冠心病心绞痛中医证候量化辨证标准及络气虚滞型动脉粥样硬化大鼠模型病理生理学基础研究
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摘要
目的:
本研究以生物—心理—社会医学模式下社会心理应激因素致病作用日益受到重视为背景,以中医络病理论为指导,通过临床流行病学调查建立定性与定量相结合的冠心病心绞痛证候辨证标准,基于证候组合与分布规律探讨络气变化在冠心病心绞痛病机演变中的作用;依据证候辨证标准建立络气虚滞证候模型及络气虚滞型动脉粥样硬化(Atherosclerosis,As)大鼠模型,并综合评价,探讨其代谢物谱特征;研究模型大鼠神经内分泌免疫(neuro-endocrine-immunity,NEI)网络相关因子变化规律及其与As病变的关系;离体观察与络气虚滞证候因素相关的血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)促大鼠血管平滑肌细胞(vascular smooth musclecells,VSMCs)增殖的信号通路机制及通络药物干预作用。基于上述临床与基础研究探讨络气虚滞证候因素对As病变的影响,为研究社会心理因素作用提供实验依据,并初步揭示络气虚滞证候的病理生理学基础,同时佐证络病理论科学价值。
方法:
本文包括四部分研究:
1冠心病心绞痛中医证候量化辨证标准研究
1.1冠心病心绞痛临床流行病学调查
以文献研究和专家咨询为基础,确定调查与冠心病心绞痛相关的中医证候信息并予以综合分级量化,制定临床调查表,选择北京、上海、广州、济南、沈阳、长春等地8个调查中心,开展多中心大样本冠心病心绞痛患者中医证候临床流行病学现况调查。调查过程中严格质量控制,保证调查数据真实可靠。
1.2冠心病心绞痛量化辨证标准的建立
对收集的符合入选与排除标准的403例冠心病心绞痛患者,按3:1分为运算组(302名)和考核组(101名)。采用基于熵的复杂系统分划方法(entropy-based complex systems partition method,ECSPM)提取运算组302例患者症状信息,计算症状贡献度,运用诊断性试验受试者工作特征曲线(receiver operator characteristic curve,ROC)分析建立证候辨证阈值,形成量化辨证标准。以考核组101例患者进行辨证标准的前瞻性检验。
1.3冠心病心绞痛中医证候组合与分布规律研究
以ECSPM进行冠心病心绞痛证候关联度分析,计算各证候在全部冠心病心绞痛患者中所占比例,根据关联度分析及证候分布情况探讨其病机特点。
2络气虚滞型As大鼠模型的建立及其评价
以建立的络气虚滞证辨证标准为依据,根据中医“劳则气耗”理论,以“基础饮食+负重游泳”建立络气虚滞证候模型,以维生素D_3灌胃、高蛋氨酸和高脂饲料喂养建立As模型,以两种造模因素叠加建立络气虚滞型大鼠As模型(复合模犁)。Wistar大鼠随机分为7组:①正常对照组。②As模型组。③络气虚滞组(负重游泳组)。④络气虚滞型As模型组(复合模型组)。⑤通心络大剂量组。⑥通心络小剂量组。⑦辛伐他汀组。以一般状况观察及表征半定量评分、力竭游泳时间、悬杆及旷场实验、电生理检测客观评价络气虚滞主要证候信息(乏力、气短、神疲、心悸症状)在模型大鼠的表现;以丰动脉光镜及电镜形态学观察、免疫组化技术α-肌动蛋白(α-actin)标记染色、血中总胆固醇(total cholesterol,TC)、低密度脂蛋白(low density lipoprotein,LDL)和同型半胱氨酸(homocysteine,HCY)检测评价As病变程度;以代谢组学技术分析大鼠血浆代谢物谱变化。
3络气虚滞型As大鼠NEI网络相关因子变化及通心络干预研究
以建立的络气虚滞模型、As模型及复合模型大鼠为研究对象,探讨络气虚滞状态下NEI网络相关因子变化规律及其对As病变的影响。动物分组及干预同上,实验结束后麻醉大鼠经颈动脉插管采集血液标本,分离血浆或血清,检测下丘脑—垂体—肾上腺轴指标(hypothalamo-pituitary-adrenal axis,HPAA):下丘脑组织促肾上腺皮质激素释放激素(corticotropin releasing hormone,CRH)、血浆促肾上腺皮质激素(adrenocorticotrop(h)ic hormone,ACTH)和血清皮质酮(corticosterone,CORT)(放射免疫分析法),交感肾上腺髓质:血清去甲肾上腺素(norepinephrine,NE)、肾上腺素(epinephrine,E)(ELISA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)、醛固酮(aldosterone,ALD)、血浆及组织AngⅡ(放射免疫分析法),细胞因子:血清白介素(interlcukin,IL)-6(放射免疫分析法)、干扰素(interferon,IFN)-γ(ELISA法);留取大鼠胸主动脉常规制作石蜡组织切片,免疫组化技术检测主动脉增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达。
4 AngⅡ对大鼠VSMCs增殖影响的信号转导机制及通心络干预作用研究
选择络气虚滞证候因素干预引起NEI网络紊乱的主要效应因子AngⅡ,探讨其对体外培养大鼠VSMCs增殖影响的信号转导机制及通心络干预作用,为证候因素干预血管病变的作用机制提供实验依据。采用组织贴块法培养原代大鼠VSMCs,通过MTT法检测细胞增殖能力,流式细胞术检测细胞周期,激光共聚焦显微镜观察STAT_3核转位,Westernblot检测Janus激酶(Janus Kinase,JAK)2、信号转导和转录激活因子(signaltransducer and activator of transcription,STAT)3蛋白及其磷酸化蛋白表达,以JAK_2特异性抑制剂AG490和辛伐他汀做对照,探讨AngⅡ对VSMCs增殖影响的信号转导机制及通心络干预作用。
结果:
1.冠心病心绞痛中医证候量化辨证标准建立
1.1冠心病心绞痛证候提取及其量化辨证标准
基于症状无监督分析共提取出与冠心病心绞痛相关的8个证候,其症状对证候的贡献度(症状名右下角数字)及证候辨证阈值(证候名右下脚数字)分别为:①络气郁滞证_5:、心胸憋闷_4、善太息_3、情志抑郁_3、烦躁_2、脉弦_1;②络气虚滞_6:心胸隐痛_1、心悸_2、气短_2、神疲_3、乏力_3、懒言_2、舌淡_1、脉弱_1;③痰浊_4:头重如蒙_2、口粘腻_2、痰多_3、胸脘痞闷_2、体胖_1、肢体困重_2、苔白腻_2、脉弦滑_1;④血瘀_3:胸痛如刺如绞痛有定处_3、面色晦暗_2、口唇青紫_3、舌紫暗或有瘀斑瘀点_3、脉涩_2;⑤阴虚_7:五心烦热_3、失眠_3、多梦_3、潮热盗汗_3、腰膝酸软_2、少苔或无苔_1、脉细数_1;⑥郁热_6:面红_2、口干_2、口苦_3、溲赤_2、便秘_2、舌红_1、苔黄_1、脉数_2;⑦阳虚_5:面色白光白_2、小便清长_1、面浮肢肿_1、畏寒_3、肢冷_3、舌淡胖_1、脉沉迟无力_1;⑧痰热_3:口粘腻_2、苔黄腻_2、脉滑数_1咳吐黄痰_2。
1.2冠心病心绞痛证候组合与分布规律
证候关联度分析结果:络气郁滞证可与郁热证、血瘀证、痰热证、痰浊证、阴虚证相关联,络气虚滞证可与痰浊证、阳虚证、血瘀证、阴虚证相关联。
证候分布比例:全部冠心病心绞痛患者均有络气郁滞或络气虚滞证候表现,络气虚滞证患者232例,占403例调查患者的57.6%,络气郁滞证占31.5%(127例),兼有络气虚滞与郁滞证者占10.9%(44例),而既往研究较多的血瘀证占60.3%(243例),痰浊(热)证占43.4%(175例),其次为阴虚证40.4%(163例)、郁热证32.8%(132例)、阳虚证14.9%(60例)。
1.3基于证候组合与分布规律提出冠心病中医病机观点
络气郁滞或虚滞为冠心病心绞痛的始动因素并贯穿全过程,痰、瘀既是继发于络气郁滞或络气虚滞基础上的病理产物又成为继发性致病因素,络气郁而化火伤阴,出现气郁→郁热→阴虚的病机变化,气虚日久又可发展为阳虚。表明络气郁滞、络气虚滞为冠心病心绞痛的基础证候,在此基础上形成血瘀、痰浊(热)、郁热、阴虚、阳虚等为兼夹证候,从络气变化切入研究冠心病心绞痛把握住了其病机根本,有助于更深刻揭示冠心病心绞痛的病理机制。
2络气虚滞型As大鼠模型的建立及其评价
2.1络气虚滞证候模型评价
与正常对照组相比,络气虚滞模型组大鼠出现精神不振、眯眼懒动、皮毛少泽、体重减轻、缩肩拱背、鼠尾淡白等表现,生物学表征评分明显升高(P＜0.01);力竭游泳时间与游泳第一天比较明显减少(P＜0.01);悬杆时间也明显缩短(P＜0.01);旷场实验中央格停留时间明显延长(P＜0.05),水平运动及垂直运动得分均明显减少(P＜0.05);呼吸频率和心率明显加快(P＜0.0 1)。以上表现反映了络气虚滞证乏力、神疲、气短、心悸等临床症状与络气虚滞大鼠具有较好一致性。
2.2 As模型评价
As模型组除体重下降明显外,生物学表征改变均不明显,旷场实验和生理功能指标均无异常(P＞0.05)。光镜观察显示内皮细胞脱失,内膜增厚,VSMCs排列紊乱,胶原纤维增生,局部管壁向管腔突出;电镜观察显示内皮受损,表面有沉积物,平滑肌细胞胞质内线粒体溶解,内质网扩张;免疫组化染色显示中膜平滑肌细胞α-actin阳性染色细胞明显减少,血中TC、LDL和HCY明显升高(P＜0.01)。以上结果显示该模型表现出As早期纤维增生性斑块的病理特点。
2.3复合模型评价
复合模型表现出精神萎顿、反应迟钝、皮毛枯槁、被毛脱落等,生物学表征评分明显升高(P＜0.01);力竭游泳时间明显缩短(与同组第1天比较P＜0.01);悬杆时间明显缩短(与正常对照组和As模型组比较均P＜0.01);旷场实验中央格停留时间明显延长(P＜0.05),水平运动和垂直运动得分明显减小(P＜0.01),垂直运动与As模型组比较亦有显著性差异(P＜0.05);呼吸、心跳加快(与正常对照组及As模型组比较均P＜0.01)。同时形态学观察显示复合模型组病理学改变较As模型组不同程度加重,免疫组化显示α-actin阳性细胞明显减少,血脂紊乱,血中同型半胱氨酸升高。表明复合模型组既具备络气虚滞证候表现,又具备As病理表现,两种表现均不同程度加重。
2.4代谢组学结果
与正常对照组比较,As模型组、络气虚滞组、复合模型组血浆代谢物谱均存在明显差异,初步鉴定出不同模型相对于正常对照组的潜在生物标志物。经分析发现,胆汁酸类和磷脂类代谢物在As模型呈升高趋势,而络气虚滞模型则呈下降趋势,前者在复合模型亦呈上升趋势,而后者则呈下降趋势;氨基酸类和肉碱类在三种模型中均呈下降趋势。
2.5药物干预作用
通心络明显改善与证候干预相关的生物学表征评分(P＜0.01),延长力竭游泳时间和悬杆时间(P＜0.01),缩短中央格停留时间,改善水平运动和垂直运动评分(P＜0.05),降低心率和呼吸频率(P＜0.01);同时通心络也可显著改善主动脉形态与结构改变,促进VSMCs表型由合成型向收缩型转变,降低TC、LDL和HCY(P＜0.05,P＜0.01)。辛伐他汀以改善主动脉局部病理改变为主,虽对力竭游泳时间和悬杆时间表现出一定作用,但作用不及通心络,对复合模型与证候相关的生物学表征、电生理、旷场实验变化没有改善作用。
3络气虚滞型As大鼠NEI网络相关因子变化及通心络干预研究
3.1 HPAA变化
模型变化:与正常对照组比较,As模型组下丘脑组织CRH、血浆ACTH、血清CORT均无统计学差异(P＞0.05);络气虚滞组ACTH和CORT明显升高(P＜0.01),CRH表现出下降趋势,但无统计学差异(P＞0.05);复合模型组CRH明显下降(P＜0.01),ACTH和CORT明显升高(P＜0.01)。
药物干预:与复合模型组比较,通心络大剂量组能显著升高CRH水平(P＜0.05),降低ACTH水平(P＜0.05),通心络大小剂量组均能显著降低CORT水平(P＜0.05),辛伐他汀组与复合模型组比较变化无显著差异(P＞0.05)。
3.2交感—肾上腺髓质指标结果
模型变化:与正常对照组比较,As模型组E、NE指标有上升趋势但无统计学意义(P＞0.05);络气虚滞组E、NE明显升高(P＜0.01),与As模型组比较亦显著升高(P＜0.01);复合模型组E、NE亦明显升高(P＜0.05)。
