Research On The Effects Of Mitophagy On The Process Of Compression-induced Nucleus Pulposus Cell Programmed Necrosis And The Correlated Mechanisms

Part Ⅰ Compression induced mitochondrial autophagy in rat nucleus pulposus cellsObjective: To observe whether compression induced mitochondrial autophagy(mitophagy)in rat nucleus pulposus cells.Methods: Lumbar intervertebral discs from Spraguee-Dawley rats(aged 2 months,250-300 g)were harvested immediately after intraperitoneal injection of chloride hydrate anesthesia(350-400 mg/kg).The nucleus pulposus(NP)was extracted with tweezers after rat lumbar discs were horizontally cut open and the second generation was used for subsequent experiments.Rat NP cells were cultured in a pressure apparatus of 1.0 MPa for0,12,24 and 48 h.After mechanical load treatment,intracellular ROS levels were measured using H2DCFH-DA assay(Beyotime,China).JC-1 probe was used to explore the transition of MMP.ATP test kit was used to detect mitochondrial ATP changes.NAO staining was used to detect the change of mitochondrial quality.TOM20 expression was detected by western blot with a anti-TOM20 antibody.Furthmore,the levels of mitophagy was observed by TOM20 and LC3 B staining under laser confocal microscopy.Transmission electron microscopy was used to observe the ultrastructural changes of nucleus pulposus cells under mechanical load.RT-PCR and western blot were used to detect the gene and protein expression of mitophagy related proteins(PINK1,Parkin)in rat nucleus pulposus cells under compression.The changes of PINK1 and Parkin expression in normal intervertebral discs and degenerative intervertebral discs were detected by immunohistochemistry.Results: NP cells subjected to compression showed collapse of MMP,elevation of ROS and decrease of ATP levels.Those results indicates that compression induced NP cell mitochondrial dysfunction.Furthmore,the levels of NAO were decreased in a time-dependent manner in NP cells exposed to compression and the expression of TOM20 showed the same tendency.Those results indicates that compression induced the loss of mitochondrial mass in NP cells under mechanical load.Furthmore,we found that the co-localization of TOM20 and LC3 B increased with the prolongation of compression time.In addition,the mitochondria of the nucleus pulposus cells under compression are encircled by the autophagic structure.Baf-A1,an inhibitor of autophagy,prevented the decrease of mitochondria quality conducted by compression.PCR and western blot showed that the gene and protein expression of mitochondrial autophagy-related proteins(PINK1,Parkin)was significantly increased in NP cells under compression than the control group.Results of immunohistochemistry showed that the expression of PINK1 and Parkin in the degenerative intervertebral discs increased significantly compared with the normal intervertebral discs.Conclusion: Mechanical load induced PINK1/Parkin mediated mitopaghy in NP cells,which may play an important role in the degeneration of intervertebral disc.These findings may provide new insight for the prevention and treatment of intervertebral disc degeneration.Part ⅡExperimental study on the effects of compression-induced mitophagy to programmed necrosis in rat nucleus pulposus cellsObjective: To discuss the effects of compression-induced mitophagy to programmed necrosis in rat nucleus pulposus cellsMethods: Lumbar intervertebral discs from Spraguee-Dawley rats(aged 2 months,250-300 g)were harvested immediately after intraperitoneal injection of chloride hydrate anesthesia(350-400 mg/kg).The nucleus pulposus(NP)was extracted with tweezers after rat lumbar discs were horizontally cut open and the second generation was used for subsequent experiments.We studied the effect of mitophagy on programmed necrosis in compression-treated NP cells by using mitochondrial autophagy inhibitors Mdivi-1,Cs A and Parkin Si RNA.After treatment,the rate of cell necrosis was detected by PI staining with flow cytometry.Furthermore,the release of HMGB1 was determined by western blot and LDH release activity was detected by a LDH Assay Kit.Mitophagy inhibitor Cs A was used to detect the changes of mitochondrial function in NP cells exposed to compression:Intracellular ROS levels were measured using H2DCFH-DA assay.JC-1 probe was used to explore the transition of MMP.ATP test kit was used to detect mitochondrial ATP changes.In addition,the expression AIF in compression-treated NP cells in the presence or absence of Mdivi-1 was measured by western blot and laser confocal microscope.Rat nucleus pulposus cells were treated with 1 MPa for 0 h,12 h,24 h and 48 h.A fluorescence probe Fluo-3AM was used to observe the changes of intracellular calcium levels in nucleus pulposus cells by flow cytometry and laser confocal microscope.The levels of Ca2+ in NP cells under compressive conditions were observed in the presence or absence of extracellular calcium chelating agent EGTA,endoplasmic reticulum stress inhibitor salubrinal,endoplasmic reticulum IP3 R channel inhibitor 2-APB or RYR channel inhibitor dantrolene.Next,BAPTA-AM,a pharmacological calcium inhibitor,was used to inhibit calcium levels in NP cells and then cell necrosis was determined by PI uptake,HMGB1 release assay.Furthermore,cell mitophagy was detected by examining the expression levels of LC3 B and TOM20.Meanwhile,in order to detect the molecular mechanisms of intracellular calcium-mediated mitophagy,western blot was used to detect the effects of BAPTA-AM on the expression of PINK1 and Parkin in nucleus pulposus cells under mechanical load.Results: Compression induced necrosis in NP cells.Mitophagy inhibitor and Parkin Si RNA can significantly reduce the increasing PI uptake in NP cells.Furthermore,inhibition of cell mitophagy attenuated release of LDH and HMGB1 into the extracellular medium.We also found that inhibition of mitophagy can inhibit NP cell mitochondrial dysfunction in the condition of compression.Moreover,mitophagy inhibitor Mdivi-1significantly prevented the redistribution of AIF from mitochondria to nucleus.The levels of intracellular calcium in nucleus pulposus cells under compression were significantly higher than those of the control group.Moreover,salubrinal and 2-APB significantly inhibited the increase of calcium levels in the compression-treated NP cells.Calcium inhibitor BAPTA-AM can inhibit the intracellular calcium levels and reduce the necrosis rate of NP cells conducted by compression.Meanwhile,the levels of mitophagy in NP cells were significantly decreased in compression-treated NP cells in the presence of BATPA-AM.In addition,BAPTA-AM also reduced the expression of mitophagy related proteins(PINK1,Parkin)in NP cells.Conclusion: Mitophagy took part in the programmed necrosis of nucleus pulposus cells exposed to mechanical load.Inhibition of mitophagy can inhibit programmed necrosis of nucleus pulposus cells induced by compression.AIF translocation from mitochondria to nuclear and mitochondrial dysfunction may be involved in mitophagy-mediating NP cell programmed necrosis.Additionally,mechanical load leads to the opening of IP3 R calcium channel and the release of calcium from endoplasmic reticulumto the cytoplasm.Inhibition of intracellular calcium can inhibit the expression of mitophagy related proteins(PINK1and Parkin),thus inhibits mitophagy and slows down cell necrosis.