Effect And Mechanism Of PINK1/Parkin Mediated Mitophagy On Oxidative Stress Induced Senescence In Human Nucleus Pulposus Cells

Objective To investigate the regulatory effect of PINK1 on oxidative stress induced senescence in human NPCs and its relative mechanisms.Methods The nucleus pulposus tissues obtained from LVF and DDD patients were collected,and in situ PINK1 expression of the two groups was detected by immunohistochemistry.The NPCs were isolated and cultured from the nucleus pulposus tissues.Cells at passage II were used in subsequent experiments.Then,intracellular production of ROS was evaluated by DCFH-DA.Senescent rate of NPCs was detected by SA-?-Gal staining.Relatively normal NPCs were obtained from nucleus pulposus samples of patients with LVF.Then NPCs at passage II were subsequently incubated with 150?M H₂O₂ for 1 h to induce sublethal oxidative stress.The cell cycle and cell apoptosis were detected by flow cytometer.The mitochondria membrane potential was observed by confocal microscopy through JC-1 staining.The autophagosomes were detected by TEM and the mitophagic protein?PINK1?Parkin?LC3,and P62?expression levels were analyzed by Western blot.For depletion of PINK1 in NPCs,PINK1-shRNA was transfected into NPCs by using recombinant lentiviral vector.After transfected with PINK1-shRNA,NPCs were subsequently incubated with or without 150?M H₂O₂ for 1h to induce oxidative stress.Then,NPCs were randomly divided into control group,H₂O₂ group,PINK1-shRNA group and PINK1-shRNA+H₂O₂ group.Then the expression levels of senescence-related and mitophagic biomarkers were analyzed by Western blot.Results Immunohistochemistry of human NP tissues showed that PINK1 were expressed in all tissues.Compared with the LVF group,the DDD group showed an increased number of PINK1-positive cells?P<0.05?.Intracellular ROS production and percentage of SA-?-Gal-positive cells of DDD group were significantly higher than LVF group?P<0.05?.Flow cytology results showed that administration of 150?M H₂O₂ did not cause increased levels of the early-and late-stage apoptosis in the NPCs?P>0.05?.However,the H₂O₂-induced oxidative stress did increase the population of G1 phase cells and the percentage SA-?-Gal-positive cells?blue?,and staining intensity?P<0.05?.After treated with H₂O₂,the green/red fluorescence ratio of NPCs markedly increased,suggesting mitochondria membrane potential reduced by the treatment of H₂O₂?P<0.05?.TEM observation found increased number of autophagosomes and shrink mitochondrion in NPCs under H₂O₂ treatment.Western blot analysis also revealed that the expression levels of mitophagic proteins were significantly increased in H₂O₂ group?P<0.05?.And the level of PINK1protein expression was reduced after treatment of PINK1-shRNA.Simultaneously,the ratio of LC-3 II/I decreased and expression of Parkin,P62 increased markedly in NPCs with PINK1-shRNA transfection?P<0.05?.The results of Western blot analysis revealed that NPCs suffered PINK1-shRNA transfection accompanying with H₂O₂ treatment had the highest expression level of senescence-related proteins?P<0.05?.However,down-regulating PINK1 alone didn't increase the expression of senescence-related proteins comparing with the control group?P>0.05?.Conclusion PINK1 protects against oxidative stress induced senescence of human NPCs via regulating PINK1/Parkin mediated mitophagy,and these findings may provide a better understanding in pathomechanism of IDD and potential therapeutic approaches for DDD treatment.