药物干预:与复合模型组比较,通心络大小剂量组E、NE均显著下降(P＜0.05),辛伐他汀组与复合模型组比较无差著性差异(P＞0.05)
3.3 RAAS变化
模型变化:与正常对照组比较,As模型组血中PRA、AngⅡ、ALD变化均无显著差异(P＞0.05);络气虚滞组PRA、AngⅡ明显升高(P＜0.05),ALD变化无显著性差异(P＞0.05);复合模型组PRA、AngⅡ和ALD均显著升高(P＜0.01,P＜0.05,P＜0.01)。
与正常对照组比较,As模型组主动脉组织AngⅡ明显升高(P＜0.05),络气虚滞组主动脉组织AngⅡ具有上升趋势但无统计学意义(P＞0.05),复合模型组主动脉组织AngⅡ亦明显升高(P＜0.01)。
药物干预:与复合模型组比较,辛伐他汀能显著降低PRA和AngⅡ水平(P＜0.05),通心络大小剂量均能显著降低PRA、AngⅡ和ALD水平(P＜0.05,P＜0.05,P＜0.01)。辛伐他汀和通心络大小剂量均显著降低组织AngⅡ水平(P＜0.01,P＜0.01,P＜0.05)
3.4细胞因子结果
模型变化:与正常对照组比较,As模型组和络气虚滞组IFN-γ明显下降(P＜0.05,P＜0.01),IL-6变化无统计学差异(P＞0.05),复合模型组IFN-γ明显下降(P＜0.01),IL-6明显升高(P＜0.05)。
药物干预:与复合模型组比较,辛伐他汀能显著升高IFN-γ水平(P＜0.05),降低IL-6水平(P＜0.05),通心络小剂量组能明显升高IFN-γ水平(P＜0.01),通心络大剂量组既能明显升高IFN-γ水平(P＜0.05),又能降低IL-6水平(P＜0.05)。
3.5各组主动脉组织PCNA免疫组化结果
正常对照组和络气虚滞组未见PCNA阳性细胞,As模型组和复合模型组血管中膜可见大量PCNA阳性细胞,PCNA阳性细胞计数,复合模型组显著高于As模型组(P＜0.01)。各用药组干预后PCNA阳性细胞数均显著减少(P＜0.01)。
4 AngⅡ对大鼠VSMCs增殖影响的信号转导机制及通心络干预作用研究
4.1 AngⅡ对大鼠VSMCs增殖的影响
与正常对照组比较,10~(-8)、10~(-7)、10~(-5)、10~(-6)mol/L的AngⅡ均能显著促进平滑肌细胞增殖(P＜0.05,P＜0.01);与10~(-8)mol/L的AngⅡ相比,10~(-7)、10~(-6)、10~(-5)mol/L的OD值显著升高(P＜0.01);10~(-7)、10~(-6)、10~(-5)mol/L的AngⅡOD值无统计学意义。
4.2药物对AngⅡ诱导的大鼠VSMCs增殖的干预作用
AG490(10、20、50、100、200μmol/L)、辛伐他汀(0.01、0.1、1、10μmol/L)、通心络(100、200、500μg/ml)均不同程度抑制10~(-7)mol/LAngⅡ诱导的大鼠VSMCs增殖(P＜0.05,P＜0.01),具有一定的剂量依赖性。
4.3 AngⅡ对大鼠VSMCs细胞周期影响及药物干预作用
与正常对照组比较,10~(-7)mol/L的AngⅡS期细胞比例显著增加,G_0/G_1期细胞周期所占比例明显减少,(P＜0.01),各用药组干预后S期细胞比例显著下降,G_0/G_1期细胞比例明显增加(P＜0.01),各用药组之间无差著差异(P＞0.05)。
4.4 AngⅡ对大鼠VSMCs STAT_3核转位的影响及药物干预作用
正常对照组STAT_3胞浆、胞核均表达,AngⅡ组120 min时STAT_3明显由胞浆向胞核聚集,即发生核转位,10 min时核转位不明显,各用药组均能抑制AngⅡ作用120min引起的STAT_3核转位。
4.5 AngⅡ对大鼠VSMCs JAK_2/STAT_3信号通路蛋白影响及药物干预作用与正常对照组比较,AngⅡ组10 min时p-JAK_2表达明显增加(P＜0.05),p-STAT_3表达没有明显变化(P＞0.05);AngⅡ组120 min时p-JAK_2和p-STAT_3表达均明显增加(P＜0.05,P＜0.01)。
与AngⅡ组比较,AG490组10min、120min p-JAK_2和p-STAT_3表达明显下降(P＜0.05);通心络大剂量组和中剂量组120min时p-JAK_2和p-STAT_3表达均明显下降(P＜0.01,P＜0.05);辛伐他汀组和通心络小剂量仅120min时p-STAT_3表达显著下降(P＜0.05)。实验过程中各组JAK_2和STAT_3蛋白总量无显著差异(P＞0.05)。
结论:
1本研究以络病理论为指导,以文献研究、专家咨询为基础,通过多中心、大样本临床流行病学调查建立冠心病心绞痛证候辨证标准,运用基于熵的复杂系统分划方法从原始症状无监督分析入手,建立量化证候辨证标准,避免单纯依靠文献研究或专家经验所致主观偏倚;由于构成证候的症状之间存在着非线性复杂关系,该方法较之既往研究中采用的处理线性关系方法更为准确的反映了症状间相互作用的复杂关系,为建立证候模型并探讨其病理生理学基础奠定基础。
2基于证候组合与分布规律探讨冠心病心绞痛病机特点,研究结果显示络气郁滞或络气虚滞与血瘀、痰浊(热)、郁热、阴虚、阳虚等证候形成较强的关联关系,而络气郁滞或络气虚滞存在于所有冠心病心绞痛患者,所占比例高于血瘀、痰浊(热)、郁热、阴虚、阳虚等证候所占比例,显示络气变化所致郁滞或虚滞为冠心病心绞痛的始发病因并贯穿病变全过程,而国内医学界几十年来所重视和研究的血瘀、痰浊(热)等因素为冠心病心绞痛的继发致病因素。从络气切入研究冠心病心绞痛不同于已有从血瘀、痰浊切入开展的研究,显示了气血相关的络病理论特色,对生物—心理—社会医学模式下探讨各种社会心理因素的致病作用具有重要指导意义。
3以建立的络气虚滞证候辨证标准为依据,以“基础饮食+负重游泳”建立络气虚滞大鼠模型,从大鼠一般状况观察、生物学表征半定量评分、力竭游泳时间、悬杆实验、行为学观察、电生理检测客观评价模型大鼠络气虚滞证主要症状乏力、神疲、气短、心悸与标准一致性,也是本研究的特色之一。以As大鼠模型叠加“基础饮食+负重游泳”建立病证复合动物模型,模型大鼠既具有早期纤维增生性As的局部病理特点,又表现出络气虚滞宏观证候,代谢组学方法分析显示3种模型代谢物谱存在明显差异,这为探讨其病理生理学基础及络气虚滞证候因素对As病变的影响奠定了基础。
4探讨络气虚滞型As大鼠模NEI网络相关因子变化规律,络气虚滞证候状态下下丘脑—垂体—肾上腺轴调节功能紊乱、交感肾上腺髓质与肾素—血管紧张素—醛固酮激活,而As模型没有显著变化。络气虚滞证候因素使复合模型NEI网络相关因子变化紊乱且不同程度加重的同时,又促使As病理造模因素引起的组织AngⅡ升高,总体效应表现为复合模型反映平滑肌细胞增殖的PCNA表达明显高于As模型,揭示了络气虚滞证候因素可通过引起NEI网络内相关因子调控失常影响As病变。
5以络气虚滞证候因素作用相关的AngⅡ为干预因子,探讨其对体外培养大鼠主动脉平滑肌细胞增殖作用的影响及其相关信号转导机制。结果表明AngⅡ可明显促进大鼠平滑肌细胞增殖,与其促进细胞由G_0/G_1期进入S期有关。同时AngⅡ可促进平滑肌细胞p-JAK_2和p-STAT_3表达,促进STAT_3核转位,而上述作用可被JAK_2的特异性阻断剂AG490阻断,显示其促平滑肌细胞增殖作用与JAK_2/STAT_3信号通路有关。
6复方通心络和辛伐他汀对叠加证候造模因素和病理造模因素的复合模型的作用显示:二者均可改善局部病理改变,并通过作用于JAK_2/STAT_3信号通路抑制AngⅡ引起的血管平滑肌细胞增殖;通心络同时显著改善与络气虚滞证候因素相关的大鼠行为学、电生理等宏观表现,而辛伐他汀对证候变化基本无改善作用;特别是通心络改善络气虚滞证候因素干预之后引起的NEI网络相关因子紊乱表现出辛伐他汀不具备的整合调节作用,这对于现代生活条件下各种社会心理因素与病理因素叠加造成人体复杂病理生理现象的防治具有更广泛意义。
Objective:More and more scholars were paying attention to the pathogenesis of social psychological stress factors under the bio-psycho-social medical model.Based on the background,guided by collateral desease theory of Traditional Chinese Medicine(TCM),and through the clinical epidemiological survey on patients of Angina Pectoris of Coronary Heart Disease(APCHD),our study was to extract essential elements of syndromes using complicated system analysis method;to establish the standard about TCM differentiation of syndroms which combined qualitation with quantitation;to reveal the characters of pathogenesis according to the combining and distributing rules about syndromes;to establish the rat model of atherosclerosis(As)with collateral-qi deficiency according to standard of differentiation of syndroms,and evaluate objectively the concordance of syndromes of model animals with standard of differentiation of syndroms;to research the metabolic characters in model rats;to study the change rules of NEI network correlation factors and the relationship with As in model rats;to observe signaling pathway mechanisms of angiotensinⅡ(AngⅡ),which was the main factor of NEI network change caused by syndrome factor of collateral-qi deficiency,and which promoted vascular smooth muscle cells (VSMCs)proliferation;to evaluate Tongxinluo's effect on rat model of As with collateral-qi deficiency and on VSMCs proliferation promoted by AngⅡ. We also explored the influence of syndrome factor of collateral-qi deficiency to As according to the results from clinic patients and animals combined with cells,and illuminated initially the pathophysiologic basis on the rat model of As with collateral-qi deficiency,provided experiment evidences to support the research of the mechanisms about effect of social psychological factors on As. Meanwhile,we testified the scientific value of collateral desease theory.
