热量限制预防及延缓动脉硬化发生发展机制的研究
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摘要
心脑血管疾病是发达国家人口死亡的主要原因,在我国的死亡率也呈逐年增高的倾向。而动脉粥性硬化(atherosclerosis, AS)性心血管疾病则被称为发达国家的“头号杀手”,它在发展中国家发病率也越来越高(Shimizu T,2008)。其中冠状动脉粥样硬化性心脏病(coronary atheroselerosis heart disease)简称冠心病(coronary heart disease, CHD)是一种常见疾病,已成为欧美国家最多见的心脏病种。1990年全球人口为53亿,死亡5000万,其中630万死于冠心病,占12.4%。1991年美国心脏协会公布,1988年冠心病死亡人数占总死亡的45.3%,约1/5的美国人在65岁前死于心血管病。作为亚洲国家,我国目前冠心病发病率和死亡率仍处于较低发国家的行列,但随着人们生活方式的改变,近年来有增高的趋势。在住院心脏病患者中,本病所占的比例也不断增加。而冠心病的基本病因为动脉粥样硬化(atherosderesis, As),而AS斑块的不稳定,引起局部血小板聚集,急性血栓形成,管腔急性闭塞是急性冠脉综合征(acute coronary syndrome, ACS)发生的共同机制。随着我国经济社会的发展,人民生活水平提高和饮食水平习惯改变,以及随之而来的人口的老龄化,动脉粥性硬化引发的疾病也逐渐成为我国主要死因(牟一鸣等,2001)。世界卫生组织预测,在世界范围内的传染性疾病病死率得到有效控制之后,到2020年心管管疾病将成为全世界第一死因。
动脉粥样硬化(atherosclerosis, AS)是心脑血管疾病的重要病理基础,防治AS是防治心脑血管病的根本措施。动脉粥样硬化(atherosclerosis, AS)是以富含脂质的斑块在大动脉壁聚集为特征的系统疾病,是心脑血管病的主要病理基础,它是一种多因素所致的动脉血管慢性疾病,集中表现为全身性血管内皮功能障碍(endothelialdysfunction)、血管内膜慢性炎症、纤维增生、管腔狭窄及血栓形成等。其主要临床表现是心肌梗塞、中风和外周血管疾病,常伴有高血压、高胆固醇血症或糖尿病等。AS病因病理复杂,目前尚未完全阐明,但可以确定的是AS是一类涉及到多种遗传因素和环境因素的复杂疾病,这些遗传因素和环境因素之间的相互作用最终导致斑块形成和出现随后的一系列临床事件。AS性血管疾病病因众多,其中不少与生活方式密切相关,包括吸烟、高糖高脂饮食、缺乏体力活动等习惯。从生物学角度来看,AS是一种长期慢性炎症疾病,与血脂紊乱密切相关,其发生发展是一个十分复杂的过程。AS性疾病临床事件主要为冠心病和中风。
因此,对AS发生机制的研究日益受到关注,血脂增高和紊乱是大多数AS发生的前提和基础,血管内皮功能障碍是AS发生的始发事件,多种因素相互作用,导致了AS的发生、发展。其中主要因素包括：血管内皮细胞功能障碍,血管平滑肌细胞增生,血小板和血液凝固状态异常,单核细胞、巨噬细胞浸润,自由基损伤,病毒及细菌感染,细胞凋亡,炎症和免疫反应,钙离子沉积,基因异常等。目前普遍认认同的发病机制是“损伤应激学说”(Ross R,1999)。该学说认为AS的过程包括以下几个步骤：内皮细胞(vascular endothelial cell, VEC)受各种刺激而损伤；单核细胞通过损伤处进入内膜,转变为巨噬细胞；巨噬细胞在与协同的T淋巴细胞分泌的细胞因子的作用下转变为巨噬细胞源性泡沫细胞；动脉中膜的血管平滑肌细胞迁入内膜,吞噬脂质形成平滑肌源性泡沫细胞,并增生迁移形成纤维帽；纤维帽被巨噬细胞分泌的基质金属蛋白酶和T淋巴细胞分泌的Y-干扰素削弱,使斑块破裂而移动,形成动脉粥样理化的栓子。
关于AS的形成,近年来免疫炎症学说越来越受到重视。研究认为AS是对血管内皮局部损伤的一种保护性炎症—纤维增殖性回应。如果损伤持续一段时间,这种回应则变得过度,最终成为疾病即斑块形成。在斑块的形成过程中脂质沉积是最重要的因素,也是损伤反应最早期的表现之一。伴随着脂质的沉积,氧化低密度脂蛋白胆固醇(oxLDL-C)的形成,循环中的白细胞和单核细胞被激活,并迁移到病变处,后者在oxLDL-C作用下变成活化的巨噬细胞,通过他们的清道夫受体,摄取oxLDL-C变成泡沫细胞,泡沫细胞的不断产生和堆积导致脂质条纹的形成。泡沫细胞死亡时则释放大量的胆固醇酷与血浆脂蛋白的沉积构成斑块下的脂质核心。炎症应答继续发展,T细胞活化,则引发纤维增殖反应最终形成纤维帽。在斑块形成的早期脂质核心小,纤维帽厚,斑块呈稳定状态,伴随着泡沫细胞的不断死亡和血浆脂类的沉积,斑块下的脂质核心不断增大,另一方面大量巨噬细胞浸润释放大量水解酶,尤其是金属蛋白酶系列,通过降解纤维帽以及抑制胶原纤维的生成使纤维帽逐渐变薄,从而使稳定斑块转为不稳定斑块,后者在内、外因素作用下,最终发生破裂导致急性冠脉事件发生。病理学家亦发现在斑块的破裂部位可见大量巨噬细胞浸润,炎性标记物表达增加。由此可见,斑块的不断进展与炎症反应的程度密切相关。斑块是否发生破裂与斑块内的炎症反应强度及巨噬细胞的浸润程度有密切的关系。动脉壁内脂纹或斑块形成是AS的特征性病变,而动脉内膜的巨噬细胞以及由中膜向内膜迁移的平滑肌细胞经由清道夫受体(scavenger receptor, SR)摄取氧化低密度脂蛋白(oxidizedLDL, oxLDL),从而形成巨噬／平滑肌细胞源性泡沫细胞,是AS特征性病变发生发展过程中的一个核心事件。
人们对衰老与长寿的研究逾近百年,但至今为止,控制人类寿命之迷仍未被揭开。尽管衰老本身不是一种疾病,但很多疾病与衰老相关,如肿瘤,老年痴呆(Alzheimer's disease),动脉硬化,代谢紊乱(metabolic disorders)等。心脏病和中风引起的死亡率随年龄呈指数增加,65-74岁死亡率超过40%,85岁及以上的死亡率将近60%。越来越多的研究发现,即使没有高血压,糖尿病及吸烟等各种危险因子存在,心血管系统随年龄增加亦趋于易发疾病和受损,这可能和衰老血管的细胞内稳态相关(Ungvari et al.,2010).在实验动物和人的身上都已证实,氧化损伤产生的活性氧(reactive oxygen species, ROS)激活NAD(P)H氧化酶及下调NO的活性,导致衰老进程的内皮细胞功能障碍,从而引发老年人冠状动脉疾病和中风。
