【作者】 任宝娣；

【导师】 李振彬；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2007， 硕士

【摘要】 目的:近年来,随着皮质激素、免疫抑制剂治疗的应用,类风湿关节炎(rheumatoid arthritis,RA)患者的生存期得到明显延长,与此同时心血管并发症也逐渐增多。心血管疾病成为RA患者的首要死亡原因。因此探索其发病机理及防治措施成为RA临床治疗中亟待解决的重要问题之一。本课题拟研究清心饮对佐剂性关节炎(adjuvant arthritis,AA)大鼠离体血管内皮依赖性舒张功能及其对同型半胱氨酸(homocysteine, Hcy)、血管细胞间粘附分子-1(vascular cell adhesion molecule-1,VCAM-1 )、内皮素-1 ( endothelin-1, ET-1)、肿瘤坏死因子(tumor necrosis factor-α, TNF-α)、白介素-1β(interleukin-1β, IL-1β)表达的影响,研究其血管保护作用机制,为临床用药提供实验依据。方法:选择体重150-180g的Wistar大鼠,雌雄各半。测量双侧足爪容积后,于右后足跖部常规消毒,皮内注射完全弗氏佐剂(0.1ml/只)建立AA大鼠模型。其中10只大鼠用相同方法注入等量生理盐水。造模后第19天将出现二次反应的大鼠随机分为模型组、甲氨喋呤(MTX)组、MTX+清心饮(MTX+QXY)组,每组10只。注入生理盐水组为空白对照组。当日测量大鼠体重、用自制容积测量仪测量各组大鼠足爪容积。各治疗组连续灌胃给药(模型组灌胃给予等量生理盐水)21天,于给药结束后第2天测量各组大鼠足爪容积、体重后,3%戊巴比妥麻醉,腹主动脉取血,分别留取血清、血浆。放射免疫法检测大鼠血浆ET-1,血清TNF-α、IL-1β,ELISA法检测血清VCAM-1,循环酶法检测血清Hcy;取血后立即打开胸腔,取出胸主动脉,分离周围脂肪及结缔组织,保留血管内皮,截取长为3-4mm的主动脉环,主动脉环浴皿法检测内皮依赖性舒张功能;剩余部分动脉,经固定、脱水、包埋、染色,免疫组织化学法检测血管内皮细胞VCAM-1的表达。统计学处理:所有数据均采用SPSS for windows 13.0软件包进行统计学处理,所得结果均以X±S表示。各组间均数比较采用单因素方差分析。结果:1 QXY对AA大鼠一般情况的影响与模型组比较,MTX组、MTX+QXY组一般情况均有所改善。造模后,模型组、MTX组与MTX+QXY组体重明显低于空白对照组(P<0.01),但模型组、MTX组与MTX+QXY组间无差异(P>0.05);灌胃完毕,模型组、MTX组与MTX+QXY组体重明显低于空白对照组(P<0.01),模型组与MTX组明显低于MTX+QXY组(P<0.01),模型组明显低于MTX组(P<0.01);MTX、模型组体重增加明显低于空白对照组(P<0.01),MTX、模型组体重增加明显低于MTX+QXY组(P<0.01),MTX+QXY组体重增加值与空白对照组比较无显著性差异(P>0.05),模型组体重增加明显低于MTX组(P<0.01)。2 QXY对AA大鼠足爪肿胀的抑制作用给药3周后,MTX组、MTX+QXY组造模侧足爪平均肿胀度明显低于模型组(P<0.01),MTX组与MTX+QXY组比较无明显差异(P>0.05),但MTX+QXY组对造模侧肿胀足爪的抑制率稍优于MTX组(58.20% VS 54.7%)。(2)MTX组、MTX+QXY组非造模侧平均肿胀度明显低于模型组(P<0.01),MTX+QXY组与MTX组比较无显著差异(P>0.05),但MTX+QXY组对非造模侧肿胀足爪的抑制率稍优于MTX组(48.49% VS 44.20%)。3 QXY对AA大鼠内皮依赖性舒张功能的影响主动脉环对乙酰胆碱(acetylcholine, Ach)引起的内皮依赖性舒张作用(%)各个浓度(10-9 mmol/L、10-8 mmol/L、10-7 mmol/L、10-6 mmol/L)中模型组、MTX组、MTX+QXY组均明显低于空白对照组,差异有显著性(P<0.01、0.05),模型组、MTX组明显低于MTX+QXY组(P<0.01);从10-8 mmol/L开始,模型组明显低于MTX组,差异有显著性(P<0.05、0.01)。主动脉环对硝普钠(sodiumnitroprusside, NaNP)引起的非内皮依赖性舒张作用(%)中前两个浓度(即10-9mmol/L、10-8mmol/L)各组间存在明显差异,显著性水平从0.05到0.01不等,后两个浓度(10-7 mmol/L、10-6 mmol/L)各组间均无明显差异(P>0.05)。4 QXY对AA大鼠对血清TNF-α、IL-1β,血浆ET-1,血清Hcy,血清及血管内皮细胞VCAM-1的影响4.1血清TNF-α、IL-1β水平比较,模型组、MTX组明显高于空白对照组(P<0.01),模型组、MTX组明显高于MTX+QXY组(P<0.01),MTX+QXY组与空白对照组比较无明显差异(P>0.05),模型组明显高于MTX组(P<0.01);4.2血浆ET-1水平比较,模型组、MTX组与MTX+QXY组明显高于空白对照组(P<0.01),模型组、MTX组明显高于MTX+QXY组(P<0.05),模型组与MTX组比较无明显差异(P>0.05)。4.3.1血清VCAM-1水平比较,模型组、MTX组明显高于空白对照组(P<0.01),模型组、MTX组明显高于MTX+QXY组(P<0.01),MTX+QXY组与空白对照组无显著差异(P>0.05),模型组明显高于MTX组(P<0.01)。4.3.2动脉内皮细胞VCAM-1(灰度值)比较,模型组、MTX组明显高于空白对照组,差异有显著性(P<0.01),模型组、MTX组明显高于MTX+QXY组(P<0.05),模型组明显高于MTX组,差异有显著性(P<0.01),MTX+QXY组与空白对照组比较无明显差异(P>0.05)。4.4血清Hcy水平比较,模型组、MTX组与MTX+QXY组明显高于空白对照组,差异有显著性(P<0.01),模型组、MTX组明显高于MTX+QXY组,差异有显著性(P<0.01),模型组与MTX组比较无明显差异(P>0.05)。结论:1 AA大鼠血清Hcy、VCAM-1及血浆ET-1水平明显升高,主动脉内皮依赖性舒张功能明显下降,说明AA大鼠存在心血管损害的相关因素。2清心饮具有显著抗炎作用。清心饮联合MTX可明显降低AA大鼠足爪肿胀度及血清炎性细胞因子TNF-α、IL-1β水平。3清心饮对AA大鼠血管内皮损伤具有保护作用。清心饮可明显改善AA大鼠主动脉内皮依赖性舒张功能,降低血浆ET-1,血清VCAM-1、Hcy水平,减少胸主动脉内皮细胞VCAM-1的表达,从而改善AA大鼠血管内皮损伤。更多还原

