佐剂性关节炎模型大鼠滑膜组织凋亡因子Fas／FasL及Bcl-2／Bax表达与青蒿琥酯的干预

背景:类风湿关节炎的发病与细胞凋亡过程异常密切相关,其滑膜细胞过度增生源于滑膜细胞凋亡的相对不足,诱导滑膜细胞凋亡对治疗类风湿关节炎有积极意义。青蒿素及其衍生物可诱导多种细胞凋亡。目的:观察青蒿琥酯对佐剂性关节炎滑膜组织中凋亡因子Fas/FasL及Bcl-2/Bax表达的影响。设计:随机对照观察。单位:三峡大学第一临床医学院。材料:实验于2005-09/2005-11在三峡大学免疫实验室及形态动物学实验室完成,选用50只生后8周雄性Wistar大鼠,体质量(150±21)g,清洁级,由华中科技大学同济医学院动物实验中心提供。完全Freud佐剂为美国SIGMA公司产品,青蒿琥酯注射液购自桂林南药股份有限公司。兔抗鼠Fas(SC-716)、兔抗鼠FasL多抗(SC-834)、山羊抗小鼠IgG抗体-HRP多聚体、兔抗鼠P53(M3566)多抗、Bcl-2(sc-7382)多抗、兔抗鼠Bax(sc-7480)多抗以及ABC复合物试剂盒和DAB显色试剂盒均为美国SantaCruz公司产品;甲氨蝶呤由上海华联制药有限公司生产;德国Leica生物应用显微镜及图象分析系统;德国Leica切片机。方法:摸球法随机将大鼠分为6组:空白组(n=8),模型组(n=8),青蒿琥酯高剂量组(n=10),青蒿琥酯低剂量组(n=8),青蒿琥酯加甲氨蝶呤组(n=8),甲氨蝶呤组(n=8)。①实验干预:按照文献叙述的模型构建方法,除空白组外,余5组每只右足跖处给予注射0.1mL完全Freud佐剂,致炎成佐剂性关节炎模型,空白组注射生理盐水0.1mL。致炎后第13天开始给药,青蒿琥酯高剂量组、青蒿琥酯低剂量组分别注射40,20mg/(kg·d)青蒿琥酯注射液,青蒿琥酯加甲氨蝶呤组注射20mg/(kg·d)青蒿琥酯注射液,0.4mg/(kg·3d)甲氨蝶呤注射液。甲氨蝶呤组给予注射0.4mg/(kg·3d)甲氨蝶呤注射液,模型组腹腔注射1mL/d生理盐水。各组给药方式均为腹腔注射。②实验评估:给药前及给药后13d分别评价大鼠关节肿胀指数;免疫组织化学法检测给药后13d大鼠滑膜组织中Fas/FasL、Bcl-2及Bax的表达情况。主要观察指标:各组关节肿胀指数及滑膜组织中凋亡相关因子Fas/FasL,Bcl-2/Bax的表达。结果:纳入大鼠50只均进入结果分析。①给药后13d,各实验组关节肿胀指数明显低于给药前,差异有统计学意义(P<0.01),各实验组关节肿胀指数均明显低于模型组,差异有统计学意义(P<0.01)。②青蒿琥酯高剂量组、青蒿琥酯低剂量组和青蒿琥酯加甲氨蝶呤组滑膜组织中FasL及Fas均上调,与模型组比较差异有统计学意义(P<0.01),甲氨蝶呤组与模型组比较差异无统计学意义(P>0.05);青蒿琥酯高剂量组、青蒿琥酯低剂量组、青蒿琥酯加甲氨蝶呤组及甲氨蝶呤组Bcl-2表达下调,Bax上调,与模型组比较差异有统计学意义(P<0.05)。结论:实验证实青蒿琥酯具有诱导佐剂性关节炎病情缓解的作用,上调滑膜组织中Fas/FasL及Bax,下调Bcl-2的表达而诱导滑膜细胞凋亡可能是其机制之一。

Effect of artesunate on the expression of Fas/FasL and Bcl-2/Bax in synoviocytes of rats with adjuvant arthritis

