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sks803

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目的：
以外周血、滑膜组织和以CIA大鼠的膝关节滑膜组织，外周血,为主要研究对象，以关节组织的病理变化，关节积分，外周血中的IL-1，IL-6，SOD,MDA.GSH-Px含量的高低，滑膜组织中血管内皮生长因子（VEGF）中的两个受体FLT-4和FLK-1,以及缺氧诱导因子-1α（HIF－1α）等细胞因子的变化为主要的检测指标，并与雷公藤多苷做对照，探讨祛痹方对RA的治疗作用，并从其治疗的效果中揭示RA的作用机理，从而寻找治疗RA的更为有效的治疗方案。
方法:
选用清洁级健康雄性SD大鼠72只，适应性喂养1周后，随机选取12只作为正常对照组，其余60只采用尾根部皮下注射Ⅱ型胶原与弗氏完全佐剂的方法诱导出关节炎模型，具体方法为：取适量CⅡ溶液（浓度为2mg/ml）逐滴加入至等容积的不完全弗氏佐剂中，CⅡ终浓度为1mg/ml。冰浴中用匀浆器充分乳化，以滴加水中不扩散为度，取乳化后的混合物按0.2ml /只，即200μg CⅡ/只，于尾根部皮下注射。7d后按0.1ml /只，即100μg CⅡ/只，于尾根部皮下加强免疫一次，10天后即可诱导出关节炎模型。免疫15d以后，将60只CIA大鼠随机分为4组：CIA模型组、雷公藤多苷组、祛痹方高剂量组、祛痹方低剂量组，每组各15只。并开始灌胃给药，每天1次，连续4 w。各组给药量如下：祛痹方高剂量组：6.66 g/kg•d；祛痹方低剂量组：3.33 g/kg•d；雷公藤多苷组：9.68 mg/kg•d；正常对照组和CIA模型组大鼠灌服等体积纯净水。观察动物的关节炎指数，关节肿胀度。治疗四周后动物处死，光镜下观察大鼠踝关节的病理损伤情况；采用ELISA方法测定动物血清中抗Ⅱ型胶原抗体、IL-1，IL-6的水平；采用比色法测定血清中SOD、MDA 和GSH-Px 活性；采用免疫组化的方法检测动物膝关节滑膜组织中血管内皮生长因子（VEGF）中的两个受体FLT-4和FLK-1,以及缺氧诱导因子-1α（HIF－1α）等细胞因子的变化。
结果：
关节损伤的结果显示：与正常对照组相比，免疫后15 d大鼠关节炎指数显著增加（P<0.01），而各组造模大鼠关节炎指数无明显差异。给药后，与模型组比较，雷公藤多苷组和祛痹方组大鼠关节炎指数有降低趋势，但无统计学意义。CIA模型组大鼠关节肿胀度明显高于正常对照组（P<0.01）；与CIA模型组相比，雷公藤多苷组、祛痹方高剂量组大鼠关节肿胀度明显降低（P<0.01），且祛痹方高剂量组大鼠关节肿胀度明显低于雷公藤多苷组（P<0.05）
光镜下可见可见明显的滑膜细胞增生，排列紊乱，滑膜组织充血水肿，毛细血管增生，并可见炎细胞浸润；增生的滑膜组织形成绒毛状，可伸向关节腔深处，或向软骨面爬行形成血管翳。在血管翳覆盖下的软骨表层可见明显的软骨表层组织的变性、坏死。关节软骨面剥脱，可见关节腔内有脱剥的关节软骨及滑膜组织。
治疗后滑膜细胞增生减轻，滑膜组织充血水肿明显减轻，血管增生和浸润炎细胞数量减少，血管翳形成显著减少。关节软骨面可见少量扁平层剥脱，剥脱软骨面平整，软骨下骨的骨小梁大小、排列基本正常。与模型组比较，祛痹方高剂量组与雷公藤多苷组大鼠关节损伤均明显减轻（P< 0.01，P< 0.05）；祛痹方高剂量组与雷公藤多苷组相比，病理积分明显降低（P<0.05）
采用ELISA方法检测血清中细胞因子水平的结果表明：与正常对照组比较，CIA模型组大鼠血清抗II型胶原抗体、IL-1β、IL-6水平明显升高；雷公藤多苷组和祛痹方高剂量组大鼠血清抗II型胶原抗体、IL-1β、IL-6水平较模型组明显降低；祛痹方低剂量组大鼠血清抗II型胶原抗体水平与CIA模型组比较无显著性差异。与雷公藤多苷组比较，祛痹方高剂量组抗II型胶原抗体、IL-1β含量明显降低，祛痹方低剂量组大鼠血清IL-1β水平明显下降（P<0.05），而IL-6水平明显下降，但无统计学意义。
采用比色法检测血清中GSH-Px、SOD和MDA活性的影响表明：与正常对照组比较，CIA模型组大鼠血清GSH-Px、SOD 活性明显下降（P<0.05，P<0.01），MDA活性明显升高（P<0.01）。雷公藤多苷和高低剂量祛痹方均能使大鼠血清GSH-Px活性明显升高、MDA活性明显下降（P<0.05，P<0.01），雷公藤多苷和高剂量祛痹方亦能使大鼠血清SOD活性明显升高（P<0.01）。
通过免疫组化检测滑膜组织中血管内皮生长因子（VEGF）中的两个受体FLT-4和FLK-1,结果表明：与正常对照组比较，模型组大鼠血清FLK-1、FLT-4水平明显升高（P<0.01），而雷公藤多苷组和祛痹方高剂量组大鼠血清FLK-1水平较模型组明显降低（P<0.05，P<0.01）；且祛痹方高剂量组大鼠血清FLK-1较雷公藤多苷组比较明显降低，雷公藤多苷组和祛痹方高剂量组大鼠血清FLK-1水平较模型组明显降低（P<0.01，P<0.05）；祛痹方低剂量组大鼠血清FLK-1、FLT-4水平明显下降，但无统计学意义。
缺氧诱导因子-1α（HIF－1α）的检测结果表明：与正常对照组比较，模型组大鼠血清HIF－1α水平明显升高（P<0.01），而雷公藤多苷组和祛痹方高剂量组大鼠血清HIF－1α水平较模型组明显降低（P<0.01）；祛痹方低剂量组大鼠血清HIF－1α水平明显下降，但无统计学意义。
结论：
袪痹方可以显著减轻CIA大鼠踝关节肿胀程度和关节炎指数，对CIA大鼠具有明显的治疗作用，其疗效要优于雷公藤多苷。
祛痹方可以显著降低血清中抗II型胶原抗体水平，减少免疫复合物的形成，抑制CIA大鼠异常的自身免疫反应，并通过下调血清中IL-1、IL-6、MDA的含量，上调GSH-Px、SOD的水平，调节机体免疫状态，改善关节滑膜炎性病变，改善体内氧自由基代谢，减少骨破坏，从而达到治疗CIA的作用。
袪痹方可以通过降低滑膜组织中血管内皮生长因子（VEGF）中的两个受体FLT-4和FLK-1以及HIF－1α的含量，从而抑制新生血管形成和滑膜细胞增生，改善关节滑膜炎性病变，达到治疗CIA的作用。

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wangzhengyun

