【作者】 江丽霞；

【导师】 许能贵；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 高脂血症(Hyperlipidemia)是指人体内脂肪代谢异常,使血中动脉粥样硬化的脂质总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、甘油三酯(TG)、载脂蛋白B(apo-B)和(或)脂蛋白(a) [(Lp(a)]增高,高密度脂蛋白胆固醇(HDL-C)和(或)载脂蛋白A (apo-A)降低。高脂血症是动脉粥样硬化形成和发展的易患因素,而动脉粥样硬化是导致心脑血管疾病的病理基础,大量的研究资料表明,高脂血症是脑卒中、冠心病、心脏猝死独立而重要的危险因素,因此有效地控制高脂血症是防治心脑血管疾病的重要途径。针灸作为一种自然疗法,因其简、便、验、廉等优势在几千年的中医传统诊疗中起着十分重要的作用,且众多的研究也证实针灸具有良好的调节体内脂质代谢的作用,在治疗诸多现代疾病中已逐渐显示出优势。虽然目前针刺治疗高脂血症等临床研究工作开展得较多,但实验研究较少,从实验角度深入探讨针刺对高脂血症的影响作用,务求进一步揭示针刺防治高脂血症的作用机制,为针刺更好地应用于防治这一临床重要综合征提供重要的依据。本研究的开展可为针刺防治高脂血症提供系统的依据,为针刺这一具有我国中医特色的治疗方法治疗重大疾病提供重要的科学依据,也为中医药防治某些重大疾病的基础研究提供方法学的借鉴。本研究是前人研究的基础上,以高脂饮食复制高脂血症动物模型,结合现代检测技术,深入探讨针刺治疗高脂血症的作用机制。全文分文献研究、实验研究和小结三大部分进行。1.文献研究通过对大量关于高脂血症的文献综合研究,从高脂血症概念、高脂血症与相关疾病关系、高脂血症发生机理及防治等多方面阐述了高脂血症的最新研究进展,并对中西医药防治高脂血症的研究现状进行了分析,特别对针灸治疗高脂血症的国内外研究进展进行了综述,提出今后的研究方向,认为针刺不失为改善高脂血症的有效方法之一,应加强对其作用机理的深入研究和探讨。2.实验研究2.1方法2.1.1实验分组将30只雄性SD大鼠[体重(200±20g]以普通饲料进行适应性饲养1周,按体重随机分为3组,每组10只,分别为空白对照组、高脂血症模型组、针刺组。空白对照组以普通饲料喂养,其他各组均以高脂饲料饲养。从第6周开始,每隔1周空白对照组和高脂血症模型组大鼠从眼眶静脉窦采血1次,检测TG(甘油三酯)、TC(总胆固醇)、HDL-C(高密度脂蛋白)、LDL-C(低密度脂蛋白),并计算动脉粥样硬化指数进行对比,确定造模是否成功。第8周造模成功后,随后的3周,空白对照组以普通饲料喂养,模型组和针刺组以高脂饲料继续喂养,其中针刺组开始针刺治疗,1次／d。2.1.2取穴与针刺方法取穴：穴位选用“脂三针”(手厥阴心包经“内关”、足阳明胃经“足三里”、足太阴脾经“三阴交”)以及丰隆、肝俞。其定位参照大鼠的常用针灸穴位：“内关”位于前肢内侧,离腕关节约3mm左右的尺挠骨缝间；“足三里”位于大鼠后肢膝关节外下方当腓骨小头下约5mm处；“三阴交”位于大鼠后肢内踝尖直上10mm处；“丰隆”位于大鼠小腿骨的中点腓骨后缘,相当于丰隆穴肌肉中,“肝俞”位于大鼠后背第9胸椎棘突下两侧旁开5mm。刺法：内关、足三里、三阴交、丰隆均以30号1寸毫针(华佗牌,苏州医疗用品厂生产),沿皮斜刺8mm,针尖朝向肢体近端,肝俞直刺5mm,在内关、足三里、三阴交、丰隆穴,接电针治疗,将针柄分别连接至电针仪的电极上,电针仪采用华佗牌,苏州医疗用品厂生产的SDZ-II模型,采用疏密波,频率5-25Hz,强度以肌肉轻微抖为度,时间持续30分钟。每天1次,连续治疗21天。2.1.3样本处理及检测方法各组大鼠均于各阶段治疗结束后按规定的时间禁食12h过夜,检测甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C),并计算动脉粥样硬化指数进行对比。最后一次于全部干预治疗结束后以戊巴比妥钠(35 mg/kg)腹腔麻醉,于腹腔静脉取血。检测相关指标：TG、TC、HDL-C、LDL-C,血液流变学相关指标、内皮素(ET)、一氧化氮(NO),超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)。脱臼法处死大鼠,取一定量肝脏组织测定肝组织SOD、MDA、GSH-Px含量,留取肝脏左叶置10%福尔马林溶液中,酒精递度脱水,石蜡包埋,制成厚度为3mm切片,进行HE染色,光镜下观察形态结构。2.2结果2.2.1针刺对高脂血症大鼠体重及一般状况的影响在实验过程中,空白对照组大鼠体质量增长稳定,精神良好,反应灵敏,动作自如,毛发有光泽。第5周开始见,模型组、针刺组、大鼠性情变温顺,嗜睡少动,大鼠毛发蓬乱无光泽,大便量较正常组偏少,色灰,质较软,体重增长较快,较肥胖。模型组第8周后体重仍有增长,但增长速度小于前8周,且精神萎靡,反应迟钝,动作迟缓。针刺组从第9周开始给予针刺治疗,但仍给予高脂饲料喂养,可观察到大鼠体重仍有增长,但增长速度小于前8周,表现出精神萎靡,反应迟钝,动作迟缓。经针刺治疗3周后大鼠精神萎靡,反应迟钝,动作迟缓等较前有所好转,体重增长速度明显降低。2.2.2针刺对高脂血症大鼠动物粥样硬化指数(AI)及脂质代谢的影响在经高脂饲料喂养八周后,模型组及针刺组(未接受针刺干预前)脂质代谢已经出现紊乱,与空白组比较,模型组及针刺组(未接受针刺干预前)血清TG、TC、LDL-C水平都显著高于空白组(P<0.05-0.01),血清HDL-C水平显著低于空白组(P<0.01)；AI指数显著高于空白组(P<0.01)。这一结果提示,经高脂饲料喂养八周后,模型组和针刺组(未接受针刺干预前)动物脂质代谢紊乱程度及动脉硬化程度已经达到了高脂血症动物模型的要求。在经针刺干预治疗3周以后,模型组大鼠血清TG、TC、LDL-C水平显著都高于空白组(P<0.05-0.01),血清HDL-C水平显著低于空白组(P<0.01),AI指数显著高于空白组(P<0.01)；针刺组在经针刺干预三周后,针刺组大鼠血清TC、TC、LDL-C水平显著都低于模型组(P<0.05-0.01),血清HDL-C水平显著高于模型组(P<0.05),AI指数显著低于模型组(P<0.01)。2.2.3针刺对高脂血症大鼠肝脏形态学的影响空白对照组：肝小叶结构清晰,中央静脉居中,界面平滑,肝索排列规则,向四周呈放射状排列；窦内皮细胞、储脂细胞及枯否氏细胞未见增生。肝细胞胞浆红染,核居中。汇管区结构清晰,胆管未见破坏,纤维结缔组织未见增生,未见脂肪变性及肝内炎症现象(见附录3图1)。模型组：可见弥漫性肝细胞脂肪变性,以小空泡脂肪变为主,肝小叶内有炎细胞浸润,但未见肝纤维化。脂变肝细胞极度肿胀成圆形,体积较正常明显增加,胞质内充满大量脂肪空泡(见附录3图2)。针刺组：可见散在肝细胞脂肪变性,以小空泡脂肪变为主,肝小叶内少许炎细胞浸润(见附录3图3)。2.2.4针刺对高脂血症大鼠血液流变学的影响高脂血症模型大鼠血液呈高黏高凝状态：血液黏滞度增高,血细胞聚集性增强在经针刺干预治疗3周后,与空白组比较,模型组的高切变率、低切变率、血浆比粘度、红细胞压积、红细胞电泳时间、红细胞聚集指数都显著高于空白组(P<0.05或P<0.01)。