Methods:4 parts included:
1 The study of quantitive standard for differentiation of syndrome to APCHD
1.1 The clinical epidemiological survey in patients of APCHD
Based on the study of literature and the consultants to experts,we bolted and classified the four physical examinations' information of TCM,instituted the clinical questionnaire,selected eight of GradeⅢ-A general hospitals in Beijing,Shanghai,Guangzhou,Ji'nan,Shenyang and Changchun as our investigating centers,carried out large sample clinical epidemiological survey in patients of APCHD from multicentres.During the investigation,we carried out strict quality control following principle of clinical epidemiology to ensure the reality and reliability of the data.
1.2 The establishment of quantitive standard for differentiation of syndromes to APCHD
The study enrolled 403 subjects who visited during our investigation.They were suitable to the standard of APCHD.The 403 subjects were divided randomly into two groups by 3:1.One group,302 subjects,as the calculating group was to establish quantitive standard of differentiation of syndrome. Another group,101 subjects,as the examining group was to test prospectively diagnostic code of syndromes' quantization.
The data of 302 subjects were filled in Matlab software,syndromes information was exercted,the contribution of symptoms to syndromes was calculated by entropy-based complex systems partition method(ECSPM).The thresholds on the differentiation of syndromes were set up by the analysis of receiver operator characteristic curve(ROC).The quantitive standards of differentiation of syndrome to APCHD was established successfully when those results of above research were obtained.Then the differentiation of tandards of syndromes prospectively was tested in another group.
1.3 The research of combining and distributing rules of syndromes in APCHD.
We analysed correlative grade of syndromes by ECSPM,and calculated the proportion of syndromes in 403 cases patients based on the established differentiation standard of syndrome,and then analysed the characters of pathogenesis according to the combing and distributing rules of the syndromes in patients.
2 The establishment and evaluation of the rat model of As with collateral-qi deficiency
The differentiation standard of syndromes in collateral-qi deficiency was established based on clinical epidemiological survey.According to the TCM theory of "excess fatigue consuming qi",basic diet plus loading swimming was applied to induce the syndromes of collateral-qi deficiency.Rats were given VitaminD_3,high-methionine and high-grease diet to induce As model. And then the rat model of As with collateral-qi deficiency was induced by the two above factors' superposition.
One hundred and five Wistar rats were divided into 7 groups randomly:①Control group.②As group.③Collateral-qi deficiency group(loading swimming group).④As with collateral-qi deficiency(complex model group) group.⑤Large dose Tongxinluo group.⑥Small dose Tongxinluo group.⑦Simvastatin group.
After the establishment of rat models,general condition,score of biology superficial syndrome,climbing-time,open-field experiment,and electrophysiology were detected to evaluate objectively the major syndromes' information(debilitation,short breath,fatigue,palpitation)in model rats with collateral-qi deficiency.
At the same time,the HE staining,Masson staining,electron microscope, immunohistochemical staining of arota were carried out.Total cholesterol(TC), low density lipoprotein(LDL)and homocysteine(HCY)were detected.The changes of metabolin were analyzed by metabonomics.All those indexes were to evaluate the degree of As.
3 The NEI network changes of the As model rats with collateral-qi deficiency and the intervening effect of Herbs
The collateral-qi deficiency model,As rats model and complex model had been instituted as the study objects to investigate the change rules of indexes related to NEI network and the influence of the indexes to As.The dividing of rat groups and the drugs observed were as above.At the finished time point of the model,the rats were anesthetized and blood preparation was collected through arteria carotis cannula,plasma or blood serum was separated.The indexes were detected as following:
Hypothalamo-pituitary-adrenal axis(HPAA):corticotropin releasing hormone(CRH)of hypothalamus,adrenocorticotrop(h)ic hormone(ACTH)in plasma,corticosterone(CORT)in serum were detected by Radio-immunity Approach.Norepinephrine and epinephrine in serum was detected by ELISA Approach.Renin-angiotensin-aldosterone system(RAAS):plasma renin activity(PRA),aldosterone(ALD)in plasma,AngⅡin aorta and plasma were detected by Radio-immunity Approach.Cytokine:interleukin(IL)-6 in serum was detected by Radio-immunity Approach,and interferon(IFN)-γin serum was detected by ELISA Approach.Paraffin sections of aorta pectoralis was used to detect the expression of proliferating cell nuclear antigen(PCNA)by Envision immunohistochemistry kit.
4 The signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it
AngⅡwas selected,which was the main effect factor of NEI network derangement induced by the intervention of collateral-qi deficiency.It was studied by us that the signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it.The research could also provide the experimental evidence of the mechanism of the syndrome factor influncing vascular lesion.The primary culture VSMCs of rat by the tissue sticking mass method was used for the study.The proliferation capability of VSMCs was detected using MTT method.The cell cycle of VSMCs was detected using flow cytometry.The nuclear translocation of signal transducer and activator of transcription(STAT)3 was observed with laser confocal microscopy.The expression of proteins of Janus Kinase(JAK)2, STAT3,phospho -JAK_2 and phospho-STAT_3 was detected with Westernblot technology.Such drugs as AG490,an special inhibitor of JAK2,Tongxinluo and Simvastatin were selected.
Result:
1 The establishment of quantitive standards of differentiation of syndrome to APCHD
1.1 The extraction of syndromes and the establishment of the quantitive standard of APCHD
Based on the analyses of syndrome without supervision,eight syndromes were obtained in the patients with APCHD.The contribution of symptoms to syndromes(the number at the low right of symptoms)and thresholds(the nmuber at the low right of syndroms)as followings:①collateral-qi stasis_(5): oppressed feel in heart and chest_(4),preference for sighing_(3),depressed emotion_(3),restlessness_(2),viry pulse_(1);②collateral-qi deficiency_(6):vague pain in heart and chest_(1),cardiopalmus_(2),short breath_(2),mind lassitude_(3), debilitation_(3),lazy to speak_(2),pale tongue_(1),weakened pulset_(1);③phlegm turbid_(4):cerebaria_(2),muco-greasy in mouth_(2),excessive phlegm_(3),muffled in chest and gastric cavity_(2),body fat_(1),distress in limbs_(2),whiteness greasy of musci_(2),viry and slide pulse_(1);④blood stasis_(3):localized pain in chest as acanthus and angor_(3),eclipse of facial expression_(2),cyanochroia in labia oris_(3),purplish tongue with ecchymosis or petech_(3),unsmooth pulse_(2);⑤yin asthenia_(7):feverish of five centres_(3),insomnia_(3),multi-dream_(3),hectic fever and night sweat_(3),sour soft in waist and knee_(2),few musci or no musci(1), pulse fine and cipher_(1);⑥heat accumulation_(6):flushing_(2),dry mouth_(2),bitter taste of mouth_(3),suitable red_(2),constipation_(2),red tongue with yellowish fur_(1),yellow tongue fur_(1)pulse cipher_(2);⑦yang asthenia_(5):facial expression white_(2),urine schedule length_(1),mild facial edema and swollen limbs_(1), chilly_(3),limbs cold_(3),pale tongue and fat_(1),pulse heavy slow and powerless_(1);⑧phlegm fever_(3):mouth sticking and greasy_(2),pulse slide and cipher_(1), yellow tongue fur with greasy dirt_(2),cough yellow phlegm_(2).
1.2 The combining and distributing rules of syndroms in APCHD
The results of correlative grade of syndromes:the syndrome of collateral-qi stasis had the strong correlation with heat accumulation,blood stasis,phlegm fever,phlegm turbid,yin asthenia,and the syndrome of collateral-qi deficiency had the strong correlation with phlegm turbid,yang asthenia,blood stasis,yin asthenia.
The distributing proportion of syndromes:all the patients have either collateral-qi stasis syndrome or collateral-qi deficiency syndrome.The collateral-qi deficiency syndrome accounts for 57.6%(232 cases)of all investigated patients(403 cases),collateral-qi stasis accounts for 31.5%(127 cases),10.9%(44 cases)has both collateral-qi deficiency and the collateral-qi stasis.However,blood stasis accounts for 60.3%(243 cases),which was thought the major syndrome in previous study,phlegm turbid(heat)accounts for 43.4%(175 cases),gasthenia accounts for 14.9%(60 cases).
1.3 To raise the viewpoint about the pathogenesis of APCHD based on the combining and distributing rules of syndromes
Based on the above results,either collateral-qi stasis or collateral-qi deficiency was the initiating agent of APCHD and breakthrough the whole process.Phlegm and blood stasis were not only the patho-products induced by collateral-qi stasis or collateral-qi deficiency,but also influenced the developing of the disease conversely.Meanwhile,collateral-qi stasis could induce pyretic generation,damage yin,depress vital energy and yin asthenia. Deficiency of vital energy for a long timemay developped to yang asthenia. Both collateral-qi stasis and collateral-qi deficiency were the basic syndromes, and heat accumulation,blood stasis,phlegm turbid(fever),yin asthenia,yang asthenia and yin asthenia,which were derived from collateral-qi stasis or collateral-qi deficiency,were the accompanied syndromes.
2 Establishment and evaluation of the rat model of As with collateral-qi deficiency
2.1 The evaluation of model rats of collateral-qi deficiency
Compared with the control group,the rats of collateral-qi deficiency group appeared low-spirited,eye-closed,superficial few gloss,and extrados appearance.The score of biology superficial signs was significantly higher than control group(P＜0.01).Fatigued swimming time was significantly decreased than that on the first day(P＜0.01).The rats of collateral-qi deficiency group also displayed the shorter climbing time,the longer stay-time in central frame and lower crossing and rearing score in open-field test(P＜0.05,P＜0.01).All the results reflected the cardiopalmus,short breath, debilitation,mind lassitude of collateral-qi deficiency syndrome had the concordance with the model rats with collateral-qi deficiency syndrome.
2.2 The evaluation of model rats of As
Rats of As model group only appeared weight breakdown.The biology superfocial signs score of As group had no significant difference.Open-field test and physiologic function also had no significant change(P＞0.05).HE staining and Masson staining showed endothelial cell partly loss, endomembrane thickened,VSMCs hyperplasy and arrange disorder,collagen fiber proliferation,local tubal walls lumina outstanding and form fiber hyperplasy As.SEM and TEM showed endothelium injured,and there was deposition on surface.Bilblast of VSMCs dissolved,and endoplasmic reticulum distended.Aortic immunityhiscochemstry showed theα-actin masc cells decreased obviously.While the TC、LDL and HCY increased obviously. All those results showed the rat model had the pathological features of plaque fiber proliferation in earlier period of As.
2.3 The evaluation of complex model rats
Rats of the complex model group appeared energetic dispirited,insensitive response,skin slacked,hair on the back falled off,et al.The score of complex model group was highest,which was significantly higher than both As group and collateral-qi deficiency group(P＜0.01).The climbing-time,respiratory frequency and heart rate,the rearing score of open-field of complex model rats had the same results as the above.At the same time,the swimming-time of complex model group was significant lower than that on the first day of the same group.The stay-time in central frame of open-field was longer and crossing score was lower than control group(P＜0.05,P＜0.01).As degree of complex model was more serious than As model according the observation of HE staining,Masson staining and ultramicrostructure.The masc cells expressing of aorticα-actin in complex model group also decreased,and the TC,LDL,HCY increased highly.All those results showed that there were both collateral-qi deficiency syndrome and As in complex model,whose degree was more seriously.
2.4 Metabonomics results
Compared with control group,As model group,collateral-qi deficiency group,complex model group metabolin spectra had obvious difference.The potential biomarkers were identified initially.According the identification results,bile acid and phospholipid increased in As model,while decreased in collateral-qi deficiency model.Bile acid increased in complex model,while phospholipid decreased in it.Amino acids and carnitine all increased in three models.