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Objective:
Peripheral blood and synovial tissue of CIA rats knee synovial tissue and peripheral blood, as the main object of study, the pathological changes of the joint tissue, joint points in the peripheral blood of IL-1, IL-6 and SOD in two in the level of MDA.GSH-Px content in synovial tissue vascular endothelial growth factor (VEGF) receptor FLT-4 and of FLK was-1, and hypoxia-inducible factor-1α (HIF-1α), cytokines changes for the major indexes, and more than Tripterygium glycosides as control Qubi side of the RA treatment, and reveal the mechanism of RA from the effects of treatment, in order to find more effective treatment for the treatment of RA programs.
Methods:
Selection of clean grade healthy male SD rats 72, one week after the feeding of the adaptability of randomly selected 12 as the normal control group, the remaining 60 of the base of the tail subcutaneous injection of type Ⅱ collagen and Freund's complete adjuvant-induced arthritis model, specific methods for: the proper amount of C II solution (concentration 2mg/ml) was added dropwise to equal volume of incomplete Freund's adjuvant, C II, a final concentration of 1mg/ml. Ice bath using emulsified with homogenizer for the degree of non-proliferation, to the dropping water emulsified in the mixture according to 0.2ml / only 200μg of C Ⅱ / only, subcutaneous injection in the base of the tail. After 7 days, 0.1ml /, subcutaneous 100μg C II / only at the base of the tail, strengthen the immune once the 10 days can be induced arthritis model. 15d after immunization, 60 of CIA rats were randomly divided into four groups: high dose group of the CIA model group, Tripterygium Glycosides group, Qubi square Qubi side low dose group, 15 in each group. And begin gavage once daily for four consecutive w. Each group administration as follows: Qubi side high dose group: 6.66 g / kg • d; Qubi side low-dose group: 3.33 g / kg • d; Tripterygium glycosides: 9.68 mg / kg • d; normal control the size of the group and CIA model rats fed with pure water. The animals were observed arthritis index, joint swelling. Animals were sacrificed after four weeks of treatment, under the light microscope to observe the pathology of the rat ankle injury; ELISA method for the determination of the anti-II collagen antibody in animal serum, IL-1, IL-6 levels; Determination of serum SOD the two vascular endothelial growth factor (VEGF) receptor FLT-4, MDA and GSH-Px in activity; by immunohistochemical method to detect knee joint synovial tissue and of FLK was 1, and hypoxia-inducible factor-1α (HIF-1α), cytokine changes.