热量限制(calorie restiction, CR)是指在提供生物体充分的营养成分如必需氨基酸、维生素等,保证生物体不发生营养不良的情况下,限制每日摄取的总热量。一般CR饮食是指减少动物随意进食量的30%—40%。热量限制功能以一科,被动的方式降低氧化和其他损伤,从而导致生命周期延长,可以调控从酵母到哺乳动物等生物体的生命周期。自1935年McCay等首次报道CR延长大鼠寿命以来,大量研究已表明CR是除遗传操作以外最强有力的延缓衰老的方法。热量限制不仅能维持许多年轻时的生理状态,还能延缓和预防一些与年龄相关疾病如心血管疾病、糖尿病、肿瘤的发生、发展。热量限制(caloric restriction, CR)是已经被证明具有抗衰老及延长寿限的作用(Russell JC2008; Fontana L,2009;),包括热量摄入限制和减少进食次数两种方式。CR对心血管系统具有多效保护作用,如保护血管(阻止动脉硬化进程),提高心肌耐受性及延缓心脏衰老(Shinmura K.2011),衰老进程中,转录因子NF-kB通过激活IL-1,IL-6,TNF-a等细胞因子介导机体长期慢性炎症反应,这种长期慢性炎症反应引发一系列老年相关疾病的关键。因此,有人提出可以通过抗氧化药物(如姜黄素(curcumin))来抑制依赖NF-kB信号的炎症反应,而延缓衰老(Sikora Eab et al,2010)。研究表明,CR可以降低炎症因子IL-6和leptin的表达(Reed JL,2010)。
本课题分为四个部分,首先依据已经发表的芯片数据结果进行生物信息学的分析和挖掘,具体为：来源于美国Miller SJ提交的GSE7281芯片数据集,Fischer344大鼠自然衰老,年龄分别为3,6,15,28个月,取大鼠动脉提取mRNA
进行全基因组芯片分析
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7281;和来源于美国Becker KG提交的GSE11845芯片数据集,选取数据集中正常对照(自由饮食),热量限制(间断饮食)及高脂(氢化椰子油,热量高于正常对照组的60%)三组样本数据,进行心脏,肝脏及肌肉相关基因分析。http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11845。挖掘动脉硬化与衰老相关基因。接下来建立Wistar大鼠模型,利用RT-PCR, ELISA等方法进行血清氧化低密度脂蛋白(OXLDL),C反应蛋白(C-reactive protein, CRP),血栓前体蛋白(TpP)等靶标的检测以及进行病理分析观察血管形态,对芯片数据挖掘的结果进行进一步的实验验证。在建立高糖饮食与热量限制的大鼠模型的基础上,进一步通过实时荧光定量PCR技术(Real-time quantitative Polymerase Chain Reaction, Real Time PCR)验证生物信息学结果并筛选得到31个同时响应热量限制与高脂高糖饮食的基因表达。通过建立HC诱导动脉硬化的细胞模型(细胞株EA.hy926),对高糖高胆固醇诱导EA.hy926细胞凋亡中对分子伴侣家族一员,HSP60蛋白的表达进行了初步的研究。
结果分述如下：
1.生物信息学挖掘动脉硬化与衰老相关基因。
通过年龄相关大鼠动脉芯片表达谱比较共获得166条探针(155个基因),其中有36个基因响应热量限制与高脂高糖饮食。其中Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt等参与糖代谢,在大鼠衰老的进程中呈下降趋势；而Gygl参与肝糖原合成,随年龄增加而持续上调。HK2, PFKP, Dlat, Pdp2, Acyl等是糖酵解与三羧酸循环的关键调控酶,Tkt, TECR与ACS15则是NADPH提供途径的关键酶。在大鼠衰老的进程中,糖代谢相关的酶表达下调,NADPH生成途径受阻,表明在个体发育成熟后,机体对能量的需求可能减少,NADPH下降可能将导致SIRT1的活性下降。有报道SIRT1对血管及在动脉硬化中具有保护作用,它在热量限制介导机体寿限的延长中起到非常重要调作用。因此,通过热量限制可能上调这些基因的表达,对机体的“燃料”进行充分燃烧,减少脂肪堆积,延长寿限。
生物信息学的分析表明,随着年龄的增长,大鼠动脉血管的细胞粘附因子Vcam1, Icam1, Postn, Fn1, Sdc1, Mfge8, Spp1, Jup, Tln1上调表达,表明内皮细胞随年龄增长功能下降,容易诱发动脉硬化。与血管发育相关的Agt, Colla1, Gja5随年龄增长而下调,而参于血管调节与重塑Bgn, Acvrl1, Fgfr1, Box等则上升。这些基因可能参与高血压的发生,推测热量限制可能抑制Bgn, Acvrll的表达。此外,参与免疫的HSPD1与抗氧化的Pex19, Cyld等基因随年龄呈下降趋势,热量限制显著提高他们的表达。随着年龄的增长,动脉硬化的易感基因表达上调,表明患动脉硬化的风险增加；而热量限制可以下调这些基因的表达,推测其可能预防或延缓动脉硬化的发生。
2.高糖高脂诱导大鼠动脉硬化模型建立以及生物信息学结果的实验验证。
将44只6月和40只11月大的SPF级的Wistar雄性大鼠进行随机分组,FC(自由饮食组)给予基本饲料和普通算来水喂养；CR(热量限制组)每天定时给予基本饲料100-150g/3只老鼠,此根据老鼠一天能刚吃完饲料重量而定；HC(高糖高脂组),饲料组成为：基本饲料+15%猪油+3%胆固醇+5%精制糖。各组大鼠进行2个月和4个月喂食后,心脏取血后,通过血清OXLDL和血浆TpP含量检测,以及血液中CRP的mRNA检测表明,本实验通过高糖高脂喂养的大鼠更有诱发动脉硬化的可能性。并且随年龄增长,长期高糖高脂饮食罹患动脉硬化的可能性更大,而热量限制饮食即可减低此风险。
在建立高糖饮食与热量限制的大鼠模型的基础上,通过Q-PCR检测生物信息学筛选得到31个同时响应热量限制与高脂高糖饮食的基因表达。结果表明,热量限制可以上调成年大鼠动脉中Aldh5al (F=5.301, P=0.022), Ldha (F=2.215, P=0.016), Hk2(F=3.556, P=0.061), Pfkp (F=3.045, P=0.085), Slc2a4(F=4.006, P=0.046), Dlat (F=3.389, P=0.068), Tkt(F=0.177,P=0.008), Gygl (F=5.831,p=0.017)等参与糖代谢相关的基因,而高糖高脂的饮食则起到相反的作用。同时,热量限制还可以有效抑制Icam1(F=3.739. P=0.045)和Tln1(1.546, P=0.050)的表达,但对Vcam1(F=0.177, P=0.267)和Fn1(F=1.495, P=0.102)作用不明显。Zou Y等报道热量限制可以有效抑制不同年龄大鼠l cam1和Vcam1的表达,但热量限制的时间持续13和31个月。本研究是在成年鼠后进行热量限制4个月进行检测,可能由于处理时间偏短,从而影响了结果。此外,热量限制还可以有效抑制Bgn(F=13.189, P=0.001), Acvrll(F=2.306, P=0.142)的表达,而上调HSPD1(F=3.191, P=0.075), Pexl9(2.474, P=0.126), Cyld(F=1.888, P=0.194)等基因的表达。高糖饮食则相反。
