Effect Of ROS-AKT-mTOR Axis On Autophagy Of MC3T3-E1 Cells

Objective: To investigate the effect of ROS-AKT-m TOR axis on autophagy,proliferation and apoptosis of MC3T3-E1 cells.Methods: MC3T3-E1 cells cultured in vitro were divided into the following groups:control group,NAC group,11.0m M glucose group, 11.0m M glucose+NAC group, 22.0m M glucose group, 22.0m M glucose+NAC group,CQ group,22.0m M glucose+CQ group.ROS production was measured with DCFH-DA fluorescent probe.Cell proliferation was measured by MTT analysis.Cells in different groups were stained with Annexin V-FITC/PI,then the apoptosis rate were detected by Flow Cytometry and nucleus morphology was observed under fluorescence microscope after loaded with Honchest33258.Protein expression were measured with western blot and immunofluorescence.Results:(1)Apoptosis of MC3T3-E1 cells in 11.0mmol/L glucose and 22.0mmol/L glucose group increased in a dose-dependent manner dramatically compared to control group(P<0.01).(2)Comparing with control group,proliferation of MC3T3-E1 cells in 11.0mmol/L glucose group slightly decreased(P>0.05),while it decreased significantly in 22.0mmol/L glucose group(P<0.01),and its decrease was in a dose-dependent manner.(3)Expression of LC3B-II and Beclin1 protein of MC3T3-E1 cells in 11.0m M glucose group were significantly increased(P<0.01;P<0.05) and P62 was decreased(P<0.01) compared to control group; When glucose concentration increased to 22.0m M,LC3B-II expression decreased dramatically(P<0.01) and Beclin1 and P62 protein expression increased compared to control group(P<0.01;P>0.05).Comparing with control group,LC3B-II expression of MC3T3-E1 cells in CQ group increased significantly(P<0.05).LC3B-II expression of MC3T3-E1 cells in 22.0mmol/L+CQ group increased dramatically compared to both 22.0mmol/L glucose and CQ group(P<0.01).( 4) ROS production of MC3T3-E1 cells in both 11.0mmol/L and 22.0mmol/L glucose group were increased significantly compared to control group(P<0.01),but not in a dose-dependent manner.NAC,as an antioxidant,could decrease ROS production in MC3T3-E1 cells and then ameliorate cell apoptosis,proliferation abnormity and increase autophagy caused by high glucose.(5)Expression of p-AKT and p-m TOR proteins of MC3T3-E1 cells in the two high glucose groups were siginificantly down-regulated.And the expression of the two proteins could be up-regulated by use of NAC in a statistical significant manner.Conclusion:(1)Apoptosis and proliferation of MC3T3-E1 cells in high glucose group increases and decreases respectively in a dose-dependent manner.(2)Autophagy significantly increases under high glucose circumstance.(3)High glucose increases autophagy and apoptosis of MC3T3-E1 cells and decreases proliferation of these cells through ROS-AKT-m TOR axis.