通痹灵对滑膜细胞趋化因子RANTES、MCP-1影响的研究

目的：探讨通痹灵对类风湿关节炎滑膜产生趋化因子RANTES、MCP-1的影响。方法：分离培养类风关患者滑膜成纤维样细胞，IL-10刺激，不同浓度通痹灵总碱、MTX作用后，ELISA法测定培养上清RANTES、MCP-1含量,取经通痹灵、MTX治疗后的CIA大鼠关节滑膜组织，提取总RNA，用RT-PCR法测定其中RANTESmRNA、MCP-1mRNA水平．结果：通痹灵总碱10mg／L可以非常明显减少RANTES产生，与IL-10组比较，差异非常显著：MTX1mg／L也能减少RANTES产生，与IL-10组比较，有显著性差异．通痹灵总碱和MTX各剂量组都能减少MCP-1产生。通痹灵能抑制滑膜组织中趋化因子MCP-1mRNA、RANTESmRNA的表迭，MTX对MCP-1mRNA、RANTESmRNA的影响作用不明显．结论：通痹灵可抑制类风关滑膜趋化因子RANTES，MCP-1产生．     

青藤碱对T淋巴细胞活化及TH1类细胞内细胞因子表达的影响

目的：深入研究青藤碱对T淋巴细胞Thl类细胞因子表达的影响。方法：分离小鼠淋巴结细胞，加入不同浓度的青藤碱作用1小时后，加多克隆刺激剂PDB和离子霉素，继续培养4小时后收获细胞，进行细胞内细胞因子染色，以流式细胞术对T细胞Th1类细胞因子TNF-γ、炎症性细胞因子TNF-α分子表达情况进行分析；同时，观察药物对T细胞活化早期标志CD59表达的影响；结果：1000μmol／L(329．24μg/ml)青藤碱能够抑制CD69表达，200μmol/L青藤碱对CD69表达无影响；200、1000、2000μmol／L青藤碱能够剂量依赖性地显著降低T细胞内细胞因子TNF-γ、TNF-α分子表达。结论：抑制T细胞活化异常可能是青藤碱治疗类风湿关节炎免疫药理机制之一，此抑制效应可能主要不是通过影响PKC而是与影响钙离子依赖的T细胞活化信号传导途径相关。     

通痹灵总碱对T细胞内Th1型细胞因子表达的影响

目的 :研究通痹灵总碱 (TBL)对T细胞中Th1型细胞因子表达的影响 ,并揭示其抗炎机制 ,为临床用药提供理论依据。方法 :分离小鼠肠系膜淋巴结细胞 ,加入不同浓度的TBL作用 1h ,再加多克隆刺激剂佛波醇酯 (PDB)和离子酶素 ,继续培养 4h收获细胞 ,进行双色荧光素标记 ,以流式细胞术对Th1型细胞因子INF γ和TNF α的表达进行分析。结果 :2 0 0mg/L 和 10 0mg/L的TBL ,能显著抑制T细胞内INF γ和TNF α的表达。结论 :TBL可抑制Th1型炎症性细胞因子的表达 ,可能是其治疗类风湿关节炎的免疫药理机制之一     

Effects of the Overall Alkaloid of a Traditional Chinese Medicine “Tongbiling” on the Cytokine Expression in Thl and Th2 Cells of Rheumatoid Arthritis and Their Signaling Pathway

To investigate the effects of overall alkali of a traditional Chinese medicine “Tongbiling” (brucine and strychnine alkaloids in main) on the cytokines expression in Th1 and Th2 cells in the synovial fluid of patients with rheumatism arthritis and their signal pathway, the mononuclear cells in the synovial fluid (SFMC) of patients were isolated by Ficoll-Hypaque gradient centrifugation, and the CD3^+ CD69^+ and CD3^+ HLA-DR antigen were analyzed by flow cytometry in comparison with those of the peripheral blood. The rest of cells were cultured after resuspension with RPMI 1640 culture medium. Phorbol 12, 13-dibutyrate (PDB) and ionomycin were added successively into the culture with various concentration of overall alkali Tongbiling (TBL). After 4 h of cultivation, the expression of IFN-γ and IL-4 in CD3^+ cells were analyzed by flow cytometry. The influence of overall alkali TBL ( 100 mg/I,) on the intracellular calcium was investigated after Fluo-3/AM labeling and stimulation with PDB and ionomycin at 1, 2, 4 and 10 min, and the influence of TBL on the expression of CD3^+ CD69^+ cells were determined with stimulation of PDB for 24 h in the whole blood lymphocytes culture. It was found that the percentage of T cells bearing CD69 was significantly up-regulated (77%), while that of T cells bearing HLA-DR was 44% in the synovial mononucleated cells. After PDB and ionomycin stimulation, the expression of IFN-7 in CD3 ~ cells were up-regulated, but there was no change on the expression of IL-4 in CD3^+ cells, indicating that ratio of Th1/Th2 was significantly increased and Th cells differentiate to Thl cells in mainly. Four concentrations of overall alkaloid of TBI, (200 mg/L, 100 mg/L, 50 mg/L, 25 mg/L) could down-regulated the expression of IFN-γ in CD3^+ cells and the Th1/Th2 ratio obviously, but all the concentrations of the overall alkaloids had no effect on the expression of IL-4 in CD3^+ cells. 100 mg/L concentration of the overall alkaloid did not down-regulate the intracellular calcium level. Each concentration of the overall alkaloid could down-regulated the expression CD69 obviously on the PDB-activated mouse T cells. It concluded from the above observations that the overall alkaloid of TBL could relieve the inflammatory and immune damages by suppressing the expression of Thl type cytokines and Th1 cell differen-tiation, regulating the imbalance of Th1/Th2 cells and inhibiting the early activation of the T lymphocytes bearing CD69. There was no remarkable influence on the intracellular calcium signaling transduction pathway. The inhibitory effected on T cells to express 1FN-γ might be due to the suppression of PKC-MAPK signaling pathway. From the standpoint of traditional Chinese medicine, this might be due to the regulation of “Yin” and “Yang” imbalance of joints to modify the pathological status in rheumatoid arthritis. This study provided an experimental basis for the application of overall alkaloids of TBL in the treatment of rheumatoid arthritis.     

