节点文献
化浊通脉方对动脉粥样硬化兔胆固醇逆向转运的影响
【作者】 王晓宁；
【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2015， 博士
【摘要】 动脉粥样硬化(AS)是导致心脑血管疾病的重要因素,目前AS引起的心血管疾病(CVD)仍居发达国家人口死亡原因之首。人民寿命的增长,人口老龄化的出现,使得AS发病率、致死率居高不下。因此,对于AS的发病原因、发病机制、预防及治疗措施一直是心血管病研究者的研究热点。AS是由多种因素共同作用引起的多系统参与疾病,作用机制复杂,故而单一作用机制的药物无法起到有效抗AS的作用。中药复方以中医理论为指导,以整体观为主导思想,着眼于宏观、综合治疗,从多角度、多层次、多靶点有效地防治AS,是目前抗AS研究的热点。“脉浊”理论的提出,丰富了中医学对AS病机的认识角度,并将中医学与现代医学相通,化浊通脉法正是针对这一证候所创立,以此为治法的中药复方在前期临床观察中取得了良好的疗效。目的：观察化浊通脉方对AS兔主动脉血管壁AS斑块形成以及对血清和主动脉血管壁细胞内脂质含量的影响,从其影响胆固醇逆向转运(RCT)过程的作用,探讨化浊通脉方抗AS的作用机制。实验方法：健康纯种雄性新西兰大耳白兔40只随机分为4组：空白对照组,模型组,辛伐他汀组,化浊通脉方组(中药复方组)。采用单纯高脂饲料喂养方式制备AS兔模型,分别在给药前及给药14周末禁食后经耳缘静脉采集血液样本,给药15周末经耳缘静脉麻醉后处死动物,取胸主动脉及肝脏样本。肉眼观察各组兔胸主动脉标本脂纹、脂斑形成情况；主动脉标本行HE染色、油红O染色,观察各组兔主动脉壁脂质沉积及斑块形成情况；酶法测定血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)水平；酶联免疫吸附法(ELISA)测定主动脉血管壁细胞内TC、游离胆固醇(FC)水平,肝脏细胞内CD36水平；蛋白印迹法(WB)测定主动脉壁内小凹蛋白-1(cav-1).亲环素A、三磷酸腺苷结合盒转运蛋白Al (ABCA1)、肝脏B类I型清道夫受体(SR-B I)蛋白表达量；实时定量聚合酶链式反应(real-time PCR)测定主动脉壁cav-1 mRNA.亲环素A mRNA、ABCA1 mRNA.肝脏SR-B I mRNA. CD36 mRNA表达量。实验结果：空白对照组兔主动脉血管壁内膜表面光滑,各层结构清晰,内皮细胞完整连续,平滑肌细胞排列整齐紧密有序,无脂质、斑块等沉积；模型组兔主动脉血管壁明显增厚,内皮层脱落,管壁满布脂质、斑块；中药复方组兔主动脉壁内膜稍有增厚,内膜下可见散在泡沫细胞沉积及脂质沉积,未见明显斑块,与辛伐他汀组无明显差异。14周末与空白对照组相比,模型组兔血清中TC、TG、LDL显著升高(P<0.01),平滑肌细胞内TC、FC明显升高(P<0.05,P<0.01)；与模型组相比,中药复方组兔血清中TC、TG、LDL明显下降(P<0.05,P<0.01),主动脉血管壁细胞内TC、FC显著升高(P<0.01)。15周末主动脉细胞测定结果显示：与空白对照组相比,模型组兔cav-1蛋白及mRNA表达量显著下降(P<0.01)；与模型组相比,中药复方组cav-1 mRNA表达量显著增加(P<0.01),cav-1蛋白表达量也有增加趋势,但差异无统计学意义(P>0.05)。与空白对照组相比,模型组兔亲环素A蛋白表达量明显增加(P<0.05),亲环素A mRNA表达量有下降趋势,但差异无统计学意义(P>0.05)；与模型组相比,中药复方组亲环素A蛋白表达量显著下降(P<0.01),亲环素A mRNA表达量显著增加(P<0.01)。与空白对照组相比,模型组兔ABCA1蛋白表达量有下降趋势,但差异无统计学意义(P>0.05),ABCA1 mRNA表达量显著增加(P<0.01)；与模型组相比,中药复方组ABCA1蛋白表达量有增加趋势,但差异无统计学意义P>0.05), ABCA1 mRNA表达量显著下降(P<0.01)。肝细胞测定结果显示：与空白对照组相比,模型组兔SR-B I蛋白及mRNA表达量有下降趋势,但差异无统计学意义(P>0.05)；与模型组相比,中药复方组SR-B I mRNA表达量有增加趋势,但差异无统计学意义(P>0.05)。与空白对照组相比,模型组兔CD36蛋白表达量有增加趋势,但差异无统计学意义P>0.05), CD36 mRNA表达量明显增加(P<0.05)；与模型组相比,中药复方组CD36蛋白表达量有减少趋势,但差异无统计学意义(P>0.05)。结论：1.化浊通脉方能够抑制AS兔主动脉血管内膜增生,减少内皮下泡沫细胞的形成及脂质沉积,从而减少AS斑块形成。2.化浊通脉方可有效降低AS兔血清TC、TG、LDL水平,显著降低AS兔主动脉血管壁细胞内TC、FC含量。3.化浊通脉方能够通过提高cav-1蛋白及基因水平、亲环素A的基因水平,促进细胞内胆固醇转运复合体生成,对ABCA1蛋白表达量有上调作用趋势,促进细胞内胆固醇流出,保障cav-1、亲环素A的正常表达,减少斑块中亲环素A的过度表达,最终达到抗AS的作用。4.化浊通脉方能够通过对肝脏SR-B I基因水平有一定的上调作用趋势、对CD36蛋白表达有一定的抑制作用趋势,可能促进血中胆固醇被肝脏选择性摄取、代谢,使脂质顺利代谢,最终达到抗AS的作用。
【Abstract】 Atherosclerosis(AS) is an important factor which leads to cardiovascular and cerebrovascular diseases ranking first among all causes of death in developed countries. The growth of people’s lifetime and the aging of population lead to AS with high morbidity and mortality rate. Therefore, it becomes a hot area of research about cause and mechanism, prevention and treatment measures of AS that medical investigator need face and solve. AS is a complex multifactorial pathological process, therefore a single mechanism of action of the drug probably cannot fully inhibit the development of AS. The Chinese