与模型组比较,针刺组的高切变率、红细胞压积、红细胞电泳时间、红细胞聚集指数都显著下降(P<0.05或P<0.01)。2.2.5针刺对高脂血症大鼠氧自由基的影响血脂升高时,脂质过氧化作用增强,丙二醛(MDA)产生增多,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)降低。在经针刺干预治疗3周后,与空白组比较,模型组血清SOD、GSH-Px水平均显著低于空白组(P<0.05或P<0.01),血清MDA水平显著高于模型组(P<0.01)。与模型组比较,针刺组血清GSH-Px水平显著高于模型组(P<0.05),血清MDA水平显著低于模型组(P<0.01)。2.2.6针刺对高脂血症大鼠血清—氧化氮(NO)及血浆内皮素(ET)的影响高脂血症可造成动脉内皮功能损害,引起内皮细胞功能障碍,或血管壁功能和结构的损伤,从而影响血管功能,而ET、NO在血管收缩及舒张中有重要作用。在经针刺干预治疗3周后,与空白组比较,模型组血清NO水平显著低于空白组(P＜0.05),模型组血浆ET水平显著高于空白组(P<0.05或P<0.01)；与模型组比较,针刺组血清NO水平显著高于模型组(P<0.05),针刺组血浆ET水平显著低于模型组(P<0.05)。3.结论高脂饮食可成功复制出高脂血症大鼠模型,该模型能够表现出高脂血症所应具备的基本特征：血脂升高、脂肪肝、动脉粥样硬化、向心性肥胖等。经本研究证实,本模型大鼠血管内皮功能有明显的受损表现,为本研究的顺利开展打下良好的基础。针刺干预可显著改善高脂血症模型的脂质代谢紊乱：降低高脂血症大鼠血脂、动脉粥样硬化指数、改善脂肪肝,与模型组对比,差异显著。针刺可干预治疗高脂血症,其机制可能与其降低血脂、改善动脉粥样硬化指数、调节失衡的体内氧化-抗氧化系统、改善血液流变学、改善氧自由基功能、改善脂肪肝有关。更多还原

【Abstract】 Hyperlipidemia refers to lipid metabolism disorder in human body, causing atherosclerosis with elevating total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), triglyceride (TG), apolipoprotein B (apo-B) and(or) lipoprotein (a) [(Lp(a)], and lowering high density lipoprotein-cholesterol (HDL-C) and (or) apolipoprotein A (apo-A). Hyperlipidemia is a risk factor in the formation and development of atherosclerosis, and atherosclerosis is the basic pathogenesis of cardiovascular and cerebrovascular disease. A vast volume of research data show that hyperlipidemia is an independent and vital risk factor leading to stroke, coronary heart disease, sudden cardiac death, therefore a vital pathway in controlling hyperlipidemia is essential in preventing cardiovascular and cerebrovascular disease.Acupuncture is a natural therapy, it plays a significant role in the traditional Chinese medicine treatment. It lasts for thousands of years because of its simple, convenient, effective and low-cost operation. Also it gradually becomes outstanding in treating a number of modern diseases as researches prove acupuncture has good result in regulating lipid metabolism in human body. Now, there is more clinical research development in treating hyperlipidemia by means of acupuncture, but less experimental research has been done. By experimental study, the effect of acupuncture in treating hyperlipidemia can be studied in depth with a view to further reveal its working mechanism. It can provide important evidence to support its application in prevention of certain clinical syndrome. This experimental research on the intervention effect of acupuncture on relative factors in hyperlipidemic rat model can provide systematic evidence in preventing hyperlipidemic syndrome. It can provide scientific evidence for treating major diseases by means of acupuncture which is a specialty in Chinese medicine treatment, also it sets a reference in methodology for basic research in Chinese medicine in prevention of major diseases.This research was carried on subsequent to the foundation built by previous researches in the past. Hyerlipidemic rat model was developed by feeding rats with high fat diet. The working mechanism of the effect of acupuncture in rat model in treating