Results:
Joint injury results showed that: compared with normal control group, 15 d after immunization, rat arthritis index was significantly increased (P <0.01), each model rat arthritis index was no significant difference. Compared with model group, after administration of Tripterygium glycosides group and Qubi of side group rat arthritis index decreased, but not statistically significant. CIA model rats swollen joints was significantly higher than the normal control group (P <0.01); compared with the CIA model group, Tripterygium Glycosides group, high-dose rats Qubi side joint swelling decreased significantly (P <0.01 ), high dose Qubi side joint swelling degree of the rats was significantly lower than Tripterygium glycosides group (P <0.05)
Light microscope visible visible synovial cell hyperplasia, disorganized, and synovial tissue congestion and edema, capillary proliferation, and visible inflammatory cell infiltration; villous proliferation of synovial tissue formation, and can stretch the depths of the joint cavity, or to cartilage surface crawling to the formation of pannus. Under the cover of the pannus cartilage surface can be seen clearly the organization of the cartilage surface degeneration and necrosis. The articular cartilage surface exfoliation, the intra-articular joint cartilage and synovial tissue off the peel.
After treatment, proliferation of synovial cells to reduce congestion and edema of the synovial tissue is significantly reduced, reduced the number of vascular proliferation and infiltration of inflammatory cells, pannus formation were significantly reduced. Articular cartilage surface of a small amount of visible flat layer exfoliation, stripping the formation of cartilage, subchondral bone, trabecular bone size, arrangement basically normal. Compared with model group, high dose group of Qubi side and joint damage of Tripterygium glycosides rats significantly reduced (P <0.01, P <0.05); Qubi side high dose group compared with Tripterygium glycosides group, pathological integral decreased significantly (P <0.05)
The serum levels of cytokines measured by ELISA results show that: compared with the normal control group, the CIA model of serum anti-type II collagen antibodies, IL-1β, IL-6 levels were significantly increased; Tripterygium glycosides group and cured Bi side high-dose group, serum anti-type II collagen antibody, IL-1β, IL-6 levels are significantly lower than in model group; Qubi side low-dose group, serum anti-type II collagen antibody levels in the CIA model group showed no significant difference. Tripterygium glycosides, anti-type II collagen antibody Qubi side high dose group, IL-1β levels decreased significantly Qubi side low-dose group, serum IL-1β levels were significantly decreased (P <0.05), while IL -6 levels were significantly decreased, but not statistically significant.
Of GSH-Px, SOD and MDA activity in the colorimetric assay of serum showed that: compared with normal control group, the CIA model group, serum GSH-Px in the activity of SOD decreased significantly (P <0.05, P <0.01), MDA, activity was significantly increased (P <0.01). Tripterygium glycosides and high and low dose Qubi party can make the serum GSH-Px activity was significantly increased MDA activity decreased significantly (P <0.05, P <0.01), Tripterygium Glycosides and the dose Qubi side also SOD activity was significantly increased (P <0.01).
Two were detected by immunohistochemistry in synovial tissue vascular endothelial growth factor (VEGF) receptor FLT-4 and of FLK was-1, the results showed that: compared with the normal control group, model group, serum levels of FLK-1, FLT- 4 levels were significantly increased (P <0.01), high dose group of Tripterygium glycosides group Qubi side serum level of FLK-1 is significantly lower than in model group (P <0.05, P <0.01); Qubi side high-dose group, serum FLK-1 compared with Tripterygium glycosides group was significantly reduced, Tripterygium Glycosides group and Qubi side high dose group, serum level of FLK-1 compared with the model group was significantly lower (P <0.01, P < 0.05); Qubi side low dose group, serum FLK-1, of FLT-4 levels were significantly decreased, but not statistically significant.
Hypoxia-inducible factor-1α (HIF-1α) test results showed that: compared with normal control group, model group, serum levels of HIF-1α levels were significantly increased (P <0.01), while Tripterygium glycosides and Qubi side high-dose group, serum levels of HIF-1α levels than in model group decreased significantly (P <0.01); remove low dose of the paralysis side serum of HIF-1α levels were significantly decreased, but not statistically significant.
Conclusion:
Eradicates paralysis side can significantly reduce the CIA rat ankle joint swelling degree and arthritis index has obvious therapeutic effect on CIA rats, its efficacy is superior to the Tripterygium glycosides.
Qubi side can significantly reduce serum anti-type II collagen antibody levels, reduce the formation of immune complexes, inhibition of CIA rats with abnormal autoimmune response, and lowered serum IL-1, IL-6, MDA, increase GSH-Px and SOD levels, modulation of immune status to improve synovitis lesions, improving the metabolism of oxygen free radicals, reduce bone destruction, so as to achieve the role of treatment of the CIA.
Eradicates paralysis can be lower in synovial tissue vascular endothelial growth factor (VEGF) in the two receptors FLT-4 and FLK-1 and HIF-1α in content, thus inhibiting angiogenesis and synovial cell proliferation, and improve joint synovitis lesions, treatment of the CIA's role.