【作者】 骆庆峰；

【导师】 孙兰；

【作者基本信息】 中国协和医科大学 ， 药理学， 2008， 博士

【摘要】 动脉粥样硬化(atherosclerosis,AS)是心脑血管疾病最重要的发病因素。在动脉粥样硬化的诱发因子中,高脂血症是一个主要的独立危险因素。所以从药用植物中寻找具有降血脂作用的天然化合物,并研究其作用机理是近年来的研究热点,具有重大的应用前景和社会效益。在对高脂血脂诱发动脉粥样硬化的研究中发现,低密度脂蛋白胆固醇(low density lipoprotein-cholestero,LDL-C)是主要的致病因子之一。近期的研究表明胆固醇的逆向转运过程(reverse cholesterol transport,RCT)在改善血脂代谢,防治动脉粥样硬化中的作用越来越重要。RCT过程能够促进外周组织胆固醇的清除,降低体内胆固醇水平,延缓动脉粥样硬化的发生和发展。本课题组在既往的研究中发现,豆类植物木豆的叶子提取物能明显降低动物的体重和血糖水平。木豆叶总提取物中主要包含芪类成分和黄酮类成分,可用于治疗妇女更年期综合症、防治骨质疏松、降低血糖。但是,关于木豆叶提取物调节胆固醇代谢的研究目前还没有报道。本课题在国内外首次以木豆叶提取物为干预药物,观察芪类成分及其合成类似物S03、黄酮类成分调节体内胆固醇代谢,促进RCT过程的机理,并探讨它们抗动脉粥样硬化形成的潜在作用。1.木豆叶提取物及其合成类似物S03对高脂血症模型动物血脂代谢的影响首先通过手术去除动物卵巢的方式建立高脂血症模型模拟绝经后女性血脂变化,初步了解木豆叶总提取物对该模型的治疗作用。研究发现木豆叶总提取物具有降低动物血脂和血糖水平的趋势。为进一步明确木豆叶总提取物中具有调节胆固醇代谢作用的有效成分,通过喂食高脂饲料的方式建立高脂血症动物模型,分别给予木豆叶芪类成分和黄酮类成分,观察两类成分调节血脂的效应和作用机理。研究结果表明黄酮类成分对于血脂代谢无明显影响。而木豆叶芪类提取物却具有明显的血脂调节功能,能够减轻动物体重,显著降低高脂模型小鼠血清总胆固醇(TC)、甘油三酯(TG)和LDL-C水平,提高血清超氧化物歧化酶(SOD)的活力。同时芪类成分还可以明显减少肝脏中TC和TG的蓄积。为探讨芪类物质的作用机制,选择肝脏中涉及胆固醇合成、吸收和代谢的三个关键蛋白-HMG-CoA R、LDL-R和CYP7A1作为观察对象,以RT-PCR的方法检测其表达水平。结果表明,芪类成分可以上调这三者的基因表达,通过促进LDL-C的吸收和增加胆固醇向胆汁酸转化来降低体内胆固醇水平。同时,在实验中还观察到芪类成分的合成类似物S03可以改善去卵巢高脂血症模型动物的血脂代谢。去卵巢手术后动物体重和血清胆固醇含量异常升高,S03可以明显减轻去卵巢动物体重和肝脏重量,减少动物血清中TC含量和LDL-C含量。此外,S03还可以降低动物体内卵泡刺激素(FSH)和促黄体生成素(LH)的浓度,纠正去卵巢后的性激素分泌失调。2.木豆叶芪类单体成分C03对胆固醇逆向转运过程的影响根据前一个实验结果,选择芪类成分中的单体成分C03作为观察药物,了解其对RCT过程的影响。巨噬细胞中胆固醇通过ABCA1流出是RCT过程的起始步骤。同时,由巨噬细胞大量摄取氧化低密度脂蛋白转化成的泡沫细胞,也是动脉粥样硬化斑块的重要组成部分。肝脏细胞中含有的LDL-R和SR-BⅠ介导了RCT过程的终止步骤。本实验确定上述在RCT过程起始和终止阶段的重要功能蛋白作为观察对象,检测C03对胆固醇逆向转运过程中ABCA1、LDL-R和SR-BⅠ蛋白功能和表达的影响,以及C03抑制巨噬细胞内胆固醇蓄积,减少泡沫细胞形成的效应。结果显示C03可以上调巨噬细胞中ABCA1的表达,促进巨噬细胞内[~3H]胆固醇的流出,有助于启动RCT过程。同时还发现C03可以抑制巨噬细胞对氧化低密度脂蛋白的吞噬摄取,降低细胞内的总胆固醇含量和胆固醇酯含量,进而抑制泡沫细胞的形成。C03能够显著上调肝脏细胞中LDL-R的表达,且该作用具有剂量依赖性和时间依赖性。对于SR-BⅠ的表达,C03的作用并不明显。研究表明C03不但可以促进RCT过程,而且具有防治动脉粥样硬化脂质斑块形成的功能,有益于减缓动脉粥样硬化的病理改变。此外,根据已有的实验结果,采用受体结合实验来了解C03与肝脏雌激素受体的结合特性,观察C03与雌激素受体结合的有关常数。为深入探讨C03的作用机制奠定基础。3.木豆叶芪类成分C03及其合成类似物S03血管保护作用的初步研究氧化损伤是血管内皮细胞损伤的重要因素之一,而内皮细胞的损伤和功能改变是动脉粥样硬化的始动因素。本部分实验采用过氧化氢损伤人脐静脉血管内皮细胞株(EAhy926细胞)建立内皮细胞过氧化损伤模型,观察C03对过氧化氢损伤的拮抗作用,同时了解C03改善血管内皮细胞功能的效应。结果表明C03可以降低过氧化氢对EAhy926细胞的损伤,提高细胞的存活率。C03抗氧化、保护血管内皮细胞的作用可能是通过提高细胞内超氧化物歧化酶的活性,同时增加细胞的NO分泌量来达到的。在动脉粥样硬化形成过程中,动脉内皮发生损伤诱导血管平滑肌细胞增殖,参于炎症反应和斑块纤维帽的形成。本实验通过将C03和S03与血管平滑肌细胞共同培养发现,S03能够剂量依赖性抑制血管平滑肌细胞的增殖,而C03的抑制作用相对较弱,可能具有减弱血管壁增厚的潜能。综上所述,本课题建立了两种不同的高脂血症动物模型,分别验证了木豆叶芪类成分及其合成类似物S03改善高脂血症的疗效,并探讨了芪类成分降低体内胆固醇含量的作用机理。然后通过体外细胞实验,进一步探讨芪类成分C03对胆固醇逆向转运过程的干预效果和减缓泡沫细胞形成的作用。同时,对于C03抗氧化、保护血管内皮细胞,以及与雌激素受体的结合特性也做了一定程度的研究。本课题在国内外首次从体内体外两个方面采用不同的模型观察木豆叶芪类成分干预胆固醇代谢的作用和血管保护功能,并明确其产生治疗效应的初步机制。研究结果为发现新型调节胆固醇代谢的药物提供了思路和选择,也将为临床治疗动脉粥样硬化等心脑血管疾病带来新的手段。更多还原

【Abstract】 Atherosclerosis is the basis of coronary artery disease, one of the most serious diseases in man. Hypercholesterolemia is a dominant risk factor for the development and progression of increasing atherosclerosis and related cardiovascular diseases. Thus, the therapeutic benefits of plant extracts without side effects have been the focus of many extensive studies. Plenty of studies indicate that high serum level of low density lipoprotein cholesterol （LDL-C） is associated with atherosclerosis and an increased risk of coronary heart disease.Recently, there are several