【作者】 高怀林；

【导师】 吴以岭；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2008， 博士

【摘要】 目的:本研究以络病理论为指导,建立血管内皮功能障碍的中医证候量化辨证分型标准,并在此基础上建立络气虚滞型血管内皮功能障碍病证复合动物模型和细胞模型,探讨“络气虚滞”状态下血管内皮功能障碍变化、神经内分泌免疫(Neuro-endocrine-immunity,NEI)网络调控紊乱及两者的内在联系,同时观察病证复合模型动物血清对内皮细胞骨架的纤维状肌动蛋白(filamentous-actin,F-actin)及单层内皮细胞通透性的影响,研究络气虚滞对内皮细胞的影响并探索不同类别通络药物的作用机制,为络病理论指导血管病变防治提供科学依据。方法:1血管内皮功能障碍中医辨证分型标准的建立通过文献调研、专家咨询、中医四诊信息的分级赋分确立“血管内皮功能障碍中医证候临床调查表”,并进行临床流行病学调查。纳入标准:①年龄40岁以上;②至少具备下列诊断中的一项:单纯高血压、血脂异常、超重或肥胖、吸烟、代谢综合征人群;③一氧化氮(nitro oxygen, NO)<59 umol/L及内皮素(endothelin , ET)>53.38 pg/ml。排除标准:①心、脑、肾、神经、眼底等靶器官损伤者;②严重肝、肾功能不全者;③妊娠或哺乳期妇女、精神病患者。调查由专人负责,用Epidata3.0建立数据库。将400例入选病人,随机分为甲、乙两组,甲组300例,乙组100例,甲组用于建立量化辨证分型标准及对量化辨证分型标准进行回顾性检验,乙组用于量化辨证分型标准的前瞻性检验。将甲组300例临床调查资料,采用基于熵的复杂系统分划方法对数据进行处理分析,按症状聚堆结果,提取血管内皮功能障碍各证型的中医症状信息,归纳各证型,并计算各证型中的症状对相应证型的贡献分值,采用诊断性试验中的接受者操作特性曲线(receiver operating characteristic curve,ROC曲线)分析方法,结合专业知识,确定各证型的诊断阈值。根据临床流行病学诊断性试验研究方法,进行量化辨证分型标准的回顾性和前瞻性检验。分析各证型之间的关联度,归纳各证型组合规律及病机转趋规律。2络气虚滞型血管内皮功能障碍模型的建立及通络药物干预研究依据上述临床流行病学调查确立的辨证分型标准,以中医“劳则气耗”、“饥则损气”理论为指导,在高蛋氨酸(homocysteine,HCY)饮食建立血管内皮功能障碍大鼠模型基础上,叠加“基础进食+强迫负重游泳”络气虚滞证候因素建模。将清洁级雄性Wistar大鼠随机分为以下7组,每组15只:(1)对照组;(2)血管内皮功能障碍模型组(HCY组);(3)复合模型组(络气虚滞证候+HCY);(4)人参(复合模型+人参);(5)四味通络组(复合模型+四味通络方);(6)通心络组(复合模型+通心络超微粉);(7)贝那普利组(复合模型+贝那普利)。从模型大鼠的生物学表征、血管内皮病理组织形态学、血管内皮的生化功能等方面对模型进行综合评价,同时运用单味药物(人参)、组分配伍药物(四味通络方)、复方通络药物(通心络)干预,并与贝那普利进行对照研究,观察不同层次通络药物对复合模型的作用。3络气虚滞型血管内皮功能障碍模型大鼠NEI网络变化及通络药物干预研究本研究应用络气虚滞型血管内皮功能障碍病证复合大鼠模型,通过检测交感—肾上腺髓质系统:血清去甲肾上腺素(norepinephrine,NE)、肾上腺素(epinephrine,E)的水平(ELISA法);下丘脑—垂体—肾上腺轴(hypothalamo-pituitary-adrenal axis,HPAA):血浆促肾上腺皮质激素释放激素(corticotropin release hormone,CRH)、促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)和血清皮质酮(corticosterone,CORT)的水平(放射免疫分析法);肾素—血管紧张素—醛固酮系统(renin-angiotensin-aldosterone system,RAAS):血浆肾素活性(plasma renin activity,PRA)、血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)、醛固酮(aldosterone,ALD)的水平(放射免疫分析法);免疫系统:血清白介素-1β(interleukin-1β,IL-1β)、白介素-2 (interleukin-2,IL-2)的水平(放射免疫分析法)等,反应NEI网络的功能变化。运用典型相关分析,对NEI网络相关因子与内皮功能相关指标: ET、NO、血管性假血友病因子(von wilebrand factor, vWF)进行相关性分析,并观察通络药物的干预作用。4病证复合模型大鼠血清培养HUVEC建立细胞模型并观察细胞骨架蛋改变及通络药物干预研究采用络气虚滞型血管内皮功能障碍病证复合模型大鼠血清培养人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)细胞株,激光共聚交显微镜观察细胞骨架蛋白F-actin的形态、分布,流式细胞仪检测F-actin含量、伊文思蓝标记牛血清白蛋白检测单层内皮通透性变化,Western blot技术检测HUVEC细胞骨架蛋白F-actin、p38、磷酸化p38(phospho-p38, p-p38)蛋白表达和p38丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号转导通路变化及通络药物干预作用。实验细胞分组:(1)对照组:无血清培养基(F12K)中加入终浓度为20%的胎牛血清;(2)正常血清组:无血清培养基(F12K)中加入终浓度为20%的正常大鼠血清;(3)复合模型血清组:无血清培养基(F12K)中加入终浓度为20%的复合模型大鼠血清;(4)人参组:于无血清培养基(F12K)中先加入人参(终浓度:1μg/ml)孵育,2h后加入20%复合模型大鼠血清;(5)四味通络组:于无血清培养基(F12K)中先加入四味通络方(终浓度:100μg/ml)孵育,2h后加入20%复合模型大鼠血清。(6)通心络组:于无血清培养基(F12K)中先加入通心络超微粉(终浓度:1μg/ml)孵育,2h后加入20%复合模型大鼠血清;(7)SB203580组:于无血清培养基(F12K)中先加入SB203580(p38阻断剂,终浓度:25umol/L)孵育,1h后加入20%复合模型大鼠血清。加入血清后各组培养时间为2h、6h、12h、24h四个时间点。结果:1血管内皮功能障碍中医辨证分型标准的建立以基于熵的复杂系统分划方法,对临床400例血管内皮功能障碍患者症状分析结果显示:血管内皮功能障碍中医证型包括络气郁滞、络气虚滞、郁热、痰湿、痰热、血瘀、阴虚、阳虚等证型;根据各症状对其证型的贡献分值,结合ROC曲线得出血管内皮功能障碍各证型的诊断阈值(证型后括号内数值)及辨证分型标准如下(每位患者症状贡献分值累加大于或等于该证型诊断阈值即可辨证为该证型,每症状后数值为其贡献分值):基本证型:络气郁滞证(5):心胸憋闷3善太息3烦躁3情志抑郁3脉弦2;络气虚滞证(6):乏力3神疲2气短2心悸2懒言1舌淡1脉弱1。兼夹证:郁热证(7):口干3口苦3面红2溲赤2便秘2舌红1苔黄1脉数1;痰湿证(5):胸脘痞闷3肢体困重3形体肥胖1痰多1苔白腻2脉滑1;痰热证(4):咳吐黄痰2口苦2苔黄腻1脉滑数1;血瘀证(1):舌暗1脉涩1;阴虚证(4):潮热盗汗2五心烦热2腰膝酸软2颧红1少苔或无苔1脉细数1;阳虚证(7):畏寒3肢冷3便溏2小便清长1舌淡胖1脉沉迟无力1。回顾性和前瞻性检验结果显示:各证型的灵敏度、特异度、符合率均大于85%,显示出各证型辨证诊断阈值有较好的诊断效能。以证型之间的关联度分析可见,络气郁滞证与郁热证、阴虚证、阳虚证、血瘀证具有较强的关联度,络气虚滞证与痰湿证、阴虚证、阳虚证、血瘀证具有较强的关联度,另外,痰湿证与痰热证、痰湿证与郁热证、郁热证与阳虚证亦有关联关系。基于证型关联度分析及证型分布规律结果:络气虚滞证45.3%,络气郁滞证55.8%,痰湿证17.8%,痰热证22%,血瘀证13.5%,阴虚证38.3%,阳虚证11.8%,可见血管内皮功能障碍中医证型包括络气虚滞、络气郁滞两大基本证型,同时可兼夹痰湿、郁热、阴虚、阳虚、痰热、血瘀等证型。2络气虚滞型血管内皮功能障碍模型建立及通络药物干预作用模型建立后,从以下几方面对模型进行评价:(1)生物学表征:复合模型组大鼠表现为倦怠懒动,精神萎顿,行动迟缓,尾、鼻颜色淡白少润,与对照组相比,生物学表征评分显著升高(P<0.01);心率、呼吸加快(P<0.01)、爬杆时间降低(P<0.01)。(2)胸主动脉内皮细胞形态学观察:光镜结果显示,复合模型组可见内皮细胞肿胀、分布不均匀、局部内皮细胞消失,内膜部分增厚、内膜内有炎性细胞浸润、内弹力板有断裂;电镜结果显示,血管内皮细胞线粒体大部分嵴和膜融合或消失,粗面内质网扩张,呈圆形或椭圆形,脱颗粒现象明显,细胞质、细胞核水肿,部分细胞膜破裂、缺失,内容物外溢。(3)免疫组化结果显示,血管内皮功能障碍模型组和复合模型组可见内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)阳性信号明显减弱,诱生型一氧化氮合酶(inducible nitric oxide synthase, iNOS)、ET1阳性信号明显增强。(4)血管内皮功能相关指标:与对照组相比,血管内皮功能障碍模型组和复合模型组大鼠血浆ET、vWF含量明显升高(P<0.05,P<0.01),血清NO含量显著降低(P<0.01)。本研究成功建立了既反映血管内皮功能障碍临床络气虚滞证的乏力、神疲、气短、心悸等症状、又表现出血管内皮损伤或功能障碍病的复合大鼠模型。通络药物的干预作用:(1)证候改善:与复合模型组相比,三种通络药物干预后,大鼠生物学表征评分、心率、呼吸均明显降低、爬杆时间明显延长,其差异均有统计学意义(P<0.01),以通心络组作用最优;而贝那普利组以上各项指标均无统计学意义(P>0.05)。