compound is based on traditional Chinese medicine theory, focusing on the macro level, comprehensive treatment, overall adjustment in theory, from multi-angle, multi-level and multi-target preventing and treating AS effectively. The presentation of "Pulse turbidity" theory innovate the comprehension of AS pathogenesis and connect with traditional Chinese medicine and modern medicine. Resolving turbidity and dredging collaterals method is established for this syndrome, which has received a good clinical efficacy.Objective:To observe the effects of Huazhuotongmai compound on plaques on arterial wall and lipid in serum and aorta vascular smooth muscle cell of rabbit models of atherosclerosis and investigate the mechanisms of Huazhuotongmai compound for against AS on reverse cholesterol transport(RCT).Experimental methods:There were 40 healthy purebred male New Zealand rabbits divided into four groups randomly:blank control group, model group, simvastatin group, and Huazhuotongmai group. The rabbits of AS were established by simple high-fat feeding method. The blood samples were collected from ear marginal veins before and after drug administration for 14 weeks after abrosia. The thoracic aortas and livers of the animals were taken after execution under anesthesia at the end of 15 weeks. The rabbit fatty streaks and fatty plaques of gross specimen were inspected by visual study. The formation of rabbit arterial plaque was observed by HE staining. The lipidosis in arterial wall was viewed by oil red o staining. The levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotcin(LDL), high-density lipoprotein(HDL) in serum were measured by enzymic method. The levels of TC and free cholesterol(FC) in vascular smooth muscle cell and CD36 in livers were measured by enzyme-linked immuno sorbent assay(ELISA). The expressions of caveolin-1(cav-1), cyclophilin A(CyPA), ATP-binding cassette transporters A1(ABCA1) in arterial wall and scavenger receptor claBs B type I (SR-B I) in liver were measured by western blot(WB). The expressions of mRNA of cav-1, CyPA and ABCA1 in arterial wall, mRNA of SR-B I and CD36 in liver were evaluated by real-time quantitative polymerase chain reaction(real-time PCR).Results:The surface of arterial wall in blank control group is smooth, the layers of structure are clear and the endothelial cells and the smooth muscle cells are continuous without lipidosis. The arterial wall in model group is obviously thickened and covered with lipid plaques obviously. The arterial wall in Huazhuotongmai group is slightly thickened with a little lipidosis. There are no differences between Huazhuotongmai group and simvastatin group. At the end of 14 weeks, TC, TG and LDL in serum in model group are significantly higher than those in blank control group(P<0.01). TC and FC in smooth muscle cells in model group are higher than that in blank control