| Rheumatoid arthritis (RA) is one of the most common osteoarthropathia which induces joint destruction. It not only affects tens of thousands people all of the world, but also brings tremendous losts to individuals, families and society. Untill now, although the pathogenesis is unclear, the target tissue it acts on is synovialis. The main factor of the joint destrucion is the tumor-like proliferation and invasion of the fibroblast-like synoviocytes (FLS) in the synovial tissue. It leads to the key point if researchs and treatments can be carried out on the base of synovialis instead of the complicated cellular and molecular network. Therefore, new drug target can be found if the molecule and mechanism that RA-FLS are suppressed can be discovered. We find that cysteine-rich 61(CYR61) is high expressed in the synovialis, FLS and joint fluid, it also can induced proliferation of FLS by IL-17. CYR61 is known as an important player in tumor cells progression and invasion, which is similar as the function of RA-FLS.RA is included in the "Bi Zheng" in Traditional Chinese Medicine with a long history. Chinese herbs play an important role in the prevention and treatment of rheumatoid diseases, there are many Chinese herbal extracts and Chinese herbal medicine have perfect therapeutic effects, low side-effects and few costs. Professor Shen Ying in Guangzhou Hospital of Guangzhou Military Command is the first who applys Compound Salvia Injection in treating rheumatoid diseases such as RA, which has achieved high curative effect. Part of the mechanism of action from the points of free radical, hemorheology, synovial pathology, apoptosis of synovial cell in animal model, cell factor and gene expression are elaborated through animal experiments, but the mechanism of its action on RA-FLS and destruction of joint cartilage is still unclear. So it is urge to make further research to explicit the role of Compound Salvia Injection in synovial proliferation and cartilage destruction with the aim to put the foundation for clinical application, drug research and development of Chinese medicine in treating rheumatoid diseases such as RA.Objection:This research is based on the work foundation and background above, aiming at exploring the biological behavior of CYR61 on proliferation, apoptosis and invasion of RA-FLS. The research also explores the intervention of Compound Salvia Injection on the process above and find the evidence on the relationship between CYR61 and invasion of RA-FLS, therefore it can provide scientific evidence on CYR61 as new target on the application of Compound Salvia Injection in treating patients with RA.Methods:1. To observe the therapeutic effect and safety of Compound Salvia Injection in treating 212 patients with RA for the course of 20 days.2. The synovial tissue of RA and healthy people are seperated and cultivated to achieve pure FLS, the cellular surface markers are identified by flow cytometer.3. To construct the lentiviral vector of CYR61 and transfect it to the FLS. To synthesize the small interference RNA(siRNA) and make transient transfection of FLS by the technology of RNA interference(RNAi), observing the transfection efficiency and expression of CYR61.4. To detect the proliferation ratio of FLS by MTS.5. To detect the apoptosis ratio of FLS by Tunel.6. To detect the invasion of FLS by Transwell method and detect the level of MMP-1,3,10,13 by ELISA.7. To investigate the interference of Compound Salvia Injection on CYR61 mRNA and protein expression of RA-FLS and proliferation, apoptosis, invasive ability of RA-FLS by the methods mentioned above.Results:1. Clinical perspecive research shows that the tender joint count, swollen joint count, time of morning stiffness, grip strength, patient's assessment of pain, patient's assessment of disease activity, physician's assessment of disease activity, patient's assessment of disability (index of the Health Assessment Questionnaire [HAQ]), Disease Activity Score in 28 joints(DAS28), erythrocyte sedimentation rate (ESR), C reactive protein (CRP) and rheumatoid factor(RF) are improved after the treatment of Compound Salvia Injection(P <0.05). Morning stiffness, grip strength, patient's assessment of pain, patient's assessment of disease activity, physician's assessment of disease activity, patient's assessment of disability, DAS28 and HAQ are improved comparing with the control group treating with conventional therapy (P<0.05). The rate of ACR20 of treated group (79.72%) is higher than control group(59. 