3.高糖高胆固醇诱导EA.hy926细胞凋亡中对HSPD1基因表达的影响
随胆固醇浓度的增加,EA.hy926细胞活性下降,呈浓度依赖性。0.8mM胆固醇+5mM葡萄糖共培养12h的细胞活性与正常对照组(5mM葡萄糖+0mM胆固醇)无显著差异(P>0.056),而0.8mM胆固醇+25mM葡萄糖共培养12h的细胞活性与正常对照组(25mM葡萄糖+0mM胆固醇)具显著性差异(F=579.023,P=0.001)。5mM或25mM葡萄糖+0.8mM胆固醇处理EA.hy926细胞不同时间后,细胞活性呈时间依赖性下调,48h后细胞大部分变圆,脱落(F=79.758,P<0.001)。流式检测表明此时,与低糖低脂处理的细胞比较,高糖高胆固醇处理的细胞大部分发生凋亡(F=127.068,P<0.001),HSPD1基因表达显著上调(F=6.959,P=0.006)。
通过上述三个部分的实验,我们得出以下结论：
1.随着年龄的增长,动脉硬化的易感基因表达上调,表明患动脉硬化的风险增加。
2.热量限制可以下调这些基因的表达,推测其可能预防或延缓动脉硬化的发生。
3.高糖高脂喂养的大鼠,长期的HC饮食可使血液中的OXLDL升高；血浆中TpP水平显著高于FC组；而CR组显著低于FC组；从动脉血管横切图看,HC组的血管内皮显著增厚,并不同程度的形成泡沫细胞。表明HC组大鼠动脉已有不同程度动脉硬化的病变,更有诱发动脉硬化的可能性。并且随年龄增长,长期高糖高脂饮食罹患动脉硬化的可能性更大,而热量限制饮食即可减低此风险。
4.热量限制可以上调成年大鼠动脉中Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gygl等参与糖代谢相关的基因,而高糖高脂的饮食则起到相反的作用。同时,热量限制还可以有效抑制Icaml, Tlnl, Bgn, Acvrll, Fgfrl, Bax的表达,而上调HSPD1, Pex19, Cyld等基因的表达。高糖饮食则相反。即并且随着年龄的增长,动脉硬化的易感基因表达上调,患动脉硬化的风险增加；而热量限制可以下调这些基因的表达,从而预防或延缓动脉硬化的发生。相反,高糖高脂饮食则起相反的作用,加快发病及病变的进程。
5.持续激活的HSP60通过激活人血管内皮细胞表达TNF-a,E-选择素,ICAM1,IL6等细胞因子,促进自身炎症发应从而诱发AS。无论是高糖,高糖高脂,或是低糖高脂处理EA.hy926内皮细胞,都可以诱导HSPD1的nRNA表达,但高糖高脂诱导表达的效果最显著。因此,适当控制饮食摄入糖与脂含量,可预防或延缓AS的发生
Cardiovascular disease is the predominant cause of death in developed countries population, mortality also showed a tendency to increase year by year in China. Atherosclerosis (AS) cardiovascular disease is called the number one killer in developed countries, and the incidence in developing countries is also high (Shimizu, T,2008). Coronary atherosclerotic heart disease referred to as coronary heart disease (CHD) is a common disease, has become a most common kinds of heart disease in the United States and Europe. In1990, The global population is5.3billion, and the death of50million, of which6.3million died of coronary heart disease, accounting for12.4%.1991American Heart Association announced that deaths of coronary heart disease account for45.3%to total deaths in1988, and about1/5of Americans die of cardiovascular disease before the age of65. As an Asian country, coronary heart disease morbidity and mortality of China is in the ranks of lower-prone countries, but with the changing life styles, it increased in recent years. In hospitalized patients with heart disease, the proportion of the disease are also increasing. The basic pathogenesis of coronary heart disease is atherosclerosis (As). The common mechanism are AS plaque instability, caused by local platelet aggregation and acute thrombosis, acute occlusion of the lumen, and acute coronary syndrome (ACS). With China's economic and social development and improved living standards and dietary level of habit change, and the consequent aging of the population, atherosclerosis sclerosis diseases gradually become China's leading causes of death (Mu Yi-ming,2001). The World Health Organization predicted that upto2020after worldwide infectious disease case fatality rate has been effectively controlled, Heart tube disease will become the world's first cause of death.