group(P<0.05,P<0.01). TC, TG and LDL in serum in Huazhuotongmai group are lower than those in model group(P<0.05,P<0.01). TC and FC in smooth muscle cells in Huazhuotongmai group are significantly lower than those in model group(P<0.01). After 15 weeks the test results of aorta cells show that compared with blank control group, the expression of cav-1 and cav-1 mRNA in model group are significantly declining(P<0.01). Compared with model group, the expression of cav-1 mRNA in Huazhuotongmai groups is significantly increasing(P<0.01), the expression of cav-1 presents a trend of increasing, which shows no statistical significance(P>0.05). Compared with blank control group, the expression of CyPA in model group is increasiong(P<0.05), the expression of CyPA mRNA presents a trend of declining, which shows no statistical significance (P>0.05). Compared with model group, the expression of CyPA is significantly declining(P<0.01), the expression of CyPA mRNA is significantly increasing(P<0.01) in Huazhuotongmai group.Compared with blank control group, the expression of ABCA1 mRNA in model group is significantly increasing(P<0.01), the expression of ABCA1 presents a trend of declining, which shows no statistical significance (P>0.05). Compared with model group, the expression of ABCA1 mRNA in Huazhuotongmai group is significantly declining(P<0.01), the expression of ABCA1 presents a trend of increasing, which shows no statistical significance (P>0.05).The test results of liver cells show that the expression of SR-B I and SR-B I mRNA in model group declines slightly than those in blank control group, which shows no statistical significance(P>0.05). And compared with model group, the expression of SR-B I mRNA in Huazhuotongmai group presents an upward trend, which shows no statistical significance(P>0.05). Compared with blank control group, the expression of CD36 mRNA in model group is increasing(P<0.05), the expression of CD36 presents an upward trend, which shows no statistical significance(P>0.05). Compared with model group, the expression of CD36 in Huazhuotongmai group presents a trend of declining, which shows no statistical significance (P>0.05).Conclusions:1. Huazhuotongmai compound can reduce lipidosis in artery wall cells and inhibit the plaque formation in artery of rabbits with AS.2. Huazhuotongmai compound can reduce the contents of TC, TG and LDL in serum, TC and FC in artery cells.3. Huazhuotongmai compound can promote the expression of cav-1, cav-1 mRNA, CyPA mRNA and ABCA1, increase transmembrane transport of cholesterol to promote cellular cholesterol efflux, ensure the normal expression of cav-1 and CyPA and reduce the over expression of CyPA, which has an effect on anti-AS.4. Huazhuotongmai compound can inhibit the expression of CD36, promote the expression of SR-B I mRNA and cholesterol metabolism in liver, which has an effect on anti-AS.