【作者】 黄梦雨；

【导师】 罗陆一；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2010， 博士

【摘要】 动脉粥样硬化(atherosclerosis, AS)是严重危害人类健康的常见病,常累及心脑血管等重要部位,有极高发病率和死亡率。因此防治动脉粥样硬化,改善患者的生活质量,减少心脑血管事件的发生率和病死率,成为亟待解决的医学难题之一本课题从文献研究、临床研究和实验研究三方面,通过对临床颈动脉粥样硬化患者观察及人脐静脉内皮细胞培养,对AS的发病机制进行分析,并对补肾活血化痰方抗AS的作用及炎症方面的作用机制进行探讨。一、文献研究中国古代医籍中没有动脉粥样硬化的病名,多将其归属于“胸痹”、“真心痛”、“中风”、“眩晕”、“头痛”、“痴呆”、“痰饮”、“瘀证”等病症。若机体脏腑功能失调,脾胃运化功能减弱,肾气渐衰,加之饮食肥甘厚味,气机失畅,易化生为痰浊,痰浊流滞于血脉日久则成痰瘀交结之证,正虚邪实相互影响,致使病变不断发展,多位学者多认为“痰浊”、“瘀血”为本病发病之关键。罗陆一教授认为,肾为人体之根本,主一身气血运行,肾虚气化无力则津聚成痰,温煦运化不足,血脉失于温运,血运无力,留而为瘀,故认为动脉粥样硬化属本虚标实之证,本虚则之于肾虚,标实在于痰瘀互结。动脉粥样硬化是一个慢性炎症过程,细胞黏附分子在动脉粥样硬化的形成和进展中起关键作用。在动脉粥样硬化形成过程中,首先是血管内皮在各种致炎刺激因子下损伤,细胞间黏附分子-1(Intercellular adhesion molecule, ICAM-1)、血管细胞黏附分子-1(Vascular cell adhesion molecule, VC AM-1)等表达增加,调节白细胞的粘附,单核细胞穿越内皮摄取氧化型低密度脂蛋白(ox-LDL),变为激活的巨噬细胞,吞噬ox-LDL成为泡沫细胞,引起巨噬细胞的清除反应和血管平滑肌细胞的增殖,从而形成动脉粥样斑块。白细胞与血管内皮细胞黏附是炎症反应的起始步骤,介导白细胞与内皮细胞相互作用的分子基础就是细胞黏附分子。二、临床研究目的：观察补肾活血化痰方对颈动脉粥样硬化患者症状积分,颈动脉内膜中层厚度、斑块Crouse积分、血脂、血清可溶性细胞黏附分子水平的影响,探讨补肾活血化痰方抗AS的作用及其作用机制。方法：选择颈动脉粥样硬化(carotid atherosclerosis, CAS)患者100例,随机分为2组,补肾活血化痰方(Bushenhuoxuehuatan Formula, BF)组50例和阿托伐他汀(Atorvastatin Calcium, AC)组50例,分别用补肾活血化痰方和阿托伐他汀钙进行治疗6个月,对两组CAS患者治疗前后的症状、颈动脉IMT、斑块Crouse积分、血脂水平、血清sIC AM-1和sVCAM-1进行检测。结果：(1)治疗后BF组与AC组均可以明显降低症状积分(P<0.01,P<0.05),两组相比差异有显著性(P<0.05),BF组在改善症状方面优于AC组；(2)治疗后BF组与AC组均能明显降低双侧颈动脉内膜中层厚度(P<0.01,P<0.01),两组差异无显著性；(3)治疗后BF组与AC组均能显著降低颈动脉粥样硬化斑块Crouse积分(P<0.01,P<0.05)；(4)治疗后BF组与AC组均可以明显降低CAS血清中的TC(P<0.05,P<0.01)、TG(P<0.05,P<0.05)水平,BF组可以明显降低LDL-C(P<0.05),升高HDL-C(P<0.01),BF组与AC组相比在升高HDL-C方面差异有显著性(P<0.05)；(5)治疗后BF组与AC组可以明显降低CAS血清中sICAM-1 (P<0.01, P<0.01)、sVCAM-1 (P<0.01, P<0.01)水平。结论：补肾活血化痰方可以改善颈动脉粥样硬化患者症状,使症状积分明显降低,降低颈动脉内膜中层厚度,降低斑块Crouse积分,明显降低CAS患者血清中的TC、TG、LDL-C水平,升高HDL-C,明显降低CAS患者血清中sIC AM-1、sVCAM-1,补肾活血化痰方可能通过调节血脂水平、减少血清可溶性细胞黏附分子水平来发挥抗AS的作用。三、实验研究目的：观察补肾活血化痰方对TNF-α诱导人脐静脉内皮细胞(HUVEC)存活率、ICAM-1、VCAM-1、NF-κB表达的影响。方法：通过SD大鼠制备补肾活血化痰方血清,0.25%胰蛋白酶分离原代HUVEC,传代后将HUVEC分为五组：①对照组：HUVEC+DMEM培养基+10%对照组血清；②TNF-α组：HUVEC+DMEM培养基+10%对照组血清+10ng/mL TNF-a;③补肾活血化痰方高剂量组：HUVEC+DMEM培养基+10%补肾活血化痰方高剂量组血清+10ng/mL TNF-a;④补肾活血化痰方中剂量组：HUVEC+DMEM培养基+10%补肾活血化痰方中剂量组血清+10ng/mL TNF-a;⑤补肾活血化痰方低剂量组：HUVEC+DMEM培养基+10%补肾活血化痰方低剂量组血清+10ng/mL TNF-a。采用CCK-8法检测HUVEC的存活率,ELISA法检测细胞ICAM-1和VCAM-1的表达,免疫细胞化学方法观察细胞NF-κB表达,半定量逆转录聚合酶链反应(RT-PCR)法检测细胞ICAM-1 mRNA和VCAM-1 mRNA表达。结果：(1)补肾活血化痰方高、中、低剂量组细胞存活率分别为87.2%、82%、80%,TNF-α组细胞存活率为74.2%,各组细胞存活率与对照相比均降低,TNF-a组的细胞存活率最低；(2)与对照组相比TNF-α组细胞ICAM-1和VCAM-1的表达明显增高(P<0.01,P<0.01),补肾活血化痰方不同剂量组均能抑制TNF-α诱导的内皮细胞ICAM-1和VCAM-1的表达,其中BF高剂量组与TNF-α组相比ICAM-1和VCAM-1的表达明显减少(P<0.01,P<0.01),BF中剂量组与TNF-a组相比ICAM-1的表达明显减少(P<0.05)；(3)TNF-α组可见细胞胞浆中有大量棕黄色颗粒,部分胞核深染,NF-κB表达阳性,补肾活血化痰方各剂量组NF-κB阳性表达信号均弱于TNF-a组,尤其以高、中剂量组明显；(4)与对照组相比TNF-a组中内皮细胞ICAM-1mRNA、VCAM-1 mRNA表达明显增高(P<0.01,P<0.01),补肾活血化痰方高、中剂量组均能下调TNF-α诱导的内皮细胞ICAM-1 mRNA表达(P<0.01,P<0.01),补肾活血化痰方高剂量组能下调TNF-a诱导的内皮细胞VCAM-1 mRNA表达(P<0.05)。结论：补肾活血化痰方对TNF-a诱导人脐静脉内皮细胞的炎症反应有抑制作用,能抑制TNF-a诱导的内皮细胞ICAM-1、VCAM-1及NF-κB的表达,以高剂量组明显。这可能是补肾活血化痰方抗AS的作用机制之一。更多还原