Atherosclerosis (As) is an important pathological basis of cardiovascular and cerebrovascular diseases, prevention AS is a fundamental measure of prevention and treatment of cardiovascular diseases. Atherosclerosis (AS) is characterized by a lipid-rich plaques in the aorta wall gathering system disease, and is the major pathological basis of cardiovascular and cerebrovascular disease.it is a multi-factor chronic arterial disease, the concentrated expression of systemic vascular endothelial function obstacles (endothelialdysfunction,), intimal chronic inflammation, fibrosis, stenosis and thrombosis formation. The main clinical manifestations of myocardial infarction, stroke and peripheral vascular disease, often accompanied by hypertension, hypercholesterolemia or diabetes. Because of the complexity of the etiology and pathology, AS has not yet been fully elucidated, but it is certain that AS is related to a variety of genetic and environmental factors of complex diseases, the interaction between these genetic factors and environmental factors ultimately lead to plaque formation and the emergence of series of subsequent clinical events. Many AS vascular disease etiology, which is closely related to many lifestyles, including smoking, high fat diet, lack of physical activity habits. From the biological perspective, AS is a chronic inflammatory disease, and dyslipidemia is closely related to its development and it is a very complex process. The clinical events of AS disease are coronary heart disease and stroke.
Therefore, the AS mechanism have become an increasing concern, elevated blood lipids and disorder are the premise and basis of most of the AS. Endothelial dysfunction is the originating events of AS; and a variety of factors interact, result in the occurrence and development of AS. The main factors include:vascular endothelial cell dysfunction, vascular smooth muscle cells, platelets and blood coagulation state of exception, monocytes, macrophage infiltration, free radical damage, viral and bacterial infections, apoptosis, inflammation and immune response, calcium deposition, and genetic abnormalities. The pathogenesis is now generally recognized as "injury stress theory (Ross, R,1999). The process involves in AS containing the following steps:a variety of stimuli and injury of endothelial cells (vascular of endothelial cell, VEC); monocytes transmit through the lesion into the intima and tranform into macrophages; macrophages together with the role of T lymphocytes secrete cytokines transmit into macrophage-derived foam cells; medial vascular smooth muscle cells move into the intima, engulfed lipid to form foam cell then proliferation and migration to form a fibrous cap; fiber cap weakened by matrix metalloproteinases and y-dry detoxification secreted by macrophage and T lymphocyte, the formation of atherosclerosis physics and chemistry of the emboli.
As to the formation of AS, There are more and more attention paid to the immune and inflammatory theory in recent years. Studies suggest that AS is a protective inflammation-fiber proliferative response to the local damage of the vascular endothelial. If the injury sustained period of time, this response becomes excessive, and eventually become diseases that plaque formation. Lipid deposition in the plaque formation process is the most important factor in the damage response and is also one of the earliest performance. Along with lipid deposition, the formation of oxidized low-density lipoprotein cholesterol (by oxLDL-C), circulating leukocytes and monocytes are activated and migrate to the lesion, which become activated by oxLDL-C under macrophages through their scavenger receptor uptake by oxLDL-C into a foam cell generation and accumulation of foam cells lead to the formation of lipid stripes. The release of the deposition of cholesterol cool plasma lipoproteins from the foam cell death constitute the plaque lipid core. Inflammatory response to the continued development of T cell activation, triggered fiber proliferative response ultimately the formation of the fibrous cap. Early lipid core plaque formation in small, fibrous cap thickness, plaque steady state, along with the continued death of foam cells and the deposition of plasma lipids, lipid core plaque growing on the other hand a large number of infiltration of macrophages to release large amounts of hydrolytic enzymes, especially metalloproteinases series, gradually thinning the fibrous cap by degrading the fibrous cap, as well as inhibit the formation of collagen fibers, so that stable plaques transmit into unstable plaques, and the latter influenced by the inner and outside factors, and the final breakdown lead to acute coronary events. The pathologist also found a large number of macrophage infiltration, and the increased expression of inflammatory markers in the plaque rupture site. Thus, the continuous progress of the plaque and the extent of the inflammatory response are closely related. The rupture of the plaque is closely related to the intensity of inflammation and macrophage infiltration in plaque. The formation of the fatty streak or plaque within the arterial wall is the characteristic lesions of AS. However, Macrophages as well as central and membrane migration to the intima of the arterial intima smooth muscle cells via the scavenger receptor (scavenger receptor, SR) uptaking of oxidized low-density lipoprotein (oxidizedLDL, oxLDL), and thus the formation of macrophage/smooth muscle cells derived foam cells, is a central event in the development of AS process.
