【作者】 李鸥；

【导师】 陈可冀； 徐浩；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2011， 博士

【摘要】 目前,动脉粥样硬化易损斑块作导致急性心血管事件方面的重要性已受到广泛重视。改善易损斑块内部成分,使其趋于稳定,成为防治急性心血管事件的重要内容。本研究在得出与急性心血管事件有关的中医证候要素的基础上,选取相应中药。采用均匀设计法探索了稳定斑块最佳中药组分配伍组合,这对于动脉粥样硬化和冠心病的防治无疑具有重要意义。1临床研究：冠心病住院患者中医证候分布规律的多中心研究1.1目的通过对1005例冠心病住院患者进行横断面调查,观察冠心病住院患者医证候要素分布的特点；探索中医证候要素与急性心血管事件发生的关系,从而为冠心病中医临床辨治及药物研发提供依据。1.2方法收集9家医院1005例冠心病住院患者,观察冠心病住院患者的中医证候要素分布的特点。并通过前瞻性调查研究方法,随访1年,观察上述患者心血管事件发生情况,通过主成分Logistic回归分析发生心血管事件患者的中医证候要素分布特点。1.3结果冠心病住院患者最常见证候要素由高到低分别为血瘀(81.4%),气虚(56.8%)、痰浊(48.5%)、阴虚(25.1%)。随访1年,其中发生急性心血管事件的患者有66例,未发生急性心血管事件的患者有939例。经主成分Logistic回归方法分析,结果显示主要由气虚、阴虚组成的主成分2在Logistic回归中有统计学差异(P=0.036)。1.4结论血瘀、气虚、痰浊、阴虚是冠心病住院患者的主要证候要素,体现出本虚标实的特点。气虚、阴虚可能是冠心病住院患者随访发生急性心血管事件相关的证侯要素,值得深入研究。2基础研究：中药配伍优化干预ApoE基因缺陷小鼠动脉粥样硬化易损斑块作用的研究2.1目的在前期研究活血解毒中药具有较好的稳定易损斑块作用基础上,结合临床冠心病患者主要证候要素,基于均匀设计方法观察中药复方对ApoE基因缺陷小鼠主动脉粥样硬化易损斑块稳定性的作用,探索最佳组方和配比关系。2.2方法选取虎杖、三七、瓜蒌、何首鸟四种中药,采用均匀设计法“4因子8水平表”分组设计,将这四种药物的提取物分成8个给药组。100只ApoE-/-,小鼠经“西方膳食”饲料喂养13周后,将小鼠随机分为10组：8个药物组、阿托伐他汀组、模型组各10只。继续高脂喂养并给予相应药物治疗13周后,处死小鼠：首先取出主动脉,进行大体病理形态观察,并进行苏丹Ⅳ染色；然后取小鼠主动脉根部的3个横切面,分别行HE染色、Movat染色以及免疫组织化学染色法观察斑块内部成分；同时取血清。观察项目：(Ⅰ)大体主动脉：计算斑块／内膜面积的比值；(2)主动脉根部横切面：计算包括斑块面积,细胞外脂质(EL)、泡沫细胞(FC)、胶原成分(CA)以及平滑肌细胞(SMC)各自占斑块面积的百分比；(3)免疫组化染色观察主动脉根部AS斑块内PPAR-γ的表达,并计算阳性面积与斑块面积的比值；(4)测定血脂含量。最后采用易损指数(Ⅵ)综合评价药物对小鼠主动脉斑块稳定性的影响。公式为：易损指数(VI)=(EL+FC)/(CA+SMC)。以易损指数为筛选指标,经均匀设计回归分析获得“最佳组方及配比关系”。2.3结果2.3.1主动脉大体标本各组小鼠动脉均可见红染的动脉粥样硬化斑块,明显的纤维斑块,散在分布。模型组主动脉管壁病变最为严重,粥样斑块和脂纹累积范围广泛,程度重。与模型组比较,药物组2、3、6、7、8及阿托伐他汀组小鼠主动脉斑块／内膜面积比值有明显下降,有显著差异(P<0.05)。2.3.2主动脉根部横切面各组小鼠主动脉根部横切片H-E染色和Movat五色套染法可看到明显的动脉粥样硬化斑块,内膜不对称增厚,被大量泡沫细胞覆盖的薄的纤维帽,细胞外基质中可见胆固醇结晶,以及富含脂质的坏死物质,显示出易损斑块特征。某些标本血管中层变薄,弹性纤维断裂,甚至消失。Movat染色下,胶原组织染成黄色。SMα-actin免疫组化染色下,阳性平滑肌即染成棕黄色。与模型组比较,各药物组小鼠主动脉横切面AS斑块面积无明显差异(P>0.05)。2.3.3 AS斑块内部成分EL含量比较,药物组5、7较模型组明显增加(P<0.05),其余各药物组及阿托伐他汀组与模型组无差异(P<0.05)。FC含量比较,与模型组相比较,各药物组及阿托伐他汀组均无显著性差异(P>0.05)。CA含量比较,与模型组比较,药物组3、4、6、7、8及阿托伐他汀组明显增加(P<0.05,P<0.01)。SMC含量比较,药物组1、2、3、4、8及阿托伐他汀组均明显增多(P<0.05,P<0.01)。2.3.4AS斑块PPAR-γ含量与模型组比较,药物组2、3、4、6、7、8及阿托伐他汀组小鼠斑块内PPAR-y蛋白阳性表达明显增加(P<0.05)。2.3.5血脂与模型组比较,各药物组血脂(包括TC、TG、HDL、LDL)均无明显差异(P>0.05)。2.3.6评估斑块稳定性与模型组相比,药物组1、3、4、6、7、8、阿托伐他汀组小鼠主动脉斑块易损指数均显著降低(P<0.05),其中以实验组8降低最为显著(P<0.01)。2.3.7中药配伍优化结果以易损指数为筛选指标,经均匀设计回归分析获得“最佳组方及配比关系”。配方1：虎杖0.3604克,瓜蒌0.3604克,何首乌0.1834克。配方2：虎杖0.3604克,何首乌0.1606克。2.4结论由虎杖、三七、瓜蒌、何首乌组成的中药复方,可不同程度地降低ApoE-/-小鼠主动脉粥样硬化斑块易损指数,具有一定稳定动脉粥样硬化斑块的作用。通过多元回归分析,具有最佳稳定斑块作用的中药组方及配比关系为：虎杖0.3604克,瓜蒌0.3604克,何首乌0.183克g,或虎杖0.3604克,何首乌0.1606克,为下一步验证奠定了基础。更多还原

【Abstract】 Nowadays, vulnerable atherosclerotic plaque (VAP) has been highlighted for its role in leading to acute cardiovascular events. Thus, it is regarded as an important strategy to prevent the incidence of acute cardiovascular events by changing the composition of VAP to make it stable. Based on the result which TCM syndrome elements are relevant to acute cardiovascular events, we choose corresponding Chinese herbs. We further explored the optimized combination and proportion of herbal extractives in stabilizing VAP by uniform design, which is of great importance for developing effective Chinese medicine in preventing and treating AS and coronary heart disease.1 Clinical research:Multi-central research of TCM syndrome elements of hospitalized patients with coronary heart disease1.1 ObjectiveTo study distribution of TCM syndrome elements in 1005 CHD patients in hospital; to explore the correlation of TCM syndrome elements and cardiovascular events, so as to provide the clinical evidence for CHD syndrome differentiation and treatment with TCM, as well as the development of new Chinese compound prescribtion.1.2 MethodBy cross-sectional research method, we analyzed the distribution of TCM syndrome elements in 1005 CHD patients from 9 hospitals; by prospective research method, we studied the distribution of TCM syndrome elements in CHD patients with cardiovascular events in 1 year and explored the correlation