Effects Of Methamphetamine On Apoptosis And Parkin-mediated Mitophagy In SH-SY5Y Cells

Objective:To observe the effects of methamphetamine on the morphology,apoptosis level,mitochondrial ultrastructure and reactive oxygen species production of SH-SY5Y cells in vitro,the level of PINK1/Parkin-mediated mitochondrial autophagy and track the process of autophagy flux,to explore the mechanism of METH-induced injury of SH-SY5Y cells in vitro,and to provide scientific basis for the prevention and treatment of METH-induced neurotoxicity.Methods:The experimental model of SH-SY5Y cells induced by METH in vitro was established and divided into control group,1.0mmol.L^-1,1.5mmol.L^-1and 2.0mmol.L^-1METH groups.After 24 hours of treatment,the level of apoptosis and production of reactive oxygen species in SH-SY5Y cells were detected by flow cytometry,the ultrastructure and mitochondrial autophagy of SH-SY5Y cells were observed by transmission electron microscope,and the number of mitochondria were counted.The activity of lysosome phagocytosis of mitochondria in SH-SY5Y cells was observed by laser confocal microscope.The expression of autophagy regulatory protein PINK1 and Parkin,the ratio of autophagy marker protein LC3-II/LC3-I and the expression level of specific autophagy receptor protein P62 were detected by western boltting.Results:Compared with the control group,the apoptosis rate of SH-SY5Y cells increased by 42%,88%and 140%with the increase of METH concentration,and positively correlated with the concentration of METH.The level of real-time ROS in SH-SY5Y cells increased,and the average fluorescence intensity of DCFH-DA increased by 99%,178%and 239%,respectively,compared with the control group.Under transmission electron microscope,the endoplasmic reticulum and golgi apparatus of SH-SY5Y cells treated with METH were dilated,the mitochondrial crest was swollen and broken,the morphology changed from normal rod shape to small ball shape,and autophagy bodies appeared in part of the visual field.The number of mitochondria in the high concentration treatment group decreased by 51.8%compared with the control group（P<0.01）.Under laser confocal microscope,the co-localization area of mitochondrial lysosomes in SH-SY5Y cells treated with METH increased by 13.1%,34.2%,55.2%,respectively,compared with the control group,and the activity of lysosome phagocytosis of mitochondria increased with the increase of treatment concentration.With the increase of METH concentration,the expression of autophagy regulatory protein Parkin protein in cultured SH-SY5Y cells increased by 81.5%,111.1%and 190.7%respectively compared with the control group（P<0.05）.The expression of PINK1 protein had no significant change.The ratio of autophagy marker protein LC3-II/LC3-I increased with the increase of METH concentration,which increased by 40.8%,121.4%and202.3%respectively compared with the control group（P<0.05）.Compared with the control group,the expression of p62 protein decreased by 37%,62.9%and 81.4%,respectively（P<0.05）.After 48 hours of culture,compared with the control group,the expression of P62 protein decreased by 37%,62.9%and 81.4%respectively（P<0.05）.Conclusion:METH can induce apoptosis and destroy the ultrastructure of SH-SY5Y cells in vitro,especially the membrane organelles such as mitochondria.METH-induced cytotoxicity of SH-SY5Y cells in vitro may be related to METH-induced hyperoxidative stress and mitochondrial dysfunction,and can mediate mitochondrial autophagy（to clear damaged mitochondria）by inducing Parkin overexpression.In this experiment,the autophagy flux is smooth,the feedback of autophagy is delayed,and the change of mitochondrial autophagy may be related to apoptosis.