(2)胸主动脉内皮细胞形态学观察:光镜结果显示,各用药组的胸主动脉内膜病理损伤较复合模型组均明显减轻;电镜结果显示,各用药组内皮细胞超微结构的改变较复合模型组均明显减轻。(3)免疫组化结果显示,各用药组iNOS、ET1阳性黄染信号强度均较复合模型组减轻,eNOS阳性黄染信号强度均较复合模型组增强。从血管内皮病理组织形态学结果证实,通心络组和贝那普利组药物干预效果较好,人参组和四味通络组次之。(4)血管内皮功能相关指标:与复合模型组相比,通心络组和贝那普利组NO均显著升高,ET、vWF均显著下降,均有统计学意义(P<0.01,P<0.05),两组比较无统计学差异(P>0.05);而人参组和四味通络组ET下降(P<0.01,P<0.05),NO和vWF无统计学差异(P>0.05)。3络气虚滞型血管内皮功能障碍模型大鼠NEI网络变化及通络药物干预作用络气虚滞型血管内皮功能障碍模型大鼠NEI网络的变化。反应交感-肾上腺髓质系统功能的各指标,与对照组比较,血管内皮功能障碍模型组与复合模型组NE含量均明显升高(P<0.05,P<0.01),复合模型组E明显升高(P<0.01)、血管内皮功能障碍模型组E无变化。反应HPAA功能的各指标,与对照组相比,血管内皮功能障碍模型组CRH显著下降(P<0.01),复合模型组CRH、ACTH、CORT均显著下降(P<0.05,P<0.01)。反应RAAS功能的各指标,与对照组相比,复合模型组PRA、AngII、Ald均升高(P<0.01,P<0.05),但血管内皮功能障碍模型组PRA、AngII、Ald均无统计学差异(P>0.05)。反映免疫系统相关细胞因子指标,与对照组相比,复合模型组IL-1β升高(P<0.01),IL-2下降(P<0.05),但血管内皮功能障碍模型组IL-1β、IL-2均无统计学差异(P>0.05)。与血管内皮功能障碍模型组相比,复合模型组交感-肾上腺髓质系统ACTH下降(P<0.01),RAAS中PRA、AngII均升高(P<0.01),Ald无统计学差异(P>0.05),IL-1β升高(P<0.05)。显示络气虚滞型血管内皮功能障碍存在NEI网络调控紊乱,证候因素(络气虚滞证)在其中起着重要作用。血管内皮功能指标与NEI网络相关因子变化的典型相关分析结果显示,第一典型相关系数有统计学意义(P<0.01),两系统的相关性主要反映为NO、VWF与ACTH、NE、CORT、IL-2、CRH指标之间的相关关系,显示NEI网络功能紊乱与血管内皮功能障碍具有密切相关性。通络药物的干预作用研究。交感-肾上腺髓质系统,与复合模型组相比,各用药组除贝那普利组NE无统计学差异(P>0.05)外,其余NE、E均明显下降(P<0.05,P<0.01)。HPAA,与复合模型组相比,通心络组CRH、ACTH、CORT均升高(P<0.01),人参组CRH、ACTH升高(P<0.05),四味通络组CORT升高(P<0.01),贝那普利对HPAA指标的影响无统计学意义。RAAS,与复合模型组相比,通心络组和贝那普利组除贝那普利组PRA无显著差异(P>0.05)外,其余PRA、AngII、Ald均下降(P<0.05,P<0.01);人参组AngII下降(P<0.05)。反应免疫因素的指标,与复合模型组相比,各用药组IL-1β均降低(P<0.01),各组间比较无差异(P>0.05);四味通络组和通心络组IL-2均升高(P<0.05);人参组和贝那普利组IL-2无统计学差异(P>0.05)。综上分析,显示复方通心络对NEI网络具有整合调节的作用优势。4病证复合模型血清培养HUVEC建立细胞模型并观察细胞骨架蛋白改变及通络药物干预作用激光共聚焦显微镜观察F-actin形态和分布。正常培养条件的HUVEC内,F-actin主要环绕于细胞周边,形成致密外周带。与对照组相比,在2h时间点,复合模型血清组F-actin无明显变化;在6h时间点,复合模型血清组可见内皮细胞F- actin解聚和重新分布即细胞周边的致密F-actin明显解聚、胞浆内F-actin明显增多、出现大量的应力纤维并达高峰;12h稍有减轻;24h逐渐恢复。流式细胞仪检测F-actin含量。与对照组相比,正常血清组F-actin含量在各时间点没有显著差异(P>0.05)。与对照组和正常血清组比较,复合模型血清组F-actin含量在6h和12h含量显著降低(P<0.05,P<0.01);但在2h和24h均无显著差异(P>0.05)。单层HUVEC通透性检测(OD值)。复合模型血清组2h内皮细胞的通透性表现出升高趋势,6h和12h通透性明显升高(P<0.01),24h呈下降趋势。HUVEC中F-actin、p38、p-p38蛋白表达的变化。与对照组及正常血清组相比,复合模型血清组F-actin蛋白表达量显著下调(P<0.05)、p-p38蛋白表达显著升高(P<0.01,P<0.05),p38蛋白表达无明显变化(P>0.05)。通络药物的干预作用。F-actin的形态与分布变化,在6h和12h时间点,与复合模型组相比,各干预组内皮细胞F- actin形态和分布均有不同程度改善,以通心络组和SB203580组效果最优。F-actin含量变化,与复合模型组相比,在6h和12h时间点,除四味通络组在12h无统计学差异外(P>0.05),其余各干预组F-actin含量均升高(P<0.01,P<0.05)。单层细胞通透性变化,与复合模型组相比,在6h和12h时间点,各干预组单层细胞通透性明显降低(P<0.01)。HUVEC中F-actin、p38、p-p38蛋白表达的变化,与复合模型组相比,各通络药物组和SB203580组能显著上调F-actin蛋白表达(P<0.01),通心络组和SB203580组能下调p-p38蛋白表达(P<0.05)。各通络药物均可保护血管内皮屏障功能,以通心络组作用最优。综上所述,络气虚滞型内皮功能障碍病证复合模型大鼠血清可导致HUVEC内F-actin形态变化和含量减少、单层内皮细胞通透性增高,p38 MAPK信号通路可能参与了这一病变过程;通络药物可以使F-actin重聚和含量升高、降低单层内皮细胞通透性。通心络可能通过抑制p38 MAPK信号通路而对内皮细胞屏障功能起保护作用的。结论:1首次建立血管内皮功能障碍中医证候量化辨证分型标准在国内首次采用临床流行病学调查方法,将基于熵的复杂系统分划方法应用于血管内皮功能障碍中医证候辨证分型标准的研究。研究结果显示,该方法适用于中医证候辨证标准的研究,为中医证候的标准化、规范化研究提供了新思路与新方法。首次建立了血管内皮功能障碍的中医证候量化辨证分型标准,本病可分为络气虚滞、络气郁滞两大基本证型,同时可兼夹痰湿、郁热、阴虚、阳虚、痰热、血瘀等证。络气郁滞或虚滞在血管内皮功能障碍中占有重要地位。2建立络气虚滞型血管内皮功能障碍病证复合动物模型依据临床流行病学调查确立的辨证分型标准,以中医“劳则气耗”、“饥则损气”理论为指导,在高蛋氨酸饮食建立血管内皮功能障碍模型基础上,叠加“基础进食+强迫负重游泳”中医证候因素,成功建立了络气虚滞型血管内皮功能障碍病证复合大鼠模型,为深入研究其病理生理机制提供了可靠的基础。3络气虚滞型血管内皮功能障碍模型大鼠存在NEI网络调控紊乱并与血管内皮功能障碍密切相关复合模型大鼠NEI网络相关因子存在异常改变,具体表现为交感神经—肾上腺髓质系统功能亢进、HPAA功能抑制、RAAS功能亢进和免疫系统细胞因子IL-1β升高、IL-2下降,提示复合模型大鼠存在NEI网络调控紊乱。络气虚滞证候因素在复合模型大鼠NEI网络调控紊乱中起着重要作用。典型相关分析显示,复合模型大鼠血管内皮功能障碍相关指标与NEI网络相关因子之间具有相关性,具体反映在NO、vWF和ACTH、NE、CORT、IL-2、CRH之间互相影响。上述研究提示,NEI网络调控紊乱可能是络气虚滞型血管内皮功能障碍发生、发展的病理生理学机制之一。4首次建立络气虚滞型血管内皮功能障碍细胞模型采用病证复合大鼠模型血清培养HUVEC,模拟病证复合损伤因素对内皮细胞的影响。结果显示,复合模型内皮细胞骨架蛋白F-actin含量降低、解聚和重新分布,单层细胞通透性增高,其机制可能与p38 MAPK信号通路激活有关。5通络药物对血管内皮功能障碍的整合调节作用不同类别的通络药物干预,均可明显改善复合模型大鼠络气虚滞证候表现,减轻血管内皮细胞的形态损伤,调整内皮细胞相关因子的变化;改善交感神经-肾上腺髓质系统、HPAA、RAAS、免疫系统的功能紊乱。其中以复方通心络作用最明显,提示不同通络药物的科学组方在保护内皮功能方面发挥出整合调节优势。体外实验研究证实,复方通心络可改善F-actin的形态与分布,提高F-actin含量,进而降低内皮通透性,保护血管内皮,其机制与抑制p38 MAPK信号通路活性有关。更多还原