【Abstract】 Atherosclerosis (AS) is a commen disease which causing serious harm to human health, often involving the major organ, such as heart, brain and blood vessel. It has very high morbidity and mortality, therefore prevention and treatment of atherosclerosis and improving the quality of life of patients and reducing the incidence of cardiovascular events and mortality become one of medical problems urgently needed to be solved.This dissertation includes three parts:literature research, clinical and experiment research. According to the observation to carotid atherosclerosis patients and human umbilical vein endothelial cells, the pathogenesis of AS is analysed, furthermore the effect of bushenhuoxuehuatan formula against AS and the inflammatory mechanism are discussed.Part one: literature researchThere have not the name of atherosclerosis in the ancient Chinese medical record, and generally atherosclerosis was called thoracic obstruction, angina pectoris, stroke, dizziness, headache, dementia, phlegm-fluid retention, blood stasis syndrome, etc. Zang-fu organ dysfunction, hypofunction of transportation and transformation of spleen and stomach, kidney-qi deficiency and excessive consumption of fatty and sweet foods may lead to disorder of qi activity and phlegm stagnation which gradually coming into being blood stasis. It is called syndrome of binding of phlegm and blood stasis. Many scholars believe the principal pathogenesis is phlegm stagnation and blood stasis. Professor Luo luoyi thinks that the kidney is considered as the foundation of human body and dominates the movement of qi and blood all over the body. If phlegm stagnation resulted from deficiency of kidney and disorder of qi activity, lack of warmth, transportation and transformation, blood flow slowly, stay and for blood stasis. So Professor Luo believe atherosclerosis is the syndrome of root deficiency and branch excess, the root is deficiency of kidney while the branch is binding of phlegm and blood stasis.Atherosclerosis is a chronic inflammatory disease in which cell adhesion molecules play a key role. In the early process of atherogenesis, first the endothelial cell is injured by many inflammatory factors and expression of cell adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 lead to increased adherence of leukocyte. Monocytes may migrate through the endothelium and situate themselves within the subendothelial layer, engulf oxidized low-density lipoprotein, then transform in activated macrophages which continuously accumulate xidized low-density lipoprotein and become foam cells. At the same time, necrotic macrophages and proliferative smooth muscle