Etiology And Therapy Of Lumbar Intervertebral Disc Degeneration

The degenerative lumbar intervertebral disc is the major etiology in causing lumbar disc herniation (LDH) , but the mechanism of lumbar intervertebral disc degeneration has not known. However, there is some evidence that apoptosis and Fas gene may be important in disc degeneration. In this study,we examined the expression of Fas and Fas ligand(FasL) in normal and degenerative lumbar intervertebral discs. The ability of anti-Fas antibody to induce apoptosis in disc cells was analyzed.At present, there is much interest in cytokines and therapeutics that modulate the regulation of apoptosis because these provide an opportunity for the treatment of certain disease. Data obtained here show that insulin-like growth factor-1 (IGF-1) can retard or prevent apoptosis in human disc cell in vitro. The present study addresses the role of IGF-1 in prevention of disc cell apoptosis and in stimulation of extracellular matrix (ECM).Nitrix oxide (NO) is a novel mediator that is being implicated increasingly in degenerative disc tissues. The exact role of NO in intervertebral disc metabolism is unknown, but it may play an important role in the degeneration of the intervertebral disc. In the present study, degenerative lumbar intervertebral disc tissue in culture was shown to produce relatively high levels of NO, but we placed NOS inhibitor L-N (sub-amonion-ethyl) lysine(L-NIL) and s-methyliso-thiourea( SMT) in culture and found that NOS inhibitor could reduce NO releaseand stimulate the ECM synthesis.Materials and MethodsThe expression of FasmRNA and FasLmRNA in degenerative lumbar inter-vertebral discs was detected by RT-PCR; The expression of Fas and FasL in degenerative lumbar intervertebral discs was detected by westernblot.Cell morphologic change induced by anti-Fas antibody was analyzed by inverted phase contrast microscopy, transmission electron microscopy and Hoechst 33258 fluorescence.Apoptosis of disc cells induced by anti-Fas antibody was detected by DNA-ladder electrophoresis, Flow cytometry and Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL).4. The MTT assay and flow cytomety were used to check if IGF-1 has any effect on differentiation and proliferation of intervertebral disc cells in vitro. With chloramines T method and Alcian method , we measured the content of hydroxyproline and chondroitin sulfate in culture medium.5. iNOSmRNA and MMP3 mRNA expression was measured by in situ hybridization.6. The release of NO and the activity of NOS on degenerative discs in culture was measured by Griess reaction and spectrophotometric methods.Results1. Expression of Fas and Fas ligand in degenerative lumbar intervertebral discsThere were overexpression of Fas and Fas ligand in degenerative lumbar intervertebral discs, which compared with normal discs, the difference was significant (P < 0.05). The expression of Fas and Fas ligand in noncontained discs was higher than that of contained discs,the difference was significant(P < 0. 05).2. The morphologic change induced by anti-Fas antibodyUnder inverted phase contract microscopy, the cell density obviously reduced , the cellar volume become smaller, cell got round and there was vacuole like structure in cell. Under transmission microscopy, the chromatin was condense and margined. Under Hoechst 33258 fluorescence, the apoptosis cells had densitive granules in nuclei.3. Degradation of the nuclei DNA of apoptosis cells was demonstrated by visualization of" DNA Ladders "on gel electrophoresis. Moreover, DNA flow cy-tometry and TUNEL of DNA breaks also demonstrated that apoptosis induced by anti-Fas antibody.4. lmmol/L concentration L-NIL and SMT evidently reduced NO release and NOS activity in degenerative discs ( p < 0. 05 ) , and inhibited completely iNOSmRNA and MMP3 mRNA expression.5. Proteoglycan contents in degenerative discs of L-NIL and SMT groups were significantly more than untreated group (p <0. 01). And hydroxyproline release was markedly less in L-NIL and SMT groups than untreat...