Over the past century, people have done some research on aging and longevity but so far, no news has been announced. Although aging itself is not a disease but many diseases associated with aging such as cancer, Alzheimer's (Alzheimer's disease), atherosclerosis, metabolic disorders (metabolic disorders). Heart disease and stroke mortality with age exponential increase in mortality at age65-74more than40percent, nearly60%of the mortality rate of85years. Growing body of research found that, even in the absence of other risk factors as hypertension, diabetes and smoking, cardiovascular system increases with age have become more prone to disease and damage, this may homeostasis and aging vascular cells related (Ungvari et al,2010). In experimental animals and human body have confirmed that the oxidative damage generated by reactive oxygen species reactive oxygen species (ROS) in activation of NAD (P) H oxidase and lowered the activity of NO, leading to endothelial cell dysfunction of the aging process, causing the elderly coronary artery disease and stroke.
Calorie restriction (calorie restiction, CR) is to provide the organism full of nutrients such as essential amino acids, vitamins, etc., to ensure that organisms do not occur malnutrition, limiting the daily intake of total calories. General CR diet is reduced by30%-40%of the animal free food intake. The function of caloric restriction in a passive way to reduce oxidation and other damage, leading to prolonged life cycle, can regulate the life cycle from yeast to mammals and other organisms. Since1935McCay, first reported in the CR to prolong rats life, a large number of studies have shown that CR is the most powerful anti-aging methods In addition to genetic manipulation. Calorie restriction not only to maintain the physiological state of many a young man, but also delay and prevent the age-related diseases such as cardiovascular disease, diabetes, tumor occurrence and development. Calorie restriction (caloric restriction, CR) has been shown to have anti-aging and extend the life of the role (Russell JC2008; Fontana, L,2009), including calorie intake limit and reduce the consumption of the number of two ways. Multi-effect protective effect of CR on the cardiovascular system, such as protection of blood vessels (to prevent atherosclerosis process) to improve myocardial tolerance and delay cardiac aging (Shinmura K,2011). In the aging process, the activation of IL-1, IL-6and TNF-a and other cytokine by the transcription factor of NF-kB, mediated body chronic inflammation, which lead to the key of a series of aging-related diseases. Therefore, it was suggested by antioxidant drugs (such as curcumin (curcumin)) to suppress the inflammatory response dependent on NF-kB signaling and aging (Sikora, the Eab et al.,2010). It is shown that the CR can reduce the inflammatory cytokine IL-6and leptin expression (Reed, JL,2010).
This topic is divided into four parts, Firstly, based on published microarray data results, we do bioinformatics analysis and data mining, as the following:derived from the GSE7281microarray data sets submitted by the United States Miller, the SJ, Fischer344rats of natural aging, aged3,6,15,28months, the artery mRNA was extracted from rat to do whole genome microarray analysis. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7281, Submitted from the United States Becker, KG the GSE11845chip data set, select the data set in normal controls (free diet), calorie restriction (discontinuous diet) and fat (hydrogenated coconut oil, heat higher than60%of the normal control group) of three groups of samplesdata, heart, liver and muscle-related gene analysis. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11845.
Mining of atherosclerosis and aging-related genes. Then set up Wistar rat model, Using the RT-PCR, ELISA methods, we do serum oxidized low-density lipoprotein (of OXLDL), C-reactive protein (C-of reactive protein, CRP), thrombus precursor protein (TpP) targets detection and pathological analysis of the observed vascular morphology, and further experimental validation of the results of the microarray data mining. High carbohydrate diet and calorie restriction in a rat model based on the real-time fluorescence quantitative PCR (Real-time quantitative Polymerase Chain Reaction and Real Time PCR), to verify the results of bioinformatics and filter31 while responding to caloric restriction gene expression and high-fat high-sugar diet. EA.hy926cell apoptosis through the establishment of the HC induced cell model of atherosclerosis (cell line of EA. Hy926), high glucose and high cholesterol induced a preliminary study of a molecular chaperone family of HSP60protein expression. The results are as follows:
1. Bioinformatics mining of atherosclerosis and aging-related genes.
The total of166probes (155genes) were obtained from the Age-related rat arterial microarray, of which36genes respond to calorie restriction and high-fat high-sugar diet. The Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, etc. involved in glucose metabolism, a downward trend in the aging process in rats; Gyg1involved in glycogen synthesis, increased with age and continued upward. HK2, PFKP, Dlat, Pdp2, Acyl, etc. are a key regulatory enzyme of glycolysis and the citric acid cycle, Tkt, TECR, and ACS15are the key enzyme of the NADPH pathway. Along with the aging process in rats, the glucose metabolism enzyme expression and NADPH formation routes blocked, suggesting that individual mature, the body may reduce the energy demand of NADPH, the decline may lead to decreased activity of SIRT1, It is reported that SIRT1has a protective effect to blood vessels and atherosclerosis, and it play a very important role in mediating the body the life extension by calorie restriction. Therefore, caloric restriction may increase the expression of these genes, the body's "fuel" burning, reduce fat accumulation, and to extend the lifespans.
Bioinformatics analysis showed that the increase with age, rat arterial vascular cell adhesion molecule Vcam1, Icaml, Postn, the Fn1, Sdcl, Mfge8, Sppl, Jup, Tln1upregulated, indicating that the decline of endothelial cells increased with agelikely to cause atherosclerosis. Agt, Collal, Gja5which related to the vascular development, will decrease, and Bgn, Acvrll, Fgfrl, Bax which are involved in vascular regulation and remodeling will rose. These genes may be involved in the occurrence of hypertension, suggesting that caloric restriction may inhibit the expression of Bgn, Acvrl1. In addition, the HSPD1involved in the immune and antioxidant genes such as Pex19, Cyld declined with aging, calorie restriction significantly increased their expression. With aging, atherosclerosis susceptibility genes were up-regulated, indicating that the increase in the risk of suffering from arteriosclerosis; calorie restriction lowered the expression of these genes, suggesting that it may prevent or delay atherosclerosis.
