Protective Effects Of PINK1/Parkin Mediated Mitophagy On ZnO Nanoparticles-induced Toxicity In SH-SY5Y Cells

Objective:With the increasing application of ZnO nanoparticles(ZnONPs)in the field of biomedicine,the neurotoxicity caused by these particles has raised serious concerns.In this study,We compared the cytotoxicity differences of three particle sizes of ZnONPs(10nm,50 nm,100 nm)in SH-SY5 Y cell model,then the most toxic 50 nm ZnONPs was selected for subsequent toxicity mechanism research.To clarify the role of autophagy and mitophagy in mechanism of ZnONPs induced cytotoxicity,we used a variety of molecular biology techniques and investigated the link between autophagy and mitophagy with cytotoxicity in ZnONPs-treated SH-SY5 Y cells.Finally,we hope that the results of our study can provide certain theoretical basis for its biological safety research.Methods:1.The cell viability of ZnONPs on SH-SY5 Y cells were detected by CCK-8 assay and LDH assay.Annexin V-FITC/PI double staining were used to detect cell apoptosis.The protein expression of caspase9,caspase3,bcl-2 and bax were examined by Western blot.2.The expression of LC3 protein in SH-SY5 Y cells were observed by Immunofluorescence.The expression levels of autophagy-related proteins such as P62,LC3 and Beclin 1 were measured by Western blot,respectively.The effect of 3-MA and rapamycin on cytotoxicity induced by ZnONPs in SH-SY5 Y cells were detected by CCK-8.The effect of 3-MA and rapamycin on apoptosis induced by ZnONPs in SH-SY5 Y cells were detected by Flow cytometry.3.Some techniques were used to detect effects of ZnONPs on mitochondria in SH-SY5 Y cells,such as NAO staining,fluorescent quantitation PCR,flow cytometry andWestern blot.Mitochondria-containing autophagosomes and autolysosomes were determined by confocal laser microscopy.The expression of PINK1 protein in SH-SY5 Y cells were observed by Immunofluorescence.The expression levels of total PINK1,total Parkin,cyto-parkin and mito-parkin were measured by Western blot.The effects of CsA on cytotoxicity and apoptosis induced by ZnONPs in SH-SY5 Y cells were detected by CCK-8and Flow cytometry.The effects of CsA on mitochondrial damage induced by ZnONPs in SY5 Y cells were determined by NAO staining and Flow cytometry.Results:1.The viability of SH-SY5 Y cells exposed to ZnONPs(10 nm,50 nm,100 nm)demonstrated a significant decreases compared with the control,and 50 nm ZnONPs showed the most obvious toxic which showed a dose-dependent manner.The expression of cleaved caspase3,cleaved caspase9 and bax were up-regulated and bcl-2 was down-regulated in the ZnONPs-treated group compared with control group.Flow cytometry result showed that ZnONPs can induce apoptosis on SH-SY5 Y cell line.2.ZnONPs significantly increased LC3 II/LC3 I ratio and Beclin 1 levels,and decreased P62 expression.The expression of fluorescently labeled LC3 II puncta were increased after treatment with ZnONPs.The combination of autophagy inhibitor 3-MA with ZnONPs significantly lessened autophagy,increased the cytotoxicity and apoptosis caused by ZnONPs.The combination of autophagy agonist rapamycin with ZnONPs rescued the cytotoxicity and apoptosis caused by ZnONPs.3.ZnONPs exposure significantly reduced mitochondrial mass and the expression of COX IV,and up-regulated ROS production levels.Real-time PCR assay showed that ZnONPs markedly reduced the MT-CO2 and MT-CO3 mRNA levels.We stained the mitochondria and lysosomes with Mito-Tracker Green and Lyso-Tracker Red,respectively,and the results showed that ZnONPs increased the colocalization of LC3 puncta with mitochondria and colocalization of mitochondria and autolysosomes.The expression of total PINK1 and mito-parkin were up-regulated and cyto-parkin was down-regulated,meanwhile the total-parkin level was almost unchanged.The combinationof mitophagy inhibitor CsA with ZnONPs significantly lessened mitophagy,increased the cytotoxicity and apoptosis caused by ZnONPs.And the combination of CsA with ZnONPs simultaneously decreased ZnONPs-induced mitochondrial mass and increased ZnONPs-induced intracellular ROS levels.Conclusion:1.The viability of SH-SY5 Y cells exposed to ZnONPs(10 nm,50 nm,100 nm)demonstrated a significant decreases compared with the control,and 50 nm ZnONPs showed the most obvious toxic.ZnONPs can cause SH-SY5 Y cytotoxicity and mitochondrial pathway-mediated apoptosis.2.ZnONPs can induce intracellular autophagy and PINK1/Parkin mediated mitophagy in SH-SY5 Y cells.3.Autophagy and PINK1/Parkin mediated mitophagy are one of the protective mechanisms of SH-SY5 Y cells against the toxicity and mitochondrial damage caused by ZnONPs,suggesting that we can reduce the neurotoxicity of ZnONPs by autophagy agoinst.