Research On The Effects Of Programmed Necrosis On The Process Of Oxidative Stress-induced Nucleus Pulposus Cell Death And The Correlated Mechanisms

Part ⅠResearch on programmed necrosis involved in the process of hydrogen peroxide-induced rat nucleus pulposus cells deathObjective:To investigate whether programmed necrosis is involved in rat nucleus pulposus(NP)cells death under oxidative stress and to explore its possible mechanisms.Methods:The second-generation rat NP cells from thoracic and lumbar intervertebral discs were subjected to different concentrations of H2O2 for various time periods.Cell viability was evaluated by cell counting kit-8(CCK-8),and then the appropriate concentration and time were selected for subsequent experiments.The morphological changes of the cells were observed by inverted phase-contrast optical microscopy.The PI positive rate were detected by flow cytometry(FCM)after Annexin V/propidium dide(PI)staining and laser scanning confocal microscope(LSM)after Hoechst 33258/PI staining.Ultrastructural changes were examined by transmission electron microscopy(TEM).The gene and protein expression changes of RIP1 and RIP3 were measured through RT-PCR and Western Blot.In addition,necroptosis inhibitor Nec-1 and gene silencing were used to further evaluate the effects of necroptosis on cell viability and mortality.Immunohistochemistry was used to detect differences in RIP1 and RIP3 protein expression in normal and degenerated intervertebral discs.Results:H2O2 reduced the viability of NP cells in dose and time dependent manner.With the rise of H2O2 concentration,the number of dead cells increased obviously,PI positive rate increased gradually,and the ultra structure of cells showed necrosis-like changes.The gene and protein expression of RIP1 and RIP3 were significantly upregulated.Nec-1 pretreatment could effectively inhibit H2O2-induced decrease in cell viability,increase in cell death,elevation in PI positive rate,and necrosis-like ultras true tural changes.Transfection of SiRNA-RIP3 reduced H2O2-induced cell death,whereas SiRNA-RIP1 had the opposite effect.The results of immunohistochemistry in human specimens confirmed that the expression levels of RIP1 and RIP3 in the degenerated NP were significantly higher than those in the normal NP tissue.Conclusion:RIP1/RIP3 mediated programmed necrosis is involved in NP cell death under oxidative stress.The effect of RIP1 may be two-sided.Moderate expression may promote cell survival,and overexpression may facilitate the occurrence of programmed necrosis.Regulating programmed necrosis is expected to reduce the oxidative stress-induced nucleus pulposus cell death,and then provide a new strategy for the prevention and treatment of disc degeneration.Part IIMechanism of programmed necrosis involved in oxidative stress-induced rat nucleus pulposus cells deathObjective:To explore the relationship between RIP 1/RIP3-cediated necroptosis and PARP-AIF signaling pathway in NP cells.Methods:The second-generation rat NP cells from thoracic and lumbar intervertebral disc were incubated with H2O2(250 μM)for 24 h.After the fluorescent probe JC-1 staining,the mitochondrial membrane potential(MMP)was detected by FCM and LSM.After the fluorescence probe H2DCF-DA staining,the reactive oxygen species(ROS)levels were detected by FCM and LSM.The programmed necrosis-related molecules poly(ADP-ribose)polymerase(PARP),poly(ADP-ribose)(PAR)and apoptosis inducing factor(AIF)in mitochondria and nucleus were determined by Western Blot.AIF translocation was observed by LSM after immunofluorescence staining.CCK-8 was used to detect cell viability.PI staining was used to detect cell death rate.In addition,the effects of programmed necrosis on cell viability and mortality were further evaluated using the RIP1 specific inhibitor Nec-1,PARP-specific inhibitor DPQ,and gene silencing.Results:With the treatment of 250 μM H2O2 for 24 h,the ROS level increased significantly and the mitochondrial membrane potential declined markedly,suggesting that oxidative stress level increased and mitochondrial function was impaired under H2O2 condition.Nec-1 could significantly inhibit H2O2-induced ROS elevation and mitochondrial membrane potential decrease,which indicated that Nec-1 could obviously reduce H2O2-induced oxidative stress and mitochondrial dysfunction.With the increase of H2O2 concentration,the expression of PARP protein gradually decreased,the expression of Cleaved-PARP protein gradually increased;and the activity of PARP enzyme and the expression of PAR protein increased significantly;and mitochondrial AIF protein expression levels gradually decreased,nuclear AIF protein expression levels gradually increased.Nec-1 and SiRNA-RIP3 significantly inhibited PARP activity and protein expression,while SiRNA-RIP1 showed the opposite effects.DPQ significantly inhibited the decrease of mitochondrial AIF and the increase of nuclear AIF.SiRNA-AIF significantly reduced cell death and increased cell viability.Conclusion:The PARP-AIF pathway,as a downstream pathway of RIP1/RIP3,may play a key role in the process of H2O2-induced NP cell programmed necrosis.Regulation of RIP1/RIP3-PARP-AIF signaling pathway is expected to reduce oxidative stress-induced NP cell death,and then provide new strategies and methods for retarding or even reversing intervertebral disc degeneration.