痹肿消汤干预胶原诱导性关节炎大鼠滑膜病变的蛋白质组学研究

Effect of bizhongxiao decoction on protein in synovitis of rats with collagen-induced arthritis

目的:观察痹肿消汤干预胶原诱导性关节炎大鼠滑膜病变的蛋白质改变,分析痹肿消汤治疗类风湿关节炎可能的作用机制。方法:实验于2004-05/2005-02在中南大学湘雅医院中西医结合研究所实验室、卫生部肿瘤蛋白质组学重点实验室完成。70只SD大鼠随机分为3组,正常组10只,其余60只大鼠采用牛Ⅱ型胶原与完全福氏佐剂诱导SD大鼠实验性关节炎模型,选取造模成功者再随机分为两组,即痹肿消汤组和模型组。痹肿消汤煎液制备:白花蛇舌草15g,肿节风30g,丹参15g,络石藤20g,骨碎补15g,苡米30g等15味中药,双蒸水煎2次,30min/次,两煎混合,G4滤器过滤,于60℃水浴中浓缩成每毫升药液含生药116g。痹肿消汤组灌服痹肿消汤煎液10mL/kg(相当于生药62.1g),2次/d灌服;模型组灌服生理盐水10mL/kg,2次/d灌服。正常组自由进水。应用双向凝胶电泳技术分别分离正常组、模型组及痹肿消汤组大鼠关节滑膜的总蛋白后,经胶体考染显色、图像分析、识别差异表达的蛋白质点,应用MALDI-TOF-MS质谱仪得到相应的肽质量指纹图谱,然后搜索数据库鉴定部分差异蛋白质点。结果:纳入实验的70只大鼠,造模不成功被淘汰7只,最终痹肿消汤组26只、模型组27只、对照组10只,共63只大鼠进入结果分析。①建立了正常组、模型组和痹肿消汤组关节滑膜的双向凝胶电泳图谱,其平均蛋白质点数分别为947±39,994±41,1031±52,其匹配率为92%,91%,94.2%。②发现并鉴定了正常组、模型组和痹肿消汤组大鼠滑膜的差异表达大于2倍的蛋白质点55个,这些差异表达蛋白质的功能涉及物质代谢、能量产生、物质转运、抗氧化应激、信号转导及细胞骨架蛋白,随即用免疫印迹的方法差异蛋白质annexin1、aldolaseA在正常组、模型组、痹肿消汤组中的表达,其结果也显示了类似的表达差异。结论:类风湿关节炎模型关节滑膜病变是一个多种蛋白质参与的复杂过程。滑膜组织的比较蛋白质组学分析是一种筛选病变相关蛋白的可靠方法之一。annexin1、aldolaseA可能与实验性关节炎的关节滑膜病变有关;痹肿消汤可能通过对实验性关节炎大鼠滑膜蛋白质表达的调控达到治疗目的。

AIM： To investigate effect of bizhongxiao decoction （BZXD） on changes of protein in synovitis of rats with collagen-induced arthritis （CIA） and analyse mechanism of BZXD on treating rheumatoid arthritis （RA）. METHODS： The experiment was completed in the Institute of Combined Traditional Chinese Medicine and Western Medicine, Xiangya Hospital of Central South University and Key Laboratory of Tumor Protein of Ministry of Public Health between May 2004 and February 2005. Totally 70 SD rats were divided randomly into 3 groups. Ten rats were regarded as normal group, and other 60 rats were modeled by type Ⅱ collagen and complete Freunds adjuvant. Successful rat models were selected and divided into BZXD group and model group. BZXD was consisted of 15 kinds of Chinese herbs such as baihuashe shecao 15g, zhongjiefeng 30 g, danshen 15 g, luoshiteng 20 g, gusuibu 15 g, yimi 30 g, etc., and fried with double distilled water for twice with 30 minutes each time. Then, liquid was mixed, filtered with G4 filter and concentrated in water bath at 600C with 116 g raw materials in each milliliter. Rats in BZXD group were perfused with 10 mL/kg BZXD （equal to 62.1 g raw materials） twice a day; rats in model group were perfused with 10 mL/kg saline twice a day; rats in normal group were drunk water freely. The total proteins of synovial tissue of rats in normal group, model group and BZXD group were separated by two-dimensional gel electrophoresis （2-DE）, respectively. Then gels were stained by Coomassie brilliant blue, scanned by Image Scanner. Relative abundance of the protein spots was calculated by comparing spots density volume on the gel. The differentially expressed protein spots were identified by poptide mass fingerprint （PMF） based on matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and database searching, then some of them were validated by western blotting. RESULTS： Among 70 rats, 7 were lost because of unsuccessful modeling, and totally 63 rats, 26 in BZXD group, 27 in model group and 10 in control group, entered the final analysis. ① Reproducible 2DE maps of synovial tissue were established in normal group, experimental arthritis model group and BZXD group respectively, the average spots of protein were 947±39, 994±41 and 1 031±52 respectively, and the match rates were 92%, 91% and 94.2%. ② Totally 55 protein, spots were twice than expression of synovial tissue in normal group, experimental arthritis model group and BZXD group. This suggested that the functions of these proteins involved in metabolism, energy generation, transportation, anti-oxidation, signal transduetion and protein folding etc. Then the different expressions of annexin 1 and aldolase A in normal group, experimental arthritis model group and BZXD group by western blotting were similar to those of proteomic results. CONCLUSION： Synovium of joint of rheumtoid arthritis is a complex process which is involved by multiple proteins. Histological analysis of proteins in synovium tissue is one of methods for screening related protein of lesion. Annexin 1 and aldolase A are related to CIA synovitis, and BZXD has a good effect on CIA by regulating protein expression of synovial tissue of rats.