化浊通脉方对动脉粥样硬化兔胆固醇逆向转运的影响

发布时间：2018-05-04 09:48

本文选题：动脉粥样硬化 + 胆固醇逆向转运　；参考：《北京中医药大学》2015年博士论文

【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(P0.01),平滑肌细胞内TC、FC明显升高(P0.05,P0.01)；与模型组相比,中药复方组兔血清中TC、TG、LDL明显下降(P0.05,P0.01),主动脉血管壁细胞内TC、FC显著升高(P0.01)。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(P0.01)；与模型组相比,中药复方组cav-1 mRNA表达量显著增加(P0.01),cav-1蛋白表达量也有增加趋势,但差异无统计学意义(P0.05)。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(P0.05),亲环素A mRNA表达量有下降趋势,但差异无统计学意义(P0.05)；与模型组相比,中药复方组亲环素A蛋白表达量显著下降(P0.01),亲环素A mRNA表达量显著增加(P0.01)。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(P0.05),ABCA1 mRNA表达量显著增加(P0.01)；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P0.05), ABCA1 mRNA表达量显著下降(P0.01)。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(P0.05)；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(P0.05)。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P0.05), CD36 mRNA表达量明显增加(P0.05)；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(P0.05)。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 leading to cardiovascular and cerebrovascular diseases. At present, the cardiovascular disease (CVD) caused by AS is still the first cause of death in the developed countries. The growth of the people's life and the emergence of the aging population make the incidence of AS and death rate high. Therefore, the causes, pathogenesis, prevention and treatment measures for the pathogenesis of AS are made. The research hotspot of cardiovascular disease researchers.AS is a multi system involved in disease caused by a variety of factors. The mechanism is complex, so the single action mechanism can not play an effective anti AS effect. Chinese medicine compound is guided by the theory of traditional Chinese medicine, guided by the concept of holism, focusing on macroscopical, comprehensive treatment, from multi angle. Degree, multi-level and multi target effectively prevent and cure AS, is the hot spot of anti AS research at present. "Pulse turbid" theory has enriched the understanding angle of Chinese medicine to AS pathogenesis, and interlinked the traditional Chinese medicine with modern medicine. Objective: To observe the effect of Hua Zhuo Tong Mai Recipe on the formation of AS plaque in the aorta wall of AS rabbit and the lipid content in the blood serum and aorta wall cells, and to explore the mechanism of anti AS in the process of RCT. The method of actual test: healthy pure breed male New Zealand white rabbit 40 Only 4 groups were randomly divided into 4 groups: blank control group, model group, simvastatin group, turbid Tongmai prescription group (Chinese medicine compound group). The rabbit model was prepared by feeding simple high fat diet. The blood samples were collected before and after the 14 weekend fasting, and the animals were killed after the ear vein anesthesia at the end of the 15 week and the thoracic aorta was taken for the 15 week. The lipid profiles and formation of fat spots in the thoracic aorta of rabbits were observed by the naked eye. The lipid deposition and plaque formation of aorta wall in each group were observed by HE staining and oil red O staining. The serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) were measured by enzyme method. Enzyme-linked immunosorbent assay (ELISA) was used to determine the intracellular TC, free cholesterol (FC) level, CD36 level in the liver cells, and Western blot (WB) for the determination of -1 (Cav-1) in the aortic wall. The expression of cyclophilin A, adenosine triphosphate binding cassette transporter Al (ABCA1), and liver B I type scavenger receptor (Cav-1) protein expression; The quantitative polymerase chain reaction (real-time PCR) was used to determine the Cav-1 mRNA. Pro cyclophilin A mRNA of the aortic wall and the SR-B I mRNA. CD36 expression of the ABCA1 mRNA.. The experimental results showed that the intima surface of the aorta wall of the rabbit aorta was smooth, the structure of each layer was clear, the endothelial cells were complete and continuous, the smooth muscle cells were arranged neatly and orderly, without lipid. In the model group, the aorta wall of the rabbit aorta was thickened obviously, the endothelial layer fell off, the wall of the tube was covered with lipid and plaque. The intima of the aorta was slightly thickened in the compound group of Chinese medicine, and the deposition of foam cells and lipid deposition were found under the intima, and there was no obvious plaque in the intima. The.14 weekend was compared with the blank control group, compared with the blank control group. The serum levels of TC, TG and LDL in the rabbit serum were significantly increased (P0.01), and TC and FC in the smooth muscle cells were significantly increased (P0.05, P0.01). Compared with the model group, the serum TC, TG, LDL in the rabbit serum of the Chinese medicine compound group decreased significantly (P0.05, P0.01), and the aortic vascular wall cells in the aorta were significantly higher than those in the blank control group. The expression of Cav-1 protein and mRNA in the model group decreased significantly (P0.01). Compared with the model group, the expression of Cav-1 mRNA in the compound group of Chinese medicine increased significantly (P0.01), and the expression of Cav-1 protein also increased, but the difference was not statistically significant (P0.05). Compared with the blank control group, the expression of cyclophilin A protein in the model group was significantly increased (P0.05), pro ring. There was no significant difference in the expression of A mRNA, but there was no significant difference (P0.05). Compared with the model group, the expression of Pro cyclophilin A protein decreased significantly (P0.01), and the expression of cyclophilin A mRNA increased significantly (P0.01). Compared with the blank control group, the expression of ABCA1 protein in the model group had a downward trend, but the difference was not statistically significant (P0.0). (P0.0, the difference was not statistically significant (P0.0). 5), the expression of ABCA1 mRNA increased significantly (P0.01). Compared with the model group, the expression of ABCA1 protein in the Chinese medicine compound group increased, but the difference was not statistically significant P0.05), and the expression of ABCA1 mRNA decreased significantly (P0.01). The results of hepatocyte determination showed that the expression of SR-B I protein and mRNA in the model group had a decreasing trend compared with the blank control group. But the difference was not statistically significant (P0.05). Compared with the model group, the expression of SR-B I mRNA in the Chinese medicine compound group increased, but the difference was not statistically significant (P0.05). Compared with the blank control group, the expression of CD36 protein in the model group was increased, but the difference was not statistically significant P0.05), CD36 mRNA expression increased significantly (P0.05); and the model of the model group (P0.05); The expression of CD36 protein in the Chinese medicine compound group decreased, but the difference was not statistically significant (P0.05). Conclusion: 1. Hua Tong Mai Fang can inhibit the intima hyperplasia of aorta in AS rabbit, reduce the formation of the foam cells under the endothelium and the lipid deposition, and reduce the form of AS plaques form.2. turbid and Tongmai, which can effectively reduce the TC, TG, LDL in the serum of AS rabbit. Level, significantly reduced TC in AS rabbit aortic vascular wall cells, FC content.3. turbid pulse can improve the level of Cav-1 protein and gene, the gene level of cyclophilin A, promote the formation of intracellular cholesterol transport complex, the upward trend of the expression of ABCA1 protein, promote intracellular cholesterol outflow, guarantee Cav-1, and procyclin A Normal expression, reduce the overexpression of cyclophilin A in plaque, and finally achieve the anti AS effect of.4. turbid vein, which can have a certain trend of up regulation of the liver SR-B I gene, and can inhibit the expression of CD36 protein, which may promote the selective uptake and metabolism of cholesterol in the liver and the smooth metabolism of the lipid. Finally, the effect of anti AS is achieved.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285.5

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