Experimental Study Of Effects Of MMPs Inhibitor And Massage Treating On Rat Lumbar Intervertebral Disc Degeneration

Intervertebral disc degeneration (IDD) are an important cause of neck, shoulder, back, leg pain. Annulus fibrosus broken is caused by a variety of incentives such as acute sprains, chronic fatigue and cold etc.So liquid-like nucleus pulposus spills, diffuses along the channel between the disc and nerve root. And chemical radicular neuritis is induced by glycoprotein andβprotein within the nucleus pulposus which are strong chemically irritant and the following released histamine nucleus pulposus. Because of inflammation, a lot of inflammatory protein which exudes from high permeable vascular, accumulates in the intima and outima of nerve root and vertebral sinus nerve. Protein exudation causes local ischemia and electrolyte disorders and results in stimulation of nerve root and vertebral sinus nerves, causing corresponding innervation zone pain. The pain causes local muscle contraction and protective lumbar scoliosis , and the physiological biomechanical changes takes place and aggravate pain, so resulting in a vicious cycle. Furthermore, muscle spasm can aggravate vertebral facet joint dysfunction, spinal canal stenosis and so on. IDD pathophysiological mechanism is still under investigation. With development of biomechanics and molecular "biology and tissue engineering , growth factor therapy, gene therapy, cell therapy and tissue engineering etc. provide some choice for the treatment of IDD.In the past, it is well accepted that IDD is a result of the biomechanical change, but recent studies have shown that biochemical factors also play an important role. MMPs are often reported in the development of IDD and provide a new sight in the pathological process. Intervertebral disc has a similar matrix components comparing with articular cartilage. It is reported that expression of MMPs and TIMP is similar in the normal cartilage , but the balance of MMPs and TIMP is interrupted in osteoarthritis and rheumatoid arthritis with a significantly higher MMPs activity and mRNA expression, and this imbalance results in cartilage damage . Tetracyclines and their derivatives are very effective inhibitors of MMPs, can inhibit MMP-1, MMP-3, MMP-13, MMP-2 and MMP-9 activity through different mechanisms. Studies have shown that tetracycline and their derivatives can inhibit collagenase and other MMPs effects and slow down the disc degeneration. And a large number of experimental study found that intervertebral disc degeneration are closely related with COL-2a , Col-1a , Aggrecan, CILP, TGF-β1, VDR, MMP-3, MMP-13, Timp-3, Timp-1 gene expression.The present investigation focused on the effects of intervention using the MMPs inhibitors and traditional Chinese medicine massage on rat IDD model. The general morphology was investigated by imaging and pathology, and real time PCR was used to study gene expression of degenerated intervertebral disc before and after the MMPs inhibitors and massage, respectively. This research aimed to reveal the mechanism of intervention and provide theoretical basis to quantify and standardize the treatment approach. PartⅠ: morphological impact of exogenous MMP inhibitors tetracycline and massage on lumbar IDD in rat modelAbstract Objective: To investigate the morphological impact of exogenous MMPs inhibitor tetracycline and massage on lumbar IDD in rat model, to elucidate mechanism of their inhibition in terms of imaging and morphology. Methods: 36 male SD rats were randomly divided into four groups: normal group, control group, tetracycline group and massage group. All the 36 rats were single-cage feed and the control, tetracycline,model group were modeled.4 weeks after modeling, subcutaneous injection of normal saline was administrated to control group daily, subcutaneous injection of tetracycline 25 mg to tetracycline group, and 10 minutes massage to the massage group daily, Respectively. All sustained two weeks. 3 rats were randomly selected in each group for CR-Xray before and 2weeks, 8 weeks after modeling.4 weeks, 3 rats were randomly selected in each group for lumbar CR-Xray before first injection of massage, anesthetized by 10% chloral hydrate through intraperitoneal injection. 