Nicotinamide Phosphoribosyl Transferase Inhibitor APO866 Prevents Intervertebral Disc Degeneration Via Autophagy

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Role of Nampt in the Pathogenesis of Intervertebral Disc DegenerationBackgroundLow back pain(LBP)is very common in clinic.More than 80% of people are suffering from low back pain during their lifetime,which is the primary cause of human disability.Intervertebral disc degeneration and a series of related diseases are the most important causes of LBP.Study showed that inflammatory cytokines are significantly up-regulated in the degenerated discs.Inflammation is the most important pathophysiological process in the intervertebral disc degeneration.Inflammatory factors can not only induce extracellular matrix degrading enzyme expression,decrease synthesis of extracellular matrix gene expression,but also induce nerve and blood vessels,which lead to intervertebral disc degeneration and LBP.Nicotinamide phosphoribosyl transferase(Nampt),also known as Visfatin and Pre-B cell colony-enhancing factor(PBEF),was first discovered in 1994.Nampt,which exists widely in the organism,is the the rate limiting enzyme of nicotinamide adenine dinucleotide(NAD+)salvage pathway.Studies showed that nampt,just like a preinflammatory factor,is involved in many cellular activities,such as,cell metabolism,apoptosis,or autophagy process,etc.Nampt also plays an important role in the autoimmune disease,inflammatory diseases,cell metabolism and other related diseases.It has been proved that Nampt can mediate the degradation of extracellular matrix through the proinflammatory action and participate in the pathogenesis of osteoarthritis and rheumatoid arthritis.However,the role of Nampt in intervertebral disc degeneration has not been reported yet.Considering the similar pathophysiology mechanism between intervertebral disc degeneration and osteoarthritis,rheumatoid arthritis,in this study,we will explore the specific mechanism of Nampt in the intervertebral disc degeneration.Objectives1.To investigate the expression of Nampt during the process of intervertebral disc degeneration.2.To explore the effect of inhibiting Nampt on the extracellular matrix metabolism of intervertebral discExperimental procedures1.The intervertebral disc specimens were obtained from patients who underwentsurgery due to lumbar spondylolisthesis,lumbar spinal stenosis,lumbar disc herniation(50cases),The degeneration degree is assessed according to Pfirrmann grade.Grade II and grade III are regard as mild degeneration group,grade IV and grade V as severe degeneration group.quantitative real-time PCR(qRT-PCR),Western blot,and immunofluorescent staning were used to evaluated the expression Nampt in the intervertebral disc tissues.2.The intervertebral disc specimens were digested using enzyme and the nucleus pulposus cells were cultured and treated with different concentrations of IL-1? for 48 hours intervention or treated at different time using IL-1?(10 ng/mL).QRT-PCR and western blot were used to evaluated the expression of Nampt.The NAD activity assay kit was used to detected the NAD activity after intervention.3.Nampt enzyme inhibitor APO866(10 nmol/L)was used to pretreat nucleus pulposus cells,and then the IL-1?(10 ng/mL)was treated for 48 h.QRT-PCR and western blot were used to evaluated the expression of Nampt.The NAD activity assay kit was used to detected the NAD activity after intervention.4.The nucleus pulposus cells were treated with IL-1?(10 ng/mL),or APO866(10nmol/L),or APO866(10 nmol/L)pretreatment combined with IL-1?(10 ng/mL).QRT-PCR and western blot were used to evaluated the expression of ADAMTS-4 and ADAMTS-5,MMP-3 and MMP-13,proteoglycan and collagen type II.Cell immunofluorescent staining was used to detect the degradation of proteoglycan and type II collagen.5.Three different shRNA lentivirus knockout vectors(shRNA1#,shRNA2# and shRNA3#)were constructed to transfect nucleus pulposus cells.QRT-PCR and Western blot were used to detect the expression of Nampt.The NAD activity assay kit was used to detected the NAD activity after intervention.6.The empty Src,shRNA1#,shRNA2#,and shRNA3# were transfected before the IL-1?