【作者】 吴相春；

【导师】 吴以岭；

【作者基本信息】 河北医科大学 ， 中医内科学， 2008， 硕士

【摘要】 目的:以络病理论为指导,依据临床流行病学调查建立的“脉络-血管系统病”辨证诊断标准,应用慢性束缚法复合高蛋氨酸饮食建立络气郁滞型血管内皮功能障碍病证复合动物模型,从大鼠一般状况、生物学表征评分、行为学及血管内皮结构与功能等方面对模型进行综合评价,并观察神经-内分泌-免疫(NEI)网络相关因子的改变,应用通络复方-组分配伍-单味药物对动物模型进行不同层次的干预。阐明络气郁滞型血管内皮功能障碍的生物学基础,探讨中医证候因素在血管内皮功能障碍发病中的作用,并对通络药物干预作用做出综合评价。方法:1络气郁滞型血管内皮功能障碍动物模型建立与评价采用慢性束缚法建立络气郁滞大鼠证候模型,以高蛋氨酸饮食建立内皮功能障碍大鼠病理模型,基于束缚应激即刻引起血清同型半胱氨酸(HCY)增高、一氧化氮(NO)明显下降,将慢性束缚法与高蛋氨酸饮食复合建立络气郁滞型血管内皮功能障碍病证复合动物模型。将清洁级健康雄性Wistar大鼠40只按体重随机分为4组:①正常对照组:正常饮食;②络气郁滞证候模型组(络气郁滞组):正常饮食,束缚6小时/日;③血管内皮功能障碍病理模型组(病理模型组):3%蛋氨酸饮食;④络气郁滞型血管内皮功能障碍病证复合模型组(复合模型组):3%蛋氨酸饮食+束缚6小时/日。造模时间为6周。记录大鼠的一般状况(精神、皮毛、摄食量、体重);实验前后行1%蔗糖水摄取量试验,实验末进行生物学表征评分、旷场试验、尾悬挂试验。放免法检测血浆内皮素(ET),硝酸还原酶法检测血清NO;光镜观察胸主动脉形态学变化,透射电镜观察主动脉组织内皮细胞(EC)超微结构变化;免疫组化法检测主动脉组织中内皮素-1(ET-1)的表达;循环酶法测定血清HCY水平。2络气郁滞型血管内皮功能障碍模型NEI网络相关因子变化实验动物、分组、造模方法同上,实验末检测NEI网络相关因子:放免法检测血浆促肾上腺皮质激素释放激素(CRH)、促肾上腺皮质激素(ACTH)、糖皮质激素(CORT)、肾素(PRA)、血管紧张素II(AngII)、醛固酮(ALD)及血清中白介素1β(IL-1β)、白介素2(IL-2)、白介素6(IL-6)、肿瘤坏死因子(TNF-α);ELISA法检测去甲肾上腺素(NE)、肾上腺素(E)、γ干扰素(γ-INF)、5羟色胺(5-HT)、多巴胺(DA);计算胸腺和脾脏指数。3通络药物对复合模型干预作用研究将清洁级健康雄性Wistar大鼠50只按体重随机分为复合模型组、复方通络(通心络)、组分配伍(薤白四味)、单味药物(薤白有效部位)、贝那普利组共5组,造模方法及造模时间同上,通心络组为1.2g生药/Kg/d,薤白四味组为3.47g生药/Kg/d,薤白组为1.2g生药/Kg/d,贝那普利组为10mg/Kg/d,连续灌胃给药6周;复合模型组按体重灌服0.5%羧甲基纤维素钠(CMC-Na)溶液。观察大鼠的生物学表征、血管内皮功能、主动脉组织形态学及内皮细胞超微结构、NEI网络相关因子变化,检测方法同上。4络气郁滞型血管内皮功能障碍大鼠主动脉组织葡萄糖调节蛋白78(GRP78)表达及通络药物干预作用将清洁级健康雄性Wistar大鼠80只按体重随机分为正常对照组、络气郁滞组、病理模型组、复合模型组、通心络组、薤白四味组、薤白单味组、贝那普利组共8组,造模方法、造模时间及药物干预同上,实验末无菌取主动脉组织,利用RT-PCR和Western blot方法检测各组大鼠主动脉组织葡萄糖调节蛋白78的基因和蛋白表达。结果:1络气郁滞型血管内皮功能障碍动物模型建立与评价1.1一般状况及生物学表征评分与正常组比较,络气郁滞组大鼠表现精神萎靡不振,扎堆及饮食减少,反应迟钝,毛色光泽性差,体重增长缓慢,饮水量减少等体征和行为变化,但动物毛色较病理模型组略轻;病理模型组与正常组比较,无明显精神萎靡,但摄食量减少,体重增长缓慢。复合模型组较络气郁滞组、病理模型组体征变化更为明显,出现明显懒动,蜷卧嗜睡,精神萎靡不振,攻击性行为减少等外观表征。与正常对照组比较,络气郁滞组、复合模型组生物学表征评分明显增高(P<0.01)。复合模型组较病理模型组明显增高(P<0.05)。与正常对照组比较,络气郁滞组、病理模型组、复合模型组日摄食量减少(P<0.01),体重增长缓慢(P<0.01),复合模型组较络气郁滞组、病理模型组更明显(P<0.01)。1.2行为学检测与正常对照组比较,络气郁滞组、病理模型组、复合模型组旷场试验得分无明显差异(P>0.05)。与正常对照组比较,络气郁滞组、复合模型组尾悬挂试验不动时间明显延长,挣扎次数明显减少。病理模型组较正常对照组无明显差异,而较络气郁滞组具有明显差异;复合模型组较病理模型组不动时间明显延长,挣扎次数显著减少(P<0.01)。与正常对照组比较,络气郁滞组、病理模型组、复合模型组蔗糖水摄取量显著降低(P<0.01或P<0.05)。1.3血管内皮功能及形态络气郁滞组血浆ET含量(163.19±24.74 pg/ml)、病理模型组(161.70±13.96 pg/ml)、复合模型组(178.25±21.85 pg/ml)较正常对照组(142.91±8.72pg/ml)明显升高(P<0.05或P<0.01)。络气郁滞组大鼠血清NO(25.68±10.92 umol/L)、病理模型组(26.82±13.03 umol/L)、复合模型组(24.91±9.95 umol/L)较正常对照组(45.22±12.42umol/L)明显下降(P<0.05或P<0.01)。病理模型组与络气郁滞组之间ET、NO无明显差异(P>0.05)。复合模型组较络气郁滞组之间ET、NO无明显差异(P>0.05),较病理模型组ET增加(P<0.05),NO无明显差异(P>0.05)。络气郁滞组大鼠较正常组血清HCY明显增高(P<0.05);病理模型组、复合模型组大鼠较正常组明显增高(P<0.01),较络气郁滞组增高(P<0.05)。病理模型组与复合模型组之间无明显差异(P>0.05)。HE染色显示:络气郁滞组:内皮细胞轻微肿胀,平滑肌轻微水肿。病理模型组:内皮细胞肿胀、分布不均匀、密度增加,内膜有多量淋巴细胞附壁、内膜内有炎细胞浸润、内弹力板有断裂;平滑肌细胞水肿。复合模型组:内皮细胞肿胀、分布不均匀、密度增加,内膜有多量淋巴细胞附壁、内膜内有炎细胞浸润、内弹力板有断裂;平滑肌可见明显水肿。免疫组织化学染色结果显示:正常对照组主动脉组织未见ET-1表达。络气郁滞组、病理模型组、复合模型组主动脉组织中ET-1表达。电镜显示:络气郁滞组:内皮细胞线粒体大部分嵴和部分膜融合或消失,粗面内质网扩张。脱颗粒明显,吞饮小泡数量明显减少。病理模型组:内皮细胞线粒体大部分嵴和少部分膜融合或消失,粗面内质网轻度扩张,脱颗粒现象明显,吞饮小泡数量减少。复合模型组:内皮细胞线粒体大部分嵴和少部分膜融合或消失,几乎见不到粗面内质网和吞饮小泡。2络气郁滞型血管内皮功能障碍NEI网络相关因子变化2.1下丘脑-垂体-肾上腺皮质轴与正常对照组比较,络气郁滞组、病理模型组、复合模型组之间CRH无明显差异(P>0.05)。络气郁滞组、病理模型组、复合模型组ACTH明显下降(P<0.05),CORT明显增高(P<0.01或P<0.05),络气郁滞组与病理模型组之间无明显差异(P>0.05)。复合模型组大鼠较络气郁滞组、病理模型组ACTH无明显差异(P>0.05),CORT明显增高(P<0.01)。2.2肾素-血管紧张素-醛固酮系统与正常对照组比较,络气郁滞组、病理模型组、复合模型组大鼠PRA、AngII明显增高(P<0.01或P<0.05),络气郁滞与病理模型组之间无明显差异(P>0.05)。与正常对照组比较,病理模型组ALD有增高,但无明显差异(P>0.05),复合模型组ALD明显增高(P<0.01)。与络气郁滞组比较,复合模型组ALD明显增高(P<0.01)。2.3交感神经-肾上腺髓质系统与正常对照组比较,络气郁滞组、复合模型组大鼠NE、E水平明显增高(P<0.01);病理模型组NE、E水平无明显差异。与病理模型组比较,络气郁滞组、复合模型组NE、E水平明显增高(P<0.01或P<0.05)。2.4单胺类神经递质与正常对照组比较,络气郁滞组、病理模型组、复合模型组大鼠5-HT、DA明显下降(P<0.01),络气郁滞、病理模型组之间无明显差异(P>0.05)。与络气郁滞组比较,复合模型组5-HT、DA明显下降(P<0.01或P<0.05)。2.5脾脏、胸腺脏器指数的变化:与正常对照组比较,络气郁滞组脾脏指数、胸腺指数降低(P<0.05);病理模型组、复合模型组脾脏指数明显增高(P<0.01),胸腺指数降低(P<0.05)。复合模型较病理模型脾脏指数降低(P<0.05)。复合模型较络气郁滞组、病理模型组胸腺指数明显降低(P<0.01或P<0.05)。