2. High glucose and fat induced atherosclerosis Rat model and experimental verification of bioinformatics result.
44, six months and40, eleven months SPF male Wistar rats were randomized to FC (free diet group) to give basic feed and count runoff feeding; CR (calorie restriction group) time every day to give the basic feed100～150g/3mice, according to the mouse day had just eaten the feed weight; HC (high-sugar high-fat group), feed composition:basic feed+15%lard and3%cholesterol,5%refined sugar. Rats in each group after2months and4months feeding, cardiac blood was collected by the serum OXLDL and the plasma TpP content testing, and blood CRP mRNA detection showed that this experiment in rats fed by high glucose and fat more the possibility of induced atherosclerosis. And with aging, they are more likely to suffer from arteriosclerosis in long-term high-fat diet, however, calorie restriction diet can reduce this risk.
Q-PCR was done to verificated the bioinformatics results, Based on the high-sugar diet and calorie restriction rat model, screened31gene expression in response to calorie restriction and high-fat high-sugar diet. The results show that calorie restriction can increase the adult rat arterial Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gygl and other genes involved in glucose metabolism, high-sugar high-fat diet have the opposite effect. The same time, calorie restriction can effectively inhibit the expression of Icam1and Tln1, but not obvious effect on Vcaml and Fnl gene. Zou, Y reported that caloric restriction can effectively inhibit the expression of different ages the rat Icam1and Vcam1, but the heat restrictions are thirteen months and thirty-one months. In this study, the adult mice were calorie restriction for four months for testing, maybe due to the shorter processing time, thus affecting the results. In addition, calorie restriction can effectively inhibit the expression of Bgn, Acvrl1, Fgfr1, Bax, and activate HSPD1, Pex19, Cyld gene expression. The high carbohydrate diet is the opposite.
3. High glucose and high cholesterol induce EA.hy926cell apoptosis and have effect on HSPD1gene expression.
With increasing cholesterol concentration of EA. Hy926cell activity dropped to a concentration dependent manner. But the cell activity has no significantly different between the0.8mM cholesterol+5mM and glucose co-cultured for12h and normal control group (5mM glucose+0mM cholesterol)(P>0.05); There is significant difference between the0.8mM cholesterol+25mM and glucose co-cultured12h and normal control group (25mM glucose+0mM and cholesterol)(P<0.01).5mM or25mM glucose+0.8mM cholesterol dealing with EA.hy926cells at different times, cell activity was time-dependent decrease, most of the cells rounded off after48h. Flow cytometry showed that the majority ofcells treated with high sugar and high cholesterol treated cells undergo apoptosis (50%) contrast to low-sugar low-fat treatment, and HSPD1gene expression was significantly upregulated (F=6.959, P=0.006).
Through above three-Part test, we draw the following conclusions:
1. With aging, atherosclerosis susceptibility genes upregulated, indicating that the risk of an increased risk of atherosclerosis.
2. Calorie restriction can downregulate the expression of those genes, suggesting that it may prevent or delay atherosclerosis occurrence.
3. High sugar and high fat fed rats, the long-term HC diet can increase blood OXLDL; the plasma TpP level significantly higher than the FC group; CR group were significantly lower than the FC group; from the arterial cross-section diagram, HC group of vascular endothelial significantly is thicker, and varying degrees of formation of foam cells. So that, the HC rats artery has varying degrees of atherosclerosis lesions, the possibility of more-induced atherosclerosis. And with aging, It is more likely to suffer from arteriosclerosis in long-term high-fat diet, however, calorie restriction diet can reduce this risk.
4. Caloric restriction can increase the Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gyg1and other genes involved in glucose metabolism in the adult rat arteries, high-sugar and high-fat diet have the opposite effect. The same time, calorie restriction can effectively inhibit the expression of Icam1, Tlnl, Bgn, Acvrll, Fgfr1, Bax, and to upregulate HSPD1, Pex19, Cyld gene expression. The high carbohydrate diet is the opposite. And with aging, atherosclerosis susceptibility gene expression increased, which raised risk of atherosclerosis; calorie restriction lowered the expression of these genes, thus preventing or delaying the occurrence of atherosclerosis. In contrast, high-fat diet is counterproductive to speed up the process of onset and lesions.
5. The sustained activation of HSP60activate the expression of TNF-a, E-selectin factors, ICAM1, IL6, cytokines by the human vascular endothelial cells to promote the inflammation and induce the AS. Although high-sugar, high sugar and fat or low sugar and high fat treatment on lipid EA.hy926endothelial cells can induce HSPD1mRNA expression, but high glucose and fat-induced expression of the most significant effect. Therefore, an appropriate control of dietary intake of sugar and fat content, can prevent or delay the occurrence of AS.