2.5 Drugs intervention
Tongxinluo could improve the indexes related to the syndrome,such as improve the biology superficial signs score(P＜0.01),prolong the fatigued swimming time and climbing time(P＜0.01),shorten the stay-time in central frame,improve the crossing score and rearing score(P＜0.05),and degrade the respiratory frequency and heart rate(P＜0.01).At the same time, Tongxinluo might also improve the change of shape and structure of aorta, promote the phenotypic modulation of VSMCs from contractile phenotype to synthetic phenotype,reduce the level of TC、LDL and HCY(P＜0.05,P＜0.01).Simvastatin mainly improved the locally pathological change of aorta. Although Simvastatin had the effect on the fatigued swimming time and climbing-time,whose effect was not as good as Tongxinluo.Simvastatin had no effect on biology superficial signs score,respiratory frequency and heart rate,open-field test in combinted model.
3 The study about the changes of NEI network in collateral-qi deficiency As rats and the intervention of Tongxinluo
3.1 The changes of HPAA
Changes of model rats:compared with control group,tissue CRH,blood plasma ACTH,serum CORT in As group had statistic difference(P＞0.05), ACTH and CORT in collateral-qi deficiency group increased obviously(P＜0.01),CRH only appeared decrease tendency(P＞0.05),and CRH decreased obviously(P＜0.01),ACTH and CORT increased obviously in complex model group(P＜0.01).
The intervention of drugs:compared with complex model group,large dose Tongxinluo could increase obviously CRH level(P＜0.05),decrease ACTH level(P＜0.05).Large and small dose Tongxinluo could obviously decrease CORT level,and Simvastatin had no obvious effect(P＞0.05)。
3.2 The changes of Sympathesis-Adrenal Medulla Axis.
Changes of model rats:compared with the control group,the indexes of E,NE in As group had upgrade tendency but had no significant difference (p＞0.05).In collateral-qi deficiency group,the index of E,NE upgraded significantly(P＜0.01),which was significantly different compared with arteriosclerosis group(P＜0.01).To compare with normal group,the index of E,NE in complex model group is obviously heighten(P＜0.05).
The intervention of drugs:compared with the complex model group, E,NE of large dose and low dose tongxinluo group obviously decreased (P＜0.05).But E,NE of Simvastatin group have no significant difference
3.3 The changes of RAAS
Changes of model rats:compared with control group,the PRA,angⅡand aldosterone in blood of As group have no significant change(P＞0.05). The PRA,angⅡin blood of collateral-qi deficiency group obviously increased(P＜0.05),and ALD had no significant difference(p＞0.05).The PRA,angⅡand ALD in blood of the complex model group obviously increased(P＜0.05,P＜0.01)
Compared with the control group,AugⅡin aorta of As group obviously increased(P＜0.05).AngⅡin aorta of collateral-qi deficiency group had upgrade tendency,but no significant difference(P＞0.05).AngⅡin aorta of the complex model group obviously increased(P＜0.01).
The intervention of drugs:compared with the complex model group, Simvastatin could lower the level of PRA and angⅡ(P＜0.05).large and small dose Tongxinluo could lower the level of PRA,angⅡand ALD(P＜0.05,P＜0.01)。
Compared with the complex model group,AngⅡin aorta of simvastatin, large and low dose Tongxinluo group obviously decreased(P＜0.05,P＜0.01).
3.4 The changes of cytokines
Changes of model rats:compared with normal control group,IFN-γin As and collateral-qi deficiency group obviously decreased(P＜0.05),while IL-6 had no significant difference(P＞0.05).IFN-γin the complex model group obviously decreased(P＜0.01),and IL-6 obviously increased(P＜0.05).
The intervention of drugs:compared with the complex model group, Simvastatin could obviously raise the level of IFN-γ(P＜0.05)and decrease the level of IL-6(P＜0.05).The low dose Tongxinluo could obviously raise the level of IFN-γ(P＜0.01),while the large dose Tongxinluo could obviously both raise the level of IFN-γ(P＜0.05)and decrease the level of IL-6(P＜0.05)。
3.5 The expressing of PCNA in aorta
PCNA masc cells In the control group and collateral-qi deficiency group had been found.While plenty of PCNA masc cells in As group and complex model group had been found.The count of masc cells in the complex model group was obviously higher than that in As group(P＜0.01).All drugs could reduced the masc cells(P＜0.01)。
4.The signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it
4.1 The influence of AngⅡto the proliferation of rat VSMCs
Compared with DMEM group,10~(-5)、10~(-6)、10~(-7)、10~(-8)mol/L of AngⅡcould obviously promote VSMCs proliferation(p＜0.05,P＜0.01).Compared with 10~(-8)mol/L of AngⅡgroup,the OD value of 10~(-5)、10~(-6)、10~(-7)mol/L group obviously rised(P＜0.01).The OD value of 10~(-5)、10~(-6)、10~(-7)mol/L of AngⅡhad no significant difference.
4.2 The interventional effect of drugs on proliferation of rat VSMCs induced by AngⅡ
AG490(10、20、50、100、200μmol/L)、Simvastatin(0.01、0.1、1、10μmol/L)、Tongxinluo(100、200、500μg/ml)could inhibitted proliferation of rat VSMCs induced AngⅡat the concentration of 10~(-7)mol/L(P＜0.05,P＜0.01),which was dose dependent.
4.3 The effect of AngⅡon the cell cycle of VSMCs and the interventional effect of drugs on it
Compared with DMEM group,the percentage of cells in phase S significantly increased after the incubation of AngⅡat the concentration of 10 ~(-7)mol/L,while the percentage of cells in phase G_0/G_1 bviously decrease(P＜0.01).All drugs could decrease the percentage of cells in phase S,and increased the percentage of cells in phase G_0/G_1(P＜0.01)
4.4 The influence of AngⅡto nuclear translocation of STAT_3 induced by AngⅡand effect of drugs on it
In the DMEM group,STAT_3 was detected well-distributedly in the kytoplasm and nucleus.After 120min incubated by AngⅡ,STAT_3 was detected mainly in the nucleus,which showed us the nuclear translocation of STAT_3.However,After 10min incubated by AngⅡ,the nuclear translocation of STAT_3 was not observed.All drugs could inhibited the nuclear translocation of STAT3 after 120min incubated by AngⅡ.
4.5 The influence of AngⅡto the protein expression of JAK2/STAT3 signal passway and effect of drugs on it
Compared with DMEM group,the protein expression of p-JAK_2 increased obviously after 10 min incubated by AngⅡ(P＜0.05),while the protein expression of p-STAT_3 had no significant difference.The protein expression of both p-JAK_2 and p-STAT_3 increased obviously after 120 min incubated by AngⅡ(P＜0.05,P＜0.01)
Compared with AngⅡgroup,AG490 could reduce the protein expression of p- JAK_2 and p- STAT_3 after 10 min and 120 min incubated by AngⅡ(P＜0.05).Both large dose and median dose Tongxinluo could reduced the protein expression of p- JAK_2 and p- STAT+3 after 120 min incubated by AngⅡ(P＜0.01,P＜0.05).Simvastatin and samll dose Tongxinluo only reduced the protein expression of p- STAT_3 after 120 min incubated by AngⅡ(P＜0.05).The whole protein of JAK_2 and STAT_3 had no significant difference(P＞0.05)
Conclusion:
1 Guided by the theory of collateral disease,based on literature study, experts consultant and large sample clinical epidemiological survey in patients of APCHD from multicentres,the study was to establish quantitive differentiation of syndromestandard,on base of the data analysis without supervision using ECSPM,which avioded the subjective bias of simple literature study and experts'experience.Because the nonlinear and complex relationship existed in symptoms of syndrome,ECSPM could reflect the complex interactive relationship between symptoms more precise,compared with the method of dealing with liner relationship in previous study,which makes a found to establish the syndrom model and study the pathophysiologic basis.
2 Studying the pathogenesy of TCM about APCAD on basis of the combining and distributing rules of syndromes,Our results showed that collateral-qi stasis or collateral-qi deficiency existed in all patients of APCAD, which was higher than the percentage of blood stasis,phlegm muddy(heat), heat accumulation,yin asthenia,yang asthenia etc.The results showed that stagnancy or deficiency caused by the changes of collateral-qi might be initiating agent of APCAD.Phlegm muddy(heat)and blood stasis were the patho-products derived of collateral-qi stasis or collateral-qi deficiency, which had been paid attention and researched for several decades by domestic medical society.Studying the change of collateral-qi of APCAD was different from that that study such blood stasis and phlegm muddy,etc,which showed the feature of qi related to blood in collateral desease theory,and had the guidance significance to explore the pathomechanism of various kinds of social-psycho factors under the bio-psycho-social medical model.
3 Based on the differentiation standard of syndromes established by clinical epidemiological survey,we eatablished syndrome model of collateral-qi deficiency,using basic diet plus forced loading swimming.The model rats were evaluated objectivly by the methods of the general condition observation,semi-quantity score of biology superficial signs,fatigued swimming-time,climbing-time,ethology,electrophysiology,according to main symptoms of collateral-qi deficiency.The complex model was established using As rat model overlain basic diet plus forced loading swimming.The model rats had both local pathological feature in the earlier period of As,and the representation of collateral-qi deficiency syndrome.The three models showed difference in the metabolism spectra using the metabonomics technology.This study also lain the foundation for exploring pathophysiologic basis of collateral-qi deficiency syndrome and it's effect on AS.
4 This study had explore the changes rules of factors related to NEI network in the rat model of As with collateral-qi deficiency.The function of HPAA was in the chaos in the condition of collateral-qi deficiency.Adrenergic nerve -adrenal medulla system and RAAS were activated.However AS model had not such significant changes.The syndrome factor of collateral-qi deficiency not only caused the indexes related to NEI network to change more significantly in complex model rats,but also promoted more increase of AngⅡin aorta,which caused by pathological factors.Total effect was that PCNA which reflected the VSMCs proliferation in the complex model rats was obviously higher than simple As model,which revealed that syndome factors of collateral-qi deficiency could cause the factors related to the NEI network to be disorder and then influence As.
5 The study selected AngⅡas the interventional factor,which was the main factor related to the NEI network influenced by the syndrome factor of collateral-qi deficiency,and explored the influence of AngⅡto the proliferation of rat VSMCs,which would provide the experimental evidence about the pathomechanism of syndrome of collateral-qi deficiency.The results showed that AngⅡcould obviously promote rats proliferation of VSMCs, which was related to the influence to the cell circle.Meanwhile,AngⅡalso promoted protein expression of p-JAK_2 and p-STAT_3 of VSMCs,while the effect above could be stocked by AG490,which was specific inhibitor of JAK_2.Those results showed that the effect of AngⅡpromoting the proliferation of VSMCs was related to signaling pathway of JAK_2/STAT_3.
6 The effect of Tongxinluo and Simvastatin on the complex model rats showed that they could both improve local pathological change,and inhibit proliferation of VSMCs caused by AngⅡthrough blocking JAK_2/STAT_3 signaling pathways.Tongxinluo also significantly improved the pathological changes caused by syndrome factor of collateral-qi deficiency,such as ethology,electrophysiology etc,while Simvastatin had no such effect. Especially Tongxinluo showed the integrated regulation effect in improving the disorder of NEI network caused by intervention of syndrome factor of collateral-qi deficiency,which was better tha Simvastatin.These results manifested that Tongxinluo showed double-effect on improving both local and global pathological change,which was different from Simvastatin.These results also possessed much more widespread meaning to prevent and care human body complex pathological and physiological phenomenon,which caused by various kinds of social psychological stress factors and biological pathogenic factors under modern living conditions and pathology factors.