2.6γ-INF、TNF-α、IL-1β、IL-2、IL-6的变化:与正常对照组比较,络气郁滞组大鼠γ-INF下降,但无明显差异(P>0.05),病理模型组、复合模型组γ-INF明显下降(P<0.01)。络气郁滞与病理模型组之间无明显差异(P>0.05)。复合模型组较络气郁滞组大鼠明显下降(P<0.05)。各组之间TNF-α、IL-1β、IL-2、IL-6无明显差异(P>0.05)。3通络药物对络气郁滞型血管内皮功能障碍模型干预作用3.1生物学表征评分及行为学给药6周后,与复合模型组比较,贝那普利组生物学表征评分无显著差异(P>0.05)。薤白组、薤白四味组、通心络组可明显降低模型大鼠的生物学表征评分(P<0.01或P<0.05),较贝那普利组有明显差异(P<0.01)。各通络药物组之间无明显差异(P>0.05)。与复合模型组比较,贝那普利组大鼠的体重无明显差异(P>0.05)。薤白组、薤白四味组、通心络组大鼠体重明显增加(P<0.05或P<0.01),与贝那普利组比较具有明显差异(P<0.01)。与复合模型组比较,各用药组可不同程度的增加模型大鼠的日摄食量。与复合模型组比较,贝那普利组蔗糖水摄取量无明显差异,薤白组、薤白四味组、通心络组蔗糖水摄取量明显增加(P<0.01),与贝那普利组比较有明显差异(P<0.01)。与复合模型组比较,贝那普利组大鼠挣扎次数明显增加(P<0.01),不动时间缩短,但无明显差异(P>0.05);各通络药物组大鼠不动时间明显缩短,挣扎次数增加,具有显著性差异(P<0.01)。薤白、薤白四味组较贝那普利组有明显作用(P<0.05)。3.2血管内皮功能与形态与复合模型组比较,各治疗组血浆ET水平明显降低(P<0.01或P<0.05),各治疗组之间无明显差异(P>0.05)。贝那普利组、薤白四味组、通心络组NO水平升高,但无明显差异(P>0.05);薤白组NO明显升高(P<0.01),较贝那普利组具有明显差异(P<0.05)。与复合模型组比较,各用药组可不同程度的改善模型大鼠的主动脉大体形态,以通心络组较为明显。与复合模型组比较,各治疗组可不同程度的改善模型大鼠主动脉组织内皮细胞的超微结构,以通心络组较为明显。3.3 NEI网络相关因子单胺类神经递质的变化:与复合模型组比较,贝那普利组5-HT水平无明显差异(P>0.05),薤白组、薤白四味组、通心络组明显增高(P<0.01)。各治疗组可明显升高DA的水平(P<0.01)。下丘脑-垂体-肾上腺皮质轴:与复合模型组比较,贝那普利组、薤白四味组、通心络组血清CORT明显降低( P<0.05或P<0.01),薤白组无明显差异(P>0.05);薤白四味组、通心络组较贝那普利组有显著性差异( P<0.01);薤白四味较薤白组有显著性差异(P<0.01)。交感神经-肾上腺髓质系统:与复合模型组比较,贝那普利组、薤白组、薤白四味组、通心络组血清NE水平明显降低( P<0.05或P<0.01);薤白四味组优于贝那普利组(P<0.05),薤白四味组优于薤白组(P<0.01)。各治疗组血清E水平明显降低(P<0.01),薤白四味、通心络组优于贝那普利组( P<0.05或P<0.01)。肾素-血管紧张素-醛固酮系统:与复合模型组比较,各治疗组血浆PRA水平明显降低(P<0.01),各治疗组之间无明显差异(P>0.05)。各治疗组血浆AngII水平明显降低( P<0.05或P<0.01),其中薤白组优于贝那普利组(P<0.05)。各治疗组ALD水平降低,其中薤白四味组、通心络组作用明显( P<0.05),薤白四味组优于贝那普利组(P<0.05),薤白四味组、通心络组优于薤白组( P<0.05或P<0.01)。细胞因子:与复合模型组比较,各治疗组γ-INF水平明显增高( P<0.05或P<0.01);薤白组、薤白四味组、通心络组与贝那普利组之间无明显差异(P>0.05);通心络组优于薤白口服组(P<0.05)。与复合模型组比较,各治疗组TNF-α水平有下降趋势,但无明显差异(P>0.05);各治疗组之间无明显差异(P>0.05)。与复合模型组比较,各治疗组IL-1β、IL-2、IL-6无明显差异(P>0.05)。胸腺、脾脏指数:与复合模型组比较,各治疗组胸腺指数有增大趋势,但无明显差异(P>0.05)。与复合模型组比较,各治疗组脾脏指数无明显差异(P>0.05)。4络气郁滞型血管内皮功能障碍大鼠主动脉组织GRP78基因和蛋白相对表达的变化与正常组比较,络气郁滞组、病理模型组、复合模型组GRP78基因的相对表达量均明显增强(P<0.05),三组间无明显差异(P>0.05)。复合模型组GRP78蛋白表达量较正常组、络气郁滞组、病理模型组明显增强(P<0.05)。与复合模型组比较,各治疗组可明显抑制GRP78基因和蛋白的相对表达(P<0.01或P<0.05),各治疗组之间无明显差异(p>0.05)。结论:1络气郁滞型血管内皮功能障碍动物模型建立及评价依据通过临床流行病学调查建立的络气郁滞型内皮功能障碍临床辨证诊断标准,采用慢性束缚法建立络气郁滞大鼠证候模型,以高蛋氨酸饮食建立内皮功能障碍大鼠病理模型,基于束缚应激即刻引起血清HCY增高,NO明显下降,以“束缚+蛋氨酸饮食”首次建立络气郁滞型血管内皮功能障碍病证复合大鼠模型。采用一般状况观察、生物学表征评分、行为学指标和血管内皮形态和功能检测等半定量和定量检测方法对上述模型进行综合评价。研究表明,络气郁滞证候模型既表现出明显的精神萎靡,反应迟钝,摄食量减少、体重增长缓慢等证候表现,又表现出明显的内皮形态和功能的改变,显示出络气郁滞证候因素在血管内皮功能障碍发病中发挥重要作用。病理模型仅有内皮形态和功能改变而无明显的精神萎靡等症状。复合模型大鼠表现出络气郁滞证候特征性的同时出现内皮功能障碍,更充分地体现出临床疾病特点和证候特征信息。2络气郁滞型血管内皮功能障碍大鼠NEI网络的变化特征通过对正常对照组、络气郁滞组、病理模型组、复合模型组大鼠NEI网络相关因子检测探讨其变化特征,对比分析络气郁滞证候因素在血管病变中的重要作用。结果表明与正常对照组比较,络气郁滞组、病理模型组、复合模型组均不同程度存在下丘脑-垂体-肾上腺轴、肾素-血管紧张素-醛固酮系统激活、单胺类递质下降,免疫系统能力下降;络气郁滞组与复合模型组交感神经-肾上腺髓质系统激活,而病理模型组此系统变化不明显。与络气郁滞组和病理模型组比较,复合模型显示出络气郁滞证和病理模型的非线性叠加效应。揭示既往未被重视的证候因素是血管病变发生的重要影响因素。NEI网络相关因子的紊乱可能是络气郁滞型血管内皮功能障碍的生物学基础之一。3通络药物对络气郁滞型血管内皮功能障碍大鼠的干预作用以通络复方(通心络)、通络药物组分配伍(薤白四味)、单味通络药物(薤白)分别干预络气郁滞型血管内皮功能障碍模型,三个层次通络药物均可以改善大鼠的一般状况及生物学评分,提高蔗糖水摄取量,改善大鼠的情志抑郁症状,改善内皮功能,不同程度抑制下丘脑-垂体-肾上腺皮质轴、肾素-血管紧张素-醛固酮系统、交感神经-肾上腺髓质系统的激活,升高单胺类神经递质,增强免疫功能,调节NEI网络。各通络药物组改善行为学、内皮功能、NEI网络指标优于对照药贝那普利,尤其在改善一般状况和行为学方面,三个层次类通络药物作用明显优于贝那普利,其中通心络作用更为明显,可能与复方通络药物针对血管病变共性病理环节配伍有关,在动物模型实验层面佐证了辨证论治及病、证、法、药的关联性与科学性。通络干预络气郁滞型血管内皮功能障碍模型呈现出通过调节疾病状态下NEI网络多个系统的紊乱状态,重建机体内多个系统之间的自稳调控机制,以达到动态平衡的效应目标,为揭示通络复方的效应规律提供思路。4络气郁滞型血管内皮功能障碍大鼠主动脉组织葡萄糖调节蛋白78表达的变化近来的研究发现内质网应激,是线粒体应激、核应激的共同通路。内质网应激反应是细胞本身的一种自我保护性机制,但是反应功能障碍或者过强的和长时间的内质网应激都可以引起细胞功能失调,甚至细胞死亡等病理现象。本实验发现证候模型及复合模型大鼠主动脉组织可见GRP78蛋白、mRNA的表达增强,三个层次通络药物均可明显降低模型大鼠主动脉组织的GRP78蛋白及其mRNA的表达,与贝那普利比较无差异。提示内质网应激可能是络气郁滞、复合模型的共同发病机制之一,薤白、薤白四味及通心络可能通过降低模型大鼠主动脉组织的GRP78蛋白及其mRNA的表达来改善血管内皮功能的。更多还原

