Role Of Mitochondrial Dynamics In Methamphetamine-induced Injury Of SH-SY5Y Cells Cultured In Vitro

Objective:1.To evaluate the proliferative capacity,mitochondrial membrane potential(MMP),mitochondrial ultrastructure and related proteins of human neuroblastoma cells(SH-SY5Y cells)cultured in vitro by methamphetamine(METH)-induced.To investigate the role of mitochondrial protein 1(Fisson 1,Fisl)in METH-induced injury of SH-SY5Y cells cultured in vitro.To elucidate the neurotoxic mechanism of METH-induced SH-SY5Y cells and provide scientific evidence for the prevention and treatment of METH neurotoxicity.Methods:1.To observe the effect of METH on the proliferative capacity,MMP,mitochondrial ultrastructure and related proteins of SH-SY5Y cells cultured in vitro:SH-SY5Y cells were cultured in vitro and establishment of METH-induced SH-SY5Y model cell cultured in vitro;The effect of METH on the proliferative capacity of SH-SY5Y cells was analyzed using CCK-8 cytotoxic proliferation assay;The effect of METH on the MMP level of SH-SY5Y cells was detected by mitochondrial membrane potential detection kit(JC-1);The effect of METH on mitochondrial ultrastructure of SH-SY5Y cells by transmission electron microscopy;Western blot analysis of the effect of METH on the expression of mitoch-ondrial fusion protein 1(Mitonus 1,Mfhl)and Fisl protein in SH-SY5Y cells.2.To investigate the role of Fisl in METH-induced injury of SH-SY5Y cells cultured in vitro:The pre-synthesized siRNA-NC and siRNA-Fisl were transfected into SH-SY5Y cells by liposome-mediated gene transfection;The SH-SY5Y cells were divided into unsilent groups(METH),silent negative groups(siRNA-NC+METH)and silent groups(siRNA-Fis1+METH),0.0,1.0 and 2.0 mmol·L-1 METH-induced SH-SY5Y cells cultured in vitro for 24h;The proliferative capacity of SH-SY5Y cells in each group were analyzed CCK-8 cytotoxic proliferation assay;The other methods are the same as above.Results:1.Successful construction of METH-induced SH-SY5Y cell model cultured in vitro.METH induced SH-SY5Y cells for 3,6,12,24 h,compared with the control group,the survival rate of SH-SY5Y cells in METH treatment group decreased(P<0.05).MMP levels in SH-SY5Y cells of METH treatment group showed a significant decrease(P<0.05);METH induced SH-SY5Y cells for 24 h,the mitochondria of the control group showed a rod-like bilayer membrane structure,and the mitochondrial cristae were normal and clear.The mitochondrial rod-like double-layer membrane structure of the METH-induced group was divided into small globular structures,and the mitochondrial inner and outer membranes were damaged,the mitochondria cristae were distorted and reduced or disappeared in part,partially vacuolated,and mitochondrial autophagosomes and autophagosomes were found.Compared with the control group,the mitochondrial small globular structure increased and the mitochondrial division level in the METH-induced group was increased(P<0.0167).METH induced SH-SY5Y cells for 3,6,12,24 h,compared with the control group,the expression of Mfnl protein was decreased(P<0.05)and the expression of Fisl protein was increased(P<0.05)in the METH-induced group.2.After testing,the siRNA transfection efficiency reached 75%;In the unsilent group,silent negative group and silent group,in each group compared with the control group(0.0 mmol·L-1 METH),the expression level of Fis1 protein was increased(P<0.05),the proliferative capacity was weakened(P<0.05)and the MMP level was decreased(P<0.05)in SH-SY5Y cells of each METH-induced group;Compared with the unsilent group and the silent negative group,the expression level of Fisl protein was decreased in the silent group(P<0.05),the proliferative capacity in the silent group(2.0 mmol·L-1 METH)was increased(P<0.05),and the MMP level in the silent group was increased(P<0.05).Under transmission electron microscopy,compared with the control group,the mitochondrial small globular structure increased and the mitochondrial division level increased in the unsilent group,the silencing negative group and the silent group(P<0.01).Conclusion:METH can induce the proliferative capacity of SH-SY5Y cells cultured in vitro,decrease MMP,mitochondrial ultrastructural changes,mitochondria tend to divide,initiate mitochondrial autophagy,and find abnormal expression of Mfnl and Fisl proteins.These mitochondrial morphological and functional changes may be related to Mfnl and Fisl regulated mitochondrial dynamics.Silencing Fisl gene expression may reduce the mitochondri-al division of METH-induced SH-SY5Y cells cultured in vitro and stabilize MMP,inhibit cell damage,and partially restore the proliferative capacity of SH-SY5Y cells.Mitochondrial dysfunction may be one of the important mechanisms of METH-induced neurotoxicity of SH-SY5Y cells,Fisl may play a key role in METH-induced mitochondrial morphology and dysfunction in SH-SY5Y cells.