本研究以生物—心理—社会医学模式下社会心理应激因素致病作用日益受到重视为背景,以中医络病理论为指导,通过临床流行病学调查建立定性与定量相结合的冠心病心绞痛证候辨证标准,基于证候组合与分布规律探讨络气变化在冠心病心绞痛病机演变中的作用;依据证候辨证标准建立络气虚滞证候模型及络气虚滞型动脉粥样硬化(Atherosclerosis,As)大鼠模型,并综合评价,探讨其代谢物谱特征;研究模型大鼠神经内分泌免疫(neuro-endocrine-immunity,NEI)网络相关因子变化规律及其与As病变的关系;离体观察与络气虚滞证候因素相关的血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)促大鼠血管平滑肌细胞(vascular smooth musclecells,VSMCs)增殖的信号通路机制及通络药物干预作用。基于上述临床与基础研究探讨络气虚滞证候因素对As病变的影响,为研究社会心理因素作用提供实验依据,并初步揭示络气虚滞证候的病理生理学基础,同时佐证络病理论科学价值。
方法:
本文包括四部分研究:
1冠心病心绞痛中医证候量化辨证标准研究
1.1冠心病心绞痛临床流行病学调查
以文献研究和专家咨询为基础,确定调查与冠心病心绞痛相关的中医证候信息并予以综合分级量化,制定临床调查表,选择北京、上海、广州、济南、沈阳、长春等地8个调查中心,开展多中心大样本冠心病心绞痛患者中医证候临床流行病学现况调查。调查过程中严格质量控制,保证调查数据真实可靠。
1.2冠心病心绞痛量化辨证标准的建立
对收集的符合入选与排除标准的403例冠心病心绞痛患者,按3:1分为运算组(302名)和考核组(101名)。采用基于熵的复杂系统分划方法(entropy-based complex systems partition method,ECSPM)提取运算组302例患者症状信息,计算症状贡献度,运用诊断性试验受试者工作特征曲线(receiver operator characteristic curve,ROC)分析建立证候辨证阈值,形成量化辨证标准。以考核组101例患者进行辨证标准的前瞻性检验。
1.3冠心病心绞痛中医证候组合与分布规律研究
以ECSPM进行冠心病心绞痛证候关联度分析,计算各证候在全部冠心病心绞痛患者中所占比例,根据关联度分析及证候分布情况探讨其病机特点。
2络气虚滞型As大鼠模型的建立及其评价
以建立的络气虚滞证辨证标准为依据,根据中医“劳则气耗”理论,以“基础饮食+负重游泳”建立络气虚滞证候模型,以维生素D_3灌胃、高蛋氨酸和高脂饲料喂养建立As模型,以两种造模因素叠加建立络气虚滞型大鼠As模型(复合模犁)。Wistar大鼠随机分为7组:①正常对照组。②As模型组。③络气虚滞组(负重游泳组)。④络气虚滞型As模型组(复合模型组)。⑤通心络大剂量组。⑥通心络小剂量组。⑦辛伐他汀组。以一般状况观察及表征半定量评分、力竭游泳时间、悬杆及旷场实验、电生理检测客观评价络气虚滞主要证候信息(乏力、气短、神疲、心悸症状)在模型大鼠的表现;以丰动脉光镜及电镜形态学观察、免疫组化技术α-肌动蛋白(α-actin)标记染色、血中总胆固醇(total cholesterol,TC)、低密度脂蛋白(low density lipoprotein,LDL)和同型半胱氨酸(homocysteine,HCY)检测评价As病变程度;以代谢组学技术分析大鼠血浆代谢物谱变化。
3络气虚滞型As大鼠NEI网络相关因子变化及通心络干预研究
以建立的络气虚滞模型、As模型及复合模型大鼠为研究对象,探讨络气虚滞状态下NEI网络相关因子变化规律及其对As病变的影响。动物分组及干预同上,实验结束后麻醉大鼠经颈动脉插管采集血液标本,分离血浆或血清,检测下丘脑—垂体—肾上腺轴指标(hypothalamo-pituitary-adrenal axis,HPAA):下丘脑组织促肾上腺皮质激素释放激素(corticotropin releasing hormone,CRH)、血浆促肾上腺皮质激素(adrenocorticotrop(h)ic hormone,ACTH)和血清皮质酮(corticosterone,CORT)(放射免疫分析法),交感肾上腺髓质:血清去甲肾上腺素(norepinephrine,NE)、肾上腺素(epinephrine,E)(ELISA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)、醛固酮(aldosterone,ALD)、血浆及组织AngⅡ(放射免疫分析法),细胞因子:血清白介素(interlcukin,IL)-6(放射免疫分析法)、干扰素(interferon,IFN)-γ(ELISA法);留取大鼠胸主动脉常规制作石蜡组织切片,免疫组化技术检测主动脉增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达。
4 AngⅡ对大鼠VSMCs增殖影响的信号转导机制及通心络干预作用研究
选择络气虚滞证候因素干预引起NEI网络紊乱的主要效应因子AngⅡ,探讨其对体外培养大鼠VSMCs增殖影响的信号转导机制及通心络干预作用,为证候因素干预血管病变的作用机制提供实验依据。采用组织贴块法培养原代大鼠VSMCs,通过MTT法检测细胞增殖能力,流式细胞术检测细胞周期,激光共聚焦显微镜观察STAT_3核转位,Westernblot检测Janus激酶(Janus Kinase,JAK)2、信号转导和转录激活因子(signaltransducer and activator of transcription,STAT)3蛋白及其磷酸化蛋白表达,以JAK_2特异性抑制剂AG490和辛伐他汀做对照,探讨AngⅡ对VSMCs增殖影响的信号转导机制及通心络干预作用。
结果:
1.冠心病心绞痛中医证候量化辨证标准建立
1.1冠心病心绞痛证候提取及其量化辨证标准
基于症状无监督分析共提取出与冠心病心绞痛相关的8个证候,其症状对证候的贡献度(症状名右下角数字)及证候辨证阈值(证候名右下脚数字)分别为:①络气郁滞证_5:、心胸憋闷_4、善太息_3、情志抑郁_3、烦躁_2、脉弦_1;②络气虚滞_6:心胸隐痛_1、心悸_2、气短_2、神疲_3、乏力_3、懒言_2、舌淡_1、脉弱_1;③痰浊_4:头重如蒙_2、口粘腻_2、痰多_3、胸脘痞闷_2、体胖_1、肢体困重_2、苔白腻_2、脉弦滑_1;④血瘀_3:胸痛如刺如绞痛有定处_3、面色晦暗_2、口唇青紫_3、舌紫暗或有瘀斑瘀点_3、脉涩_2;⑤阴虚_7:五心烦热_3、失眠_3、多梦_3、潮热盗汗_3、腰膝酸软_2、少苔或无苔_1、脉细数_1;⑥郁热_6:面红_2、口干_2、口苦_3、溲赤_2、便秘_2、舌红_1、苔黄_1、脉数_2;⑦阳虚_5:面色白光白_2、小便清长_1、面浮肢肿_1、畏寒_3、肢冷_3、舌淡胖_1、脉沉迟无力_1;⑧痰热_3:口粘腻_2、苔黄腻_2、脉滑数_1咳吐黄痰_2。
1.2冠心病心绞痛证候组合与分布规律
证候关联度分析结果:络气郁滞证可与郁热证、血瘀证、痰热证、痰浊证、阴虚证相关联,络气虚滞证可与痰浊证、阳虚证、血瘀证、阴虚证相关联。
证候分布比例:全部冠心病心绞痛患者均有络气郁滞或络气虚滞证候表现,络气虚滞证患者232例,占403例调查患者的57.6%,络气郁滞证占31.5%(127例),兼有络气虚滞与郁滞证者占10.9%(44例),而既往研究较多的血瘀证占60.3%(243例),痰浊(热)证占43.4%(175例),其次为阴虚证40.4%(163例)、郁热证32.8%(132例)、阳虚证14.9%(60例)。
1.3基于证候组合与分布规律提出冠心病中医病机观点
络气郁滞或虚滞为冠心病心绞痛的始动因素并贯穿全过程,痰、瘀既是继发于络气郁滞或络气虚滞基础上的病理产物又成为继发性致病因素,络气郁而化火伤阴,出现气郁→郁热→阴虚的病机变化,气虚日久又可发展为阳虚。表明络气郁滞、络气虚滞为冠心病心绞痛的基础证候,在此基础上形成血瘀、痰浊(热)、郁热、阴虚、阳虚等为兼夹证候,从络气变化切入研究冠心病心绞痛把握住了其病机根本,有助于更深刻揭示冠心病心绞痛的病理机制。
2络气虚滞型As大鼠模型的建立及其评价
2.1络气虚滞证候模型评价
与正常对照组相比,络气虚滞模型组大鼠出现精神不振、眯眼懒动、皮毛少泽、体重减轻、缩肩拱背、鼠尾淡白等表现,生物学表征评分明显升高(P＜0.01);力竭游泳时间与游泳第一天比较明显减少(P＜0.01);悬杆时间也明显缩短(P＜0.01);旷场实验中央格停留时间明显延长(P＜0.05),水平运动及垂直运动得分均明显减少(P＜0.05);呼吸频率和心率明显加快(P＜0.0 1)。以上表现反映了络气虚滞证乏力、神疲、气短、心悸等临床症状与络气虚滞大鼠具有较好一致性。
2.2 As模型评价
As模型组除体重下降明显外,生物学表征改变均不明显,旷场实验和生理功能指标均无异常(P＞0.05)。光镜观察显示内皮细胞脱失,内膜增厚,VSMCs排列紊乱,胶原纤维增生,局部管壁向管腔突出;电镜观察显示内皮受损,表面有沉积物,平滑肌细胞胞质内线粒体溶解,内质网扩张;免疫组化染色显示中膜平滑肌细胞α-actin阳性染色细胞明显减少,血中TC、LDL和HCY明显升高(P＜0.01)。以上结果显示该模型表现出As早期纤维增生性斑块的病理特点。
2.3复合模型评价
复合模型表现出精神萎顿、反应迟钝、皮毛枯槁、被毛脱落等,生物学表征评分明显升高(P＜0.01);力竭游泳时间明显缩短(与同组第1天比较P＜0.01);悬杆时间明显缩短(与正常对照组和As模型组比较均P＜0.01);旷场实验中央格停留时间明显延长(P＜0.05),水平运动和垂直运动得分明显减小(P＜0.01),垂直运动与As模型组比较亦有显著性差异(P＜0.05);呼吸、心跳加快(与正常对照组及As模型组比较均P＜0.01)。同时形态学观察显示复合模型组病理学改变较As模型组不同程度加重,免疫组化显示α-actin阳性细胞明显减少,血脂紊乱,血中同型半胱氨酸升高。表明复合模型组既具备络气虚滞证候表现,又具备As病理表现,两种表现均不同程度加重。
2.4代谢组学结果
与正常对照组比较,As模型组、络气虚滞组、复合模型组血浆代谢物谱均存在明显差异,初步鉴定出不同模型相对于正常对照组的潜在生物标志物。经分析发现,胆汁酸类和磷脂类代谢物在As模型呈升高趋势,而络气虚滞模型则呈下降趋势,前者在复合模型亦呈上升趋势,而后者则呈下降趋势;氨基酸类和肉碱类在三种模型中均呈下降趋势。
2.5药物干预作用
通心络明显改善与证候干预相关的生物学表征评分(P＜0.01),延长力竭游泳时间和悬杆时间(P＜0.01),缩短中央格停留时间,改善水平运动和垂直运动评分(P＜0.05),降低心率和呼吸频率(P＜0.01);同时通心络也可显著改善主动脉形态与结构改变,促进VSMCs表型由合成型向收缩型转变,降低TC、LDL和HCY(P＜0.05,P＜0.01)。辛伐他汀以改善主动脉局部病理改变为主,虽对力竭游泳时间和悬杆时间表现出一定作用,但作用不及通心络,对复合模型与证候相关的生物学表征、电生理、旷场实验变化没有改善作用。
3络气虚滞型As大鼠NEI网络相关因子变化及通心络干预研究
3.1 HPAA变化
模型变化:与正常对照组比较,As模型组下丘脑组织CRH、血浆ACTH、血清CORT均无统计学差异(P＞0.05);络气虚滞组ACTH和CORT明显升高(P＜0.01),CRH表现出下降趋势,但无统计学差异(P＞0.05);复合模型组CRH明显下降(P＜0.01),ACTH和CORT明显升高(P＜0.01)。
药物干预:与复合模型组比较,通心络大剂量组能显著升高CRH水平(P＜0.05),降低ACTH水平(P＜0.05),通心络大小剂量组均能显著降低CORT水平(P＜0.05),辛伐他汀组与复合模型组比较变化无显著差异(P＞0.05)。
3.2交感—肾上腺髓质指标结果
模型变化:与正常对照组比较,As模型组E、NE指标有上升趋势但无统计学意义(P＞0.05);络气虚滞组E、NE明显升高(P＜0.01),与As模型组比较亦显著升高(P＜0.01);复合模型组E、NE亦明显升高(P＜0.05)。
药物干预:与复合模型组比较,通心络大小剂量组E、NE均显著下降(P＜0.05),辛伐他汀组与复合模型组比较无差著性差异(P＞0.05)
