Involvement Of PINK1/Parkin Mediated Mitophagy In Zno Nanoparticle-induced Toxicity In BV-2 Cells

BackgroundZinc oxide nanoparticles(ZnO NPs)has been widely used in dentistry.Recent studies have confirmed that ZnO NPs can enter the CNS through BBB or neurotransmitter after respiratory tract inhalation,digestive tract absorption,skin contact,drug injection,implant release,etc.,leading to neurotoxic effects,such as change neurotransmitter levels,neuronal cell apoptosis,reduced number of neuron which finally caused the damage to learning and memory ability in animals.Even the specific mechanism is uncertain at present,mitochondrial damage caused by nano-zinc oxide is widely accepted as early pathological phenomena.Mitophagy is a selective way to remove excess or damaged mitochondria,which plays an important role in regulating mitochondrial quantity and maintaining normal function of mitochondria.So,whether the mitophagy is involved in toxicity induced by ZnO NPs?At present,there are no relevant research to explain it.ObjectiveTo construct the PINK 1 gene silencing BV-2 cell model through RNA interference,then compare the excitation of mitophagy the changes of mitochondrial membrane potential and the expression of mitophagy related protein after ZnO NPs treatment.To clarify the role of PINK1/Parkin mediated mitophagy in the defensing ability of BV-2 cells against ZnO NPs.Materials and methodsTEM,DLS analyzer,Zeta potential meter,BET surface area measuring instrument and X-ray energy dispersive spectrometer(EDS)were used to determin the characterization of Zinc oxide nanoparticles;the PINK1 gene of BV-2 cells was silenced by RNA interference technique.The BV-2 cells were cultured with different concentrations of ZnO NPs suspension.Then,CCK8 cell proliferation assay was used to find suitable treatment concentration of ZnO NPs in the following experiment.Then both PINK1 silenced BV-2 cell clones and wildtype BV-2 cells were divided into five groups according to the duration of treatment:ie control group,4 h group,8 h group,12 h group and 24 h group.Then,CCK8 cell proliferation assay,ROS intracellular reactive oxygen species detection and JC-1 mitochondrial membrane potential assay were used to observe the pathological changes of BV-2 cells.The expression levels of autophagy-related proteins,such as LC3,P62,Beclinl,total Parkin,cyto-parkin,mito-parkin and total PINK1 were measured by Western Blot respectively.TEM,Immunofluorescence and GFP-LC3 plasmid transfection were used to determin the damaged mitochondria and autophagysome.Results1.The shape of ZnO NPs was hexagonal prism,the original particle were about 50nm.Nano-zinc oxide particles has a certain agglomeration in the deionized water solution,the average hydration particle size was about 500 nm or so.The potential of the ZnO NPs in the aqueous solution was 32.9 mV,with a positive charge.The results of XRD showed that the pattern of ZnO NPs was consistent with that of standard control.2.Compared with the control group,the cell viability of was decreased to 50%and 80%in 20 μg/mL and 25 μg/mL ZnO NPs treated group,respectively,there was no significant difference in the cell viability in the concentrations below 10μg/mL(P>0.05).3.The mitochondrial membrane potential was decreased after BV-2 cells treated with 10 μg/mL ZnO NPs for 4 h,8 h,12 h and 24 h,the difference was statistically significant(P<0.01).The fluorescence intensity of MDC increased,and the difference was statistically significant(P<0.01).4.After BV-2 cells were treated with ZnO NPs for 4h,8h,12h,24h.Cellular immunofluorescence showed that the number of intracellular LC3Ⅱ spots increased in different degrees(P<0.05).Western Blot showed that the ratio of LC3Ⅱ/LC3Ⅰ was increased,the expression of Beclin 1 was upregulated and the expression of P62 was downregulated,all these changes were statistically significant(P<0.05)compared with the control group.The results of Western Blot showed that there was no significant difference in the total Parkin level(P>0.05),but the expression of Parkin in the cytoplasm was decreased at 8 h,12 h and 24 h after treated with ZnO NPs(P<0.05,P<0.01,P<0.01).The expression of PINK1 was significantly increased in all groups(P<0.05).5.The growth curves of wild type BV-2 cells,BV-2 transfected with empty vectors and BV-2 transfected with PINK1 siRNA were compared by CCK8.The results showed that the growth curves of the three types of cells were basically the same.The cell viability of BV-2 cells transfected with PINK1 was lower than that of wild-type BV-2 cells after the same ZnO NPs treatment(P<0.05),and the difference was statistically significant(P<0.01).6.The results of JC-1 showed that the mitochondrial membrane potential of PINK 1 silenced cells decreased more significantly than that of wild type cells after ZnO NPs treatment,the difference was statistically significant(P<0.05).Western Blot showed that mitochondrial Parkin in PINK1 silenced BV-2 cells content was significantly lower than that in wild-type BV-2 cells(P<0.05),while Parkin level in cytoplasm was significantly higher than that in wild-type BV-2 cells(P<0.05).Conclusion1.ZnO NPs can cause oxidative stress in BV-2 cells,leading to mitochondrial damage and increased intracellular autophagy.2.ZnO NPs can induce the translocation of Parkin from cytoplasm to mitochondria in BV-2 cells,which clearly demonstrate that zinc oxide can induce mitophagy in BV-2 cells..3.PINK1/Parkin pathway plays a key role in the regulation of mitophagy induced by ZnO NPs.Mitophagy is one of the important protective mechanisms of BV-2 cells against the toxicity of ZnO NPs.