investigates reveale that reverse cholesterol transport （RCT） is one key mechanism in antiatherosclerosis research. Atheroprotective effect of RCT is attributed to its ability to remove macrophage or peripheral organs redundant cholesterol into liver for degradation and eccrisis. The RCT accelerates the cholesterol cleaning in peripheral organs, decreases the serum cholesterol contents, so it is benefit to the prevention and cure of atherosclerosis.Cajanus cajan L.Millsp is the subtropical brush plant, which belongs to adjouns genus of popilionaccae family, grows in the southern part of China. There are two active components in the Cajanus cajan L, i.e. the flavonoids and the stilbenes. The stilbenes component possesses the estrogen-like property, it can been used to treat the osteoporosis, diabetes, climacteric symptom of menopause women. In our previous researches, we find the stilbenes containing extract-fraction from Cajanus cajan L （sECC） could decrease the body weight and blood sugar level. But there has been no report on the effect of sECC on cholesterol metabolism.Our research team initiately investigated the hypocholesterolemic effect of medicative extract-fraction from Cajanus cajan L. and synthetical analog （S03） on hypercholesterolemia animal models. We observed the hypocholesterolemic effects of flavonoids component and stilbenes component, respectively. Furtherly, we investigated the auxoaction of stilbenes component on reverse cholesterol transport. Based on the experimental evidences, we evaluated the potential antiatherosclerosis effectes of these compoundes. These resultes will help us to extend the drug actions of medicative extract-fraction from Cajanus cajan L in modify cholesterol metabolism and preservation atherosclerosis.1. The hypolipidemic effects of medicative extracts of Cajanus cajan L and synthetical analog on hypercholesterolemia animal modelOvariectomy induces obesity and hypercholesterolemia by removing the catabolic action of estrogen. We simulated the hypercholesterolemia in post- menopause women with the ovariectomized mice. We finded the descending tendency in the serum cholesterol and glucose content according with the dosage of the medicative extract-fraction from Cajanus cajan L.Diet-induced hypercholesterolemia mice had been used for the assessment of flavonoids extract-fraction or stilbenes extract-fraction with beneficial effects on cholesterol metabolism. The serum lipid profile, the hepatic total cholesterol and triglyceride, and the changes in the gene expressions of the key enzymes responsible for cholesterol homeostasis had been detected. After 4 weeks treatment, the increased serum and hepatic total cholesterol and triglyceride levels were markedly attenuated by sECC, the serum LDL cholesterol content also decreased accompanying with the activity of serum superoxide dismutase was increased in sECC treated mice. The mRNA expressions of HMG-CoA reductase, CYP7A1, and LDL-receptor were significantly enhanced in the mice administered with sECC. sECC’s hypocholesterolemic effect may involved the enhancement of hepatic LDL-receptor and CYP7A1 