(10 ng/mL)intervention.QRT-PCR and western blot were used to evaluated the expression of ADAMTS-4 and ADAMTS-5,MMP-3 and MMP-13,proteoglycan and collagen type II.Cell immunofluorescent staining was used to detect the degradation of proteoglycan and type II collagen.Results1.Fifty patients were enrolled in the study,with 26 cases in the mild degeneration group and 24 cases in severe degeneration group.The results of qRT-PCR showed that thethe expression level of Nampt in severe degeneration group was significantly higher than the mild degeneration group;Western blotting and immunohistochemistry showed that the protein expression level of Nampt in severe degeneration group was also significantly higher than the mild degeneration group.2.The expression of Nampt in mRNA and protein level were increased in a dose-dependent manner after different concentrations of IL-1? intervention.The expression of Nampt in mRNA and protein level were increased in a time-dependent manner after different times of IL-1? intervention.3.The expression of Nampt remained unchange after intervention of APO866,and APO866 did not affect the upregulation of Nampt expression induced by IL-1?.However,APO866 significantly decreased NAD activity in nucleus pulposus cells,and significantly inhibited the effect of IL-1? on NAD activity.4.The expressions of ADAMTS-4,ADAMTS-5,MMP-3,and MMP-13 were significantly upregulated by IL-1? intervention.The expressions of proteoglycan and type II collagen were significantly downregulated by intervention.APO866 did not affect the expression of the above genes,but could significantly inhibit the upregulation of ADAMTS-4,ADAMTS-5,MMP-3,and MMP-13 induced by IL-1?,and also inhibit the downregulation of proteoglycan and type II collagen induced by IL-1?.5.The expression of Nampt in nucleus pulposus cells was significantly knocked out by three kinds of lentivirus vectors,both at mRNA and protein levels.The NAD activity was also significantly inhibited.6.Nampt knocked-out did not affect the expressions fo ADAMTS-4,ADAMTS-5,MMP-3,and MMP-13,as well as the proteoglycan and type II collagen.However,the upregulation of ADAMTS-4,ADAMTS-5,MMP-3,and MMP-13 induced by IL-1?,and the downregulation of proteoglycan and type II collagen induced by IL-1?,were significantly reversed after transfected with shRNA1# and shRNA3#.Conclusion1.The expression of Nampt was increased in the degenerated discs,and was positively correlated with the severity of intervertebral disc degeneration,indicating that Nampt is involved in the process of intervertebral disc degeneration.2.The expression of Nampt was induced by IL-1?.Inhibiting the expression of Nampt or Nampt knocked-out could significantly inhibit the extracellular matrix gegradation induced by IL-1?,indicating that Nampt is involved in the intervertebral discdegeneration induced by IL-1?.This study provides new ideas and strategies for the treatment of intervertebral disc degeneration and low back pain.Part ? The role of autophagy induced by APO866 in the pretection of intervertebral disc degenerationBackground Autophagy is a way of catabolism in mammalian.Under the stress of cellular metabolism increased or under the stress of hypoxia and nutritional deficiency,cells will transport damaged organelles or proteins to autophagosome and degrade them by autophagy.Autophagy is an important way of cell development and survival.If cell autophagy lasts too long or stays too strong,and beyond the ability of cell self-regulation,cell metabolism will be disrupted and cells will be degraded by apoptosis.Therefore,autophagy plays an important role in maintaining cell homeostasis.At present,studies have confirmed that autophagy is closely related to the occurrence and development of many diseases,such as inflammatory and immune diseases,neurological and cardiovascular diseases,and cancer.Intervertebral disc degeneration is closely related to cell aging,hypoxia and nutritional deficiency.The main reasons for intervertebral disc degeneration are apoptosis,aging or dysfunction of intervertebral disc.Recently,it has been