复方丹参注射液治疗RA疗效及其干预CYR61对RA-FLS生物学行为的研究
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摘要
类风湿性关节炎(Rheumatoid Arthritis, RA)是最常见和致残率最高的骨关节疾病之一,正侵袭着全球所有种族数以千万计的人口,造成个人、家庭和社会的巨大损失。迄今为止,虽然RA的发病机制还不十分明确,但是它的作用“终端”靶组织很明确,就是滑膜。滑膜组织中的成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)的类肿瘤样生长及其侵蚀性是关节破坏的主要因素。如果能够从RA滑膜入手去研究和治疗,就可以避开纷乱繁杂的细胞和分子网络,直达“病处”。因此,如果能寻找到抑制RA-FLS的增生和侵蚀性的关键分子和重要机制,可以为RA的有效治疗提供新的药物靶点。通过前期的研究发现,高半胱氨酸蛋白61 (cysteine-rich61, CYR61)在RA患者的滑膜组织、FLS和关节液中均高表达,并且能促进IL-17介导FLS增殖。CYR61能调控肿瘤细胞的增殖和侵袭力,而这些功能恰与RA-FLS增殖性和侵蚀性相似。
RA属中医“痹证”范畴。中医药治疗痹证历史悠久,几千年来在风湿病的防治方面收到了一定的疗效,很多中药和中成药在治疗RA不仅有着非常好的疗效,而且副作用较少,费用较低。广州军区广州总医院沈鹰教授在国内首次将复方月参注射液用于治疗RA等风湿性疾病,取得明显的临床疗效。并通过动物实验,初步从自由基、血流变、滑膜病理、动物模型的滑膜细胞凋亡、细胞因子及基因表达等多角度阐述了复方月‘参注射液治疗RA的部分机制,但复方丹参注射液对于RA-FLS和RA关节软骨侵蚀方面的作用和机制尚不明确。因此亟待能够在基础方面进一步研究,明确复方丹参注射液在RA滑膜增生和软骨侵蚀中的作用,为中医药治疗RA等风湿病的临床推广和药物研发奠定基础。
目的：
本课题正是基于以上工作基础和背景进行立题,旨在研究复方丹参注射液治疗RA的临床疗效和其干预CYR61对于RA-FLS增殖、凋亡和侵袭力的生物学行为影响,旨在获得CYR61直接参与RA-FLS侵袭性的可靠证据及复方丹参注射液的干预机制,为复方丹参注射液治疗RA的应用和确立CYR61作为RA研究和治疗的新靶点提供科学依据。
方法：
1.应用随机对照方法观察了212例复方丹参注射液治疗活动期类风湿关节炎20天的临床疗效及安全性。
2.获取RA和正常人滑膜的活组织,然后进行分离和培养,获得纯化的FLS,并应用流式细胞仪对细胞表面标志物进行鉴定。
3.构建CYR61慢病毒载体,然后进行CYR61慢病毒载体的包装和FLS转染。同时应用RNA干扰(RNA interference, RNAi)技术,合成CYR61特异性小干扰RNA(smallinterference RNA, siRNA)瞬时转染RA-FLS。观察以上转染效率和FLS中CYR61表达情况。
4.应用MTS法检测各组FLS增殖率；
5.应用Tunel法检测各组FLS凋亡率；
6.应用Transwell法检测各组FLS侵袭能力,并应用ELISA检测各组细胞液中MMP-1,3,10,13水平；
7.应用以上方法观察复方丹参注射液对RA-FLS中CYR61基因和蛋白表达以及RA-FLS增殖、凋亡和侵袭力的干预作用。
结果：
1.临床前瞻性研究结果显示,复方丹参注射液治疗组治疗后20天与治疗前比较,触痛关节数、肿胀关节数、晨僵时间、双手握力,患者疼痛程度评估、患者对疾病的总体评估、医生对疾病的总体评估、患者的躯体活动功能(HAQ)、28关节病情活动度评分(DAS28)、血沉(ESR)、C反应蛋白(CRP)和类风湿因子(RF)均有明显改善(P<0.05)。复方丹参注射液治疗组患者治疗后晨僵时间、双手握力、患者疼痛程度评估、患者对疾病的总体评估、医生对疾病的总体评估、DAS28和HAQ比常规治疗组患者均有明显的改善(P<0.05)；治疗组患者ACR20改善率(79.72%)明显高于对照组患者(59.43%)(P<0.05)；两组患者的不良反应及安全性指标无显著性差异(P>0.05)。