【Abstract】 Objectives: Directed by the theory of collateral disease,according to diagnostic standard of vessel collateral-vascular system disease established through the clinical epidemiological investigation, to establish the rat model of endothelial dysfunction with collateral-qi stasis applied by chronic constraint stress and homomethionin diet. The rat model was evaluated systematically from different aspects of general condition of health, score of biological superficial syndrome,ethology, and the construction and function of blood vessel endothelium. At the same time, the changes of main indicatrix related to nerve–endocrine-immunization (NEI) network were observed, and the effect of different level of Tongluo drugs including compounds of Chinese herbal, compounds of components and single effective component on the rat model was explored. The purpose of our research was to elucidate the pathomechanism of the model with endothelial dysfunction and collateral-qi stasis, explore the effect of syndrome factor of TCM on the endothelial dysfunction, and evaluate systematically the effect of Tongluo drugs.Methods : 1 The establishment and evaluatement of the rat model of endothelial dysfunction with collateral-qi stasisIn this experiment, the rat model of collateral-qi stasis was established by chronic constraint. The rat model of pathology was established by homomethionin diet. On the basis of the result of experiment (data not shown in the paper) that acute constraint stress could raise blood serum HCY level and decrease NO level, the combinated rat model with endothelial dysfunction and collateral-qi stasis was established by chronic constraint stress and homomethionin diet. Forty healthy male Wistar rats of sanitary degree were randomly divided into 4 groups according to their body weight,①the normal control group, fed with normal diet.②the collateral-qi stasis group,fed with normal diet and given to repeated restraint stress for 6 hours every day.③the pathological model group,fed with 3% methionine diet.④the combinated model group (rat animal models of endothelial dysfunction and collateral-qi stasis), given to repeated restraint stress for 6 hours every day and fed with 3% methionine diet simultaneously. The whole experimental time is 6 weeks. The general condition of health including spirit, coat, appetite, weight was recorded. The test of intake of 1% cane sugar water at the beginning and endpoint of the experiment was measured. At the endpoint of experiment, the score of biology superficial syndrome, open-field and tail swing were carried out. The concentration of plasma endothelian (ET)was detected with radioimmunoassay, and serum nitro oxygen (NO) was detected with nitrate reductase. The morphological changes of aorta thoracica observed by light microscope and the ultramicrostructure changes of aorta observed by transmission electron microscope. Immunohistochemistry method was used to analyze the expression of endothelin-1(ET-1) in aorta tissue.2 The changes of NEI network of the rat model of endothelial dysfunction with collateral-qi stasisExperimental animal, the method of dividing groups and establishing model were the same as aboves. The concentrations of CRH, ACTH, CORT, PRA, AngII, ALD and serum IL-1β, IL-2, IL-6, TNF-αwere detected with radioimmunoassay. The concentration of NE, E,γ-INF, 5-HT, DA were detected with ELISA. The index number of thymus and spleen were calculated.3 Effect of Tongluo drugs on the combinated model ratsFifty healthy male Wistar rats of sanitary degree were randomly divided into 5 groups according to their body weight,①the combinated model group ,②Benazepril group ,administered at 10mg/Kg/d.③Xiebai group, administered at 1.2g crude drug /Kg/d.④Xiebaisiwei group, administered at 3.47g crude drug /Kg/d.