mRNA expression levels. Whereas, there was no conspicuous change occurred in flavonoids component groups. In the hypercholesterolemia of the ovariectomized rats, we investigate the effects of the synthetical analog of stilbenes component （S03）. The results revealed the S03 decreased the serum total cholesterol and LDL cholesterol contents. Meanwhile, there were marked reduces in the body weight and liver weight in the S03 treated rats. The S03 also decreased the abnormal contents of luteotrophic hormone and follicle-stimulating hormone induced by ovariectomized operation.2. The effect of monomer compound of sECC （C03） on the reverse cholesterol transportWe investigated the effect of monomer compound of sECC （C03） on the reverse cholesterol transport. The usage of C03 could up-regulation the protein expression of the ATP binding cassette transporter A1, following facilitate the [³H] label cholesterol effluent from the macrophage. C03 also prevented the formation of foam cell by depleted the total cholesterol and cholesterol esters in the macrophage.The investigations data indicated that C03 could enhance the protein expression of LDL-receptor in liver cell, this effect displayed the characteristic of dose dependent and time dependence. But, there was no conspicuous change occurred in the expression of scavenger receptor class B type I. So we conclude the C03 may promote the reverse cholesterol transport mainly through accelerating the initial phase.We have evaluated the binding property of C03 and the estrogen receptor with the competive receptor binding assay.3. The protective effects of C03 and S03 on cultured vascular endothelial cell and vascular smooth muscle cellOxidative damage is an important risk factor for the development and progression of vascular dysfunction. Then, vascular dysfunction is the initiating agent of atherosclerosis. We used an injury model induced by H₂O₂ in EAhy926 cells （human umbilical vein endothelial cell） and assessed the protective effect of C03. The injuries of endothelial cell induced by H₂O₂ were indicated as cell viability, release of NO and the activity of SOD. The results exhibited that C03 could significantly increase cell viability, the increased activity of SOD and secretion of NO also been observed.Proliferation of vascular smooth muscle cell （VSMC） represents an essential event in the development of atherosclerosis. The S03 represented significant inhibitory effect on VSMC proliferation. However, C03 has showed lower inhibition on it. These findings suggested that S03 and C03 may arrest the atherosclerosis via the inhibitory effect on VSMC proliferation.In a summary, we used different hyperlipoidemia animal models to evaluated the hypolipidemic effect of medicative extract-fraction from Cajanus cajan L and it’s synthetical analog. Then, we investigate the accelerating effect of C03 on the reverse cholesterol transport and the effects of C03 and S03 on cultured vascular endothelial cell and vascular smooth muscle cell protection. These results indicated that sECC will be a promising drug for the hypercholesterolemia and atherosclerosis.更多还原