The Role Of Syndecan-4 In Intervertebral Disc Degeneration

Part One. The expression of Syndecan-4 protein in degenerativeintervertebral disc of patients Objective: To explore the expression of Syndecan-4 in degenerative lumbar intervertebral disc, and their relationship with lumbar disc degeneration. Methods: 18 disc specimens were collected from the patients of posterior lumbar decompression and fusion. All specimens were graded according to Pfirrmann disc degeneration grading system, Grade IV- V as degeneration group(10 cases), Grade I- III as control group(10 cases). All of the discs were divided into two parts. One for RT-PCR detection, and the other for histological detection. Results: Immunohistochemical staining showed that the Syndecan-4 positive expressions existed in the both control and degeneration groups. However Syndecan-4 expression was significantly higher in the degeneration group than that of control group. The average optical density value of Syndecan-4 in the degeneration group were higher than that of the control group, with significant difference(p<0.05). RT-PCR results also show that Syndecan-4 m RNA expression was higher in the degenerative group than that of control group, with significant difference(p<0.05). Conclusion: Degenerative intervertebral discs showed higher expression of Syndecan-4, which may be involved in the process of intervertebral disc degeneration. The result of Syndecan-4 could provide a reference for future therapies for biological treatment of intervertebral disc degeneration.Part Two. Effect of Syndecan-4 on the biological behavior of human nucleus pulposus cells Objective: To explore nucleus pulposus cells morphology and proliferation and function by changes of Syndecan-4 expression, and provide the experimental basis for the further study on the mechanism of intervertebral disc degeneration.Methods: Constructed virus of Syndecan-4 overexpression and knockdown infected human nucleus pulposus cells respectively. The morphological changes of cells were recorded. Cells proliferations were detected by CCK-8 method. The levels of both m RNA and protein were detected by RT-PCR and Western Blot for nucleus pulposus related functional factor. Results: There is no obvious change on the cells morphology. CCK-8 analysis also showed that the expression of Syndecan-4 had no effect on cells proliferation. Overexpressing Syndecan-4, protein and m RNA levels of Aggrecan, Collagen II and SOX-9 were decreased, while the levels of Collagen X were elevated(p<0.05). Knocking down Syndecan-4, the results were just the opposite. Conclusion: The expression of Syndecan-4 did not affect the morphology and proliferation, but could affect the function of nucleus pulposus cells, which involved in the degeneration of intervertebral disc. Overexpression of Syndecan-4 can promote the nucleus pulposus cells degeneration.Part Three. Effect of Syndecan-4 on the JNK/p53 pathway Objective: To explore the effect of JNK pathway and downstream p53 pathway in the nucleus pulposus degenerative process induced by Syndecan-4.Methods: The levels of JNK, p JNK, p53 protein and its target genes MDM2 and p21 were detected by Western Blot in the Syndecan-4 overexpression and knockdown nucleus pulposus cells. The nucleus pulposus related functional factors were also measured when the p53 overexpression and the JNK pathway inhibition. Results: Overexpression of Syndecan-4 significantly increased JNK protein phosphorylation, and the inhibition of Syndecan-4 can obviously reduce it. Similarly, The Role of Syndecan-4 in Intervertebral Disc Degeneration Abstract Syndecan-4 overexpression increased the p53 and its target genes MDM2 and p21 significantly in nucleus pulposus cells(p<0.05). p53 overexpression can significantly reduce the Aggrecan, Collagen II protein and m RNA level. Blocking the JNK pathway, inhibiting the expression of p53, related functional factor of nucleus pulposus were increased(p<0.05). Conclusion: Syndecan-4 could activate JNK signal pathway, and downstream p53 pathways, promote nucleus pulposus cells degeneration. Inhibiting JNK pathway, down-regulating p53 expression, can improve the degeneration of nucleus pulposus cells.Part Four. The role of Syndecan-4 in the rabbit intervertebral discdegeneration Objective: To explore the effect of Syndecan-4 in recovering rabbit intervertebral disc degeneration, and the potential mechanism. Methods: The discs of New Zealand white rabbits were punctured by 18 G needle. After confirmation of disc degeneration with MRI, Syndecan-4 si RNA, control si RNA and saline were injected to the degenerated discs, respectively. All of the animals were sacrificed, and conducted histological and immunohistochemical analysis. Results: After the injection of Syndecan-4 si RNA to the degenerated rabbit’ intervertebral discs, the disc signal improves significantly(p<0.05), while after the injection of control si RNA and saline, the improvement is not obvious. Immunohistochemistrical results show that injection of Syndecan-4 si RNA within the nucleus lead to the significantly increase of type II collagen content in the extracellular matrix, and JNK and p53 positive expressions were significantly decreased(p <0.05). Conclusion: Syndecan-4 si RNA injection can stop the development of rabbit’s disc degeneration, and even has a reverse effect, in which JNK/p53 plays a role.