⑤Tongxinluo group, administered at 1.2g crude drug/Kg/d. The time and method of established model were the same as aboves. The time of intragastric administration is 6 weeks. The combinated model group was given with 0.5%CMC-Na. The changes of biology superficial syndrome, the morphological changes and the ultramicrostructure changes of aorta, blood vessel endothelial function and the changes of NEI nethwork were detected.4 The research of expression of glucose regulating protein78(GRP78) mRNA and protein in aortic tissue of the combinated model rats and effect of Tongluo drugs on itEighty healthy male Wistar rats of sanitary degree were randomly divided into 8 groups according to their body weight,①the normal control group,②the collateral-qi stasis group,③the pathological model group,④the combinated model group,⑤Benazepril group,⑥Xiebai group,⑦Xiebaisiwei group,⑧Tongxinluo group. The method of establishing model and intragastric administration were the same as aboves. The expression of GRP78 mRNA and protein in aortic tissue of every group rats was detected by RT-PCR and WesternblotResults:1 The establishment and evaluatement of the rat model of endothelial dysfunction with collateral-qi stasis1.1 The general condition of health and score of superficial syndromeCompared with normal control group, the rats of collateral-qi stasis group showed depressed, reduction of diet, torpidity, lost of color pattern, increasing slowly weight, while the rats of pathological model group showed reduction of diet, lost of color pattern and increasing slowly weight except with obvious depressed. The rats of combinated model showed lazy, drowsiness, depressed and reduction of aggressive behavior.Compared with normal control group, the score of biology superficial syndrome of the collateral-qi stasis group and the combinated model group increased significantly (P<0.01). Compared with the pathological model group, the score of the combinated model group increased (P<0.05).Compared with normal control group, the appetite of the collateral-qi stasis group, the pathological model group and the combinated model group were significantly more lowered (P<0.01). And the body weight increased slowly (P<0.01). Compared with the pathological model group and collateral-qi stasis group, the changes of the combinated model group was obviously lowered (P<0.01).1.2 The result of ethologyCompared with the normal control group, the score of the open-field of the collateral-qi stasis group, the pathological model group and the combinated model group had no difference.Compared with normal control group, the immobility time of the tail swing of the collateral-qi stasis group and the combinated model group were prolonged significantly and the struggling frequency were reduced significantly (P<0.01), the pathological model group had no difference.Compared with normal control group, the intake of cane sugar water of the collateral-qi stasis group, the pathological model group and the combinated model group were significantly lowered (P<0.01 or P<0.05).1.3 The changes of function and morphous of blood vessel endothelium in every groupCompared with the content of ET(142.91±8.72pg/ml)in plasma of rats in normal control group, the content (163.19±24.74 pg/ml) of the collateral-qi stasis group, the content (161.70±13.96 pg/ml)of the pathological model group and the content(178.25±21.85 pg/ml) of combinated model group were significantly increased (P<0.05). Compared with the content of NO(45.22±12.42umol/L) in serum of rats in normal control group, the content (25.68±10.92 umol/L) of the collateral-qi stasis group, the content(26.82±13.03 umol/L) of the pathological model group and the content(24.91±9.95 umol/L) of combinated model group were significantly lowered (P<0.05 or P<0.01). The difference between