8 weeks after modeling, rats were executed by overdose 10% chloral hydrate intraperitoneal injection. vertebral column were excided through dorsal meso-incision. the removed vertebral column were put on ice as soon as possible andL1/2, L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were procured. Half of L1/2 intervertebral disc tissue was fixed in 4% paraformaldehyde, decalcified in 5% nitric acid, embedded in paraffin, 5um thick section was obtained, and went on HE staining. OLYMPUSBH optical microscope was used for observation. The remaining intervertebral disc tissue were used for other experiments. Results: Two months after modeling, lumbar spine X-ray shown: tetracycline group had narrower intervertebral space, smaller foraminal, worse spine curvature and more obvious bone sclerosis than the other two groups after the intervention; massage group had better spine curvature, but narrower intervertebral space, smaller foraminal in some upper segment than the other two groups after the intervention; the control group showed no changes in physiological curvature, bone sclerosis in some anterior ventro-rvertebrae. No significant difference was found among the normal group, the tetracycline group and the massage group under optical microscope: nucleus pulposus is at the center, no shrinkage, enveloped by the peripheral annulus f ibrosus; the control group showed a slight shrinkage of nucleus pulposus. Conclusion: two months after modeling, X-ray imaging showed significantly changes and mild degeneration and indicated a trend of degeneration. From the X-ray and morphological study, the exogenous MMPs inhibitor-tetracycline, massage can inhabit the early stages of disc degeneration. PartⅡ: gene expression research of the impact of MMP inhibitors - tetracycline and massage on lumbar intervertebral disc degeneration in ratsAbstract Objective : To investigate gene expression of degenerated intervertebral disc treated the exogenous MMPs inhibitors and massage, to reveal mechanism of intervention from the genetic level and provide theoretical basis to further quantify and standardize the treatment approach. Methods: 36 male SD rats were randomly divided into four groups: normal group, control group, tetracycline group and massage group. All the 36 rats were single-cage feed and the control , tetracycline, model group were modeled. 4 weeks after modeling, subcutaneous injection of normal "saline was administrated to control group daily, subcutaneous injection of tetracycline 25 mg to tetracycline group, and 10 minutes massage to the massage group daily, Respectively.All sustained two weeks. 3 rats were randomly selected in each group for CR-Xray before and 2weeks, 8 weeks after modeling. 4 weeks, 3 rats were randomly selected in each group for lumbar CR-Xray before first injection of massage , anesthetized by 10% chloral hydrate through intraperitoneal injection. 8 weeks after modeling, rats were executed by overdose 10% chloral hydrate intraperitoneal injection. Vertebral column were excided through dorsal meso-incision. the removed vertebral column were put on ice as soon as possible and L1/2, L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were procured. The procured L2/3, L3/4, L4/5, L5/6 intervertebral disc tissue were stored in liquid nitrogen. Gene expression level of COL-2a, COL-1a, Aggrecan, CILP, IGF-(β1, VDR, MMP-3, MMP-13, Timp-3, Timp-1 in intervertebral disc were tested using real time PCR assay after treatment of MMPs inhibitors-tetracycline and massage.DPS9.5 statistical software package were used to do variance analysis and correlation coefficient analysis between scoliosis angle in posterior-anterior X-ray and associated gene expression. Results: 1. Statistical analysis was performed with statistical package DPS9. 50. Completely random designed one-way ANOVA ( LSD method ) was used for comparison between groups. (1) The expression of COL-1a showed no difference in normal group, chirismus group and tetracycline group (P > 0. 05) and significantly higher in control compared with other groups (P < 0. 01). (2) The expression of COL-2a was significantly higher in normal group compared with other groups (P < 0. 01). There was no difference between chirismus group and tetracycline group (P> 0. 05). The expression of COL-2a was significantly lower in control compared with chirismus group and tetracycline group (both P < 0.01). (3) The expression of aggrecan was significantly higher in normal group compared with other groups (P < 0.01) , significantly lower in control than those in chirismus group and tetracycline group (both P < 0.01), and significantly higher in chirismus group compared with tetracycline group (P<0. 01). (4) The expression of CILP was significantly lower in normal group compared with other groups (P< 0. 01) and showed no difference in control, chirismus group and tetracycline group (P>0. 05). (5) The expression of TGF-β1 was significantly higher in normal group compared with tetracycline group and control (both P<0. 