通痹灵总碱对活化的小鼠T淋巴细胞CD69表达的影响

目的:研究通痹灵(TBL)总碱对活化的小鼠T淋巴细胞表达CD69的影响及其可能的免疫调控机制。方法:培养小鼠淋巴细胞,加入不同浓度的TBL总碱预孵1h后,再加佛波醇酯(PDB)或刀豆蛋白(ConA),24 h后,用流式细胞术检测T淋巴细胞CD69的表达率。结果:不同质量浓度的TBL总碱对PDB或ConA激活的T淋巴细胞表达CD69,均有明显的下调作用(对ConA激活的T淋巴细胞的作用要强于PDB激活的T淋巴细胞),呈明显的量效关系。结论:TBL总碱可明显抑制 活化的小鼠T淋巴细胞CD69的表达,为其用于类风湿性关节炎的治疗提供了实验依据。

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 。

通痹灵总碱对T淋巴细胞活化CD71表达的影响

目的 :通痹灵总碱 (TBL)是从通痹灵中提取出来的总生物碱部位。本研究旨在探索TBL对T淋巴细胞活化CD71表达的影响 ,探讨通痹灵免疫调控作用的机理。方法 :对清洁级小鼠T淋巴细胞进行培养 ,体外加入通痹灵总碱 ,采用流式细胞仪检测T淋巴细胞CD71的表达率。结果 :TBL(10 0 μg mL)对ConA或PDB激活的大鼠T淋巴细胞活化表达CD3+ CD71+ 抗原有明显的下调作用 (P <0 0 1) ,并且随着剂量的增加 ,其抑制作用加强 (P <0 0 1) ,呈明显的量效关系 ;对ConA活化组的作用要强于PDB。结论 :TBL对ConA和PDB诱导活化T细胞的CD71表达率均有明显的抑制作用 ,而且呈明显的量效关系。提示对TBL在T细胞活化信号传导过程有明显作用 ,在治疗RA的过程中可能可以通过影响上游T淋巴细胞的活化 ,调节其细胞免疫机制来达到治疗RA的目的。

通痹灵总碱对LPS(脂多糖)刺激后的巨噬细胞产生炎性细胞因子的影响

目的:观察中药通痹灵(TBL)的有效部位-总生物碱对LPS(脂多糖)刺激后的巨噬细胞产生炎性细胞因子IL-1β、TNF-α的影响。方法:培养人类单核白血病细胞系———THP-1细胞,在PMA(Phorbol12-myristate13-acetate)作用下分化为巨噬细胞。体外用LPS(Lipopolysaccharide)刺激分化的巨噬细胞,以MTX(甲氨喋呤)作为对照,培养有药物作用的细胞24小时后检测细胞上清液中炎性细胞因子IL-1β、TNF-α含量并用PR-PCR(逆转录-聚合酶链式反应)半定量测定IL-1βmRNA、TNF-αmRNA的表达。结果:LPS刺激PMA分化后的THP-1细胞,其IL-1β、TNF-α含量明显升高(与正常组比较P<0.01),不同浓度的通痹灵总碱(200、100、50、25mg/L)及MTX明显抑制IL-1β、TNF-α的产生(P<0.01),并下调IL-1βmRNA、TNF-αmRNA的表达。结论:通痹灵总碱抑制巨噬细胞分泌炎性细胞因子IL-1β、TNF-α,进而抑制了一系列免疫炎症反应造成的病理损伤。

通痹灵总生物碱抑制关节破坏的细胞免疫作用机制

目的:证实通痹灵总生物碱抑制胶原诱导型关节炎(CIA)大鼠关节炎症和关节破坏的药效,揭示其调节细胞免疫的作用机制。方法:牛II型胶原混合弗氏不完全佐剂于SPF级Wistar大鼠尾根部注射,足肿后随机分为造模加生理盐水组,甲氨蝶呤(MTX)治疗组和通痹灵总生物碱(以马钱子碱和士的宁为主的生物碱,TBL总碱)治疗组,容积法评价后肢肿胀度;给药后35d,乳腺钼靶机大鼠全身摄片,计分法评价各组大鼠X光片四肢96块骨侵蚀和100个关节间隙变化;处死动物,取左前肢和右后肢近端第3足趾关节苏木素-伊红(HE)染色,评价中性粒、淋巴细胞、浆细胞浸润、滑膜细胞增生的情况;MTT法检测TBL总碱对Jurkat细胞增殖的影响;Western blot检测TBL总碱对Jurkat细胞ERK1/2磷酸化的影响。结果:造模成功后,MTX和TBL总碱给药35d都可明显抑制大鼠关节肿胀和关节破坏,与造模加生理盐水组有统计学差异(P<0.01);TBL总碱可明显抑制CIA淋巴细胞、浆细胞的浸润和滑膜增生(P<0.05),MTX可抑制滑膜增生。TBL总碱可显著抑制T细胞增殖以及ERK1/2的磷酸化。结论:TBL总碱具有抑制关节免疫炎症和关节破坏的作用,其机制可能是通过干预T细胞MAPK信号转导,抑制T细胞的活化和增殖来实现的,为TBL总碱治疗RA提供了实验依据。

通痹灵对于白细胞介素2及其受体α链影响的体内外实验(英文)

背景:通痹灵是由《金匮要略》中桂枝芍药知母汤加减而成,运用于临床治疗类风湿关节炎已十多年,拟从白细胞介素2及白细胞介素2受体α链(CD25)的角度,从体内和体外两个方面,阐明其调控细胞免疫以及抗类风湿关节炎的作用机制。目的:从体内体外两个方面,研究通痹灵对于胶原诱导性关节炎大鼠关节滑膜原代细胞分泌白细胞介素2,以及通痹灵总碱对于白细胞介素2受体的α链CD25表达的影响。设计:以实验动物为研究对象的完全随机设计,对照实验研究。单位:一所大学的金匮教研室,及另一所大学的生命科学院组织移植与免疫中心。材料:实验于2001-04/07在广州中医药大学第一附属医院中心实验室完成。清洁级Wistar大鼠36只,雄性,购自中山大学医学院实验动物中心,动物合格证号2000A050。体质量(110g±20)g。分为正常组,造模组,甲氨喋呤高剂量组,甲氨喋呤低剂量组,复方通痹灵高剂量组,复方通痹灵低剂量组,每组6只。方法:体内实验,采用胶原诱导性关节炎大鼠动物模型,容积法测量足肿胀度,原代滑膜细胞培养,放免法检测培养液上清,观察通痹灵对滑膜内白细胞介素2的影响;体外实验,分离大鼠淋巴细胞,佛波醇酯或刀豆蛋白体外刺激,双色免疫荧光标记,流式细胞仪检测,观察通痹灵总碱对CD3+,CD25+表达的影响。主要观察指标:复方通痹灵?

Effects of the Overall Alkaloid of a Traditional Chinese Medicine “Tongbiling” on the Cytokine Expression in Th1 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/L) 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-γ 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 Th1 cells in mainly. Four concentrations of overall alkaloid of TBL (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 Th1 type cytokines and Th1 cell differentiation, 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 IFN-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.