【作者】 李兆福；

【导师】 范永升；

【作者基本信息】 浙江中医药大学 ， 中医内科学， 2014， 博士

【摘要】 目的探讨温阳散寒除湿法(蠲痹颗粒)治疗类风湿关节炎(RA)的临床疗效、作用机制及其安全性,为温阳散寒除湿法治疗RA提供依据,丰富中医药防治RA理论。方法①引用国际公认的DAS28. CDAI、SDAI疾病活动指数疗效评价方法,将102例寒湿痹阻型RA患者,随机分为治疗组68例和对照组34例,分别采用蠲痹颗粒(45g/d)、正清风痛宁缓释片(120mg/d)治疗8周后,考察其临床疗效和安全性。②建立角叉菜胶致大鼠急性足跖肿胀炎症模型和棉球异物植入诱导大鼠肉芽组织增生慢性炎症模型,随机分为模型组、阳性药组(DEX)、蠲痹颗粒低中、高剂量组。药物干预后,检测蠲痹颗粒灌胃给药对急、慢性炎症反应的影响。③建立CIA大鼠模型,随机分为空白对照组、模型组、阳性药组(MTX)及蠲痹颗粒低、中、高剂量组。药物干预后,关节炎指数(AI)评分法评价关节炎发病程度,石蜡切片HE染色观测踝关节滑膜组织炎症,ELISA法检测血清IL-1p水平。④建立CIA小鼠模型,按临床评分平均分为模型组,阳性药组(MTX),蠲痹颗粒组。药物干预后,关节炎临床评分法评价关节炎发病严重程度,X线钼靶摄片、石蜡切片HE染色分别观测小鼠足爪关节和关节炎病理损伤程度,3H-thymidine掺入法检测CIA小鼠胶原特异性免疫应答水平,流式微珠阵列法(CBA)、ELISA法分别检测胶原诱导的脾淋巴细胞IL-6、TNF-α、IL-17A及IFN-γ,IL-10产生水平,流式细胞术(FACs)检测脾淋巴细胞表面(CD4、CD8、B220比例)及胞内细胞因子(IFN-γ、IL-17A)的表达。结果①临床观察结果显示,治疗组在总体疗效,证候积分、CRP、ESR及DAS28、 CDAI、SDAI评分等改善方面优于对照组(P<0.01,P<0.05),且安全性良好。②温阳散寒除湿法对大鼠角叉菜胶所致足跖肿胀和棉球异物植入引起的大鼠慢性炎症性肉芽组织增生均有显著抑制作用(P<0.05)。③能改善CIA大鼠关节炎AI评分(P<0.05)及关节滑膜炎症评分(P<0.01),抑制大鼠关节滑膜组织炎细胞浸润、组织细胞增生,降低大鼠血清IL-1p水平(P<0.01)。④能改善CIA小鼠关节炎临床评分(P<0.01),减轻关节炎细胞浸润及骨质损伤,抑制小鼠胶原特异性免疫反应(P<0.01),下调胶原诱导的脾淋巴细胞胞内、胞外IL-17A水平,上调脾淋巴细胞IL-10产生水平(P<0.01),一定程度促进Treg细胞的表达,而对IFN-γ、TNF-α、IL-6及淋巴细胞亚群(CD4、CD8、B220比例)无明显影响。结论①温阳散寒除湿法是临床治疗RA安全有效的方法。②温阳散寒除湿法具有显著的抗炎作用。③对CIA大鼠、CIA小鼠关节炎具有防治作用。④调控Th17细胞/Treg平衡,下调IL-17A表达,诱导IL-10释放,可能是温阳散寒除湿法治疗RA的作用机制之一。更多还原