3.3 RAAS变化
模型变化:与正常对照组比较,As模型组血中PRA、AngⅡ、ALD变化均无显著差异(P＞0.05);络气虚滞组PRA、AngⅡ明显升高(P＜0.05),ALD变化无显著性差异(P＞0.05);复合模型组PRA、AngⅡ和ALD均显著升高(P＜0.01,P＜0.05,P＜0.01)。
与正常对照组比较,As模型组主动脉组织AngⅡ明显升高(P＜0.05),络气虚滞组主动脉组织AngⅡ具有上升趋势但无统计学意义(P＞0.05),复合模型组主动脉组织AngⅡ亦明显升高(P＜0.01)。
药物干预:与复合模型组比较,辛伐他汀能显著降低PRA和AngⅡ水平(P＜0.05),通心络大小剂量均能显著降低PRA、AngⅡ和ALD水平(P＜0.05,P＜0.05,P＜0.01)。辛伐他汀和通心络大小剂量均显著降低组织AngⅡ水平(P＜0.01,P＜0.01,P＜0.05)
3.4细胞因子结果
模型变化:与正常对照组比较,As模型组和络气虚滞组IFN-γ明显下降(P＜0.05,P＜0.01),IL-6变化无统计学差异(P＞0.05),复合模型组IFN-γ明显下降(P＜0.01),IL-6明显升高(P＜0.05)。
药物干预:与复合模型组比较,辛伐他汀能显著升高IFN-γ水平(P＜0.05),降低IL-6水平(P＜0.05),通心络小剂量组能明显升高IFN-γ水平(P＜0.01),通心络大剂量组既能明显升高IFN-γ水平(P＜0.05),又能降低IL-6水平(P＜0.05)。
3.5各组主动脉组织PCNA免疫组化结果
正常对照组和络气虚滞组未见PCNA阳性细胞,As模型组和复合模型组血管中膜可见大量PCNA阳性细胞,PCNA阳性细胞计数,复合模型组显著高于As模型组(P＜0.01)。各用药组干预后PCNA阳性细胞数均显著减少(P＜0.01)。
4 AngⅡ对大鼠VSMCs增殖影响的信号转导机制及通心络干预作用研究
4.1 AngⅡ对大鼠VSMCs增殖的影响
与正常对照组比较,10~(-8)、10~(-7)、10~(-5)、10~(-6)mol/L的AngⅡ均能显著促进平滑肌细胞增殖(P＜0.05,P＜0.01);与10~(-8)mol/L的AngⅡ相比,10~(-7)、10~(-6)、10~(-5)mol/L的OD值显著升高(P＜0.01);10~(-7)、10~(-6)、10~(-5)mol/L的AngⅡOD值无统计学意义。
4.2药物对AngⅡ诱导的大鼠VSMCs增殖的干预作用
AG490(10、20、50、100、200μmol/L)、辛伐他汀(0.01、0.1、1、10μmol/L)、通心络(100、200、500μg/ml)均不同程度抑制10~(-7)mol/LAngⅡ诱导的大鼠VSMCs增殖(P＜0.05,P＜0.01),具有一定的剂量依赖性。
4.3 AngⅡ对大鼠VSMCs细胞周期影响及药物干预作用
与正常对照组比较,10~(-7)mol/L的AngⅡS期细胞比例显著增加,G_0/G_1期细胞周期所占比例明显减少,(P＜0.01),各用药组干预后S期细胞比例显著下降,G_0/G_1期细胞比例明显增加(P＜0.01),各用药组之间无差著差异(P＞0.05)。
4.4 AngⅡ对大鼠VSMCs STAT_3核转位的影响及药物干预作用
正常对照组STAT_3胞浆、胞核均表达,AngⅡ组120 min时STAT_3明显由胞浆向胞核聚集,即发生核转位,10 min时核转位不明显,各用药组均能抑制AngⅡ作用120min引起的STAT_3核转位。
4.5 AngⅡ对大鼠VSMCs JAK_2/STAT_3信号通路蛋白影响及药物干预作用与正常对照组比较,AngⅡ组10 min时p-JAK_2表达明显增加(P＜0.05),p-STAT_3表达没有明显变化(P＞0.05);AngⅡ组120 min时p-JAK_2和p-STAT_3表达均明显增加(P＜0.05,P＜0.01)。
与AngⅡ组比较,AG490组10min、120min p-JAK_2和p-STAT_3表达明显下降(P＜0.05);通心络大剂量组和中剂量组120min时p-JAK_2和p-STAT_3表达均明显下降(P＜0.01,P＜0.05);辛伐他汀组和通心络小剂量仅120min时p-STAT_3表达显著下降(P＜0.05)。实验过程中各组JAK_2和STAT_3蛋白总量无显著差异(P＞0.05)。
结论:
1本研究以络病理论为指导,以文献研究、专家咨询为基础,通过多中心、大样本临床流行病学调查建立冠心病心绞痛证候辨证标准,运用基于熵的复杂系统分划方法从原始症状无监督分析入手,建立量化证候辨证标准,避免单纯依靠文献研究或专家经验所致主观偏倚;由于构成证候的症状之间存在着非线性复杂关系,该方法较之既往研究中采用的处理线性关系方法更为准确的反映了症状间相互作用的复杂关系,为建立证候模型并探讨其病理生理学基础奠定基础。
2基于证候组合与分布规律探讨冠心病心绞痛病机特点,研究结果显示络气郁滞或络气虚滞与血瘀、痰浊(热)、郁热、阴虚、阳虚等证候形成较强的关联关系,而络气郁滞或络气虚滞存在于所有冠心病心绞痛患者,所占比例高于血瘀、痰浊(热)、郁热、阴虚、阳虚等证候所占比例,显示络气变化所致郁滞或虚滞为冠心病心绞痛的始发病因并贯穿病变全过程,而国内医学界几十年来所重视和研究的血瘀、痰浊(热)等因素为冠心病心绞痛的继发致病因素。从络气切入研究冠心病心绞痛不同于已有从血瘀、痰浊切入开展的研究,显示了气血相关的络病理论特色,对生物—心理—社会医学模式下探讨各种社会心理因素的致病作用具有重要指导意义。
3以建立的络气虚滞证候辨证标准为依据,以“基础饮食+负重游泳”建立络气虚滞大鼠模型,从大鼠一般状况观察、生物学表征半定量评分、力竭游泳时间、悬杆实验、行为学观察、电生理检测客观评价模型大鼠络气虚滞证主要症状乏力、神疲、气短、心悸与标准一致性,也是本研究的特色之一。以As大鼠模型叠加“基础饮食+负重游泳”建立病证复合动物模型,模型大鼠既具有早期纤维增生性As的局部病理特点,又表现出络气虚滞宏观证候,代谢组学方法分析显示3种模型代谢物谱存在明显差异,这为探讨其病理生理学基础及络气虚滞证候因素对As病变的影响奠定了基础。
4探讨络气虚滞型As大鼠模NEI网络相关因子变化规律,络气虚滞证候状态下下丘脑—垂体—肾上腺轴调节功能紊乱、交感肾上腺髓质与肾素—血管紧张素—醛固酮激活,而As模型没有显著变化。络气虚滞证候因素使复合模型NEI网络相关因子变化紊乱且不同程度加重的同时,又促使As病理造模因素引起的组织AngⅡ升高,总体效应表现为复合模型反映平滑肌细胞增殖的PCNA表达明显高于As模型,揭示了络气虚滞证候因素可通过引起NEI网络内相关因子调控失常影响As病变。
5以络气虚滞证候因素作用相关的AngⅡ为干预因子,探讨其对体外培养大鼠主动脉平滑肌细胞增殖作用的影响及其相关信号转导机制。结果表明AngⅡ可明显促进大鼠平滑肌细胞增殖,与其促进细胞由G_0/G_1期进入S期有关。同时AngⅡ可促进平滑肌细胞p-JAK_2和p-STAT_3表达,促进STAT_3核转位,而上述作用可被JAK_2的特异性阻断剂AG490阻断,显示其促平滑肌细胞增殖作用与JAK_2/STAT_3信号通路有关。
6复方通心络和辛伐他汀对叠加证候造模因素和病理造模因素的复合模型的作用显示:二者均可改善局部病理改变,并通过作用于JAK_2/STAT_3信号通路抑制AngⅡ引起的血管平滑肌细胞增殖;通心络同时显著改善与络气虚滞证候因素相关的大鼠行为学、电生理等宏观表现,而辛伐他汀对证候变化基本无改善作用;特别是通心络改善络气虚滞证候因素干预之后引起的NEI网络相关因子紊乱表现出辛伐他汀不具备的整合调节作用,这对于现代生活条件下各种社会心理因素与病理因素叠加造成人体复杂病理生理现象的防治具有更广泛意义。
Objective:More and more scholars were paying attention to the pathogenesis of social psychological stress factors under the bio-psycho-social medical model.Based on the background,guided by collateral desease theory of Traditional Chinese Medicine(TCM),and through the clinical epidemiological survey on patients of Angina Pectoris of Coronary Heart Disease(APCHD),our study was to extract essential elements of syndromes using complicated system analysis method;to establish the standard about TCM differentiation of syndroms which combined qualitation with quantitation;to reveal the characters of pathogenesis according to the combining and distributing rules about syndromes;to establish the rat model of atherosclerosis(As)with collateral-qi deficiency according to standard of differentiation of syndroms,and evaluate objectively the concordance of syndromes of model animals with standard of differentiation of syndroms;to research the metabolic characters in model rats;to study the change rules of NEI network correlation factors and the relationship with As in model rats;to observe signaling pathway mechanisms of angiotensinⅡ(AngⅡ),which was the main factor of NEI network change caused by syndrome factor of collateral-qi deficiency,and which promoted vascular smooth muscle cells (VSMCs)proliferation;to evaluate Tongxinluo's effect on rat model of As with collateral-qi deficiency and on VSMCs proliferation promoted by AngⅡ. We also explored the influence of syndrome factor of collateral-qi deficiency to As according to the results from clinic patients and animals combined with cells,and illuminated initially the pathophysiologic basis on the rat model of As with collateral-qi deficiency,provided experiment evidences to support the research of the mechanisms about effect of social psychological factors on As. Meanwhile,we testified the scientific value of collateral desease theory.