the combinated model group and the collateral-qi stasis group was not obvious. Compared with the normal control group, the content of HCY in serum of rats in the collateral-qi stasis group, the pathological model group and the combinated model group increased significantly (P<0.05).HE staining showed that:The collateral-qi stasis group: endothelial cells were lightly engorge. Smooth muscle were lightly edema.The pathological model group: endothelial cells were engorge, maldistribution. The density increased. There were many lymph-cell and inflammatory cells in the tunica intima. Smooth muscle were edema.The combinated model group: endothelial cells were engorge, maldistribution. The density increased. There were many lymph-cell and inflammatory cells in the tunica intima. Smooth muscle were obvious edema.Immunohistochemical staining showed that:The expression of ET-1 was not founded in the arterial tissue of the control group.The expression of ET-1 in the arterial tissue of the collateral-qi stasis group, the pathological model group and the combinated model group were enhanced.Transmission electron microscope showed that:The collateral-qi stasis group: some crests of mitochondria of vascular endothelial cells fused with membrance, and even disappeared. Rough endoplasmic reticulum was broaden. The phenomenon of degranulation was obvious. The number of pinocytosis vesic was significantly decreased.The pathological model group: some crests of mitochondria of vascular endothelial cells fused with membrance, and even disappeared. Rough endoplasmic reticulum was lightly broaden. The phenomenon of degranulation was obvious. The number of pinocytosis vesic was decreased.The combinated model group: some crests of mitochondria of vascular endothelial cells fused with membrance, and even disappeared. Rough endoplasmic reticulum and the pinocytosis vesic could not be founded almost. 2 The changes of NEI network of the rat animal models of endothelial dysfunction with stasis of collateral-qi2.1 Hypothalamic-Pituitary-Adrenal Axis:Compared with normal control group, the CRH level of the collateral-qi stasis group, the pathological model group and the combinaed model group had no statistical difference (P>0.05). The ACTH level reduced and the CORT level increased significantly(P<0.01). Compared with the collateral-qi stasis group and the combinated model group, the ACTH level of the combinated model group had no difference(P>0.05). And the CORT level increased significantly (P<0.01).2.2 Renin-Angiotensin-Aldosterone System:Compared with normal control group, the PRA and AngII level of the collateral-qi stasis group and the combinated model group increased significantly(P<0.01or P<0.05). The difference between the collateral-qi stasis group and the pathological model group was not obvious(P>0.05). Compared with the collateral-qi stasis group and the combinated model group, the ALD level of the combinated model group increased significantly(P<0.01).2.3 Trisplanchnic Nerve -Adrenal Medulla System:Compared with normal control group, the NE, E level of the collateral-qi stasis group and the combinated model group increased significantly(P<0.01). the difference between the pathological model group and the control group was not obvious(P>0.05). Compared with the pathological model group, the NE, E level of the collateral-qi stasis group and the combinated model group increased(P<0.01 or P<0.05).2.4 Monoamine neurotransmitter:Compared with normal control group, the 5-HT, DA level of the collateral-qi stasis group, the pathological model group and the combinated model group decreased significantly (P<0.01). The difference between the pathological model group and the collateral-qi stasis