The Study On The Effect Of New Compound Traditional Chinese Medicine On Lumbar Ligament Cells In Rabbits

Low back pain has emerged as a popular problem faced by humans and more and young people are involved in this problem.Low back pain basically results from intervertebral disc degeneration,and lumbar segment instability is the main factors leading to intervertebral disc degeneration.Low back pain anatomically involves bone,ligament and intervertebral disc,and ligaments act to maintain the stability of spine.The clinical treatment mostly uses surgical methods.These methods may damage such soft tissues as ligaments around the intervertebral disc.Moreover,these methods just relieve the patient’s pain by removing the herniation so as to reduce the compression to nerve roots.In a long run,the surgical removal of the intervertebral disc destroys the mechanical environment of the entire spine and may bring a series of problems such as instability of the lumbar spine and prominent recurrence.To solve the problem of low back pain from the root cause requires a systematic perspective.The Chinese medicine theory emphasizes systematically curing the diseases.It believes that the root causes for low back pain include blood stasis,stagnation,cold,and function defects of liver and kidney.Based on this theory,a compound traditional Chinese medicine（CTCM）was self-formulated to cure the low back pain through nourishing liver and kidney,strengthening muscles and bones,dispelling cold and dampness,promoting blood circulation and removing blood stasis.Our previous study has verified the effectiveness of this CTCM in restoring the functions of nucleus pulposus and the mechanical properties of ligaments.The purpose of this study is to clarify the underlying mechanism at a cellular level.To this aim,CTCM was applied to lumbar interspinous ligament fibroblasts（ILFs）and hypoxic-damaged ILFs and then the effects of CTCM on ILFs and the protective effect of CTM on hypoxic-damaged ILFs were investigated.A commercialized Chinese medicine Shu Jin Jian Yao Wan was employed as a positive.The main works and conclusions are summarized as follows:（1）Extraction,culture and identification of rabbit ILFsRabbit ILFs were isolated from the rabbit lumbar interspinous ligament tissue using the tissue block culture method.Immunofluorescence staining of collagen type I,type III and vimentin were conducted on ILFs of the third generation.The results indicate that the cells strongly expressed the three proteins,suggesting that the isolated cells are ILFs and can be employed for the following cell experiments.（2）The effects of compound Chinese medicine on ILFs secretion of extracellular matrix and its degrading enzymes,endogenous growth factors and cell migration were detectedThrough cytotoxicity experiment,the concentration of CTCM and Shu jin jian yao wan were screened out,and the effective concentration of CTCM and Shu jin jian yao wan were 200μg/mL and 30μg/mL respectively.Using RT-PCR,Western blot and immunofluorescence staining to detect the changes of extracellular matrix and its degrading enzymes and growth factors in ILFs after treating with CTCM and Shu jin jian yao wan.Cell scratch experiment was used to detect the changes of ILFs migration after treatment.The treatment of CTCM and Shu jin jian yao wan can obviously promote the secretion of collagen typeⅠ,Ⅲand elastin in the extracellular matrix of ILFs.The growth factors EGF,bFGF and MGF mRNA in ILFs have a significant up-regulation effect and can promote the cells Phosphorylation of mTOR and cell migration.In the later period,CTCM and Shu jin jian yao wan can significantly increase the expression levels of MMP-2 and MMP-9 mRNA in ILFs.（3）Establish hypoxia injury modelThere were 6 groups:the control group and other 5 groups treated with CoCl₂medium containing final concentrations of 30,50,100,200,and 500μM for 24 hours to detect the expression of HIF-1α,MMP-2,MMP-9 and VEGF.Then,the same group was used to test the cell ability by CCK-8.It was concluded that 100μM CoCl₂ is the appropriate concentration for establishing a hypoxic injury model.（4）To detect the effect of CTCM on CoCl₂-induced hypoxia injury of ILFsThere were 4 groups:control group（DMEM medium）,CoCl₂ hypoxic injury group（100μM CoCl₂）,CTCM group（100μM CoCl₂+final concentration of 200μg/mL CTCM）and the Shu jin jian yao wan group（100μM CoCl₂+30μg/mL Shu Jin Jian Yao Wan）.The mRNA expression of MMP-9,TIMP-1 and VEGF was detected,and the ability of cell migration was detected by wound healing test.After stimulated by hypoxia,HIF-1αprotein is low or not expressed,and VEGF is highly expressed.Hypoxia stimulation significantly increases the mRNA expression of MMP-2 and MMP-9 in ILFs,and reduces the expression of TIMP-1.CTCM and Shu Jin Jian Yao Wan can effectively regulate the imbalance of MMPs/TIMPs and promote angiogenesis of ILFs under hypoxic conditions.In conclusion,the result of this study is that the new compound Chinese medicine can upregulate the secretion of extracellular matrix and growth factors of lumbar ligament cells and regulate the imbalance of the expression of extracellular degradation enzymes and its inhibitors caused by hypoxia,promoting its angiogenesis,which may be the mechanism of the new compound Chinese medicine to repair the intervertebral disc and ligament at the cellular and molecular level in animal experiments.Thua,the fusion of Chinese and Western medicine for the disease has taken an important step forward.