BACKGROUND: The morbility of rheumatoid arthritis is closely associated with cell apoptosis process. Hyperplasia of synoviocyte lies in the relatively insufficiency of synoviocyte apoptosis. Therefore, induction of synoviocyte apoptosis has clinical significance in the treatment of rheumatoid arthritis. Arteannuin and its derivatives can induce the apoptosis of various cells. OBJECTIVE: To observe the effect of artesunate on the expression of Fas/FasL and Bcl-2/Bax, which are correlated with apoptosis in synoviocyte of adjuvant arthritis. DESIGN: Randomized and controlled observation. SETTING: First College of Clinical Medical Science, Three Gorges UniversityMATERIALS: The experiment was conducted in the laboratories of Immunology and Morphology, Three Gorges University from September 2005 to November 2005. Fifty 8-week-old male Wistar rats of clean grade and (150±21) g were provided by the Animal Experimental Center of Tongji Medical College, Huazhong University of Science and Technology; Complete Freud adjuvant by SIGMA, U.S., and artesunate solution by Guilin Pharmaceutical Corporation.rabbit anti-mouse Fas (SC-716), rabbit anti-mouse FasL and Technology (No. SCXK 2004-2007).Complete Freud adjuvant (SIGMA, U.S. No. 093K8932); artesunate solution (Guilin Pharmaceutical Cor); rabbit anti-mouse Fas (SC-716), rabbit anti-mouse FasL multi-antibody (SC-834), goat anti-mouse IgG-HRP multimer, rabbit anti-mouse P53 (M3566) multi-antibody, Bcl-2 (sc-7382) multi-antibody, rabbit anti-mouse Bax (sc-7480) multi-antibody, ABC compound reagent kit and DAB coloring reagent all acquired from Santa Cruz biotechnology (Santa Cruz, USA); methotrexate (MTX) purchased from Shanghai Hualian Pharmaceutical Co., Ltd.; microscope and image analysis system obtained from Leica (Leica,Germany); microtome (Leica, Germany).METHODS: Fifty rats were randomly divided into six groups: normal group (n =8), model group (n =8), high dose artesunate group (n =8), low dose artesunate group (n =8), artesunate plus methotrexate (MTX) group (n =8), and MTX group (n =8). ①Model construction was referred to literature: Except the normal group with 0.1 mL normal saline, all rats were injected with 0.1 mL complete Freund's adjuvant into the right voix pedis to establish the models of adjuvant arthritis. Since the 13th day after modeling, high and low dose artesunate groups were intraperitoneally injected with 40 and 20 mg/kg artesunate, respectively everyday; artesunate plus MTX group was intraperitoneally injected with 20 mg/kg artesunate everyday and 0.4 mg/kg MTX every three days; MTX group with 0.4 mg/kg MTX solution every three days;model group with 1 mL normal group everyday. ②The arthrosis index (Al) of each group was evaluated before and 13 days after administration; the expressions of Fas/FasL, Bcl-2, and Bax in synovial membrane tissue were examined by immunohistochemistry.MAIN OUTCOME MEASURES : Al and the expression of Fas/FasL, Bcl-2, and Bax in synovial membrane tissue of each group.RESULTS: Fifty rats were involved in the result analysis. ①Thirteen days after administration, the Al of each experiment group (including model control) was remarkably lower than that before treating (P ＜ 0.01). The Al of eachexperiment group was significantly lower than that in model control group (P ＜ 0.01). ②The expression of Fas/FasL in high and low dose artesunate groups and artesunate plus MTX group was up-regulated significantly compared with that in model group (P ＜ 0.01). There was no statistically significant difference in the expression between MTX group and model group (P ＞ 0.05); the expression of Bcl-2 was significantly down-regulated but Bax up-regulated in two artesunate groups,artesunate plus MTX group and MTX group compared with that in model group (P ＜ 0.05).CONCLUSION: The findings suggest that artesunate could alleviate adjuvant arthritis, up-regulate the expression of Fas/FasL and Bax, but down-regulate that of Bcl-2, in which the induction of synoviocyte apoptosis may be one of the mechanisms.