【Abstract】 Objective To study the clinical effects, efficacy mechanism and security of WenYangSanH-anChuShi method(Juan Bi particle) on the treatment of rheumatoid arthritis(RA), to provide theoretical basises for WenYangSanHanChuShi method in RA and enrich theories of treating RA with traditional Chinese medicine.Methods①Clinical studies cited internationally recognized DAS28, CDAI, SDAI disease activity index to evaluating the curative effect.There are totally102cases of RA patients with cold-dampness syndrome in this study, which are randomly divided into treatment group of68cases and control group of34cases. Treatment group use Juanbi particle(45g/d),and the control group use Zhengqing Fengtongning Retard tablets(120mg/d). Each group take a8weeks treatment, after that,observing the clinical efficacy and safety.②The models of carrageenan-induced edema of rat plantar and formation of cotton ball granulom in rats were adopted to investigate anti-inflammatory of WenYangSanHanChuShi method. Rats were divided into five groups, including the model group, the positive control group(DEX) and Juanbi particle groups(low, medium and high dose group). After drug intervention, effects on acute and chronic inflammation was evaluated.③Type ⅡII bovine collagen(CII)-induced arthritis (CIA) in rats were established for investigating the anti-synovitis of WenYangSanHanChuShi method. Rats were divided into six groups, including the blank control group, model group, the positive control group(MTX) and Juanbi particle groups(low, medium and high dose group).After drug intervention, arthritis disease degree was evaluated by arthritis index (AI) score method. The histological damage of synovial tissues was assessed by histological examination (H&E).Serum level of IL-1β was detected by enzyme-linked immunosorbent assay(ELISA).④Type Ⅱ bovine collagen(CII) induced arthritis (CIA) in mice were established for evaluating the inhibitory effect and mechanism of WenYangSanHanChuShi method. Mice were divided into three groups, including the model group, the positive control group(MTX) and Juanbi particle groups by the clinical score method. After drug intervention, arthritis disease degree was evaluated by the clinical score. The histological damage of arthrosis was assessed by histological examination (H&E) and X-ray mammography radiography. Lymphocyte proliferative ability in CIA mouse was detected by3H-thymidine incorporation assay. The inflammatory cytokine production level of IL-6,IL-17A and TNF-a in collagen(CII)-induced splenic lymphocytes was detected by Cytometric Bead Array (CBA) and IFN-y, IL-10was detected by ELISA. Surface marks of spleen lymphocyte(CD4, CD8, B220ratio) and the level of IFN-y, IL-17A in spleen lymphocytes was detected by fluorescence activated cell sorter(FACs).Results①Clinical observation results show that the syndrome curative effect of treatment group was better than control group. About improvement of TCM syndrome integral, CRP, ESR, DAS28, CDAI, and SDAI, it was better than the control group (P<0.01, P<0.05),and did not appear obvious side effects and adverse reactions.②WenYangSanHanChuShi method has significant inhibitory effect on swelling of paw and the formation of cotton ball granuloma(P<0.05).③Improving arthritis AI score(P<0.05), joint synovial inflammation (P<0.01), synovial tissue inflammatory cells infiltration and tissue hyperplasia and reducing levels of serum IL-1β(P<0.01) in CIA rats.④Improving clinical scores(P<0.01), reducing arthritis cell infiltration and bone injury, inhibiting immune response collagen induced(P<0.01), reducing the level of IL-17A in intrace-extracellular spleen lymphocytes collagen induced, raising IL-10in collagen induced splenic lymphocytes(P<0.01) and Promoting Treg cell expression in some degree. But there is no obvious influence on the level of IFN-y, TNF-a, IL-6and lymphocyte subgroups(CD4, CD8, B220ratio) in CIA mice.Conclusion①WenYangSanHanChuShi method is an effective measure in treatment of RA.②WenYangSanHanChuShi method has an obvious anti-inflammatory effect.③It has a therapeutic effect on CIA rats and CIA mice.④The upregulation of expression of IL-10and the downturn of expression of IL-17A in collagen(CII)-induced splenic lymphocytes may be one of the mechanisms in treatment of RA with WenYangSanHanChuShi method.更多还原