hyperlipidemia is studied in depth using modern testing technique.This paper is divided into 3 parts:literature review, experimental research and conclusion.1. Literature reviewA vast volume of literature review relating to hyperlipidermia were read, areas covered including latest research development in the concept of hyperlipidermia, its relationship with related diseases, its pathogenesis and prevention. Analysis on the current conditions of research development in treating hyperlipidermia with Chinese and western medicine has been done, particularly summarizing different ways of treating hyperlipidermia within the country and abroad, and proposal has been made for future research. Acupuncture is believed as one of the effective method in improving hyperlipidermia, therefore more effort should be made to study the working mechanism in depth.2. Experimental study2.1 Method2.1.1 Experimental Groups30 male SD rats [weight (200±20)g], fed by ordinary animal feeds for 1 week, randomly divided into 3 groups according to weight,10 rats a group, viz. blank control group, hyperlipidermic model group, acupuncture group. The rats in blank control group were fed by ordinary animal feeds, all rats in other 2 groups were fed by high fat diet. As from the 6th week, tests were carried out in every other week by drawing blood from the orbital venous sinus of each rat in the blank control group and the model group for checking out TG, TC, HDL-C, LDL-C, calculating the artheriosclerosis index for purpose of comparison, and determining the successful building of hyperlipidermic model. In the 8th week, the hyperlipidermic model was successfully developed. In the following 3 weeks, the rats in the blank control group continued to be fed by ordinary animal feeds, meanwhile, rats in model group and acupuncture group continued to be fed on high fat diet, and acupuncture treatment started once daily on the rats in acupuncture group.2.1.2 Selection of acupuncture points and acupuncture methodPoint selection:"3 lipid points" (Neiguan of Pericardium Meridian of Hand-Jueyin, Zusanli of Stomach Meridian of Foot-Yangming, Sanyinjiao of Spleen Meridian of Foot-Taiyin), Fenglong and Ganshu. Their location was determined by reference to the rats commonly used acupuncture points:Neiguanlocated on the medial aspect of the rat’s anterior limb,3mm from wrist joint between ulna and radius bone. Zusanli-located on the rat’s hind limb, lateral to the knee joint and 5mm inferior to the fibula small head. Sanyinjiaolocated on the medial aspect of the rat’s hind limb,10mm superior to the tip of the internal malleolus. Fenglong-located on rat’s lower limb, mid-point of the lower limb and at the posterior border of fibula. Ganshuon the back of the rat,5mm lateral to the spinous process of the ninth thoracic vertebrae.Needling method:For Neiguan, Zusanli, Sanyinjiao, Fenglong, use No.30 one-inch filiform needle (Hwato Brand, produced by the Suzhou Medical Appliance Factory) by oblique insertion for 8mm, the direction of these needle points were inserted towards the centre of the