通痹灵治疗强直性脊柱炎临床疗效评价

目的：评价通痹灵治疗活动期强直性脊柱炎（AS）的临床疗效、毒副反应。方法：采用随机对照单盲双模拟的方法，将69例活动期AS分为通痹灵治疗组（36例）和消炎痛对照组（33例）。能痹灵组口服通痹灵IA，每次6片，每天3次；消炎痛组口服消炎痛（通痹灵ⅡA），每片含消炎痛25mg ，每次6片，每天3次。结果：①总有效率两组间无显著性差异（P＞0．05），但通痹灵组控显率优于消炎痛组（P＜0．01）。②两组治疗前后疼痛积分、晨僵时间和指地距离改善明显（P＜0．01或P＜0．05），扩胸度和枕墙距均无改善（P＞0．05）。通痹灵组关节肿胀积分、Schober试验、整体功能、实验指标（ESR、CRP、PLT、IgG、IgA、IgM）和骶髂关节影像积分均有明显改善（P＜0．01）；消炎痛组关节肿胀积分、整体功能、实验指标和骶髂关节影像积分无改善（P＞0．05）。通痹能组改善肿胀积分、晨僵时间、指地距离、整体功能和实验指标（除CRP外）优于消炎痛组（两组治疗后比较，P＜0．0．01或P＜0．05）。③通痹灵组未发现明显不良反应。结论：通痹灵对活动期AS具有改善临床症状、体征和实验指标的作用，且对AS骨质损害具有保护作用，未发现不良反应。     

强直性脊柱炎中医证型与HLA—B27相关性研究

目的：探讨HLA-B27的表达、亚型与强直性脊柱炎中医证型的相关性。方法：采用FCM方法分别检测25例AS患者活动期、缓解期HLA-B27的表达，同时对15例FCM确定为HLA-B27阳性的样本采用PCR-SSP方法进行HLA-B27亚型分型。结果：男性HLA-B27的表达与女性比较，具有显著性差异，男性高于女性（P〈0．05）；活动期与缓解期、湿热组与非湿热组HLA-B27的表达比较，差异均无统计学意义；15例B27阳性样本中，湿热型8例，B2704占62．5％（5／8例），非湿热型7例，B2704占71．4％（5／7例），湿热型和非湿热型B2704所占比例统计学分析无显著差异（x^2=0．134，P〉0．05）。结论：HLA-B27的表达与性别有关，但与中医证型及病情活动无明显相关性；广东地区AS患者HLA-B27亚型以B2704为主，中医证型与各亚型的特异基因无明显相关性。     

通痹灵及3种抗类风湿药体外杀精子实验

目的:观察通痹灵及3种抗类风湿制剂正清风痛宁片、甲氨蝶呤片、雷公藤多甙片的体外杀精子活性,初步评价各药的抗生育作用。方法:按照世界卫生组织(WHO)标准方法,测定不同浓度的药物稀释液对大鼠精子3m in内的最低杀精浓度。结果:正清风痛宁片、通痹灵、甲氨蝶呤片、雷公藤多甙片提取物最低杀精浓度分别为(1234±427)、(822±387)、(478±149)、(29±22)mg/mL。结论:4种药的体外杀精活性依次为雷公藤多甙片>甲氨蝶呤片>通痹灵>正清风痛宁片。     

桂枝茯苓丸加味治疗神经根型颈椎病60例

目的观察桂枝茯苓丸加减治疗颈椎病的疗效。方法应用桂枝茯苓丸加味配合外治法治疗神经根型颈椎病60例。结果痊愈21例,好转36例,总有效率95%。结论桂枝茯苓丸加味治疗神经根型颈椎病,具有温阳通络、活血化瘀止痛的功效。