group was not obvious(P>0.05). Compared with the collateral-qi stasis group, the 5-HT, DA level of the combinated model group decreased(P<0.01or P<0.05).2.5 The index number of spleen and thymus:Compared with normal control group, the index number of spleen and thymus of the collateral-qi stasis group lowered (P<0.05), the index number of spleen of the pathological model group and the combinated model group increased (P<0.01), the index number of thymus lowered(P<0.05). The index number of thymus of the combinated model group was the lowest in four groups.2.6γ-INF, TNF-α, IL-1β, IL-2, IL-6:Compared with normal control group, theγ-INF level of the pathological model group and the combinated model group lowered(P<0.01). Compared with the collateral-qi stasis group, theγ-INF level of the combinated model group lowered (P<0.05). There were no obvious difference on the level of TNF-α, IL-1β, IL-2 and IL-6 among the four groups (P>0.05).3 Effect of Tongluo drugs on the combinated model rats 3.1 Score of biological superficial syndrome ethology:After 6 weeks of administration, compared with the combinated model group, the score of biological superficial syndrome of Benazepril group had no obvious change(P>0.05). The score of Xiebai group, Xiebaisiwei group and Tongxinluo group decreased significantly(P<0.01 or P<0.05). Compared with Benazepril group, the three groups decreased(P<0.01). There were no obvious difference in three groups(P>0.05).Compared with the combinated model group, the body weight of Benazepril group had no obvious difference(P>0.05). Compared with the combinated model group and Benazepril group, the weight of Xiebai group, Xiebaisiwei group and Tongxinluo group increased significantly(P<0.05 or P<0.01 ).Every drug group could increase the appetite of the combinated model rats.Compared with the combinated model group, the intake of cane sugar water of Benazepril group had no obvious difference(P>0.05). Compared with the combinated model group and Benazepril group, the intake of Xiebai group, Xiebaisiwei group and Tongxinluo group increased significantly(P<0.01).Compared with the combinated model group, the struggling frequence of Benazepril group increased(P<0.01), the immobility time decreased, but it had no difference(P>0.05). The immobility time of three Chinese medicine shortened, the struggling frequence increased(P<0.01). They had obvious difference with Benazepril group(P<0.05).3.2 Endothelial function and morphous:Compared with the combinated model group, ET level of every medicine groups decreased significantly(P<0.01 or P<0.05). There were no obvious difference in every medicine groups. NO level of Benazepril group, Xiebaisiwei group and Tongxinluo group increased, but it had no obvious difference(P>0.05). NO level of Xiebai group increased(P<0.01). Compared with Benazepril group, NO level of Xiebai group increased(P<0.05).Compared with the combinated model group, every medicine group could improve the general structure and endothelial cell ultramicrostructure of aorta throacica, especially Tongxinluo group did.3.3 The indexs related to NEI networkMonoamine neurotransmitter: Compared with the combinated model group, 5-HT level of every drug group increased, especially Xiebai group, Xiebaisiwei group and Tongxinluo group increased significantly (P<0.01). There was no obvious difference between Benazepril group and the combinated model group(P>0.05). DA level of every drug group increased obviously(P<0.01).Hypothalamic-Pituitary-Adrenal Axis: Compared with the combinated model group, CORT level of Benazepril group, Xiebaisiwei group and Tongxinluo group decreased significantly(P<0.01 or P<0.05), Xiebai group had no obvious difference(P>0.05). Compared