Effects Of Autophagy On The Function Of Osteoblasts In High Glucose

Objective:To investigate the effects of high glucose and glucose fluctuation on MC3T3-E1 osteoblasts and it's molecular mechanism.Methods:MC3T3-E1 osteoblasts cultured in vitro were divided into control(Ctr)group,high glucose(HG)group,glucose fluctuation(GF)group,N-acetylcysteine(NAC)group and chloroquine(CQ)group.NAC group included the NAC group,the HG+NAC group,and the GF+NAC group;CQ group included the CQ group,the HG+CQ group,and the GF+CQ group.We measured cell proliferation by CCK-8 analysis,the apoptosis rates of cells by flow cytometry,reactive oxygen stress(ROS)levels of cell and mitochondrial by DCFH-DA probe and MitoSOX probe,mitochondrial membrane potential(MMP)by JC-1 dye,protein expressions by WB and IF,and autophagosome formation by transmission electron microscopy.Results:(1)In HG group and GF group,cell proliferation decreased and apoptosis increased compared to the control group,and it was more significant in GF group compared to HG group.Compared with the control group,Bcl-2 was decreased and the expression of Bax,Caspase3,Cleaved caspase3 and Cytochrome c were increased in HG group and GF group,and the results of GF group were more remarkable than HG group.Compared to the control group,the production of ROS and mitochondrial ROS were increased,and mitochondrial membrane potential(??m)was decreased in HG group and GF group,which were more pronounced in GF group.(2)High glucose increased LC3-II,Beclinl and Bnip3,and the formation of autophagosome in MC3T3-E1 osteoblasts,but decreased P62,and it was more obvious in GF group.(3)NAC increased Bcl-2,LC3-II,Beclinl and Bnip3,and decreased the generation of ROS,Bax,Caspase3,Cleaved caspase3 and Cytochrome c and the apoptosis rates.(4)Compared with the control group,p-Akt and p-mTOR were decreased in HG group and GF group,and the decrease in GF group was more significant than that in HG group,but NAC increased expression of p-Akt and p-mTOR.(5)Compared with the control group,high glucose resulted in the increased apoptosis.CQ significantly increased the apoptosis and LC3-II,and decreased Bnip3.Conclusion:(1)High glucose induced decreased proliferation and increased apoptosis of MC3T3-E1 osteoblasts,which in GF group were more significant than in HG group.(2)The effects of high glucose on MC3T3-E1 osteoblasts may be related to oxidative stress,mitochondrial damage and autophagy.