Expression And Role Of Sphingosine 1-Phosphate Receptors In Human Intervertebral Disc Degeneration

Background and ObjectiveChronic low back pain(LBP)is a widespread problem all over the world [1].Numerous studies have confirmed that LBP is due in large part to intervertebral disc(IVD)degeneration[2,3].Related study is the hotspot of research in spine surgery.The intervertebral disk degeneration relates to multiple factors,such as stress,aging,inflammation,nutrition pathway and so on.Various histological changes occur with the degeneration,such as fissures in the AF,dehydration in the NP along with vessels and nerves growing into the inner disc,which break the micro-environment homeostasis in disc;pathologic calcification in EP,leading to blockage of nutrition pathway [4].Obviously,these histological changes,as the major pathogenic characterization of IVD degeneration,may also promote the progress of degenerationFurthermore,alterations in the extracellular matrix and expression profile of the NP cells,which is the base of function of cells or tissue,usually have been considered as the marker of degeneration[5].It has been confirmed that decreases in proteoglycan and type II collagen synthesis,increases in type I collagen synthesis and aggrecan fragment accumulation with IVD degeneration[6].Accordingly,degradation enzymes or proinflammatory cytokines,such as IL-1?/ TNF-?/MMP /ADAMT have also been reported [7,8].Recently,some other genes or proteins,which is not only a marker in the degeneration,but also have effects on the metabolism or the degeneration attract more attention of the researchers.Similar researches can deepen the understanding of IVD degeneration,which is helpful to finding better measures for treatment.Sphingosine 1-phosphate(S1P)is an important lipid mediator,formed by phosphorylation of sphingosine and catalyzed by sphingosine kinase.It has been confirmed to be implicated in many biological processes,including cell migration,differentiation,inflammation and angiogenesis.[9-11].S1 P exerts its various functions by binding to specific G protein-coupled receptors,and 5 functionally different isoforms(termed S1 PR 1-5)have been identified.Strikingly,previous researches have shown that in human chondrocytes,S1 P can inhibit the catabolic response induced by IL-1? via activation of the S1 P receptor[12,13],thus the activation of the receptor may be a therapeutic target for OA.In the light of similar biological property of nucleus pulposus cells,S1 P and its receptors reflect a certain application prospect in the treatment and control of intervertebral degeneration.While,to the extent of our knowledge,the expression of S1 P receptors or the effect of S1 P in IVD degeneration have not been reported..In the current study,we investigates the expression of S1 P receptors in nucleus pulposus,and compared the expression in NP cells with different degree of degeneration in order to verify the relation between the S1 PRs and IVD degeneration.Finally,we preliminary explored the effects of S1 P on intervertebral disk degeration.Materials and methods1.Samples acquirement and assessment of the degree of degeneration.Study protocols were approved by the Ethics Committee of our institution,and the informed consent was signed.Inclusion criteria:Experimental group: patients with intervertebral disc degenerative diseases(including lumbar disc / lumbar spinal stenosis / lumbar spondylolisthesis);Control group: the young patients with Vertebral body fracture,without intervertebral disc degeneration imaging findings or history of low back pain.The intervertebral disk tissue were obtained by surgical operation.Exclusion criteria:Patients with infection,tumor,immunological and endocrine disease were excluded from the current study.Sample acquired from each patient,is divided into two parts,used for histomorphology experiments(including HE,Saf-O staining and IHC)and cytology experiments(including isolation and culture of nucleus pulposus cells,Real – time PCR and western-blot)and western blot experiments.We histologically assess the grade of degeneration in IVD tissue via according to published standard.[14]And then,we analyzed the consistency between the histologically scores and the Priffmann classification.2.Expression of S1 PRs in IVDExpression of S1 PRs in IVD was confirmed by immunohistochemistry,and the location in NP cells was illustrated via immunocytochemistry.To compare the expression in NP cells with different degree of degeneration,the expression was evaluated using real-time PCR and western blot.3.The effect of S1 PR in intervertebral disk degeneration.The NP cells was stimulated by IL-1? with or without the ligand,Sphingosine 1-phosphate(S1P).Then the expression of degradation enzymes,MMP-3 and ADAMTS4 was evaluated by Real-time PCR and western blotting;NP cell was treated with S1 P,and the aggrecan content was measured by ELISA and Alcian blue staining.Results1.The histologic scoring system we used can effectively evaluate the degree of intervertebral disc degeneration,and the result is well accord with Priffmann classification.2.The immunohistochemical and immunocytochemistry shows that the NP express the S1PR1/2/3,located in the membranes and cytoplasm of cells;and the positive percentage of S1PR2 is higher than S1PR1/3.3.Real-time PCR and western-blot shows that the expression of S1 PRs decreases inthe severe degenerated NP.4.IL-1? up-regulate the expression of MMP-3 and ADAMTS4 in NP cells,while that in cells treated with IL-1? and S1 P is lower than that treated only with IL-1?;NP cell treated with S1 P down-regulated the expression of AGG.Conclusion1.The NP express the S1PR1/2/3,especially the S1PR2.2.The expression of S1 PRs down-regulated with degeneration,implicating the possible relationship between the S1P/S1 PR and IVD degeneration3.The signal of S1P/S1 PR can inhibit the catabiosis effect of IL-1? in NP cells,and attenuated the synthesis of proteoglycan AGG,implicating the role of them in NP cell and IVD degeneration.