动脉粥样硬化(atherosclerosis, AS)是心脑血管疾病的重要病理基础,防治AS是防治心脑血管病的根本措施。动脉粥样硬化(atherosclerosis, AS)是以富含脂质的斑块在大动脉壁聚集为特征的系统疾病,是心脑血管病的主要病理基础,它是一种多因素所致的动脉血管慢性疾病,集中表现为全身性血管内皮功能障碍(endothelialdysfunction)、血管内膜慢性炎症、纤维增生、管腔狭窄及血栓形成等。其主要临床表现是心肌梗塞、中风和外周血管疾病,常伴有高血压、高胆固醇血症或糖尿病等。AS病因病理复杂,目前尚未完全阐明,但可以确定的是AS是一类涉及到多种遗传因素和环境因素的复杂疾病,这些遗传因素和环境因素之间的相互作用最终导致斑块形成和出现随后的一系列临床事件。AS性血管疾病病因众多,其中不少与生活方式密切相关,包括吸烟、高糖高脂饮食、缺乏体力活动等习惯。从生物学角度来看,AS是一种长期慢性炎症疾病,与血脂紊乱密切相关,其发生发展是一个十分复杂的过程。AS性疾病临床事件主要为冠心病和中风。
因此,对AS发生机制的研究日益受到关注,血脂增高和紊乱是大多数AS发生的前提和基础,血管内皮功能障碍是AS发生的始发事件,多种因素相互作用,导致了AS的发生、发展。其中主要因素包括：血管内皮细胞功能障碍,血管平滑肌细胞增生,血小板和血液凝固状态异常,单核细胞、巨噬细胞浸润,自由基损伤,病毒及细菌感染,细胞凋亡,炎症和免疫反应,钙离子沉积,基因异常等。目前普遍认认同的发病机制是“损伤应激学说”(Ross R,1999)。该学说认为AS的过程包括以下几个步骤：内皮细胞(vascular endothelial cell, VEC)受各种刺激而损伤；单核细胞通过损伤处进入内膜,转变为巨噬细胞；巨噬细胞在与协同的T淋巴细胞分泌的细胞因子的作用下转变为巨噬细胞源性泡沫细胞；动脉中膜的血管平滑肌细胞迁入内膜,吞噬脂质形成平滑肌源性泡沫细胞,并增生迁移形成纤维帽；纤维帽被巨噬细胞分泌的基质金属蛋白酶和T淋巴细胞分泌的Y-干扰素削弱,使斑块破裂而移动,形成动脉粥样理化的栓子。
关于AS的形成,近年来免疫炎症学说越来越受到重视。研究认为AS是对血管内皮局部损伤的一种保护性炎症—纤维增殖性回应。如果损伤持续一段时间,这种回应则变得过度,最终成为疾病即斑块形成。在斑块的形成过程中脂质沉积是最重要的因素,也是损伤反应最早期的表现之一。伴随着脂质的沉积,氧化低密度脂蛋白胆固醇(oxLDL-C)的形成,循环中的白细胞和单核细胞被激活,并迁移到病变处,后者在oxLDL-C作用下变成活化的巨噬细胞,通过他们的清道夫受体,摄取oxLDL-C变成泡沫细胞,泡沫细胞的不断产生和堆积导致脂质条纹的形成。泡沫细胞死亡时则释放大量的胆固醇酷与血浆脂蛋白的沉积构成斑块下的脂质核心。炎症应答继续发展,T细胞活化,则引发纤维增殖反应最终形成纤维帽。在斑块形成的早期脂质核心小,纤维帽厚,斑块呈稳定状态,伴随着泡沫细胞的不断死亡和血浆脂类的沉积,斑块下的脂质核心不断增大,另一方面大量巨噬细胞浸润释放大量水解酶,尤其是金属蛋白酶系列,通过降解纤维帽以及抑制胶原纤维的生成使纤维帽逐渐变薄,从而使稳定斑块转为不稳定斑块,后者在内、外因素作用下,最终发生破裂导致急性冠脉事件发生。病理学家亦发现在斑块的破裂部位可见大量巨噬细胞浸润,炎性标记物表达增加。由此可见,斑块的不断进展与炎症反应的程度密切相关。斑块是否发生破裂与斑块内的炎症反应强度及巨噬细胞的浸润程度有密切的关系。动脉壁内脂纹或斑块形成是AS的特征性病变,而动脉内膜的巨噬细胞以及由中膜向内膜迁移的平滑肌细胞经由清道夫受体(scavenger receptor, SR)摄取氧化低密度脂蛋白(oxidizedLDL, oxLDL),从而形成巨噬／平滑肌细胞源性泡沫细胞,是AS特征性病变发生发展过程中的一个核心事件。
人们对衰老与长寿的研究逾近百年,但至今为止,控制人类寿命之迷仍未被揭开。尽管衰老本身不是一种疾病,但很多疾病与衰老相关,如肿瘤,老年痴呆(Alzheimer's disease),动脉硬化,代谢紊乱(metabolic disorders)等。心脏病和中风引起的死亡率随年龄呈指数增加,65-74岁死亡率超过40%,85岁及以上的死亡率将近60%。越来越多的研究发现,即使没有高血压,糖尿病及吸烟等各种危险因子存在,心血管系统随年龄增加亦趋于易发疾病和受损,这可能和衰老血管的细胞内稳态相关(Ungvari et al.,2010).在实验动物和人的身上都已证实,氧化损伤产生的活性氧(reactive oxygen species, ROS)激活NAD(P)H氧化酶及下调NO的活性,导致衰老进程的内皮细胞功能障碍,从而引发老年人冠状动脉疾病和中风。
热量限制(calorie restiction, CR)是指在提供生物体充分的营养成分如必需氨基酸、维生素等,保证生物体不发生营养不良的情况下,限制每日摄取的总热量。一般CR饮食是指减少动物随意进食量的30%—40%。热量限制功能以一科,被动的方式降低氧化和其他损伤,从而导致生命周期延长,可以调控从酵母到哺乳动物等生物体的生命周期。自1935年McCay等首次报道CR延长大鼠寿命以来,大量研究已表明CR是除遗传操作以外最强有力的延缓衰老的方法。热量限制不仅能维持许多年轻时的生理状态,还能延缓和预防一些与年龄相关疾病如心血管疾病、糖尿病、肿瘤的发生、发展。热量限制(caloric restriction, CR)是已经被证明具有抗衰老及延长寿限的作用(Russell JC2008; Fontana L,2009;),包括热量摄入限制和减少进食次数两种方式。CR对心血管系统具有多效保护作用,如保护血管(阻止动脉硬化进程),提高心肌耐受性及延缓心脏衰老(Shinmura K.2011),衰老进程中,转录因子NF-kB通过激活IL-1,IL-6,TNF-a等细胞因子介导机体长期慢性炎症反应,这种长期慢性炎症反应引发一系列老年相关疾病的关键。因此,有人提出可以通过抗氧化药物(如姜黄素(curcumin))来抑制依赖NF-kB信号的炎症反应,而延缓衰老(Sikora Eab et al,2010)。研究表明,CR可以降低炎症因子IL-6和leptin的表达(Reed JL,2010)。
