The Role Of SDF-1/CXCR4 Pathway In Regulating MMPs Expression In Intervertebral Disc Endplate Cartilage Degeneration

Objective The aim is to investigate that chemokine stromal cell-derived factor 1(SDF-1)and its receptor CXCR4 expression in the intervertebral discs(IVD),and SDF-1 induce the MMPs expression in endplate chondrocytes and accelerate the degradation of extracellular matrix in endplate.Methods ①SDF-1 and CXCR4 levels in human IVD and rat motion segment of L5/6 were quantified by enzyme-linked immunosorbent assay.The staining of SDF-1 was quantified using a microscope and Image-Pro Plus software,and integrated optical density(IOD)served as the measurement parameter.The number of immunoreactive cells of CXCR4 was expressed as a percentage of the total number of cells.②Endplate chondrocytes were isolated from the cartilage endplates of young patients with spine-fresh burst fractures,and the MRIs of the IVDs showed Pfirrmann grade Ⅰ-Ⅱ.Chondrocytes were incubated with SDF-1(50,100,200ng/ml),or AMD3100(500ng/ml),or AMD3100+SDF-1(100ng/ml)for 24 h.For examination of the downstream signaling pathways involved in SDF-1 treatment,endplate chondrocytes were pretreated with AMD3100 for 30 min before SDF-1 administration.RT-PCR,western blots and zymography analysis were used to ensure the MMPs induced by SDF-1 in endplate chondrocytes.③ Cartilage endplates were harvested from young patients with fresh burst fractures.The explants were placed in a 24-well culture dish with fresh serum-free medium containing different concentration of SDF-1(50,100,200ng/ml),or 500 ng/ml AMD3100(with or without 100 ng/ml SDF-1)and cultured for a further 2 days.Specimens were then embedded in paraffin blocks and sliced into 5-μm sections,and investigated by Safranin-O staining.Results①SDF-1 and CXCR4 were both expressed in IVD,and the levels of SDF-1 and CXCR4 were both significantly higher in the degeneration group than those in the normal group of human(or rat)discs.Both Nucleus pulposus cells and cartilaginous endplate cells are able to express CXCR4 protein.Furthermore,in nucleus pulposus and cartilaginous endplate,there were both a positive correlation between the value of IOD of SDF-1 and the percentage of CXCR4-positive disc cells.The values of IOD of SDF-1 in outer annular fibrosus and bone/endplate junction region were significantly higher than those in nucleus pulposus and cartilaginous endplate in the rat specimens.②This study found that SDF-1 enhanced CXCR4 mRNA and protein expression in human endplate chondrocytes in a dose-dependent manner.The results from RT-PCR,Western blot,and zymography indicated that SDF-1 increased mRNA and protein expression of MMP-1,-3,-13 in human endplate chondrocytes in a dose-dependent manner.The zymography results suggested that SDF-1 increased MMP-2,9 protein expression in a dose-dependent manner.AMD3100,as CXCR4-specific chemical inhibitor,significantly decreased the MMP-1,2,3,9,13 expression.③In human cartilage explants culture model,it was showed that SDF-1 accelerated the degradation of extracellular matrix,and AMD3100 decreased the cartilage cleavage.Conclusions Our findings suggest the up-regulated expression of SDF-1 and its receptor CXCR4 in the degenerated IVD,and MMPs mRNA and protein expression induced by the interaction of SDF-1 and CXCR4,which could accelerated the degradation of extracellular matrix of cartilage endplate.