Effect Of Mitophagy On The Cytotoxicty Of Manganese And Regulatory Mechanism Of BNIP3 In SH-SY5Y Cells

Objectives : To investigate whether manganese can induced mitophagy and the role and regulatory mechanism of BNIP3 on mitophagy in SH-SY5 Y cells.Method:We investigated the effect of MnCl2 on cytotoxicity of SH-SY5 Y cells were treated with different doses and time of Mn Cl2 was detected by CCK-8,and alteration on mitophagy related protein,such as BNIP3,LC3 by western-blotting.Mitophagy was observed by electron microscopy and co-localisition of mitochondria and acidic lysosomes,Further,We explore the role of mitophagy in Mn-induced neurotoxicity by inhibiting the mitophagy by 3-MA(mitophagy inhibitors).Finally,We transfection of SH-SY5 Y cells with BNIP3 SiRNA to reduce the expression of BNIP3 to investigated the regulatory of and the possible mechanism ofBNIP3 on mitphagy by co-immunoprecipitation.Results: CCK-8 detection showed that the survival rate of cells in each manganese group was significantly lower than that of the control group(P < 0.05),which was dose and time-dependently.Western blot showed that the expression of BNIP3,LC3-II/LC3-I were higher than that in control group,on the contrary,the expression of mitochondrial membrane protein TOMM20 was decreased.The difference reached statstical significance(P < 0.05).The TEM analysis demonstrated that the mitochondria in the cells of the Mn group were swollen,deformed,ridge disappearance and vacuolization,display the typical structure of mitophagy.In contrast,the cell structure of the control group was relatively complete,and the typical double membrane mitochondria were seen,autophagosomes,mitophagosomes and autolysosomes rarely.The results of confocal microscopy showed that the co-localization of Mito and Lyso in the Mn group,while the cells in the control group were relatively less.The same experimental method found:After treatment with the inhibitor of mitochondrial autophagy inhibitor 3-MA,which inhibit the occurrence of mitophagy and increase the survival rate of manganese cells.the cells treated with BNIP3 siRNA was reduce the expression of BNIP3 could inhibit the mitophagy,we also found that BNIP3 could interact with LC3 by co-immunoprecipitation,The extent of their interaction was positively correlated with the expression of BNIP3,and was closely related to mitophagy.Conclusion: manganese can induce mitophagy in SH-SY5 Y cells,which lead to excessive mitophagy caused cell death.BNIP3 involved in the regulation of the mitophagy and its possible mechanism is the combination of BNIP3 and LC3 promote mitophagy.This process is associated with the pathogenesis of manganese poisoning Parkinson syndrome.