The Mechanism Research Of Salubrinal's Suppressive Affection's On IL-17-induced Intervertebral Disc Degeneration In Human Nucleus Pulposus Cells

BackgroundsIntervertebral disc degeneration disease is a common clinical disease of orthopedics,which main features are extracellular matrix(ECM)components decrease,water loss and change of collagen ratio.Matrix metalloproteinases(MMPs)play an important role in this process which are usually produced by neutrophils,fibroblasts,macrophages and tumor cells.They are a family of Ca and Zn-dependent proteolytic enzymes involved in degrading the extracellular matrix components of various tissues in the body.Matrix metalloproteinase-13(MMP-13),mainly derived from chondrocytes,degrades type ? collagen and aggrecan,which plays a dual role in matrix degradation.Previous studies have shown that,MMP-13 played a significant role in the process of human intervertebral disc degeneration mediated by inflammatory mediators such as interleukin-1?(IL-1?),tumor necrosis factor-a(TNF-a)and so on.However,the relationship between interleukin-17(IL-17)and MMP-13 in the process of intervertebral disc degeneration has not been reported.Salubrinal,an inhibitor of eIF2a dephosphorylation,could promote the phosphorylation of eIF2a,thereby inhibiting endoplasmic reticulum stress-induced apoptosis.Recent studies have shown that salubrinal may have positive effects on bone tissues such as promoting bone healing,preventing osteoporosis and apoptosis of osteoblasts and osteocytes,and inhibiting osteoarthritiS?However,the effects of salubrinal on the preservation of intervertebral discs are still poorly understood,this experiment will investigate whether salubrinal inhibits IL-17-mediated degeneration of the intervertebral disc.Objective1.This study will investigate the regulation of matrix MMP-13 and extracellular matrix by IL-17 during intervertebral disc degeneration.2.To investigate whether salubrinal,an inhibitor of eukaryotic initiation factor 2a dephosphorylation,inhibits IL-17-mediated degeneration.Methods1.Patients enrollment tool place between February 2014 and September 2014 at the Shandong University Qilu hospital diagnosed as lumbar disc herniation patients.We selected 20 discs from patients undergoingtransforaminal lumbar interbody fusion(TLIF)and posterior lumbar interbody fusion(PLIF).All discs were classified as non-degenerated by Pfirrmann score.After disc nucleus pulposus separation,the primary nucleus pulposus cells were isolated and subcultured2.Collagen type ?(COL2A1),collagen type ?(COL1A1)and aggrecan(ACAN)in nucleus pulposus cells were stimulated by IL-17 at different concentrations and at different intervals by western blot and RT-PCR.3.The activation of NF-?B pathway was detected by western blot and immunofluorescence following IL-17 treatment.Furthermore,the expression of eIF2a was detected by western blot following IL-17 and salubrinal treatment.4.The expression of MMP-13,COL2A1,COL1A1 and aggrecan was examined by western blot and RT-PCR,followed by incubation of salubrinal or NF-?B inhibitor BAY11-7082 with or without IL-17 treatment,as well as NF-?B pathway.Results1.The results of western blot showed that IL-17 increased the expression of MMP-13 and COL1A1 and decreased the expression of COL2A1 and aggrecan in nucleus pulposus cells.RT-PCR results showed that IL-17 increased the expression of MMP-13 and reduced the expression of COL2A1,COL1A1 and aggrecan.2.Western blot and immunofluorescence showed that IL-17 activated the NF-?B pathway.In addition,immunoblotting experiments showed that IL-17 also increased the expression of eIF2aphosphorylation(p-eIF2a).Further experiments showed that the effects of BAY11-7082 on the level of p-eIF2a was not detectable(P>0.05).3.Salubrinal and NF-?B pathway inhibitor BAY 11-7082 can inhibit IL-17-driven activation of NF-?B pathway.Meanwhile,western blot showed that salubrinal and BAY11-7082 inhibited the IL-17-induced upregulation of MMP-13 and COL1A1,and downregulation of COL2A1 and aggrecan.RT-PCR showed that salubrinal and BAY 11-7082 inhibited the IL-17-induced upregulation of MMP-13,and downregulation of COL2A1,COL 1A1 and aggrecan.ConclusionsThe results showed that IL-17 increased the production of MMP-13 and decreased expression of COL2A1 and ACAN via the NF-?B pathway.Furthermore,salubrinal can inhibit this process through inhibition of NF-?B activation that is not directly linked to eIF2? phosphorylation,suggesting a potential therapeutic role in IDD.