body. For Ganshu, insert needle perpendicularly for 5mm. After needles insertion, turned on the power of the electronic acupuncture treatment instrument and connected pulse current to the Neiguan, Zusanli, Sanyinjiao, Fenglong by clipping output wires to each needle handle of the point group concerned (2 points make up a group) respectively. The electronic acupuncture treatment instrument Model SDZ-II, Hwato Brand, produced by Suzhou Medical Appliance Factory, was used. In application, sparse and dense wave at frequency 5-25 Hz was selected. Proper intensity of stimulation was marked by a slightly muscular twitch around the point. Acupuncture treatment was done continuously for 21 days at 30min per day.2.1.3 Sample handling and testing methodAfter treatment, each group of rats were on fasting for 12 hours overnight as stipulated, for the purpose of testing TG, TC, HDL-C, LDL-C, and calculating atherosclerosis index for future comparison. At the end when all intervention treatments were completed, rats were sedated with sodium pentobarbital (35mg/kg), and then blood was drawn from peritonovein. Blood lipid related indicators (TG, TC, HDL-C, LDL-C), hemorheology related indicators, ET, NO, SOD, MDA, GSH-Px were determined. Rats were executed by dislocated method, a section of liver tissues was removed to test for SOD, MDA, GSH-Px content, and the remaining left hepatic lobule was preserved in 10% formalin solution. They were dehydrated, embedded in paraffin, cut into sections of 3mm thick and stained with hematoxylin and eosin (HE), and then examined under optical microscope for morphological structure.2.2 Result2.2.1 The effect of acupuncture on the weight and general conditions in hyper lipidemic rat model.During the process of experiment, rats in the blank control group were seen to have weight increased steadily in good physical state; they were responsive, able to move freely, and their hair grown with luster. As from the observation obtained from the 5th week, the rats in model group and acupuncture group, when compared with the blank control group, were seen to appear tame, sleepy and with less movement, their hair became fluffy and lost luster, less feces were discharged, their hair turned grey and softer, they looked fatter and weight increased faster.For rats in the model group, their weight increase could still be seen as from the 8th week but growth rate slowed down, they appeared apathetic, less responsive and acted slowly. For rats in the acupuncture group, they were given acupuncture treatment as from the 9th week, and were still fed with high fat diet; their weight increase could still be seen but the rate of increase less than that in the previous weeks. They appeared apathetic, less responsive and acted slowly. After acupuncture treatment for 3 weeks, conditions of rats in acupuncture model had turned better and the rate of weight gain reduced dramatically.2.2.2 The effect of acupuncture on atherosclerosis index (Al) and lipid metabolism in hyper lip idemic rat model.After feeding with high fat diet for 8 weeks, the rats