【Abstract】 Objective: Using with glucocorticoid and immunodepressive drug in these years, the life of Rheumatoid Arthritis(RA) patients has been prolonged. At the same time, the number of RA patients with cardiovascular disease is increasing. And cardiovascular disease has became the first reason in RA patients’death. Thus, investigating the mechanism in it and the precautionary measure became one of the cardinal problem in RA clinical treatment. The aim of the study is to investigate the effect of QingXinYin to relaxant response depend on endothelium of isolated aortas, homocysteine(Hcy), vascular cell adhesion molecule-1(VCAM-1), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and endothelin-1 (ET-1) to investigate the protective mechanism and afford experimental evidence for clinical work.Methods: Choose Wistar rats weight from 150g to 180g, the number of male and female is equal. Injecting Freund’s complete adjuvant(FCA) into the subcutis of their right rear paws after sterilizing regularly is to found AA rat models and ten of rats are injected equivalent volume of saline. On the 19th day after injecting, rats that presenting secondary response are divided randomly into Model group, MTX group, MTX&QXY group, and each group includes 10 samples. The rats injected saline were as control group. After measuring the paw volumes and weights of rats in each group, the treatment rats are given drug for successive 21 days. On the second day after treatment is over, after measuring the paw volumes and the weights, rats are sacrificed by withdraw of blood by abdominal aortas under 3% pentobarbital sodium anesthesia to get serum and plasma. ET-1, TNF-α, IL-1βare determined by radioimmunoassay, VCAM-1 by ELISA, Hcy by cyclical zyme technique. After drawing blood, thorax was cut open and removed thoracic aortas quickly. Then, it was cleared of adherent fatty and connective tissue, and was scissored rings of 3-4mm length. Determining relaxant response depend on endothelium by technique of aortas in organ chambers. The remanent part of aortas was fixed, dehydrated, wrapped and dyed to determine VCAM-1 in vascular endothelium cell by immunohistochemical.Data Statistics: All data is analysed by SPSS for windows 13.0 and the results are expressed as X±S. The comparison of mean among groups is evaluated by one-way analysis of variance(ANOVA)Results:1 The effect of MTX&QXY to general condition of AA rats Comparing with model group, MTX group and MTX&QXY group are increased in general condition. After model founded, the weight of model group, MTX group and MTX&QXY group is lower than control group significantly(P<0.01), but there is no significant difference among the AA three groups. At the end of the experiment, the weight of model group, MTX group and MTX&QXY group is still lower than control group significantly(P<0.01), model group and MTX group is lower than MTX&QXY group significantly(P<0.01), and model group is lower than MTX group significantly(P<0.01). The augment of MTX group and model group is lower than control group and MTX&QXY group in weight significantly(P<0.01), model group is lower than MTX group significantly(P < 0.01), but there is no significant difference between control group and MTX&QXY group(P>0.05).2 The depressive effect of QXY to paw swelling of AA rats At the end of experiment, the average swelling degree(%) of FCA-injected paws of MTX group and MTX&QXY group is lower than model group significantly(P<0.01), and