Effects Of Manganese On BNIP3-mediated Mitochondrial Dynamic In SH-SY5Y Cells

Objective:To investigate the dysfuction mechanism of manganese on SH-SY5Y cells and the effect of manganese on neurons from the perspective of mitochondrial dynamic balance,and the regulatory role of BNIP3 in this process.In order to further clarify the mechanism of manganese neurotoxicity and to provide a scientific basis for the prevention and treatment of manganese poisoning.Methods:①Human neuroblastoma SH-SY5Y cells were used as the model of dopaminergic neurons in vitro.A cell model of silencing BNIP3 gene and overexpression of BNMP3 gene was established.② The effects of manganese on the viability and growth and proliferation of SH-SY5Y cells were analyzed by CCK-8 kit.③The effect of manganese on dopamine secretion in SH-SY5Y cells was detected by dopamine enzyme-linked immunosorbent assay kit.④Western blot assay was used to detect the effects of manganese on mitochondrial fusion proteins Mfn1 and Mfn2,mitochondrial fission protein Drp1 and autophagy labeled protein LC3 in SH-SY5Y cells.⑤The effects of manganese on the expression of BNIP3 protein,mitochondrial fusion protein Mfn1,Mfn2,mitochondrial fission protein Drpl and autophagy labeled protein LC3 in SH-SY5Y cells were detected by immunocytochemistry staining.Results:①The results of CCK-8 showed that the survival rate of cells in manganese exposed groups was significantly lower than that in control group(P<0 05),and showed a dose-time dependent effect.②By ELISA assay,dopamine secretion was decreased after manganese intervention.The secretion of dopamine increased after silencing BNIP3.③Western blot analysis showed that the expression of Mfnl,Mfn2,LC3-Ⅱ protein increased and the expression of Drp1 protein decreased after manganese intervention.After silencing BNIP3 gene,the expression of Mfnl,Mfn2,LC3-Ⅱ protein decreased and the expression of Drp1 protein increased.After overexpression of BNIP3 gene,the expression of Mfnl,Mfn2,LC3-Ⅱ protein increased and the expression of Drpl protein decreased.④Immunocytochemistry staining showed that BNIP3,Mfnl,Mfn2,LC3,Drp1 protein was expressed in cytoplasm.After manganese intervention,the expression of BNIP3,Mfnl,Mfn2,LC3 protein increased while the expression of DrpD protein decreased.After silencing the BNIP3 gene,the expression of Mfn1,Mfn2,LC3-Ⅱ protein decreased and the expression of Drp1 protein increased.After overexpression of BNIP3 gene,the expression of Mfn1,Mfn2,LC3 protein increased and the expression of Drp1 protein decreased.Conclusion:①Manganese can induce excessive mitophagy in SH-SY5Y cells,in a dose-time dependent manner,resulting in neuronal dysfunction and apoptosis.②BNMP3 can regulate manganese-induced mitophagy,promote mitochondrial fusion,,reduce mitochondrial division and cause mitophagy.③The silencing of BNIP3 gene can effectively reduce the mitophagy.④BNIP3 is involved in the regulation of mitophagy and is related to the pathogenesis of manganese-induced Parkinson’s disease.