The Role And Regulatory Mechanism Of Reactive Oxygen Species On Pyroptosis Of Nucleus Pulposus Cells In Human Degenerative Intervertebral Disc

Intervertebral disc degeneration(IDD)is a common cause of low back pain(LBP),which often causes great mental and economic burden to patients and society.The intervertebral disc consists of nucleus pulposus,annulus fibrosus and cartilage endplate.The nucleus pulposus of intervertebral disc has the function of bearing load and cushioning in the process of spinal movement.During IDD,the degenerative changes were first showed in the nucleus pulposus,and the cascade reaction led to the aggravation of the lesions.Nucleus pulposus degeneration is characterized by the decrease of nucleus pulposus cells(NPCs)and extracellular matrix(ECM),and ECM is mainly synthesized by NPCs.The pathophysiological mechanism of the decrease of NPCs is not fully understood.Exploring the underlying mechanism of NPCs death under different stress states is helpful to find new targets for the treatment of IDD.Pyroptosis is a newly discovered form of cell death and significantly different from apoptosis in molecular mechanism and cell morphology.Oxidative stress is a commonfactor inducing the senescence and autophagy of NPCs in intervertebral disc.With the increase of the degree of degeneration,the content of reactive oxygen species(ROS)in nucleus pulposus tissues increases significantly.However,the effect of ROS on the pyroptosis of NPCs in human degenerative intervertebral disc and its regulatory mechanism have not been elucidated.Nuclear factor E2 related factor 2(NFE2L2)is a transcription factor in NPCs that regulates the transcription and translation of various proteins,and decreases as the degree of intervertebral disc degeneration increases.However,the potential effect of NFE2L2 on the ROS-induced pyroptosis of NPCs is still unclear.In this study,the project was divided into three parts as following: ROS induced pyroptosis of NPCs through NLR family pyrin domain containing 3(NLRP3)/ PYD and CARD domain containing(PYCARD)signal axis;increased ROS level also increased autophagy of NPCs to establish negative regulation on pyroptosis;ROS also upregulated transcription factor NFE2L2 which also negatively regulated the pyroptosis of NPCs.PART 1.ROS INDUCES PYROPTOSIS OF NPCS THROUGH NLRP3/PYCARD SIGNAL AXISObjective:To explore whether ROS induces the pyroptosis of NPCs through NLRP3/PYCARD pathwayMethods:The expression of CASP1 in nucleus pulposus tissues from patients with IDD of different degrees was identified by immunohistochemistry.Human NPCs were extracted and cultured from IDD patients during percutaneous transforaminal endoscopic discectomy(PTED).RT-qPCR and immunofluorescence techniques were used to detect the expressions of CD24,type II collagen(COL2A1)and aggrecan(ACAN).After treatment with different concentrations of hydrogen peroxide,the intracellular reactive oxygen species(ROS)content and PI positive NPCs were detected by flow cytometry,and the cell viability was detected by cell counting kit-8(CCK-8).After treatment with inhibitors or siRNA,the expressions of pyroptosis-related proteins were detected by western blot(WB),and damage to cell membrane was detected by Hochest33342/PI double fluorescence staining.Results:CASP1,CD24,COL2A1 and ACAN were positively expressed in theextracted NPCs.Compared with the NPCs of grade IV patients,the expressions of cleaved CASP1(p20),cleaved IL-1? and cleaved IL-18 and ROS level were higher in those of grade V patients.Treatment with hydrogen peroxide of 200?M for 3h significantly increased the pyroptosis-related indexes,which were attenuated by the pretreatment with NAC,NLRP3-shRNA or PYCARD-shRNA.Conclusion:ROS induces the pyroptosis of degenerated NPCs through NLRP3/PYCARD pathway.PART 2.AUTOPHAGY PROTECTED NPCS FROM PYROPTOSIS INDUCED BY ROSObjective:To explore the role of autophagy in ROS-induced pyroptosis of NPCsMethods:The expressions of autophagy-related proteins microtubule associated protein 1 light chain 3(LC3)and sequestosome 1(SQSTM1)in NPCs of control group and hydrogen peroxide treated group were detected by WB.The effect of hydrogen peroxide(200?M,3h)on the cell viability of NPCs pretreated with different concentrations of rapamycin(RAP)was tested with CCK-8 assay.The pyroptosis-related indexes of NPCs were redetected when the autophagy was activated or inhibited before treatment with hydrogen peroxide.The expressions of autophagy-related proteins SQSTM1 and LC3 and pyroptosis-related proteins p20,cleaved IL-18 and cleaved IL-1? were detected utilizing WB.The PI positive NPCs were detected by Hochest33342/PI double staining.Results:After treatment with 200?M hydrogen peroxide for 3 hours,the expression of LC3-II of NPCs was increased,while SQSTM1 was decreased significantly(p<0.05).The results of CCK-8 showed that pretreatment with RAP alleviated the inhibitory effect of hydrogen peroxide on the viability of NPCs in a certain concentration range.Pretreatment with RAP of 500 n M significantly upregulated LC3-II,downregulated SQSTM1,and inhibited hydrogen peroxide-induced increase of pyroptosis-related proteins and PI positive cells.However,pretreatment with 10 m M 3-MA showed opposite effect.Conclusion:ROS increases the autophagy of NPCs,which attenuates the pyroptosis.PART 3.NEGATIVE REGULATION OF TRANSCRIPTION FACTOR NFE2L2 ON ROS-INDUCED PYROPTOSIS OF NPCSObjective:To explore the effect of transcription factor NFE2L2 on ROS-induced pyroptosis of NPCsMethods:The effects of different concentrations of NFE2L2 inhibitor ML385,melatonin and luzindole on the viability of NPCs were detected by CCK-8assay to find the appropriate intervention concentration.The expressions of pyroptosis-related proteins and NFE2L2 in NPCs with or without treatment of hydrogen peroxide,pretreated with ML385 or melatonin before treatment of hydrogen peroxide,pretreated with ML385 or luzindole before treatment with melatonin and hydrogen peroxide were detected by WB.The PI positive NPCs of each group were detected by Hochest33342/PI double staining.Results:ROS upregulated the expression level of NFE2L2,an intracellular transcription factor of NPCs,when treated with hydrogen peroxide.Pretreatment with ML385 effectively inhibited the expression of NFE2L2 and aggravated the ROS-induced pyrolysis of NPCs.Pretreatment with melatonin upregulated the expression of NFE2L2 and inhibited ROS-induced pyrolysis of NPCs,while melatonin receptor blockers,luzindole or ML385 could significantly reduce the protective effect of melatonin on ROS-induced pyrolysis of NPCs.Conclusion:The transcription factor NFE2L2 negatively regulates ROS-induced pyrolysis of NPCs;melatonin protects NPCs from ROS-induced pyrolysis by upregulating NFE2L2.