with Benazepril group, the CORT level of Xiebaisiwei group and Tongxinluo group decreased (P<0.01). The difference between Xiebaisiwei group and Xiebai group was obvious(P<0.01).Trisplanchnic Nerve -Adrenal Medulla System: Compared with the combinated model group, NE and E level of every drug group decreased(P<0.01 or P<0.05). Compared with Benazepril group, NE level of Xiebaisiwei group decreased(P<0.01). E level of Xiebaisiwei group and Tongxinluo group decreased (P<0.01 or P<0.05). The effect of Xiebaisiwei group in decreasing NE level was better than Xiebai group (P<0.01).Renin-Angiotensin-Aldosterone System: Compared with the combinated model group, PRA, AngII and ALD level of every drug group decreased. Compared with the Benazepril group, AngII level of Xiebai group decreased(P<0.05), ALD level of Xiebaisiwei group decreased( P<0.05). Compared with Xiebai group, ALD level of Xiebaisiwei group and Tongxinluo group decreased(P<0.05 or P<0.01).Cell factor, thymus and spleen index number: Compared with the combinated model group,γ-INF level of every drug group increased(P<0.05 or P<0.01). The effect of increasingγ-INF of Tongxinluo group was better than Xiebai group( P<0.05). Compared with the combinated model group, TNF-αlevel of every drug group decreased and the index number of thymus increased , but they had no obvious difference (P>0.05). Compared with the combinated model group, the index number of spleen, IL-1β, IL-2 and IL-6 of every drug group had no obvious difference(P>0.05).4 The expression of GRP78 in aortic tissue of rats with endothelial dysfunction and collateral-qi stasis and effect of Tongluo drugs on itCompared with the control group, the expression of GRP78 mRNA of the collateral-qi stasis group, the pathological model group and the combinated model group strengthened significantly (P<0.05). There was no obvious difference among three groups.Compared with the control group, the collateral-qi stasis group and the pathological model group, the expression of GRP78 protein of the combinated model group strengthened significantly (P<0.05).Compared with the combinated model group, the expression of GRP78 mRNA and protein of every medicine group were repressed obviously(P<0.05 or P<0.01). There was no difference among drug groups(P>0.05).Conclusion:1 Establishment and evaluation of rat model with endothelial dysfunction and collateral-qi stasisAccording to diagnostic standard of vessel collateral-vascular system disease established through the clinical epidemiological investigation, The combinated rat animal model of endothelial dysfunction and collateral-qi stasis was established successfully with the method of chronic constraint stress and homomethionin diet on the basis of the fact that acute constraint stress could raise blood serum HCY as foundation. The model rats were evaluated synthetically by many semi-quantitative and quantitative methods including the general condition of health, the score of biology superficial syndrome, indicatrixes of ethology, the construction and function of blood vessel endothelium. The study indicated that the combinated model could show macroscopic syndrome of collateral-qi stasis and the changes of construction and function of blood vessel endothelium. This showed that the establishment of combinated model was successful. The study showed that syndrome of collateral-qi stasis of TCM played an important role influencing endothelial function.2 The changing characteristics of NEI network of rat model with endothelial dysfunction and collateral-qi stasisOn the basis of analysis and investigation of NEI network in rats of normal control group, collateral-qi stasis model, pathology model and combinated model of