found that autophagy also participates in the process of intervertebral disc degeneration,and plays a protective role in intervertebral disc degeneration in most cases.Nampt is involved in the processes of cell metabolism,apoptosis and autophagy.Previous studies have shown that APO866 can induce autophagy and playa an important role in relieving acute liver injury and inhibiting tumor growth.However,it is not clear whether APO866 has the role of inducing autophagy in the nucleus pulposus cells and in the degeneration of intervertebral disc.Objective1.To investigate the effect of APO866 on autophagy in nucleus pulposus cells2.To explore the effect autophagy induced by APO866 on the degradation of the extracellular matrix mediated by IL-1?.Experimental procedures1.The nucleus pulposus cells were treated with different concentrations of APO866(0,1,5,10,20,100 nmol/L)for 48 hours or treated with APO866(10 nmol/L)at different times(0,24,48,72,96h).The cell viability was evaluated using CCK-8 assay.The formation of autophagosome was evaluated by monodansylcadaverine(MDC).The expressions of Beclin-1 and LC3-II/LC3-I ratio were evaluated by Western blot.The level of autophagosomal membrane-bound LC3 were evaluated by immunofluorescent staining.2.The nucleus pulposus cells were treated with autophagy inhibitor 3-MA,or nicotinamide mononucleotide(NMN)or recombinant human Nampt(r Nampt)at first,and then treated with IL-1?.The formation of autophagosome was evaluated by monodansylcadaverine(MDC).The expressions of Beclin-1 and LC3-II/LC3-I ratio were evaluated by Western blot.The level of autophagosomal membrane-bound LC3 were evaluated by immunofluorescent staining.3.The nucleus pulposus cells were treated with IL-1?(10 ng/m L)or APO866(10nmol/L)alone or pretreated with APO866 for 2 h then co-treated with IL-1?,or pretreated with APO866 for 2 h then co-treated with 3-MA(10 mmol/L),or pretreated with APO866 for 2 h then co-treated with 3-MA(10 mmol/L)and IL-1?(10 ng/m L)for 48 h.Untransfected NP cells were used as a control.QRT-PCR and western blot were used to evaluated the expression of ADAMTS-4 and ADAMTS-5,MMP-3 and MMP-13,proteoglycan and collagen type II.Cell immunofluorescent staining was used to detect the degradation of proteoglycan and type II collagen.The apoptosis of nucleus pulposus cells was evaluated by Tunel staining.Results1.The nucleus pulposus cells viability were significantly decreased as the concentration of APO866 above 20 nmol/L.The results of MDC staining showed that the number of autophagosome was increased in a dose-dependent manner after different concentrations of APO866 intervention.Western blot showed that the LC3-II/LC3-I ratio and Beclin-1 expression were increased in a dose-dependent manner after different concentrations of APO866 intervention.The level of autophagosomal membrane-bound LC3 was increased in a dose-dependent manner after different concentrations of APO866 intervention.The autophagosome,LC3-II/LC3-I ratio and Beclin-1 expression,and autophagosomal membrane-bound LC3 level reached the peak after treating with APO866 for 48h.2.The increased of the LC3-II/LC3-I ratio and Beclin-1 expression,and the level of autophagosomal membrane-bound LC3 induced by APO866 were significantly inhibited by autophagy inhibitor 3-MA.The increased of the LC3-II/LC3-I ratio and Beclin-1expression,and the level of autophagosomal membrane-bound LC3 induced by APO866 were also significantly inhibited by NMN and r Nampt intervention.3.Autophagy inhibitor 3-MA did not affect the expression of nampt induced by IL-1 ?.The inhibiting effects of APO866 on the expression of ADAMTS-4,ADAMTS-5,MMP-3,MMP-13,proteoglycan and type II collagen induced by IL-1? were significantly reversed by 3-MA.The increased apoptosis induced by IL-1? was inbihited by APO866,and this effect was also reversed by 3-MA.Conclusion APO866 could induce autophagy in nucleus pulposus cells.the extracellular matrix breakdown mediated by IL-1? could be inhibited by APO866 via autophagy.These results suggested that APO866 may be a new molecular target and drug for the treatment of intervertebral disc degeneration.