Methods:4 parts included:
1 The study of quantitive standard for differentiation of syndrome to APCHD
1.1 The clinical epidemiological survey in patients of APCHD
Based on the study of literature and the consultants to experts,we bolted and classified the four physical examinations' information of TCM,instituted the clinical questionnaire,selected eight of GradeⅢ-A general hospitals in Beijing,Shanghai,Guangzhou,Ji'nan,Shenyang and Changchun as our investigating centers,carried out large sample clinical epidemiological survey in patients of APCHD from multicentres.During the investigation,we carried out strict quality control following principle of clinical epidemiology to ensure the reality and reliability of the data.
1.2 The establishment of quantitive standard for differentiation of syndromes to APCHD
The study enrolled 403 subjects who visited during our investigation.They were suitable to the standard of APCHD.The 403 subjects were divided randomly into two groups by 3:1.One group,302 subjects,as the calculating group was to establish quantitive standard of differentiation of syndrome. Another group,101 subjects,as the examining group was to test prospectively diagnostic code of syndromes' quantization.
The data of 302 subjects were filled in Matlab software,syndromes information was exercted,the contribution of symptoms to syndromes was calculated by entropy-based complex systems partition method(ECSPM).The thresholds on the differentiation of syndromes were set up by the analysis of receiver operator characteristic curve(ROC).The quantitive standards of differentiation of syndrome to APCHD was established successfully when those results of above research were obtained.Then the differentiation of tandards of syndromes prospectively was tested in another group.
1.3 The research of combining and distributing rules of syndromes in APCHD.
We analysed correlative grade of syndromes by ECSPM,and calculated the proportion of syndromes in 403 cases patients based on the established differentiation standard of syndrome,and then analysed the characters of pathogenesis according to the combing and distributing rules of the syndromes in patients.
2 The establishment and evaluation of the rat model of As with collateral-qi deficiency
The differentiation standard of syndromes in collateral-qi deficiency was established based on clinical epidemiological survey.According to the TCM theory of "excess fatigue consuming qi",basic diet plus loading swimming was applied to induce the syndromes of collateral-qi deficiency.Rats were given VitaminD_3,high-methionine and high-grease diet to induce As model. And then the rat model of As with collateral-qi deficiency was induced by the two above factors' superposition.
One hundred and five Wistar rats were divided into 7 groups randomly:①Control group.②As group.③Collateral-qi deficiency group(loading swimming group).④As with collateral-qi deficiency(complex model group) group.⑤Large dose Tongxinluo group.⑥Small dose Tongxinluo group.⑦Simvastatin group.
After the establishment of rat models,general condition,score of biology superficial syndrome,climbing-time,open-field experiment,and electrophysiology were detected to evaluate objectively the major syndromes' information(debilitation,short breath,fatigue,palpitation)in model rats with collateral-qi deficiency.
At the same time,the HE staining,Masson staining,electron microscope, immunohistochemical staining of arota were carried out.Total cholesterol(TC), low density lipoprotein(LDL)and homocysteine(HCY)were detected.The changes of metabolin were analyzed by metabonomics.All those indexes were to evaluate the degree of As.
3 The NEI network changes of the As model rats with collateral-qi deficiency and the intervening effect of Herbs
The collateral-qi deficiency model,As rats model and complex model had been instituted as the study objects to investigate the change rules of indexes related to NEI network and the influence of the indexes to As.The dividing of rat groups and the drugs observed were as above.At the finished time point of the model,the rats were anesthetized and blood preparation was collected through arteria carotis cannula,plasma or blood serum was separated.The indexes were detected as following:
Hypothalamo-pituitary-adrenal axis(HPAA):corticotropin releasing hormone(CRH)of hypothalamus,adrenocorticotrop(h)ic hormone(ACTH)in plasma,corticosterone(CORT)in serum were detected by Radio-immunity Approach.Norepinephrine and epinephrine in serum was detected by ELISA Approach.Renin-angiotensin-aldosterone system(RAAS):plasma renin activity(PRA),aldosterone(ALD)in plasma,AngⅡin aorta and plasma were detected by Radio-immunity Approach.Cytokine:interleukin(IL)-6 in serum was detected by Radio-immunity Approach,and interferon(IFN)-γin serum was detected by ELISA Approach.Paraffin sections of aorta pectoralis was used to detect the expression of proliferating cell nuclear antigen(PCNA)by Envision immunohistochemistry kit.
4 The signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it
AngⅡwas selected,which was the main effect factor of NEI network derangement induced by the intervention of collateral-qi deficiency.It was studied by us that the signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it.The research could also provide the experimental evidence of the mechanism of the syndrome factor influncing vascular lesion.The primary culture VSMCs of rat by the tissue sticking mass method was used for the study.The proliferation capability of VSMCs was detected using MTT method.The cell cycle of VSMCs was detected using flow cytometry.The nuclear translocation of signal transducer and activator of transcription(STAT)3 was observed with laser confocal microscopy.The expression of proteins of Janus Kinase(JAK)2, STAT3,phospho -JAK_2 and phospho-STAT_3 was detected with Westernblot technology.Such drugs as AG490,an special inhibitor of JAK2,Tongxinluo and Simvastatin were selected.
Result:
1 The establishment of quantitive standards of differentiation of syndrome to APCHD
1.1 The extraction of syndromes and the establishment of the quantitive standard of APCHD
Based on the analyses of syndrome without supervision,eight syndromes were obtained in the patients with APCHD.The contribution of symptoms to syndromes(the number at the low right of symptoms)and thresholds(the nmuber at the low right of syndroms)as followings:①collateral-qi stasis_(5): oppressed feel in heart and chest_(4),preference for sighing_(3),depressed emotion_(3),restlessness_(2),viry pulse_(1);②collateral-qi deficiency_(6):vague pain in heart and chest_(1),cardiopalmus_(2),short breath_(2),mind lassitude_(3), debilitation_(3),lazy to speak_(2),pale tongue_(1),weakened pulset_(1);③phlegm turbid_(4):cerebaria_(2),muco-greasy in mouth_(2),excessive phlegm_(3),muffled in chest and gastric cavity_(2),body fat_(1),distress in limbs_(2),whiteness greasy of musci_(2),viry and slide pulse_(1);④blood stasis_(3):localized pain in chest as acanthus and angor_(3),eclipse of facial expression_(2),cyanochroia in labia oris_(3),purplish tongue with ecchymosis or petech_(3),unsmooth pulse_(2);⑤yin asthenia_(7):feverish of five centres_(3),insomnia_(3),multi-dream_(3),hectic fever and night sweat_(3),sour soft in waist and knee_(2),few musci or no musci(1), pulse fine and cipher_(1);⑥heat accumulation_(6):flushing_(2),dry mouth_(2),bitter taste of mouth_(3),suitable red_(2),constipation_(2),red tongue with yellowish fur_(1),yellow tongue fur_(1)pulse cipher_(2);⑦yang asthenia_(5):facial expression white_(2),urine schedule length_(1),mild facial edema and swollen limbs_(1), chilly_(3),limbs cold_(3),pale tongue and fat_(1),pulse heavy slow and powerless_(1);⑧phlegm fever_(3):mouth sticking and greasy_(2),pulse slide and cipher_(1), yellow tongue fur with greasy dirt_(2),cough yellow phlegm_(2).
1.2 The combining and distributing rules of syndroms in APCHD
The results of correlative grade of syndromes:the syndrome of collateral-qi stasis had the strong correlation with heat accumulation,blood stasis,phlegm fever,phlegm turbid,yin asthenia,and the syndrome of collateral-qi deficiency had the strong correlation with phlegm turbid,yang asthenia,blood stasis,yin asthenia.
The distributing proportion of syndromes:all the patients have either collateral-qi stasis syndrome or collateral-qi deficiency syndrome.The collateral-qi deficiency syndrome accounts for 57.6%(232 cases)of all investigated patients(403 cases),collateral-qi stasis accounts for 31.5%(127 cases),10.9%(44 cases)has both collateral-qi deficiency and the collateral-qi stasis.However,blood stasis accounts for 60.3%(243 cases),which was thought the major syndrome in previous study,phlegm turbid(heat)accounts for 43.4%(175 cases),gasthenia accounts for 14.9%(60 cases).
1.3 To raise the viewpoint about the pathogenesis of APCHD based on the combining and distributing rules of syndromes
Based on the above results,either collateral-qi stasis or collateral-qi deficiency was the initiating agent of APCHD and breakthrough the whole process.Phlegm and blood stasis were not only the patho-products induced by collateral-qi stasis or collateral-qi deficiency,but also influenced the developing of the disease conversely.Meanwhile,collateral-qi stasis could induce pyretic generation,damage yin,depress vital energy and yin asthenia. Deficiency of vital energy for a long timemay developped to yang asthenia. Both collateral-qi stasis and collateral-qi deficiency were the basic syndromes, and heat accumulation,blood stasis,phlegm turbid(fever),yin asthenia,yang asthenia and yin asthenia,which were derived from collateral-qi stasis or collateral-qi deficiency,were the accompanied syndromes.
2 Establishment and evaluation of the rat model of As with collateral-qi deficiency
2.1 The evaluation of model rats of collateral-qi deficiency
Compared with the control group,the rats of collateral-qi deficiency group appeared low-spirited,eye-closed,superficial few gloss,and extrados appearance.The score of biology superficial signs was significantly higher than control group(P＜0.01).Fatigued swimming time was significantly decreased than that on the first day(P＜0.01).The rats of collateral-qi deficiency group also displayed the shorter climbing time,the longer stay-time in central frame and lower crossing and rearing score in open-field test(P＜0.05,P＜0.01).All the results reflected the cardiopalmus,short breath, debilitation,mind lassitude of collateral-qi deficiency syndrome had the concordance with the model rats with collateral-qi deficiency syndrome.
2.2 The evaluation of model rats of As
Rats of As model group only appeared weight breakdown.The biology superfocial signs score of As group had no significant difference.Open-field test and physiologic function also had no significant change(P＞0.05).HE staining and Masson staining showed endothelial cell partly loss, endomembrane thickened,VSMCs hyperplasy and arrange disorder,collagen fiber proliferation,local tubal walls lumina outstanding and form fiber hyperplasy As.SEM and TEM showed endothelium injured,and there was deposition on surface.Bilblast of VSMCs dissolved,and endoplasmic reticulum distended.Aortic immunityhiscochemstry showed theα-actin masc cells decreased obviously.While the TC、LDL and HCY increased obviously. All those results showed the rat model had the pathological features of plaque fiber proliferation in earlier period of As.
2.3 The evaluation of complex model rats
Rats of the complex model group appeared energetic dispirited,insensitive response,skin slacked,hair on the back falled off,et al.The score of complex model group was highest,which was significantly higher than both As group and collateral-qi deficiency group(P＜0.01).The climbing-time,respiratory frequency and heart rate,the rearing score of open-field of complex model rats had the same results as the above.At the same time,the swimming-time of complex model group was significant lower than that on the first day of the same group.The stay-time in central frame of open-field was longer and crossing score was lower than control group(P＜0.05,P＜0.01).As degree of complex model was more serious than As model according the observation of HE staining,Masson staining and ultramicrostructure.The masc cells expressing of aorticα-actin in complex model group also decreased,and the TC,LDL,HCY increased highly.All those results showed that there were both collateral-qi deficiency syndrome and As in complex model,whose degree was more seriously.
2.4 Metabonomics results
Compared with control group,As model group,collateral-qi deficiency group,complex model group metabolin spectra had obvious difference.The potential biomarkers were identified initially.According the identification results,bile acid and phospholipid increased in As model,while decreased in collateral-qi deficiency model.Bile acid increased in complex model,while phospholipid decreased in it.Amino acids and carnitine all increased in three models.