【Abstract】 Objective: Under the guidance of Collateral Disease Theory, the TCM quantitative standards of differentiation of symptoms and signs for classification of syndrome on vascular endothelial dysfunction (VED) were established. On the base of the above, the animal model and the cell model of collateral-QI deficiency type with VED were made. The changes of VED and the imbalance of NEI network due to collateral-QI deficiency and the internal association between them were investigated. Meanwhile, the effect of serum from combined model rats on the endothelial cytoskeletal protein, F-actin, and the permeability of cultural monoptychial endothelium were observed. The effect ofcollateral-QI deficiency on endothelial cells and the mechanism of the different kinds of Herbs dredging collaterals were researched. All of the work will provide the foundation of treatment and prevention of vascular diseases by collateral disease theory of Traditional Chinese Medicine (TCM)Methods:1 The establishment of syndrome standards for classification of TCM differentiation of symptoms and signs on VED“The clinical questionnaire of TCM syndrome of vascular endothelial dysfunction”was established on the basis of literature research, expert consultation, and grades and scores of examination by TCM four methods . Then clinical epidemiological investigations were carried out. The inclusive criteria is as follows:①the age is above 40 years old;②at least with one of diagnosis as folloiwing: simple hypertension, hyperlipidemia, overweight or obesity, smoking, and metabolic syndrome;③NO<59umol /L and ET>53.38pg/ml. The case will be eliminated with one of diagnosis as folloiwing:①target organ damages such as heart, brain, kidney, nerve, ocular fundus;②serious liver or kidney dysfunctions;③pregnancy or breast-feeding women or psychotic patients. The special messenger was responsible for the investigation. The database was established using Epidata3.0. Four hundred patients selected were randomly divided into Group A and Group B, with 300 cases in Group A and 100 cases in Group B. The data of Group A were used for the establishment the quantitative standards of differentiation of symptoms and signs and retrospective test. The data of Group B were used for the prospective test of this standard. The clinical investigating data of 300 cases in A group were analyzed with entropy-based partition method for complex systems. According to the results of symptoms clustering, the TCM information of symptoms of VED was extracted, and the syndromes were summarized. The contributing scores of each symptom to its syndrome were calculated. ROC curve analysis method was applied to determine the threshold of syndromes. The retrospective and prospective tests of quantitative standards for differentiation of syndromes were conducted according to the research methods in clinical epidemiology diagnostic test. The combination rule and pathogenesis converting rule of syndromes were summed up through analyzing the correlative values between various syndromes.2 Establishment of rat models of VED due to collateral-QI deficiency and the intervention research of Herbs dredging collaterals.According to the TCM standards established, under the guidance of TCM theory of "over-strain consuming essence" and "hunger damaging essence". rats in our study were given homomethionin diet to establish VED models, then“Basic Diet combined loaded Swimming”was applied to superimpose the syndrome of collateral-QI deficiency. As a result, the model of combining disease and syndrome, complex model, was established. Male Wistar rats were randomly divided into the following seven groups, and there were 15 rats in each group : (1) control group; (2) pathological model group (HCY group); (3) complex model group (stagnancy of collateral-QI deficiency + HCY); (4) Ginseng group (complex model + Ginseng); (5) Siweitongluo group (complex model + Siweitongluo); (6) Tongxinluo group (complex model + Tongxinluo); (7) Benazepril group (complex model + benazepril). These models were evaluated by the methods of quantitation or half-quantitation score of the biological superficial syndrome, patho-histomorphology and biochemistry function of vascular endothelium. Different kinds of dredge collaterals herbs including singal medicine-Radix Ginseng, compatibility of composition medicine-Siweitongluo,complex dredge collaterals medicine-Tongxinluo were used to treat the rats of complex model in this study. Compared with the effect of Benazepril, the effects of Herbs dredging collaterals on the complex model were observed.3. The function of NEI network in model rats with VED due to collateral-QI deficiency and the intervention research of Herbs dredging collateralsIn the complex models, Sympathoadrenomedullary systems including NE and E were detected by ELISA; hypothalamus-hypophysis cerebri-adrenal function axle including CRH and ACTH in the blood