured in vitro,and then stained with alizarin red after osteogenic induction.Osteoblasts were inoculated into 24-well cell culture plate and divided into three groups:D1 group blank control group(Control),D2 group added methylprednisolone(SANFH),D3 group osteoblasts transfected with siRNA-ATG5 followed by methylprednisolone(SANFH+siRNA-ATG5).Firstly,the changes of mRNA and protein expression of ATG5,PERK,Caspase-3,Caspase-9 and Caspase-12 in osteoblasts were detected by rT-PCR,Western Blot and immunofluorescence staining,the survival rate of osteoblasts was measured by CCK-8 method,and osteoblast apoptosis was detected by flow cytometry.Through the above methods,the relationship between the changes of mRNA and protein expression of ATG5,PERK,Caspase-3,Caspase-9,Caspase-12 at different time points and osteoblast apoptosis was analyzed under the condition of hormone and siRNA-ATG5+hormone transfection.Finally,statistical methods were used to analyze whether there were statistical differences between D2,D3 and D1 groups at each time point.Results:(1)The results of rT-PCR and WesternBlot showed that compared with D1 group,the mRNA and protein expression of ATG5,PERK,Caspase-3,Caspase-9 and Caspase-12 were significantly increased in D2 group at different time points compared with D1 group.Compared with the D2 group,the mRNA and protein expression of ATG5,PERK,Caspase-3,Caspase-9 and Caspase-12 decreased significantly at different time points in the D3 group which was transfected with siRNA-ATG5 osteoblasts with a certain concentration of methylprednisolone.(2)The results of immunofluorescence staining showed that the fluorescence intensity of ATG5,PERK,Caspase-3,Caspase-9 and Caspase-12 in D2 group was significantly higher than that in D2 group,while the fluorescence intensity or expression of ATG5,PERK,Caspase-3,Caspase-9 and Caspase-12 in D3 group was significantly lower than that in D2 group.(3)the results of CCK-8 experiment showed that the survival rate of osteoblasts in D2 group and D3 group decreased significantly with the passage of time after treatment with certain concentration of methylprednisolone for 12,24,48 and 72 hours,suggesting that the proliferation of osteoblasts was inhibited with the prolongation of hormone treatment.(4)the results of flow cytometry showed that under the action of certain concentration of methylprednisolone,the apoptosis proportion of osteocytes in D2 group and D3 group was significantly higher than that in D1 group,and the apoptosis ratio in D2 group was higher than that in D3 group.Conclusions:(1)In the simulated in vitro state of steroid-induced avascular necrosis of the femoral head,ATG5 can activate UPR pathway to regulate apoptosis through ERS pathway.(2)Under the continuous action of certain concentration of hormone,early activation of Caspase-8 endogenous apoptosis alleviated the disorder of internal environment and promoted cell survival,and then activated UPR pathway caused Caspase-12/9/3 cascade reaction to aggravate cell apoptosis until cell death.(3)Under the continuous action of methylprednisolone,the 0-72h UPR pathway and Caspase-12-9-3 cascade of osteoblasts transfected with siRNA-ATG5 were significantly weakened,which delayed the progress of apoptosis and improved the survival rate of osteoblasts in hormone environment.