of TCM syndrome elements and cardiovascular events through the principal component analysis and logistic regression analysis method.1.3 ResultThe proportion of chief TCM syndrome elements in CHD patients in descending order are blood stasis (81.4%), qi deficiency (56.8% ), turbid phlegm (48.5% ), yin deficiency (25.1%). After 1 year follow-up, there are 66 CHD patients who happen to cardiovascular events, and the other 939 CHD patients who have no cardiovascular events. The analytic results of principal component analysis and logistic regression analysis method demonstrated that the second principal component had statistical significance in logistic regression (P=0.036), and the second principal component mainly consisted of qi deficiency and yin deficiency.1.4 ConclusionBlood stasis, qi deficiency, turbid phlegm, yin deficiency were main syndrome elements of CHD patients. And that result reflected the feature of deficiency in origin and excess in superficislity. Qi deficiency and yin definciency might be the relevant TCM syndrome elements to the CHD patients who happen to cardiovascular events in 1 year follow up.2 Experimental research:Study the effect of Chinese herbs on atherosclerotic vulnerable plaque stability of ApoE gene-deficient (ApoE) mice and explor the optimized combination and proportion of chinese herbs2.1 ObjectiveBased the results of previous study that the chinses herbs of activating blood circulation and detoxicationg could stabilize atherosclerotic plaque, we combined the main sysdrome elements of CHD patients in clinic. We would study the effect of Chinese herbs on the stability of atherosclerotic vulnerable plaque in the aortic roots of fat-fed ApoE-/- mice and explore the optimized combination and proportion of Chinese herbs by uniform design.2.2 MethodWe chose giant knotweed rhizome, panax pseudo-ginseng, snakegourd fruit, and polygonum multiflorum. A uniform design with 4-factor 8-level table was used in this study. According to the table, the four Chinese herb extractive (giant knotweed rhizome, panax pseudo-ginseng, snakegourd fruit, and polygonum multiflorum) were composed for 8 medication administration groups (No.1 group-No.8 group). One hundred ApoE mice were fed a high-fat diet for 13 weeks until the mature atherosclerotic plaques formed. Thereafter, they were randomized to ten groups (10 in each group) and fed a high-fat diet for subsequent 13 weeks:8 medication administration groups, atorvastatin group, and control group. Every group was medicated with respective drug in a clinically relevant dose for another 13 weeks. Then all the mice were sacrificed at the end of experiment. For each mouse, the aorta was dissected out and stained with SudanⅣfor subsequent assessment of atherosclerotic lesion area. Cross sections of aortic roots were prepared and stained with H-E, a modified Movat pentachrome stain, or immunohistochemistry stain respectively. The blood was collected for determination of blood lipids. The observe items included:(1) en face whole of aorta: the atherosclerotic plaques (red stained)/ aortic intimal surface area in each aorta. (2) histological cross sectional aortic sample: the area of atherosclerotic plaque, the percentage of extracelluar lipids (EL), foam cells (FC), collagen (CA) and smooth muscle cells (SMC) in plaque. (3) Immunohistochemical staining for the expression of PPAR-γ, positive staining areas were measured and expressed as a percentage of the whole plaque area. (4) Determination of serum lipid levels. The effect on plaque stability was evaluated by using the vulnerability index (Ⅵ). TheⅥwas expressed as the area ratio of foam cells and extracellular lipids to collagens and smooth muscle cells. Taking theⅥas the screen index, we explored the optimized combination and proportion of Chinese herbs by regression analysis of uniform design.2.3 Result2.3.1 En