plasma, and CORT in the serum were detected by RIA; renin-angiotonin-aldosterone system including PRA, AngⅡand ALD in the blood plasma were detected by RIA ; and immune system including IL-1βand IL-2 in the serum were detected by RIA. The correlation between endothelial function system index (ET, NO, VWF) and the NEI network system correlative factors was analyzed with canonical correlative analysis method. At the same time, the effects of Herbs dredging collaterals were studied.4 The cell model was established by HUVEC cultivated with serum from rats of complex model, in which the expression of cytoskeleton protein and the effects of the Herbs dredging collaterals were investigated In the study, HUVEC cell strain was cultivated with serum from rats of complex model. The morphological character and distribution of cytoskeletal protein, F-actin, were observed by laser confocal microscopy, the contents of F-actin were observed by flow cytometry. The permeability of monoptychial endothelium was observed thorough BSA marked by Evans blue. The expression of F-actin, p38 as well as p-p38 protein in HUVEC cells were detected by Western blot. The changes of p38 MARK signal pathway were detected by Western blot. The intervention effects of three categories of Herbs dredging collaterals were observed. The experimental cells were divided into seven groups: (1) control group: fetal bovine serum with a final concentration of 20% was added to serum-free medium (F12K); (2) normal serum group: serum from normal rats with a final concentration of 20% was added to serum-free medium (F12K); (3) complex model serum group: serum from rats of the complex model with a final concentration of 20% was added to serum-free medium (F12K); (4) Tongxinluo group: Tongxinluo ultramicro-pulverization (final concentration: 1μg/ml) was added into serum-free medium (F12K) and incubated for 2h; then the serum from rats of the complex model with a final concentration of 20% was added; (5) SB203580 group: SB203580 (final concentration: 25μmol/L) was added into serum-free medium (F12K) and incubated for 1 h, then the serum from rats of the complex model with a final concentration of 20% was added; (6) Ginseng group: Ginseng (final concentration: 1μg / ml) was added into serum-free medium (F12K) and incubated for 2 h, then the serum from rat of the complex model with a final concentration of 20% was added; (7) the group of Herbs dredging collaterals : each kind of the four Herbs dredging collaterals (final concentration: 100μg/ ml) was added into serum-free medium (F12K) and incubated for 2 h, then the serum from rats of the complex model with a final concentration of 20% was added. The study of the cell culture was performed at time 2 h, 6h, 12h, and 24h after the adding of serum.Results: 1 The standards for TCM differentiation of syndroms of VED:Based on the entropy-based partition method for complex systems, the differentiation of symptoms and signs of the 400 cases of VED disease was analyzed by us. of The results were as following. TCM differentiation of syndroms of VED including stagnancy of collateral-QI, stagnancy of collateral-QI deficency, heat accumulation, phlegmatic hygrosis, phlegmatic heat, blood stasis, yin asthenia, yang asthenia, and so on. Combined the contribution scores of symptoms to its syndrom with the ROC curve, the threshold and the differentiation of symptoms and signs for classification of syndrome standard are concluded (the syndrome can be decided if the totle contribution scores of the every symptom exceed or equal the the threshold).Basic Syndromes:Syndrome with stagnancy of collateral-QI (5): chest distress 3, sigh 3, dysphoria 3, depressed emotion 3, stringy pulse 2; Syndrome of stagnancy of collateral-QI deficency (6): weakness 3, spiritlessness 2, short breath 2, palpitation 2, tiredness with talk 1, pale tongue 1, weak pulse 1. Accompanied Syndromes:Syndrome of heat retention (7): dry mouth 3, bitter taste of mouth 3, flushing 2, reddish urine 2, constipation 2, red tongue 1, yellowish fur 1, fast pulse 1; Syndrome of turbid-phlegm (5): chest distress and stomach gas pains 3, limb-exhausted 3, corpulent body 1, excessive phlegm 1, white and greasy fur 2, slippery pulse 1; Syndrome of heat-phlegm (4): cough and spit yellowish phlegm 2, bitter taste of mouth 