2.本研究通过流式细胞仪对3代RA和正常人滑膜细胞表面标记物CD90和CD14表达阳性率检测,发现CD90的阳性率分别为93.43%和96.94%,CD14的阳性率分别是0.22%和0.13%。从检测的结果看滑膜细胞CD90的阳性率接近98%,获得了实验需要的FLS。
3.FLS转染实验结果显示,两组转染效率都在90%以上。通过Q-PCR和Wosternb1ot检测来看,正常人FLS过表达CYR61后,CYR61在FLS中的mRNA和蛋白表达量均明显高于正常人FLS和RA-FLS(P<0.05);同时,通过CYR61-siRNA干扰RA-FLS后,RA-FLS表达CYR61的mRNA和蛋白非常微量,明显低于其他FLS(P<0.05),抑制效率达到89.14%。本研究对CYR61的过表达和低表达的转染效率均很高,为下一步的功能研究奠定了基础。
4.各组FLS增殖情况结果显示,RA-FLS通过转染CYR61-siRNA第5天后,细胞增殖率明显低于正常RA-FLS(P<0.05)；正常FLS转染CYR61过表达载体5天后增殖率比正常FLS明显增加(P<0.05)。结果还显示,第5天的FLS细胞增殖率与细胞CYR61的mRNA和蛋白水平均呈正相关(P<0.01),这说明FLS的增殖与CYR61的mRNA和蛋白表达水平有关。
5.本研究结果还显示,RA-FLS通过转染CYR61-siRNA后,细胞凋亡率明显高于于RA-FLS和空转染RA-FLS(P<0.05)；正常FLS转染CYR61过表达载体后,细胞凋亡率明显低于其他各组(P<0.05)。FLS细胞凋亡率与细胞CYR61的mRNA和蛋白水平均呈负相关(P<0.01),这说明FLS的凋亡与CYR61的mRNA和蛋白表达水平有关。
6.各组FLS侵袭能力结果显示,RA-FLS侵袭力明显强于正常人FLS(P<0.05),RA-FLS通过转染CYR61-siRNA后,细胞侵袭力明显低于其他各组(P<0.05)；正常FLS转染CYR61过表达载体后,细胞侵袭力明显高于其他各组(P<0.05)。结果还显示,FLS细胞侵袭力与细胞CYR61的mRNA和蛋白水平均呈正相关(P<0.01)。同时,应用ELISA对各组细胞液中MMP-1,3,10,13水平进行检测,最后发现,MMP-3与CYR61表达水平和细胞侵袭力相关(P<0.05)
7.本研究结果显示,与正常RA-FLS和小剂量复方丹参注射液比较,大剂量复方月参注射液能抑制RA-FLS增殖和CYR61mRNA和蛋白的表达,促进RA-FLS凋亡,并且具有明显抑制RA-FLS侵袭力(P<0.05)
结论：
1.复方丹参注射液在治疗活动期RA中,具有增效作用,无不良反应。
2.CYR61可能是通过促进RA-FLS增殖、抗凋亡达到滑膜增生,再通过增进RA-FLS表达MMP-3增强其侵袭力达到破坏关节软骨的作用。
3.大剂量复方丹参注射液丹参注射液能抑制RA-FLS增殖和CYR61表达,促进其凋亡,并且抑制RA-FLS侵袭力,着可能是复方丹参注射液通过这些作用能够起到抑制RA滑膜增生和关节软骨破坏的作用。
本课题的特色和创新性：
1.提出了CYR61对于RA-FLS侵袭行为影响的假说,通过慢病毒感染和RNA干扰技术,证明了CYR61基因能调控RA-FLS增殖、调亡和侵袭力,可能是RA研究和治疗的潜在靶点。
2.本研究还发现复方丹参注射液注射液能抑制RA-FLS中CYR61表达,同时干预RA-FLS增殖、凋亡和侵袭力,这可能是复方丹参注射液治疗RA滑增生和软骨侵蚀的作用机制之一。
本研究初步阐述了CYR61可能成为RA未来研究和治疗新的重要靶点,也为复方丹参注射液在保护RA关节方面的应用提供科学依据,在评价中药抗RA的药效及抑制软骨破坏机理研究提供了一个实验平台和参考模式,有助于进一步推广中医药在治疗RA等免疫疾病中的应用。
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 RA
This 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.