2.5 Drugs intervention
Tongxinluo could improve the indexes related to the syndrome,such as improve the biology superficial signs score(P＜0.01),prolong the fatigued swimming time and climbing time(P＜0.01),shorten the stay-time in central frame,improve the crossing score and rearing score(P＜0.05),and degrade the respiratory frequency and heart rate(P＜0.01).At the same time, Tongxinluo might also improve the change of shape and structure of aorta, promote the phenotypic modulation of VSMCs from contractile phenotype to synthetic phenotype,reduce the level of TC、LDL and HCY(P＜0.05,P＜0.01).Simvastatin mainly improved the locally pathological change of aorta. Although Simvastatin had the effect on the fatigued swimming time and climbing-time,whose effect was not as good as Tongxinluo.Simvastatin had no effect on biology superficial signs score,respiratory frequency and heart rate,open-field test in combinted model.
3 The study about the changes of NEI network in collateral-qi deficiency As rats and the intervention of Tongxinluo
3.1 The changes of HPAA
Changes of model rats:compared with control group,tissue CRH,blood plasma ACTH,serum CORT in As group had statistic difference(P＞0.05), ACTH and CORT in collateral-qi deficiency group increased obviously(P＜0.01),CRH only appeared decrease tendency(P＞0.05),and CRH decreased obviously(P＜0.01),ACTH and CORT increased obviously in complex model group(P＜0.01).
The intervention of drugs:compared with complex model group,large dose Tongxinluo could increase obviously CRH level(P＜0.05),decrease ACTH level(P＜0.05).Large and small dose Tongxinluo could obviously decrease CORT level,and Simvastatin had no obvious effect(P＞0.05)。
3.2 The changes of Sympathesis-Adrenal Medulla Axis.
Changes of model rats:compared with the control group,the indexes of E,NE in As group had upgrade tendency but had no significant difference (p＞0.05).In collateral-qi deficiency group,the index of E,NE upgraded significantly(P＜0.01),which was significantly different compared with arteriosclerosis group(P＜0.01).To compare with normal group,the index of E,NE in complex model group is obviously heighten(P＜0.05).
The intervention of drugs:compared with the complex model group, E,NE of large dose and low dose tongxinluo group obviously decreased (P＜0.05).But E,NE of Simvastatin group have no significant difference
3.3 The changes of RAAS
Changes of model rats:compared with control group,the PRA,angⅡand aldosterone in blood of As group have no significant change(P＞0.05). The PRA,angⅡin blood of collateral-qi deficiency group obviously increased(P＜0.05),and ALD had no significant difference(p＞0.05).The PRA,angⅡand ALD in blood of the complex model group obviously increased(P＜0.05,P＜0.01)
Compared with the control group,AugⅡin aorta of As group obviously increased(P＜0.05).AngⅡin aorta of collateral-qi deficiency group had upgrade tendency,but no significant difference(P＞0.05).AngⅡin aorta of the complex model group obviously increased(P＜0.01).
The intervention of drugs:compared with the complex model group, Simvastatin could lower the level of PRA and angⅡ(P＜0.05).large and small dose Tongxinluo could lower the level of PRA,angⅡand ALD(P＜0.05,P＜0.01)。
Compared with the complex model group,AngⅡin aorta of simvastatin, large and low dose Tongxinluo group obviously decreased(P＜0.05,P＜0.01).
3.4 The changes of cytokines
Changes of model rats:compared with normal control group,IFN-γin As and collateral-qi deficiency group obviously decreased(P＜0.05),while IL-6 had no significant difference(P＞0.05).IFN-γin the complex model group obviously decreased(P＜0.01),and IL-6 obviously increased(P＜0.05).
The intervention of drugs:compared with the complex model group, Simvastatin could obviously raise the level of IFN-γ(P＜0.05)and decrease the level of IL-6(P＜0.05).The low dose Tongxinluo could obviously raise the level of IFN-γ(P＜0.01),while the large dose Tongxinluo could obviously both raise the level of IFN-γ(P＜0.05)and decrease the level of IL-6(P＜0.05)。
3.5 The expressing of PCNA in aorta
PCNA masc cells In the control group and collateral-qi deficiency group had been found.While plenty of PCNA masc cells in As group and complex model group had been found.The count of masc cells in the complex model group was obviously higher than that in As group(P＜0.01).All drugs could reduced the masc cells(P＜0.01)。
4.The signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it
4.1 The influence of AngⅡto the proliferation of rat VSMCs
Compared with DMEM group,10~(-5)、10~(-6)、10~(-7)、10~(-8)mol/L of AngⅡcould obviously promote VSMCs proliferation(p＜0.05,P＜0.01).Compared with 10~(-8)mol/L of AngⅡgroup,the OD value of 10~(-5)、10~(-6)、10~(-7)mol/L group obviously rised(P＜0.01).The OD value of 10~(-5)、10~(-6)、10~(-7)mol/L of AngⅡhad no significant difference.
4.2 The interventional effect of drugs on proliferation of rat VSMCs induced by AngⅡ
AG490(10、20、50、100、200μmol/L)、Simvastatin(0.01、0.1、1、10μmol/L)、Tongxinluo(100、200、500μg/ml)could inhibitted proliferation of rat VSMCs induced AngⅡat the concentration of 10~(-7)mol/L(P＜0.05,P＜0.01),which was dose dependent.
4.3 The effect of AngⅡon the cell cycle of VSMCs and the interventional effect of drugs on it
Compared with DMEM group,the percentage of cells in phase S significantly increased after the incubation of AngⅡat the concentration of 10 ~(-7)mol/L,while the percentage of cells in phase G_0/G_1 bviously decrease(P＜0.01).All drugs could decrease the percentage of cells in phase S,and increased the percentage of cells in phase G_0/G_1(P＜0.01)
4.4 The influence of AngⅡto nuclear translocation of STAT_3 induced by AngⅡand effect of drugs on it
In the DMEM group,STAT_3 was detected well-distributedly in the kytoplasm and nucleus.After 120min incubated by AngⅡ,STAT_3 was detected mainly in the nucleus,which showed us the nuclear translocation of STAT_3.However,After 10min incubated by AngⅡ,the nuclear translocation of STAT_3 was not observed.All drugs could inhibited the nuclear translocation of STAT3 after 120min incubated by AngⅡ.
4.5 The influence of AngⅡto the protein expression of JAK2/STAT3 signal passway and effect of drugs on it
Compared with DMEM group,the protein expression of p-JAK_2 increased obviously after 10 min incubated by AngⅡ(P＜0.05),while the protein expression of p-STAT_3 had no significant difference.The protein expression of both p-JAK_2 and p-STAT_3 increased obviously after 120 min incubated by AngⅡ(P＜0.05,P＜0.01)
Compared with AngⅡgroup,AG490 could reduce the protein expression of p- JAK_2 and p- STAT_3 after 10 min and 120 min incubated by AngⅡ(P＜0.05).Both large dose and median dose Tongxinluo could reduced the protein expression of p- JAK_2 and p- STAT+3 after 120 min incubated by AngⅡ(P＜0.01,P＜0.05).Simvastatin and samll dose Tongxinluo only reduced the protein expression of p- STAT_3 after 120 min incubated by AngⅡ(P＜0.05).The whole protein of JAK_2 and STAT_3 had no significant difference(P＞0.05)
Conclusion:
1 Guided by the theory of collateral disease,based on literature study, experts consultant and large sample clinical epidemiological survey in patients of APCHD from multicentres,the study was to establish quantitive differentiation of syndromestandard,on base of the data analysis without supervision using ECSPM,which avioded the subjective bias of simple literature study and experts'experience.Because the nonlinear and complex relationship existed in symptoms of syndrome,ECSPM could reflect the complex interactive relationship between symptoms more precise,compared with the method of dealing with liner relationship in previous study,which makes a found to establish the syndrom model and study the pathophysiologic basis.
2 Studying the pathogenesy of TCM about APCAD on basis of the combining and distributing rules of syndromes,Our results showed that collateral-qi stasis or collateral-qi deficiency existed in all patients of APCAD, which was higher than the percentage of blood stasis,phlegm muddy(heat), heat accumulation,yin asthenia,yang asthenia etc.The results showed that stagnancy or deficiency caused by the changes of collateral-qi might be initiating agent of APCAD.Phlegm muddy(heat)and blood stasis were the patho-products derived of collateral-qi stasis or collateral-qi deficiency, which had been paid attention and researched for several decades by domestic medical society.Studying the change of collateral-qi of APCAD was different from that that study such blood stasis and phlegm muddy,etc,which showed the feature of qi related to blood in collateral desease theory,and had the guidance significance to explore the pathomechanism of various kinds of social-psycho factors under the bio-psycho-social medical model.
3 Based on the differentiation standard of syndromes established by clinical epidemiological survey,we eatablished syndrome model of collateral-qi deficiency,using basic diet plus forced loading swimming.The model rats were evaluated objectivly by the methods of the general condition observation,semi-quantity score of biology superficial signs,fatigued swimming-time,climbing-time,ethology,electrophysiology,according to main symptoms of collateral-qi deficiency.The complex model was established using As rat model overlain basic diet plus forced loading swimming.The model rats had both local pathological feature in the earlier period of As,and the representation of collateral-qi deficiency syndrome.The three models showed difference in the metabolism spectra using the metabonomics technology.This study also lain the foundation for exploring pathophysiologic basis of collateral-qi deficiency syndrome and it's effect on AS.
4 This study had explore the changes rules of factors related to NEI network in the rat model of As with collateral-qi deficiency.The function of HPAA was in the chaos in the condition of collateral-qi deficiency.Adrenergic nerve -adrenal medulla system and RAAS were activated.However AS model had not such significant changes.The syndrome factor of collateral-qi deficiency not only caused the indexes related to NEI network to change more significantly in complex model rats,but also promoted more increase of AngⅡin aorta,which caused by pathological factors.Total effect was that PCNA which reflected the VSMCs proliferation in the complex model rats was obviously higher than simple As model,which revealed that syndome factors of collateral-qi deficiency could cause the factors related to the NEI network to be disorder and then influence As.
5 The study selected AngⅡas the interventional factor,which was the main factor related to the NEI network influenced by the syndrome factor of collateral-qi deficiency,and explored the influence of AngⅡto the proliferation of rat VSMCs,which would provide the experimental evidence about the pathomechanism of syndrome of collateral-qi deficiency.The results showed that AngⅡcould obviously promote rats proliferation of VSMCs, which was related to the influence to the cell circle.Meanwhile,AngⅡalso promoted protein expression of p-JAK_2 and p-STAT_3 of VSMCs,while the effect above could be stocked by AG490,which was specific inhibitor of JAK_2.Those results showed that the effect of AngⅡpromoting the proliferation of VSMCs was related to signaling pathway of JAK_2/STAT_3.
6 The effect of Tongxinluo and Simvastatin on the complex model rats showed that they could both improve local pathological change,and inhibit proliferation of VSMCs caused by AngⅡthrough blocking JAK_2/STAT_3 signaling pathways.Tongxinluo also significantly improved the pathological changes caused by syndrome factor of collateral-qi deficiency,such as ethology,electrophysiology etc,while Simvastatin had no such effect. Especially Tongxinluo showed the integrated regulation effect in improving the disorder of NEI network caused by intervention of syndrome factor of collateral-qi deficiency,which was better tha Simvastatin.These results manifested that Tongxinluo showed double-effect on improving both local and global pathological change,which was different from Simvastatin.These results also possessed much more widespread meaning to prevent and care human body complex pathological and physiological phenomenon,which caused by various kinds of social psychological stress factors and biological pathogenic factors under modern living conditions and pathology factors.
引文
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