in the model group and acupuncture group (before acupuncture treatment) were found to have lipid metabolism disorder. The results of rats in the model group and acupuncture group (before acupuncture treatment), when compared them with the blank control group, showed a statistically significant effect with a higher TG, TC, LDL-C in serum (P<0.05-0.01), a lower HDL-C in serum (P<0.01), and a higher AI index (P<0.01). By this result, rats in the model group and acupuncture group (before acupuncture treatment) had been confirmed to have lipid metabolism disorder and atherosclerosis after feeding with high fat diet for 8 weeks.After acupuncture treatment for a further 3 weeks, the serum of rats in model group and acupuncture group were tested. The results of the rats in model group, when compared them with the blank control group, showed a statistically significant effect with a higher TG, TC, LDL-C level (P<0.05～0.01), a lower HDL-C (P<0.01), and a higher AI index (P<0.01). The results of the rats in acupuncture group, when compared them with the model group, showed a statistically significant effect with a lower TG, TC, LDL-C level (P<0.05～0.01), a higher HDL-C (P<0.05), and a lower AI index (P<0.01).2.2.3 The effect of acupuncture on liver morphology in hyper lipidemic rat model.Blank control group:the structure of hepatic lobule was clear, central vein was centrally located, border of hepatic plate was smooth, and the hepatic cords were orderly arranged and spread outwards in radius arrangement. No hyperplasia was seen in sinusoidal endothelium cells, fat-storing cells and kupffer cells. Hepacytoplasmic was red stained and hepatocyte nuclear located in central. The structure of the portal tract was seen clear, and bile duct was not damaged. No hyperplasia was seen in fibrous connective tissue. No fatty degeneration and liver inflammation phenomenon could be seen (see Figure 1, Appendix 3). Model group:Diffused hepatocellular fatty degeneration with primarily small bubble fats was visible. There was cell infiltration inside the liver lobules but could not see hepatic fibrosis. Steatotic liver cells were extremely swollen in round shape; mass size was dramatically larger than normal. Cytoplasm was filled with great amount of fat vacuoles (see Figure 2, Appendix 3). Acupuncture group:Fatty degeneration in the liver cells was visible, primarily of small bubble fat. There was slight cell infiltration inside the liver lobule (see Figure 3, Appendix 3).2.2.4 The effect of acupuncture on hemorheology in hyper lipidemic rat model.The blood of hyperlipidemia rats in model group was of high viscosity and hypercoagulability i.e. the blood viscosity increased and hemagglutination built up. After acupuncture treatment for 3 weeks, the blood of the model group and acupuncture group were tested. The result of the blood in model group, when compared with the blank control group, showed a statistically significant effect with a higher High shear rate, Low shear rate, plasma viscosity, hematocrit, and red blood cell electrophoresis time, and red blood cell aggregation index (P<0.05 or P<0.01). The result of the blood in acupuncture group, when compared with model group, showed a statistically significant effect with a lower High shear rate, hematocrit, red blood cell electrophoresis time, and red blood cell aggregation index (P<0.05 or P<0.01). 