The Role And Mechanism Of Interleukin-1 Beta On Apoptosis And Catabolism Of Nucelus Pulposus Cells In Human Degenerative Intervertebral Disc

Low back pain(LBP)is the most common cause for the dyskinesia,and severely impacts the quality of modern life,which still lacks of effective treatment in the current clinical practice.Intervertebral disc degeneration(IVD)has been considered as the main reason for LBP,whose potential mechanism still remains unclear.Previous researches have demonstrated that there is a close relationship between inflammatory factors and LBP induced by IVD,of which interleukin-1beta(IL-1?)has been most extensively studied.however,there still lacks systematic research of the role and mechanism of IL-1? on apoptosis and catabolism of nucelus pulposus cells(NPCs)in human degenerative intervertebral disc.This project will be divided into four parts as following: Part 1.IL-1? induced NPCs apoptosis through mitochondrial pathway;Part 2.The mitochondria damage induced by IL-1? promotes autophagy activation in NPCs;Part 3.Autophagy protects against NPCs apoptosis induced by IL-1?;Part 4.SIRT1 inhibits extracellular matrix degradatoin induced by IL-1? via TLR2 /NF-?B pathway.Part 1.IL-1? Induced NPCs Apoptosis Through Mitochondrial PathwayObjective To investigate whether mitochondrial pathway is involved in the NPCs induced by IL-1?.Methods Western blot,immunohistochemical(IHC)and TUNEL staining were performed to test the IL-1? expressions and NPCs apoptosis from normal and degenerative NP tissues,respectively.The degenerative NPCs were isolated and cultured by the sequential digestion method.Under different concentration of IL-1? treatment,flow cytometry analysis based on Annexin V/PI staining for cell apoptosis and CCK-8 for cell proliferation were performed to optimize the optimum concentration.Morphological observation,Hoechst 33258 staining and flow cytometry analysis were used to investigate the influence of IL-1? on NPCs apoptosis in the presence or absence of faetal bovine serum(FBS).The mitochondrial pathway related proteins,like BAX,Bcl-2 and cytochrome C(Cyt C)were determined by western blot,intracellular ROS expressions were determined by the fluorescence probe of DCFH-DA;the enzyme activity of caspase-3 and caspase-9 were determined by microplate reader.Results The IL-1? expression and cell apoptotic rate in degenerative NP tissues were obviously higher than those in normal ones(P ?0.05),suggesting there maybe a relationship between IL-1? and NPCs apoptosis.The concentration of 10 ng/ml IL-1? is optimum for the subsequent experiments.In the serum-deprivation condition,IL-1? could significantly improved the NPCs apoptosis(P?0.05),however,FBS adequate cells were not sensitive to inflammatory stress.In addition,IL-1? could obviously increase expressoin ratio of Bax/Bcl-2(P?0.05),release cytochrome c from mitochondria to cytoplasm,suggesting activating mitochondiral pathway.Furthermore,IL-1? subsequently activated downstream caspase-9 and caspase-3(P?0.05),and increased ROS level(P?0.05)in NPCs.Conclusion In the serum-deprivation condition,IL-1? could significantly induced NPCs apoptosis through mitochondiral pathway.Part 2.The Mitochondria Damage Induced by IL-1? PromotesAutophagy Activation in NPCsObjective To investigate whether IL-1? could induce the mitochondria damage,and further promote autophagy activation in degenerative NPCsMethods In the serum-deprivation condition,NPCs were treated with or without 10 ng/ml IL-1?,while 10%FBS culture condition was served as control group.Then,the mitochondria damage under IL-1? treatment were detected as following: Mitochondrial membrane potential(??m)was assessed using JC-1,intracellular ATP content was detected using an ATP assay kit,and the mitochondrial ultrastructure was observed by transmission electron microscopy(TEM).On the other hand,autophagy related proteins LC3 and P62 were tested by western blot.To directly visualize the autophagosome,cells were transfected with adenovirus containin GFP-LC3.Autophagy was evaluated by analyzing the number of green fluorescent puncta of autophagosomes under laser confocal microscopy.To assess this autophagy flux,NPCs were pretreated by a lysosomal inhibitor,baflomycin A1 for 2h.Finally,TEM observation was performed to find the typical double-membraned autophagosomes in NPCs.Results In the serum-deprivation condition,IL-1? treatment signifcantly decreased the red/green fluorescence ratio of NP cells as observed by fluorescence microscope and flow cytometry,suggesting ??m decreased by IL-1?(P ? 0.05).Serum deprivation alone seemed no influence on the ATP level,but exposure to IL1-? signifcantly decreased ATP level(P?0.05).TEM was used to further assess mitochondrial integrity and state.Serum deprivation induced a few swollen mitochondira in NP cells,but accumulation of highly damaged,electron-dense mitochondria were observed under IL-1? treatment.All these results demonstrated that IL-1? cuased mitochondrial damage in degenerative NPCs.western blot showed that IL-1? signifcantly changed the autophagic marker LC-3II and P62/SQSTM1 expressions(P?0.05).Fluorescence-based detection of LC-3 isoforms were directly observed in NP cells,IL-1? treatment resulted in a signifcant improvement in the LC3 puncta(P?0.05),indicative of increased autophagosome formation.Te addition of baflomycin A1 further increased LC3-II and p62/SQSTM1 accumulation,compared with cells treated with IL-1? only(P ? 