| ObjectiveThis experiment was conducted to investigate the role of PINK1/Parkinmediated mitochondrial autophagy and its mechanism in an in vitro oxidative stress model of noise-induced hearing damage by observing the changes of mitochondrial membrane potential,cell survival and detecting the changes of autophagy-related proteins and m RNA in HEI-OC1 cells under the contradictory effects of autophagy inhibitors and autophagy activators,and to provide new ideas for the clinical prevention and treatment of noise-induced deafness.MethodsHEI-OC1 cells were divided into control group,autophagy activator group and autophagy inhibitor group.The control group was treated with 0.8 m M H2O2 medium for 1h in the incubator and the rest two groups were treated with0.8 m M H2O2 for 1h.After 24 h,the samples were collected.CCK-8 was used to detect the viability of HEI-OCI cells;the changes of mitochondrial membrane potential in HEI-OC1 cells were detected with the Mitochondrial Membrane Potential Assay Kit;the changes of PINK1,Parkin,DRP1,MFN1 and MFN1 in HEI-OC1 cells were detected by quantitative reverse transcription polymerase chain reaction(q RT-PCR)and western blot.DRP1,MFN1,LC3,SQSTM1 m RNA and protein expression in HEI-OC1 cells by real-time fluorescence quantitative reverse transcription polymerase chain reaction and protein immunoblotting(Western Blot).Results1.HEI-OC1 cells were treated with different concentrations of H2O2 and time points we found that at the time point of 0.8 m M H2O2 1h,HEI-OC1 cell viability was about 50%,and we used this treatment condition as the treatment standard for the in vitro oxidative stress model of HEI-OC1 cells.2.The CCK-8 results showed that HEI-OC1 cell viability was close to 50%in the control group.In the autophagy activator group,HEI-OC1 cell viability was significantly higher than that of the control group(P < 0.05,);In the autophagy inhibitor group,HEI-OC1 cell viability was significantly lower than that of the control group(P < 0.05);HEI-OC1 cell viability was significantly higher in the autophagy activator group compared with the autophagy inhibitor group(P< 0.01).3.JC-1 mitochondrial membrane potential assay showed enhanced green fluorescence and diminished red fluorescence in HEI-OC1 cells in the autophagy activator group,similar to the positive control group.The green fluorescence was diminished and the red fluorescence was enhanced in HEI-OC1 cells in the autophagy inhibitor group,similar to the control group.4.PINK1,Parkin,DRP1,MFN1,LC3,SQSTM1 protein expression levels in HEI-OC1 cells: Compared with the control group,PINK1,Parkin,DRP1,LC3,SQSTM1 protein levels were upregulated and MFN1 protein levels were downregulated in the autophagy activator group(P < 0.05,P<0.01);Compared with the autophagy activator group PINK1,Parkin,DRP1,LC3,SQSTM1 protein levels were down-regulated and MFN1 protein levels were up-regulated in the autophagy inhibitor group(P < 0.05,P<0.01).5.PINK1,Parkin,DRP1,MFN1,LC3,SQSTM1 m RNA expression levels in HEI-OC1 cells: Compared with the control group,the relative expression of PINK1,Parkin,DRP1,LC3,SQSTM1 m RNA was up-regulated and that of MFN1 m RNA was down-regulated in the autophagy activator group(P<0.05).Compared with the autophagy activator group,the relative expression of PINK1,Parkin,DRP1,LC3,SQSTM1 m RNA was downregulated and the relative expression of MFN1 m RNA was upregulated in the autophagy inhibitor group(P < 0.01).Conclusion1.The ability of HEI-OC1 cells to activate autophagy in an in vitro oxidative stress model.2.By promoting PINK1/Parkin signaling pathway-mediated mitochondrial autophagy,it is sufficient to reduce mitochondrial damage,maintain mitochondrial quality,and sustain normal cellular physiological functions.It also alleviates cellular damage through multiple pathways such as regulating the expression of various mitochondria-related proteins.3.Inhibition of mitochondrial autophagy can reduce PINK1/Parkin-mediated regulation of mitochondrial autophagy,resulting in cellular damage. |
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