The Effect And Mechanism Of Sirt1 On IL-1Β Induced Pyroptosis In Degenerative Nucleus Pulposus Cells

Low back pain(LBP)is a common cause of labor loss and even disability in adults,which causes serious economic burdens and mental disorders to individuals.Intervertebral disc degeneration(IVDD)is a leading contributor to LBP.Decreased number and function of nucleus pulposus cells(NPCs)is an important feature of IVDD.Exploring the mechanism of NPCs reduction or function loss is beneficial to find potential targets for the treatment of IVDD.During the IVDD process,the nucleus pulposus tissue has obvious inflammatory environment changes.The increase of inflammatory factors such as IL-1β will lead to the degeneration and even death of NPCs,which will accelerate IVDD.Pyroptosis and NLRP3 inflammasome is involved in the process of IVDD,but their activation pathway and inhibitory mechanism in NPCs are still unclear.Mitophagy is important to remove damaged mitochondria and maintain homeostasis,but the relationship between mitophagy and pyroptosis in NPCs has not been reported.SIRT1 plays an important role in anti-inflammatory and mitochondrial homeostasis maintenance,but in IVDD process,the role of SIRT1 in pyroptosis remains unclear.SRT1720 is a selective agonist of SIRT1,which roles have not been validated in vivo in the field of intervertebral discs.To clarify the effect and mechanism of SIRT1 on IL-1β induced pyroptosis in degenerative NPCs,this topic will be elaborated in three aspects as follows: IL-1β induces human NPCs pyroptosis through mitochondrial pathway;SIRT1 alleviates IL-1β-induced NPCs pyroptosis by promoting mitophagy;SRT1720 alleviates intervertebral disc degeneration in rat by inhibiting pyroptosis.PART 1.IL-1Β INDUCES HUMAN NPCS PYROPTOSIS THROUGH MITOCHONDRIAL PATHWAYObjective : To investigate whether IL-1β induces human NPCs pyroptosis through mitochondrial pathway.Methods: According to the inclusion and exclusion criteria,human nucleus pulposus tissues of the relatively normal group and the degeneration group were collected,tissue structure and NLRP3 inflammasome expression were observed by HE staining and immunohistochemistry(IHC),respectively.Human NPCs were extracted and cultured in vitro,after intervention of different concentrations(10,20,50,100 ng/ml)of IL-1β,CCK-8 detected the viability of NPCs.To confirm whether pyroptosis occurs,Western blot detected the expression of pyroptosis-related indicators,Hoechst33342/PI staining and scanning electron microscope observation were also used in this study.Meanwhile,after IL-1β intervention,the changes of mitochondrial function in NPCs were detected through JC-1 staining,Mito SOX staining,DCFH-DA staining and transmission electron microscopy.After pretreated with Mito-Tempo,WB detected the expression of pyroptosis-related proteins,the proportion of NPCs undergoing pyroptosis was evaluated by Hoechst33342/PI staining.Results: In degenerated NP tissue,HE staining showed decreased NPCs and loss of extracellular matrix components,IHC showed that the positive cells proportion of NLRP3 and IL-1β were significantly increased.CCK-8 showed that different concentrations of IL-1β would all reduce NPCs viability;when IL-1β exceeded 50ng/ml,the expressions of NLRP3,ASC and p20 were significantly increased,and the proportion of PI-positive cells increased significantly.Scanning electron microscopy found that IL-1β treatment resulted in more hole formation and spherical protrusions in NPCs membrane.Meanwhile,after IL-1β intervention,the mitochondrial membrane potential(Δ Ψ m)decreased significantly,the mitochondrial and overall cellular ROS levels increased significantly,the mitochondrial swelled and the crest fractured in NPCs.After Mito-Tempo pretreated,the expressions of NLRP3,ASC,p20,GSDMD-N and Cleaved IL-1β were significantly decreased,Hoechst33342/PI staining showed that the proportion of PI-positive cells was also significantly decreased.Conclusion: IL-1β induces