MTX group is parallel with MTX&QXY group(P>0.05). The average swelling degrees(%) of non-FCA-injected paws of MTX group and MTX&QXY group is lower than model group significantly(P < 0.01), and MTX group is parallel with MTX&QXY group(P>0.05).3 The effect of QXY to relaxation depend on endothelium of AA ratsThe acetylcholine(Ach) induced endothelium dependent relaxation are decreased in aortas from AA rats, and model group and MTX group is lower than MTX&QXY group significantly(P<0.01), model group is lower than MTX group significantly(P<0.01 or 0.05). The sodiumnitroprusside(NaNP) induced endothelium independent relaxation has no significant difference among groups in AA rats(P>0.05).4 The effect of QXY to TNF-αand IL-1βin serum, ET-1 in plasma and VCAM-1 in serum and vascular endothelium cell of AA rats4.1 Comparing the level of TNF-αand IL-1βin serum, model group and MTX group is higher than control group significantly(P<0.01), model group and MTX group is higher than MTX&QXY group significantly(P<0.01), model group is higher than MTX group significantly(P<0.01), but there is no significant difference between MTX&QXY group and control group(P>0.05).4.2 Comparing the level of ET-1 in plasma, model group ,MTX group and MTX&QXY group is higher than control group significantly(P<0.01), model group and MTX group is higher than MTX&QXY group significantly(P<0.05), and there is no significant difference between model group and MTX group(P>0.05).4.3.1 Comparing the level of VCAM-1 in serum, model group and MTX group is higher than control group significantly(P<0.01), model group and MTX group is higher than MTX&QXY group significantly(P<0.01), model group is higher than MTX group significantly(P < 0.01), and there is no significant difference between MTX&QXY group and control group(P>0.05).4.3.2 Comparing the level of VCAM-1 in vascular endothelium cell, model group and MTX group is higher than control group significantly(P<0.01), model group and MTX group is higher than MTX&QXY group significantly(P<0.05), model group is higher than MTX group significantly(P<0.01), and there is no significant difference between MTX&QXY group and control group(P>0.05).4.4 Comparing the level of Hcy in serum, model group, MTX group and MTX&QXY group is higher than control group significantly(P<0.01), model group and MTX group is higher than MTX&QXY group significantly(P<0.01), and there is no significant difference between model group and MTX group(P>0.05).Conclusions:1 Hcy and VCAM-1 in serum and ET-1 in plasma are all increased significantly, and relaxation dependent on endothelium is decreased significantly. They all confirm that AA rats have many factors engendering cardiovascular dysfunction.2 QingXinYin can resist inflammation positively. QingXinYin combine with MTX can remedy AA significantly, decrease the paw swelling and the level of inflammatory cell factors such as TNF-αand IL-1β.3 QingXinYin can protect vascular dysfunction of AA rats. QingXinYin increase relaxation dependent on endothelium, decrease ET-1 in plasma, VCAM-1 and Hcy in serum and VCAM-1 in vascular endothelium cell to protect endothelium dysfunction in AA rats’vascular.更多还原