0.05),which indicated IL-1?-mediated autophagy is not because of reduced autophagosome turnover,but increased autophagic flux.In addition,TEM observation found typical double-membraned autophagosomes in NP cells from serum deprivation group.Moreover,mitochondrial fragmentations were observed and sections of the mitochondrion appeared to be surrounded by double-membrane profles under IL-1? treatment,suggesting IL-1? induced autophagy plays a role of eliminating the damaged mitochondria in NPCs.Conclusion IL-1? induced the mitochondria damage in degenerative NPCs,and further promote the autophagy activation.Part 3.Autophagy protects against NPCs apoptosis induced by IL-1?Objective To investigate the effect of autopahgy induced by IL-1? on the NPCs apoptosis.Methods NPCs were pretreated with the autophagy inhibitor 3-MA,then cell apoptosis was determined by flow cytometry analysis based on Annexin V/PI staining and mitochondrial membrane potential(??m)was assessed using JC-1.To further investigate whether IL-1? induced autophagy acted as the compensative mitophagy,the Parkin expressions in mitochondria and cytoplasm were tested by western blot,respectively.In addition,after treated with the autophagy activator RAP,NPCs were transfected with adenovirus containin GFP-LC3 and Ad-HBm Tur-Mito,of which GFP-LC3 signed for autophagosome and HBm Tur-Mito signed for mitochondria,the merging yellow fluorescence represented mitophagy.Results 3MA treatment signifcantly attenuated the LC3-II expression that indicated 3MA decreased the autophagy incidence by IL-1? treatment.Flow cytometric analysis revealed that obviously increased apoptotic ratio was observed under 3MA treatment,suggesting the autophagy activation induced by IL-1? may be a self-protective mechanism in NP cells.The result indicated the autophagy activation induced by IL-1? may be a self-protective mechanism in NP cells.Moreover,decreased ??m were confrmed following autophagy inhibition by 3MA.Western blot analysis showed that serum starvation had little effect on the translocation of Parkin,but IL-1? recruited Parkin from cytoplasm onto depolarized mitochondria,which indicated that IL-1?-meidiated autophagy may be a critical homeostasis mechanism,as mitophagy.To further confirmed by laser confocal microscopy,results showed that the number of yellow fluorescence punctates increased under IL-1? treatment,moreover,RAP further increase the yellow fluorescence punctates in NPCs,suggesting the autophagy activated by IL-1? is related with mitophagy?Conclusion IL-1? promote autophagy activation,to further protect NPCs against apoptosis,which is partly related with mitophagy.Part 4.SIRT1 inhibits extracellular matrix degradatoin induced byIL-1? via TLR2 /NF-?B pathwayObjective To investigate the role and mechanism of SIRT1 on the IL-1? induced ECM degradation.Methods IHC staining was performed to detected the expressions of SIRT1?MMP-3 and MMP-13 in situ,and western blot was performed to compare the different expressions of SIRT1,COL2A1 and aggrecan in normal and degenerative NP tissues,respectively.To directly regulate the SIRT1 expressions,NPCs were transfected with lentivirus containin SIRT1 overepxression plasmid or si RNA targeting for SIRT1,to further evaluate the expressions of ECM and MMPs.Finally,western blot,co-immunoprecipitation(Co-IP)and immunocytochemistry were performed to investigate whether SIRT1 regulated ECM expressions through TLR2/NF-?B pathway.Results The results of IHC and western blot indicated that SIRT1,COL2A1 and aggrecan expressions were significantly decreased in degenerative NP tissues,while the expressions of MMP-3 and MMP-13 obviously increased,suggesting SIRT1 is closely related with ECM expression in NP tissues.After directly upregulation the expression of SIRT1 by lentivirus,SIRT1 inhibits COL2A1 and aggrecan degradatoin induced by IL-1?,however,the expressions of MMP-3 and MMP-13 remarkably increased after SIRT1 inhibited by si RNA,indicating SIRT1 could 1 inhibits IL-1?-mediated ECM degradatoin.To further explore the potential mechanism,TLR2-NF-?B pathway was activated by IL-1? treatment,Co-IP showed that there was an interaction between SIRT1 and P65.The expressions of TLR2,total and acetylated P65 both decreased after SIRT1 upregulation.In addition,knockdown of p65 blocked IL-1?-induced MMP-3 and MMP-13 expression,indicating that NF-?B inhibition exerted an anti-inflammatory effect against IL-1? stimulation.Immunocytochemical analysis confirmed that IL-1?-induced nuclear translocation of p65 was not attenuated after TLR2 silencing.These results indicated that TLR2 did not directly result in the downstream activation of NF-?B,despite inhibiting SIRT1 expression after IL-1? stimulation.Conclusion SIRT1 inhibits extracellular matrix degradatoin induced by IL-1? via TLR2 /NF-?B pathway.