2, yellow and greasy fur 1, slippery and fast pulse 1: Syndrome of blood stasis (1): dark tongue 1, unsmooth pulse 1; Syndrome of Yin deficiency (4): tidal fever fever and night sweating 2, dysphoria with feverish sensation in the chest, palms and soles 2, soreness and flaccidity in waist and knees 2, flushed cheeks 1, less or moss fur on the tongue 1, small and fast pulse 1; Syndrome of Yang deficiency(7): chilly 3, cold limbs 3, loose stool 2, urination taking long time and clear urine 1, pale and fat tongue 1, sunken, infrequent and feeble pulse 1.The results of retrospective and prospective tests showed that the sensitivity, specificity and agreement rate of various syndromes were all more than 85%, which means that the threshold of differentiation of syndroms for diagnosis of syndromes have a better diagnosis efficacy. Bases on the correlation analysis among the syndromes , more intension association between the stagnancy of collateral-QI deficency and the heat accumulation, blood stasis, yin asthenia, yang asthenia was observed; and the more intension association between stagnancy of collateral-QI deficency and phlegmatic hygrosis, blood stasis, yin asthenia, yang asthenia was observed the intension association between phlegmatic hygrosis and phlegmatic heat, phlegmatic hygrosis and heat accumulation, heat accumulation and yang asthenia was also observed. Based on the correlation analysis and the distribution rule about the pattern of syndromes, the results were that Syndrome of stagnancy of collateral-QI deficiency accounted for 45.3%, Syndrome of stagnancy of collateral-QI accounted for 55.8%, Syndrome of phlegmatic hygrosis accounted for 17.8%, Syndrome of phlegmatic heat accounted for 22%, Syndrome of blood stasis1 accounted for 3.5%, Syndrome of yin asthenia accounted for 38.3%, Syndrome of yang asthenia accounted for 11.8%. All the above indicated that there were two basic patterns of syndromes, the stagnancy of collateral-QI deficency and stagnancy of collateral-QI, in TCM differentiation of syndromes of VED, in which the heat accumulation, phlegmatic hygrosis, phlegmatic heat, blood stasis, yin asthenia, and yang asthenia can exist.2 Establishment of the model and the interventing effects of Herbs dredging collateralsModel evaluation was made after the establishment of the model. (1) Biological appearance rats in complex model group were tired with movement, depressed, limb-twisted, moved slowly, and with whitish and dry tail and nose. Compared with the control group, the biological appearance score was increased significantly (P<0.01); both heart rate and respiratory rate were increased significantly (P<0.01) , whereas, climbing-time was decreased significantly (P<0.01). (2) Morphology character of the endothelial cell of thoracic aorta was observed. The results by light microscope showed that endothelial cell swelling, uneven distribution, density increasing, local endothelial cells disappearing, part of endangium thickening, inflammatory cell infiltration in endangium, fracture of inner elastic plate were observed in the complex model group. The results by electron microscope showed that the majority cristae and membrane in mitochondria of endothelial cell were fused or even disappeared, the rough endoplasmic reticulum degranulated obviously and distended to the shape of oval or circle, cytoplasm and cellular nucleus were dropsy, part membrane collapsed and content released from the cell to outside. (3) The results by immunohistochemical staining showed that both ET1 and iNOS were increased significantly, and eNOS was decreased significantly in pathological model group and complex model group. (4) The index about vascular endothelial function. Compared with the control group, the plasma ET, plasma vWF of rats in pathological model group and complex model group were increased significantly (P<0.01, P<0.05), and serum NO content was decreased significantly (P<0.01). In this study, the complex model that combined the disease of collateral-QI deficiency syndrome and the pathological character of VED was made.The effects of Herbs dredging collaterals were studied. (1) The syndromes were improved by the Herbs observed. Compared with the complex model group, all of the biological score, the heart rate, and the respiratory rate were improved at