RA属中医“痹证”范畴。中医药治疗痹证历史悠久,几千年来在风湿病的防治方面收到了一定的疗效,很多中药和中成药在治疗RA不仅有着非常好的疗效,而且副作用较少,费用较低。广州军区广州总医院沈鹰教授在国内首次将复方月参注射液用于治疗RA等风湿性疾病,取得明显的临床疗效。并通过动物实验,初步从自由基、血流变、滑膜病理、动物模型的滑膜细胞凋亡、细胞因子及基因表达等多角度阐述了复方月‘参注射液治疗RA的部分机制,但复方丹参注射液对于RA-FLS和RA关节软骨侵蚀方面的作用和机制尚不明确。因此亟待能够在基础方面进一步研究,明确复方丹参注射液在RA滑膜增生和软骨侵蚀中的作用,为中医药治疗RA等风湿病的临床推广和药物研发奠定基础。
目的：
本课题正是基于以上工作基础和背景进行立题,旨在研究复方丹参注射液治疗RA的临床疗效和其干预CYR61对于RA-FLS增殖、凋亡和侵袭力的生物学行为影响,旨在获得CYR61直接参与RA-FLS侵袭性的可靠证据及复方丹参注射液的干预机制,为复方丹参注射液治疗RA的应用和确立CYR61作为RA研究和治疗的新靶点提供科学依据。
方法：
1.应用随机对照方法观察了212例复方丹参注射液治疗活动期类风湿关节炎20天的临床疗效及安全性。
2.获取RA和正常人滑膜的活组织,然后进行分离和培养,获得纯化的FLS,并应用流式细胞仪对细胞表面标志物进行鉴定。
3.构建CYR61慢病毒载体,然后进行CYR61慢病毒载体的包装和FLS转染。同时应用RNA干扰(RNA interference, RNAi)技术,合成CYR61特异性小干扰RNA(smallinterference RNA, siRNA)瞬时转染RA-FLS。观察以上转染效率和FLS中CYR61表达情况。
4.应用MTS法检测各组FLS增殖率；
5.应用Tunel法检测各组FLS凋亡率；
6.应用Transwell法检测各组FLS侵袭能力,并应用ELISA检测各组细胞液中MMP-1,3,10,13水平；
7.应用以上方法观察复方丹参注射液对RA-FLS中CYR61基因和蛋白表达以及RA-FLS增殖、凋亡和侵袭力的干预作用。
结果：
1.临床前瞻性研究结果显示,复方丹参注射液治疗组治疗后20天与治疗前比较,触痛关节数、肿胀关节数、晨僵时间、双手握力,患者疼痛程度评估、患者对疾病的总体评估、医生对疾病的总体评估、患者的躯体活动功能(HAQ)、28关节病情活动度评分(DAS28)、血沉(ESR)、C反应蛋白(CRP)和类风湿因子(RF)均有明显改善(P<0.05)。复方丹参注射液治疗组患者治疗后晨僵时间、双手握力、患者疼痛程度评估、患者对疾病的总体评估、医生对疾病的总体评估、DAS28和HAQ比常规治疗组患者均有明显的改善(P<0.05)；治疗组患者ACR20改善率(79.72%)明显高于对照组患者(59.43%)(P<0.05)；两组患者的不良反应及安全性指标无显著性差异(P>0.05)。
2.本研究通过流式细胞仪对3代RA和正常人滑膜细胞表面标记物CD90和CD14表达阳性率检测,发现CD90的阳性率分别为93.43%和96.94%,CD14的阳性率分别是0.22%和0.13%。从检测的结果看滑膜细胞CD90的阳性率接近98%,获得了实验需要的FLS。
3.FLS转染实验结果显示,两组转染效率都在90%以上。通过Q-PCR和Wosternb1ot检测来看,正常人FLS过表达CYR61后,CYR61在FLS中的mRNA和蛋白表达量均明显高于正常人FLS和RA-FLS(P<0.05);同时,通过CYR61-siRNA干扰RA-FLS后,RA-FLS表达CYR61的mRNA和蛋白非常微量,明显低于其他FLS(P<0.05),抑制效率达到89.14%。本研究对CYR61的过表达和低表达的转染效率均很高,为下一步的功能研究奠定了基础。