01), significantly higher in chirismus group than those in tetracycline group and control (both P<0.01), and significantly higher in normal group compared with chirismus group (P<0.05). The expression of TGF-β1 showed no difference between tetracycline group and control (P>0.05). (6) The expression of VDR was significantly higher in normal group compared with other groups (P<0.01), and significantly lower in tetracycline group than those in chirismus group and control (both P<0.01). There was no difference between chirismus group and control (P>0.05). (7) The expression of MMP-3 was significantly lower in normal group compared with other groups (P<0.01) , significantly higher in control than those in chirismus group and tetracycline group (both P<0.01), and significantly higher in chirismus group compared with tetracycline group (P< 0.05). (8) The expression of MMP-13was significantly lower in normal group compared with other groups (P<0.05), significantly higher in control than those in other groups (P<0. 01), and significantly higher in tetracycline group compared with chirismus group (P < 0.05). (9) The expression of TIMP-1 was significantly lower in normal group compared with other groups (P＜0.01) , significantly higher in control than those in chirismus group and tetracycline group (both P<0.01), and significantly higher in tetracycline group compared with chirismus group (P <0.01). (10) The expression of TIMP-3 showed no difference between normal group and chirismus group (P>0.05).The expression of TIMP-3 was significantly higher in control compared with other groups (P<0.01) , and significantly lower in tetracycline group compared with other groups (P <0.01). 2. The means of Cobb angle of rat lumbar spine are 0°in normal group, 14°in control, 12°in tetracycline group, and 10°in chirismus group, respectively. Correlation analysis was performed under other grey system methods with statistical package DPS9. 50 (Table 5). Table 5 Correlation of Cobb angle of rat lumbar spine in anteroposterior radiograph with related genesConclusions: (1)Chirismus and tetracycline have a similar therapeutic effect to relieve the lumbar disc degeneration of rats via inhibiting the expression of COL-1a. The higher expression of COL-1a in control approves this point and is consistent to the literatures which report the increase of collagen I after IDD. (2)Chirismus and tetracycline can up-regulate the expression of C0L-2a and relieve the lumbar disc degeneration of rats , which is contrary to the low expression of COL-2a in control. (3)Chirismus has a stronger effect to induce the expression of aggrecan than tetracycline, which indicates chirismus has a better therapeutic effect on the lumbar disc degeneration of rats comparing with tetracycline. (4) The expression of CILP is significantly lower in normal group than other groups in which show no statistical difference. That means the animal model is valid because the expression of CILP is up-regulated after IDD. The up-regulation of TGF-β1 by chirismus indirectly shows that chirismus could inhibit the expression of CILP and relieve IDD. Tetracycline has a similar effect. (5)Chirismus could induce the expression of TGF-β1. The increase of TGF-β1 in intervertebral disc could promote cell proliferation and extracellular matrix synthesis as well as maintain the homeostasis of intervertebral disc for relieving the lumbar disc degeneration of rats. Tetracycline has no such effect. (6) The expression of VDR was significantly higher in chirismus group than tetracycline group. VDR could directly participate the differentiation, proliferation, and maturity of chondrocytes. There are abundant chondrocytes in the intervertebral disc and plenty aggrecan in the intercellular substance. Chirismus could up-regulate the expression of VDR and relieve the lumbar disc degeneration of rats via chondrocytes as well as decrease the possibility of vertebrae spur and disc stenosis, which is consistent to the imageological conclusions. (7)Tetracycline could down-regulate MMP-3 meanwhile up-regulate TIMP-1 to relieve IDD. Chirismus has a similar effect on regulation of these genes comparing with tetracycline and relieves IDD via inhibiting the degradation of extracellular matrix. (8)Tetracycline could down-regulate the expression of TIMP-3 via negative feedback and MMP-13 would be up-regulated. Chirismus could up-regulate TIMP-3 to approach normal group and MMP-13 would be down-regulated. Then chirismus inhibits the degradation of extracellular matrix and relieves IDD via down-regulating MMPs. (9) The expression of CILP is correlated highly witn Cobb angle meanwhile TGF-β1 has a low correlation with Cobb angle (Table 1). High expression of CILP and low expression of TGF-β1 inhibit matrix synthesis as well as destroy chondrocytes, which induce intervertebral joint degeneration that produce scoliosis. Here we conclude that the expressions of above genes all participate the synthesis and degradation of matrix. IDD has a close relation with extracellular matrix. MMPs inhibitors could relieve IDD via regulating related genes. Chirismus has a similar even sometimes better therapeutic effects.