face whole aortic sampleIn each group, the atherosclerotic plaques (red stained) were clearly observed and predominantly complex fibrous plaques, which were scattered in the entire aorta. Compared with control group, the No.2 group, No.3 group, No.6 group, No.7 group, No.8 group, and atorvastatin group was associated with a significant decrease in the plaque area (P<0.05).2.3.2 Histological cross sectional aortic sampleHE staining and Movat’s pentachrome staining within the aortic cross section revealed that the atherosclerotic plaques in ApoE-deficient mice were an asymmetrically thickened intima composed with a thin fibrous cap, usually buried with foam cells, and a lipid-rich necrotic core with cholesterol clefs within the extracellular matrix, that was typical morphologic features of vulnerable plaque. In some cases, the vascular media was thinned, and elastic fibers were broken, even disappeared. The collagen was yellow stained by Movat staining, and the positive of PPAR-γexpression was brown by immunohistochemical staining. As for the cross sectional area of atherosclerotic plaques within aorta, there was no significant difference among all drug treated group compared with control group (P>0.05).2.3.3 Atherosclerotic plaque compositionAs for extracellular lipids area, compared with control group, No.5 and No.7 group had a marked increase (P<0.05), the other groups and atrovastatin group had no significant difference. As for foam cell area, compared with control group, there was no significant difference among all groups (P>0.05). As for the collagen area, compared with control group, No.3 group, No.4 group, No.6 group, No.7 group, No.8 group, and atorvastatin goup was associated with a significant increase (P<0.05). As for the smooth muscle cell area, compared with control group, No.1 group, No.2 group, No.3 group, No.4 group, No.8 group, and atorvastatin group was associated with a significant increase (P>0.05).2.3.4 Expression of PPAR-γin the aortic plaqueCompared with the control group, No.2 group, No.3 group, No.4 group, No.6 group, No.7 group, No.8 group, and atorvastatin group was associated with a a marked increase in expression of PPAR-y (P<0.05).2.3.5 Plasma lipid levelsCompared with controls, there was no statistically difference among all drug treated group (P>0.05) in the level of TC, TG, LDL and HDL respectively.2.3.6 Evaluation of plaque stabilityCompared with the control group, No.1 group, No.3 group, No.4 group, No.6 group, No.7 group, No.8 group, and atorvastatin group was associated with a significant reduction in the vulnerability index (Ⅵ) (P<0.05). And the vulnerability index of No.8 group was minimal (P<0.01).2.3.7 Optimized combination and proportion of Chinese herbsTaking the vulnerability index as the screen index and multivariant regression analysis of uniform design, we got the optimized combination and proportion of Chinese herbs. The first optimized combination:giant knotweed rhizome 0.3604g, snakegourd fruit 0.3604g, polygonum multiflorum 0.1834g. The second optimized combination:giant knotweed rhizome 0.3604g, polygonum multiflorum 0.1606g.2.4 ConclusionThe Chinese medicine compound prescription (giant knotweed rhizome, panax pseudo-ginseng, snakegourd fruit, and polygonum multiflorum) had a reduction in the vulnerability index (VI) in ApoE-/- mice which indicated the Chinese medicine compound prescription had potential effect on stabilizing atherosclerotic plaque. By the multivariant regression method, the optimized combination and proportion which could stabilize the atherosclerotic plaque were that the first optimized combination: giant knotweed rhizome 0.3604g, snakegourd fruit 0.3604g, polygonum multiflorum 0.1834g; the second optimized combination:giant knotweed rhizome 0.3604g, polygonum multiflorum 0.1606g, which laid a foundation of the optimized combination verificafion.更多还原