cells result in atherosclerotic plaque. Adherence of leukocyte with endothelial cells is the initiation of atherosclerosis, whose molecular foundation is cell adhesion molecules.Part two:clinical researchObjective:To investigate the effect of Bushenhuoxuehuatan Formula on the clinical symptom score, carotid intima-media thickness, Crouse score in atherosclerotic plaque, blood lipids and the level of soluble cell adhesion molecules and explore the effect and its mechanism of Bushenhuoxuehuatan Formula on atherosclerosis.Method: 100 carotid atherosclerosis (CAS) patients were ramdomly indived into 2 groups:Bushenhuoxuehuatan Formula (BF) group and Atorvastatin Calcium (AC) group, 50 patients in each group. After two-month treatment with Bushenhuoxuehuatan Formula and Atorvastatin Calcium respectively, the clinical symptom score, carotid intima-media thickness, Crouse score in atherosclerotic plaque, blood lipids and the level of soluble cell adhesion molecules were observed before and after treatment.Results:(1) After treatment the clinical symptom score was obviously decreased in BF group and AC group (P<0.01, P<0.05). There was a significant difference between two groups (P<0.05), and compaired with AC group, BF group has better effect in improving symptom. (2) Bilateral carotid intima-media thickness was obviously decreased in BF group and AC group (P<0.01, P<0.01), and there was no significant difference between the two groups. (3) Crouse score in atherosclerotic plaque was markedly reduced in BF group and AC group (P<0.01, P<0.05). (4) The serum level of TC (P<0.05, P<0.01) and TG (P<0.05, P<0.05) in CAS patients was obviously decreased in BF group and AC group. The level of LDL-C (P<0.05) was decreased while the level of HDL-C (P<0.01) was increased in the BF group. There was significant difference in increasing HDL-C between two groups (P<0.05). (5) The serum level of soluble intercellular adhesion molecule-1 (sICAM-1) (P<0.01, P<0.01) and soluble vascular cell adhesion molecule-1 (sVCAM-1) (P<0.01, P<0.01) in CAS patients was obviously decreased in BF group and AC group.Conclusions:Bushenhuoxuehuatan Formula can be improve symptom, decrease the clinical symptom score, decrease carotid intima-media thickness, reduce Crouse score in atherosclerotic plaque, decrease the level of TC, TG, LDL-C, increase the level of HDL-C, decrease the level of sICAM-1 and sVCAM-1 in CAS patients. The effect of Bushenhuoxuehuatan Formula against AS might be related with its regulating the blood lipids and decreasing the serum level of soluble cell adhesion molecules.Part three: experimental researchObjective:To observe the effect of Bushenhuoxuehuatan Formula on the cell livability, the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and nuclear factor-kappa B (NF-κB) in human umbilical vein endothelial cells induced by tumor necrosis factor-a (TNF-a).Method: The blank serum and Bushenhuoxuehuatan Formula serum was prepared respectively by using SD rats. The primarily HUVEC isolated by 0.25% Trypsin-EDTA, then passaged, was divided into 5 groups:①Control group:HUVEC+DMEM culture medium+10% blank serum,②TNF-a group:HUVEC+DMEM culture medium +10% blank serum+10ng/mL TNF-a,③High dose BF group:HUVEC+DMEM culture medium +10% high dose BF serum+10ng/mL TNF-a,④Medium dose BF group:HUVEC+DMEM culture medium+10% medium dose BF serum+10ng/mL TNF-a,⑤Low dose BF group: HUVEC+DMEM culture medium+10% low dose BF serum+10ng/mL TNF-a. The cell livability was detected by using cell counting (CCK-8) colorimetric analysis. ICAM-1 and VCAM-1 on cell surfaces were quantified by enzyme linked immuonosorbent assays (ELISA). The expression of NF-κB was detected by immunocytochemistry analysis. The expression of ICAM-1 mRNA and VCAM-1 mRNA were examined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).Results:(1) The cell livability was 87.2%,82% and 80% in High, medium, low dose BF group respectively,74.2% in the TNF-a group. Compared with the control group, the cell livability was decreased in other four groups, especially in the TNF-a group. (2) The expression of ICAM-1 and VCAM-1 on cell surfaces were obviously increased in the TNF-a group (P<0.01, P<0.01), compared with the control group. All doses’ Bushenhuoxuehuatan Formula could inhibit the increasing expression of ICAM-1 and VCAM-1 induced by TNF-a. Compared with TNF-a group, the expression of ICAM-1 and VCAM-1 were obviously decreased in high dose BF group (P<0.01, P<0.01), and the expression of ICAM-1 was obviously decreased in medium dose BF group (P<0.05). (3) A great deal of brown-yellow particles in the cytolymph and a small quantity of deep brown particles in the cytoplasm were found in endothelial cells of TNF-a group which showed NF-κB expression was positive. Campared with TNF-a group, the positive expression of NF-κB was significantly decreased in all doses’BF group, especially high and medium dose BF group. (4) Campared with TNF-a group, the mRNA level of ICAM-1 and VCAM-1 in HUVEC was obviously up-regulated (P<0.01, P<0.01). However, high and medium dose’ Bushenhuoxuehuatan Formula could down-regulate the expression of ICAM-1 mRNA (P<0.01, P<0.01), while only high dose’Bushenhuoxuehuatan Formula could down-regulate the expression of VCAM-1 mRNA (P<0.05).Conclusions:Bushenhuoxuehuatan Formula could mediate the inflammatory response of HUVEC induced by TNF-a and inhibit the expression of ICAM-1, VCAM-1 and NF-κB in endothelial cells, more significant effect in the high dose BF group. It might be the one of mechanism of Bushenhuoxuehuatan Formula against AS.更多还原