different levels after intervention by the Herbs (P<0.01). The effect of Tongxinluo group was the best. However, no significant effect of benazepril was observed on the syndromes (P>0.05). (2) The morphological character of the endothelial cells of thoracic aorta was observed. Compared with the complex model group, the results of light microscopic examination showed that the pathological damages of intima of all the medication groups were significantly reduced, and the ultrastructural damages of aortic endothelial cells in medication groups were also significantly reduced. (3) Compared with the complex model group, the results by immunohistochemical staining showed that the expression of iNOS and ET1 were increased significantly, and the expression of eNOS was decreased significantly of each medication group. Based on the morphological character of blood vessel endothelium , the effects of Tongxinluo and benazepril were better than those of Ginseng and the four dredge collaterals Herbs. (4) The index of vascular endothelial function was observed. Compared with the complex model group, the plasma ET and plasma vWF were significantly decreased; the level of NO of serum were significantly increased in Tongxinluo group and benazepril group (P<0.01, P<0.05), there were no significant differences between the two groups ( P>0.05); ET were significantly decreased (P<0.01, P<0.05), NO and vWF had no significant differences in Ginseng and Siweitongluo groups. Above all, the effects of Tongxinluo group and benazepril group were better.3 The changes of NEI network in model rats with VED due to stagnancy of collateral-QI deficiency and the intervention effects of Herbs dredging collateralsThe changes of NEI network in model rats with VED due to stagnancy of collateral-QI deficiency were observed. The function of sympathoadrenomedullary system was revealed by the level of NE and E. Compared with the control group, the level of NE in serum of pathological model group and complex model group were increased significantly (P<0.01, P <0.05), the level of E was increased in complex model group (P<0.01), but no significant change was observed in pathological model group. The function of HPAA was revealed by the level of CRH, ACTH and CORT. All of the CRH, ACTH and CORT of complex model group were decreased significantly (P<0.01, P<0.05), however, only CRH of the pathological model group was decreased significantly (P<0.01). The function of RAAS was revealed by the level of PRA, AngII, and Ald. All of the PRA, AngII, and Ald of complex model group were increased significantly (P<0.01, P<0.05), however, no significant change was observed in the pathological model group (P>0.05). In the complex model group, immunological cytokines IL-1βwas increased (P<0.01), while IL-2 was decreased (P<0.05). Neither IL-1βnor IL-2 had significant change in the pathological model group (P>0.05). Compared with the pathological model group, ACTH was decreased significantly (P <0.01), whereas, PRA and AngII were increased (P <0.01), and no significant difference of Ald was observed (P>0.05) in the complex model group. Compared with the pathological model group, the level of IL-1βwas increased significantly (P<0.05). All the above showed that the disturbance of NEI network existed in the rats of VED due to stagnancy of collateral-QI deficiency, and the symdrom factors, collateral-QI deficiency, had an important effect on the disturbance of NEI network.The canonical correlation analysis between vascular endothelium functional parameter and correlation factor of NEI network was undertaken. The first canonical correlation coefficiency was of statistical significance (P<0.01), which indicates that the two systems are closely related to each other. The relevance of the two systems is mainly reflected by the relationship between NO, VWF and ACTH, NE, CORT, IL-2, as well as CRH. All the obove showed that there was close interrelation between the imbalance of NEI network and VED.The effects of Herbs dredging collaterals was studied. Compared with the complex model group, the levels of NE and E in serum of all treated groups were decreased in different degrees (P<0.01, P<0.05), except for that the NE had no ignificant differences in benazepri group (P >0.05).Compared with the complex model group, CRH, ACTH and CORT