human NPCs pyroptosis through mitochondrial pathway.PART 2.SIRT1 ALLEVIATES IL-1Β INDUCED NPCS PYROPTOSIS BY PROMOTING MITOPHAGYObjective:To explore the roles of SIRT1 and mitophagy in IL-1βinduced NPCs pyroptosis.Methods: After SIRT1 was overexpressed in human NPCs by lentivirus transfection,the expression levels of mitophagy-related proteins were detected by WB.After NPCs transfected with GFP-LC3 adenovirus and treated with Mito-Tracker probe,the co-localization of autophagosomes and mitochondria was observed.Meanwhile,JC-1staining and Mito SOX staining were used to detect changes in Δ Ψ m and mitochondrial ROS,respectively.WB and immunofluorescence detected the expression of pyroptosis-related proteins,and Hoechst33342/PI staining detected the proportion change of NPCs pyroptosis.Results: Following SIRT1 overexpression,WB showed that the expressions of PINK1,Parkin and LC3 were significantly increased,while the expression of p62 was significantly decreased.In IL-1β+LV-SIRT1 group,the colocalization of autophagosomes(green granules)and mitochondria(red granules)was significantly increased,the Δ Ψ m was significantly increased,the mitochondrial ROS levels was significantly decreased,the expression of NLRP3,ASC,p20,GSDMD-N and Cleaved IL-1β were significantly decreased,the proportion of PI-positive cells was significantly decreased showed by Hoechst33342/PI staining,which indicated that SIRT1 overexpression inhibited the NLRP3 inflammasome activation and pyroptosis in NPCs.In IL-1β+LV-SIRT1+3-MA group,the expression of NLRP3,ASC,p20,GSDMD-N and Cleaved IL-1β were significantly increased,the proportion of PI-positive cells was significantly increased observed by Hoechst33342/PI staining,which indicated that the protective effect of SIRT1 was also weakened after the autophagy level was inhibited.Conclusion: SIRT1 alleviates IL-1β induced NPCs pyroptosis by promoting mitophagy.PART 3.SRT1720 ALLEVIATES INTERVERTEBRAL DISC DEGENERATION IN RAT BY INHIBITING PYROPTOSISObjective:To verify the effect of SIRT1 selective agonist SRT1720 on rat intervertebral disc degeneration in vivo.Methods: The rat caudal IVDD model was established through acupuncture annulus fibrosus.SD rats were randomly divided into 4 groups:Sham,Puncture,Vehicle and SRT group.In Sham group,the intervertebral disc(IVD)was not punctured,only the subcutaneous tissue was separated and sutured;in Puncture group,the incision was sutured after IVD puncture;in Vehicle group,IVD was punctured and 10 μl DMSO was injected into the space;in SRT group,IVD was punctured and 10 μl SRT1720 was injected into the space.After operation,the imaging degeneration degree of IVDD in each group was evaluated by MRI examination and Pfirrmann grading at 4th and 8th week.At the 8th week,the histological degeneration degree in each group was evaluated by HE and Safranin O-fast green staining;the changes of pyroptosis and autophagy-related protein levels in the NP tissue were detected by IHC.Results: MRI and Pfirrmann grading showed that,at 4th and 8th week,the IVDD degree in Puncture and Vehicle group was significantly increased compared with Sham group;in SRT group,T2 image signal of IVD and Pfirrmann grade were improved compared with Puncture and Vehicle group.HE and Safranin O-fast green staining showed that,the Puncture and Vehicle group had more damage structure of the IVD,and the NP tissue was significantly reduced;in SRT groups,the IVD damage degree was relieved,the proteoglycan and matrix components were significantly recovered compared with Puncture and Vehicle group.IHC showed that,in Puncture and Vehicle group,the proportion of NLRP3,p20 and IL-1βpositive cells was significantly increased,the LC3 expression was at low level;in SRT group,the expressions of NLRP3,p20 and IL-1β were significantly decreased,the proportion of PINK1 and LC3 positive cells was significantly increased.Conclusion: SRT1720 alleviates intervertebral disc degeneration in rat by inhibiting pyroptosis.