43%) (P<0.05). The occurrence of adverse events and clinical laboratory test abnormalities showed no significant difference between the two groups (P> 0.05). Compound Salvia Injection shows good efficacy with few adverse events in treating patients with active RA.2. Flow cytometry was used to determine the positive rate of CD90 and CD40, which are cellular surface markers of synovialis of passage-3 RA and healthy people. The research finds that the positive rate of CD90 and CD40 are 93. 43% and 96.94% seperately. The FLS the experiment needs are achieved because the positive rate of CD90 of synovial cells are almost 90%.3. The experiment of FLS transfection shows that, the transfection rates of the two groups are over 90%. The results of Q-PCR and Western blot shows that mRNA and protein expression of CYR61 in FLS that the CYR61 is over-expressed is significantly higher than normal FLS and RA-FLS(P<0.05). After the interference of CYR61-siRNA to RA-FLS, the mRNA and protein expression significant lower than other FLS(P<0.05), the suppression rate is almost 89.1%. The tansfection rate is very high, which lay the foundation for the research followed.4. The proliferation of the FLS shows that, the 5th day after the RA-FLS transfecting CYR61-siRNA, the rate of cell proliferation is significant lower than common RA-FLS(P<0.05). The rate of cell proliferation is significant higher than common FLS 5 days after CYR61 over-expression vector transfecting common FLS (P<0.05). The result also shows that the cell proliferation rate of FLS is positive correlated with level of mRNA and protein of CYR61 on the 5th day(P<0.01), which illuminated that FLS proliferation relates with expression rate of mRNA and protein of CYR61. 5. The result shows that, after RA-FLS transfecting CYR61-siRNA, the rate of apoptosis is significant lower than RA-FLS and empty-transfected RA-FLS (P<0.05). The apoptosis rate of common FLS is significant higher than other groups after CYR61 transfected (P<0.05). The apoptosis rate of FLS cells is negtive-related with level of mRNA and protein of CYR61 (P<0.01), which illuminated FLS apoptosis has relation with expression level of CYR61 mRNA and protein.6. The result also shows that, the invasive ability of RA-FLS is significant stronger than common FLS (P<0.05). After transfected by CYR61-siRNA, invasive ability of common FLS is significant lower than other groups(P<0.05). After transfected by CYR61 over-expressed vector, the invasive ability of cells is significantly higher than other groups. The result also shows that, the invasive ability of FLS positively relates with level of expression of mRNA and protein of CYR61 (P<0.01), which implys invasive ability of FLS is related with expression level of mRNA and protein of CYR61. At the same time, though detecting MMP-1,3,10,13 level of cell sap in each groups by ELISA, it shows that only MMP-3 relates with express level and invasive ability of CYR61 (P<0.05)7. The research also shows that, comparing with RA-FLS and low dose Compound Salvia Injection, high dose Compound Salvia can suppress proliferation of RA-FLS, inducing apoptosis and significantly suppress the invasive ability of RA-FLS(P<0.05), which is similar with MTX.Conclusion:1. Compound Salvia Injection shows good therapeutic effects with few adverse effects.2. The mechanism of CYR61 may enhance proliferation and anti-apoptosis of RA-FLS and promoting MMP-3 expression of RA-FLS which can intensify its invasive ability to cause destruction of cartilage.3. High dose Compound Salvia Injection can suppress the proliferation and invasive ability of RA-FLS, and can induce its apoptosis to suppress the synovial proliferation and cartilage destruction of RA. The feature and innovation of this research:1. The hypothesis is brought that CYR61 influences the invasive behavior of RA-FLS. BY lentiviral vector transfection and RNA interference, it shows that CYR61 gene can regulate the proliferation, apoptosis and invasive ability of RA-FLS.2.It is found that Compound Salvia Injection can suppress the expression of CYR61 of RA-FLS and regulate the proliferation, apoptosis and invasive ability of RA-FLS. this may be one of the mechanisms of its treatment on synovial hyperplasia and cartilage erosion of patients with RAThis research illustrates that CYR61 may be the new important target of research and treatment of RA in the future, which provide scientific evidence for application of Compound Salvia Injection in preventing cartilage destruction of RA. It also provide an experimental evidence and reference pattern for estimating therapeutic effect and investigating the mechanism of suppression of cartilage destruction of Chinese medicine in treating RA, which helps improving the application of Chinese medicine in treating rheumatoid diseases.
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