of Tongxinluo group were increased significantly (P<0.05), ACTH WAS increased in Ginseng group, and CORT was increased significantly in siweitongluo group (P<0.05), benazepri had no significant effect. Compared with the complex model group, except for PRA of benazepril group, all of the PRA, AngII, and Ald weredecreased significantly to some extent in Tongxinluo group and benazepril group (P<0.01, P<0.05).. AngII of Ginseng group was decreased significantly (P<0.05). Compared with the complex model group, IL-1βwas decreased significantly in all treated groups (P<0.01), there was no statistical difference among these groups (P>0.05); the level of IL-2 was increased significantly in siweitongluo group and Tongxinluo group (P<0.05), but no significant difference was observed in Ginseng group and benazepril group (P>0.05). It can be conclued that Tongxinluo was more efficient to adjust the function of NEI network.4 The establishment of Cell model of HUVEC cultured by serum from rats of complex model, and observation the expression of cytoskeleton protein by the HUVEC and the effects of the Herbs dredging collateralsThe cytoskeletal protein, F-actin, was observed. In the control group, the F-actin surrounded endothelial cells and formed pykno-periphery. Compared with the control group, there was no significant difference of F-actin at 2h time point; Depolymerization and redistribution of F-actin in endothelial cells were observed in the complex model serum group, which reached peak at 6 h time point. The F-actin depolymerized obviously, F-actin within the cytoplasm was increased obviously, and a lot of stress fibers were observed., Then, the depolymerization and redistribution slightly reduced at 12 h time point, and gradually restored at 24 h time point.The content of F-actin was detected by flow cytometry. Compared with the control group, there was no significant change of F-actin content of normal serum group at each time point; F-actin content of complex model serum group was decreased significantly at 6h and 12h time points (P<0.05 ,P<0.01), However, no significant changes of F-actin in complex model serum group at 2h and 24h time points (P>0.05).The permeability of monolayer HUVEC was detected.. Compared with the control group, the permeability of endothelial cells of complex model serum group was increased slightly at 2h time point. The permeability was significantly increased (p<0.01) at 6h and 12h time points, and then declining trend was observed at 24h time point.. The expression of F-actin, P38, and p-p38 protein in HUVEC was obsertved. Compared with the control group and normal serum group, the expression of F-actin protein was significantly downregulated in complex model serum group (p<0.05), and P-P38 significantly increased (p <0.01),and no obvious change of p38 was observed.The effects of Herbs dredging collaterals were studied. The changes of shape and distribution of F-actin were observed. Compared with the complex model group, the shape and distribution of F-actin in endothelial cells of all medication groups and SB203580 group were improved to various extents at 6h and 12h time points. The effects of Tongxinluo group and SB203580 group were better. Compared with the complex model group, the F-actin contents at 6h and 12h time points of treated groups were increased to varying degrees (P<0.01) except siweitongluo group at 12h time point (P>0.05). Compared with the complex model group, the permeability of monolayer HUVEC of intervention groups was decreased significantly at 6h and 12h time points (p<0.01). Compared with the complex model group, the expression of F-actin protein of the three Herbs dredging collaterals groups and SB203580 group were increased significantly(P<0.05,P<0.01), the expression of p-p38 protein of tongxinluo group and SB203580 group were decreased significantly(P<0.05)at 6h and 12h time points. The function of EC was improved by every herb dredging collaterals. Tongxinluo was the best.Above all, the serum from rats of complex model group induced the redistributing of F-actin and downregulation of F-actin content in HUVEC, and increased permeability of monolayer endothelial cell. p38 MAPK signaling pathway maybe involved in the pathological process. The Herbs dredging collaterals could reaggregate the F-actin and increase