collateral-qi stasis with endothelial dysfunction, to explore the changing characteristics of NEI network, and analyze the effect of the syndrome facor of collateral-qi stasis on the vasculopathy. The study showed that: hypothalamic-pituitary-adrenal axis and renin-angiotensin-aldosterone system were activated in different degree, and Monoamine neurotransmitter and the ability of immune system inhibited in collateral-qi stasis group, pathological model group and the combinated model group. Trisplanchnic nerve -adrenal medulla system was activated both the collateral-qi stasis group and the combinated model group, while the trisplanchnic nerve -adrenal medulla system in the pathological model group was not activated. The combinated model showed nonlinear superposition effect between the collateral-qi stasis model and the pathology model. The result indicated that syndrome of collateral-qi stasis of TCM paid no attention previously was the important influcence factor. The disorder of indexes related to NEI network was possible pathological basis of rat model with endothelial dysfunction and collateral-qi stasis..3 The effect of Tongluo drugs on the combinated model with endothelial dysfunction and collateral-qi stasisThe combinated model were cured with Xiebai, Xiebaisiwei and Tongxinluo. Three species of Tongluo drugs could improve general condition of health and score of superficial syndrome, raise the intake of cane sugar water, and improve depressed emotion and endothelial function in the combinated model rat. At the same time, they could repress the activations of hypothalamic-pituitary-adrenal axis, trisplanchnic nerve -adrenal medulla system, renin-angiotensin-aldosterone system, raise Monoamine neurotransmitter and enhance the ability of immune system. The study also showed that the effect of Chinese herbs was better than Benazepril in improving praxiology, endothelial function and regulating NEI network. The effect of Tongxinluo was better than any other medicines, which was related to compatibility of Tongxinluo due to the pathological link of vasculopathy. This results authenticated determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs was scientific, and disease-syndrome-method-drug of TCM was dependent. The effect of Tongluo drug on the combinated model with endothelial dysfunction and collateral-qi stasis showed regulation mechanism of re-establishing balance of multi-system in body through the improving the disorder state of multi-system belong to the NEI network in the morbid state, which provided the method to reveal the effective regularity of compound recipe of TCM.4 The change of GRP78 in aortic tissue of the combinated model with endothelial dysfunction and collateral-qi stasisThe recent research indicated that endoplasmic reticulum stress was the common pathway both mitochondrion stress and nuclear stress. Endoplasmic reticulum stress was one self-protective mechanism of cell, while the dysfunction, over- intensity and long-time stress could cause the celluar disorder, even celluar death. The study showed the expression of GRP78 mRNA and protein of the collateral-qi stasis group and the combinated model group was strengthened significantly. There species of Tongluo drugs could reduced the expression of GRP78 mRNA and protein of combinated model group, which was not different with Benazepril. The result indicated the endoplasmic reticulum stress was the one of common pathways of syndrome of collateral-qi stasis and the combinated model with endothelial dysfunction and collateral-qi stasis. There species of Tongluo drugs might improved the endothelial function through reducing the expression of GRP78 mRNA and protein of combinated model group.更多还原