4.各组FLS增殖情况结果显示,RA-FLS通过转染CYR61-siRNA第5天后,细胞增殖率明显低于正常RA-FLS(P<0.05)；正常FLS转染CYR61过表达载体5天后增殖率比正常FLS明显增加(P<0.05)。结果还显示,第5天的FLS细胞增殖率与细胞CYR61的mRNA和蛋白水平均呈正相关(P<0.01),这说明FLS的增殖与CYR61的mRNA和蛋白表达水平有关。
5.本研究结果还显示,RA-FLS通过转染CYR61-siRNA后,细胞凋亡率明显高于于RA-FLS和空转染RA-FLS(P<0.05)；正常FLS转染CYR61过表达载体后,细胞凋亡率明显低于其他各组(P<0.05)。FLS细胞凋亡率与细胞CYR61的mRNA和蛋白水平均呈负相关(P<0.01),这说明FLS的凋亡与CYR61的mRNA和蛋白表达水平有关。
6.各组FLS侵袭能力结果显示,RA-FLS侵袭力明显强于正常人FLS(P<0.05),RA-FLS通过转染CYR61-siRNA后,细胞侵袭力明显低于其他各组(P<0.05)；正常FLS转染CYR61过表达载体后,细胞侵袭力明显高于其他各组(P<0.05)。结果还显示,FLS细胞侵袭力与细胞CYR61的mRNA和蛋白水平均呈正相关(P<0.01)。同时,应用ELISA对各组细胞液中MMP-1,3,10,13水平进行检测,最后发现,MMP-3与CYR61表达水平和细胞侵袭力相关(P<0.05)
7.本研究结果显示,与正常RA-FLS和小剂量复方丹参注射液比较,大剂量复方月参注射液能抑制RA-FLS增殖和CYR61mRNA和蛋白的表达,促进RA-FLS凋亡,并且具有明显抑制RA-FLS侵袭力(P<0.05)
结论：
1.复方丹参注射液在治疗活动期RA中,具有增效作用,无不良反应。
2.CYR61可能是通过促进RA-FLS增殖、抗凋亡达到滑膜增生,再通过增进RA-FLS表达MMP-3增强其侵袭力达到破坏关节软骨的作用。
3.大剂量复方丹参注射液丹参注射液能抑制RA-FLS增殖和CYR61表达,促进其凋亡,并且抑制RA-FLS侵袭力,着可能是复方丹参注射液通过这些作用能够起到抑制RA滑膜增生和关节软骨破坏的作用。
本课题的特色和创新性：
1.提出了CYR61对于RA-FLS侵袭行为影响的假说,通过慢病毒感染和RNA干扰技术,证明了CYR61基因能调控RA-FLS增殖、调亡和侵袭力,可能是RA研究和治疗的潜在靶点。
2.本研究还发现复方丹参注射液注射液能抑制RA-FLS中CYR61表达,同时干预RA-FLS增殖、凋亡和侵袭力,这可能是复方丹参注射液治疗RA滑增生和软骨侵蚀的作用机制之一。
本研究初步阐述了CYR61可能成为RA未来研究和治疗新的重要靶点,也为复方丹参注射液在保护RA关节方面的应用提供科学依据,在评价中药抗RA的药效及抑制软骨破坏机理研究提供了一个实验平台和参考模式,有助于进一步推广中医药在治疗RA等免疫疾病中的应用。
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 RA
This 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.
引文
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