its content and reduce endothelial permeability. The protective effects of tongxinluo on endothelial cells may be shown through inhibiting p38 MAPK signaling pathway. Conclusion:1 It is the first time that the standards for Classification of TCM differentiation of symptoms and signs of VED syndromes have been established.In China, it is the first time that the entropy-based partition method for complex systems has been applied to establish the standards for TCM differentiation of syndromes by the investigating method of clinical epidemiology. The results of the study showed that the method could be applied to the research on the standards for TCM differentiation of syndroms, which provides new ideas and approaches for the standardization and normalization of TCM syndromes. The quantitative standards for TCM differentiation of syndromes of VED were established. The disease of VED included two basic syndromes, stagnancy of collateral-QI as well as stagnancy of collateral-QI deficiency, at the same time, accompanied potentially by the heat accumulation, phlegmatic hygrosis, phlegmatic heat, blood stasis, yin asthenia, and yang asthenia. Stagnancy of collateral-QI and stagnancy of collateral-QI deficiency are important in VED.2 The rat complex model combined VED with collateral-QI deficiency was establishedAccording to the TCM standards established, under the guidance of TCM theory of“over-strain consuming essence”and“hunger damaging essence”, rats in our study were given homomethionin diet to establish VED models, then“Basic Diet combined loaded Swimming”was applied to evoke the syndrome of collateral-QI deficiency. As a result, The combined disease and syndrome animal models with VED due to stagnancy of collateral-QI deficiency were successfully established, which offered experiment model for the study of pathophysiology mechanisms of the VED.3. The relationship between the disturbance of the function of NEI network and the disfunction of blood vessel endothelium in rats of complex model combined VED with collateral-QI deficiency. The abnormal changes of NEI network in complex model rats was observed, which showed the hyperfunction of sympathoadrenomedullary system and RAAS, inhibition of HPAA, and the upregulation of IL-1βand the dwnregulation of IL-2. All the above suggested disturbance or disorder of NEI network. The syndrome factors, stagnancy of collateral-QI deficiency, played a key or special role in disturbance of NEI network of complex model. The canonical correlation analysis shows that there is a correlation between VEC disfunction and NEI network, embodied in the reltion between NO, VWF and ACTH, NE, CORT, IL-2, and CRH. It is suggested that the disturbance of NEI network may be one of the pathophysiologic mechanisms of VED developing under the state of stagnancy of collateral-QI deficiency.4. It is the first time the VED cell model of stagnancy of collateral-QI deficiency was establishedThe HUVEC was cultured by serum from rats of complex model, and the effect of syndrome combined VED and collateral-QI deficiency on endothelial cell was imitated. The result showed that the content of F-actin in complex model was decreased significantly, depolymerization and redistribution of F-actin in endothelial cells were observed, and the permeability of endothelial cells of complex group was increased significantly. The above changes maybe connected with p38 MAPK signaling pathway.5. The effect of Herbs dredging collaterals on regulating the VED Different kinds of Herbs dredging collaterals can improve the stagnancy of collateral-QI deficiency symptom of complex rat model, which lightened the damage to the shape of EC, adjusted the EC correlation factor, and improved the immunologic derangement in adrenergic nerve-adrenal medulla system, HPAA, and RAAS obviously. Tongxinlu, the complex prescription, was the best. All the above showed the superiorityof the integrating of different kinds of Herbs dredging collaterals in protecting the endothelium.Tongxinluo, the complex dredge collaterals medicine, improved the distribution of F-actin, increased the content of F-actin, and decreased the permeability of monolayer cell in the experient in vitro. The mechanism of Tongxinluo protecting endothelium maybe related to the p38 MAPK signaling pathway inhibited.更多还原