2.2.5 The effect of acupuncture on oxygen radical in hyper lipidemic rat model.When lipid protein of rats in model group were found elevated, peroxidation also increased, more MDA were generated, but SOD and GSH-Px were found reduced. After acupuncture treatment for 3 weeks, the oxygen radical in the rats of model group and acupuncture group were tested. The result of the model group, when compared with the blank control group, showed a statistically significant effect with a lower SOD and GSH-Px in serum (P<0.05 or P<0.01), and a higher MDA level in serum (P<0.01). The result of the acupuncture group, when compared with the model group, showed a statistically significant effect with a higher GSH-Px level in serum (P<0.05), and a lower MDA level in serum (P<0.01). 2.2.6 The effect of acupuncture on Nitric Oxide (NO) in serum and endothelin (ET) in blood plasma in hyperlipidemic rat model.Hyperlipidemia could cause damage to endothelium leading to endothelial dysfunction, or damage to the function and structure of vascular wall affecting the vascular function. ET and NO played a significant role in the vasoconstriction and vasorelaxation. After acupuncture treatment for 3 weeks, the NO and ET in rats of all groups were tested. The result of NO and ET in the model group, when compared with the blank control group, showed a statistically significant effect with a lower NO in serum and a higher ET in blood plasma (P<0.05 or P<0.01). The result of NO and ET in acupuncture group, when compared with the model group, showed a statistically significant effect with a higher NO in serum and a lower ET in blood plasma (P<0.05).3. ConclusionFeeding high fat diet to the rats in model group could successfully duplicate hyperlipidemic rats. The rats in model group could show all basic characteristics that the hyperlipidermic rats possessed:high lipid in blood, fatty liver, atherosclerosis, concentric obesity etc. This research study confirmed the hemoendothelial function of rats in model group had adversely been affected significantly. These findings had paved an easy way to pursue on. The effect of acupuncture on lipid metabolism disorder in hyperlipidemic rat model group showed a significant improvement. Acupuncture could reduce lipid level in hyperlipidemic rats and atherosclerosis index, enhanced the condition of fatty liver. The effect of acupuncture in rats of hyperlipidemic model showed a significant difference when compared with that of the model group. Acupuncture could show significant effect on hyperlipidemic rats, its mechanism demonstrated that it could lower lipid in blood, improve the atherosclerosis index, adjusting the imbalance of oxidization-anti-oxidant system in the body, and improve hemorrheology, oxygen radicals and condition of fatty liver.更多还原