本课题分为四个部分,首先依据已经发表的芯片数据结果进行生物信息学的分析和挖掘,具体为：来源于美国Miller SJ提交的GSE7281芯片数据集,Fischer344大鼠自然衰老,年龄分别为3,6,15,28个月,取大鼠动脉提取mRNA
进行全基因组芯片分析
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7281;和来源于美国Becker KG提交的GSE11845芯片数据集,选取数据集中正常对照(自由饮食),热量限制(间断饮食)及高脂(氢化椰子油,热量高于正常对照组的60%)三组样本数据,进行心脏,肝脏及肌肉相关基因分析。http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11845。挖掘动脉硬化与衰老相关基因。接下来建立Wistar大鼠模型,利用RT-PCR, ELISA等方法进行血清氧化低密度脂蛋白(OXLDL),C反应蛋白(C-reactive protein, CRP),血栓前体蛋白(TpP)等靶标的检测以及进行病理分析观察血管形态,对芯片数据挖掘的结果进行进一步的实验验证。在建立高糖饮食与热量限制的大鼠模型的基础上,进一步通过实时荧光定量PCR技术(Real-time quantitative Polymerase Chain Reaction, Real Time PCR)验证生物信息学结果并筛选得到31个同时响应热量限制与高脂高糖饮食的基因表达。通过建立HC诱导动脉硬化的细胞模型(细胞株EA.hy926),对高糖高胆固醇诱导EA.hy926细胞凋亡中对分子伴侣家族一员,HSP60蛋白的表达进行了初步的研究。
结果分述如下：
1.生物信息学挖掘动脉硬化与衰老相关基因。
通过年龄相关大鼠动脉芯片表达谱比较共获得166条探针(155个基因),其中有36个基因响应热量限制与高脂高糖饮食。其中Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt等参与糖代谢,在大鼠衰老的进程中呈下降趋势；而Gygl参与肝糖原合成,随年龄增加而持续上调。HK2, PFKP, Dlat, Pdp2, Acyl等是糖酵解与三羧酸循环的关键调控酶,Tkt, TECR与ACS15则是NADPH提供途径的关键酶。在大鼠衰老的进程中,糖代谢相关的酶表达下调,NADPH生成途径受阻,表明在个体发育成熟后,机体对能量的需求可能减少,NADPH下降可能将导致SIRT1的活性下降。有报道SIRT1对血管及在动脉硬化中具有保护作用,它在热量限制介导机体寿限的延长中起到非常重要调作用。因此,通过热量限制可能上调这些基因的表达,对机体的“燃料”进行充分燃烧,减少脂肪堆积,延长寿限。
生物信息学的分析表明,随着年龄的增长,大鼠动脉血管的细胞粘附因子Vcam1, Icam1, Postn, Fn1, Sdc1, Mfge8, Spp1, Jup, Tln1上调表达,表明内皮细胞随年龄增长功能下降,容易诱发动脉硬化。与血管发育相关的Agt, Colla1, Gja5随年龄增长而下调,而参于血管调节与重塑Bgn, Acvrl1, Fgfr1, Box等则上升。这些基因可能参与高血压的发生,推测热量限制可能抑制Bgn, Acvrll的表达。此外,参与免疫的HSPD1与抗氧化的Pex19, Cyld等基因随年龄呈下降趋势,热量限制显著提高他们的表达。随着年龄的增长,动脉硬化的易感基因表达上调,表明患动脉硬化的风险增加；而热量限制可以下调这些基因的表达,推测其可能预防或延缓动脉硬化的发生。
2.高糖高脂诱导大鼠动脉硬化模型建立以及生物信息学结果的实验验证。
将44只6月和40只11月大的SPF级的Wistar雄性大鼠进行随机分组,FC(自由饮食组)给予基本饲料和普通算来水喂养；CR(热量限制组)每天定时给予基本饲料100-150g/3只老鼠,此根据老鼠一天能刚吃完饲料重量而定；HC(高糖高脂组),饲料组成为：基本饲料+15%猪油+3%胆固醇+5%精制糖。各组大鼠进行2个月和4个月喂食后,心脏取血后,通过血清OXLDL和血浆TpP含量检测,以及血液中CRP的mRNA检测表明,本实验通过高糖高脂喂养的大鼠更有诱发动脉硬化的可能性。并且随年龄增长,长期高糖高脂饮食罹患动脉硬化的可能性更大,而热量限制饮食即可减低此风险。
在建立高糖饮食与热量限制的大鼠模型的基础上,通过Q-PCR检测生物信息学筛选得到31个同时响应热量限制与高脂高糖饮食的基因表达。结果表明,热量限制可以上调成年大鼠动脉中Aldh5al (F=5.301, P=0.022), Ldha (F=2.215, P=0.016), Hk2(F=3.556, P=0.061), Pfkp (F=3.045, P=0.085), Slc2a4(F=4.006, P=0.046), Dlat (F=3.389, P=0.068), Tkt(F=0.177,P=0.008), Gygl (F=5.831,p=0.017)等参与糖代谢相关的基因,而高糖高脂的饮食则起到相反的作用。同时,热量限制还可以有效抑制Icam1(F=3.739. P=0.045)和Tln1(1.546, P=0.050)的表达,但对Vcam1(F=0.177, P=0.267)和Fn1(F=1.495, P=0.102)作用不明显。Zou Y等报道热量限制可以有效抑制不同年龄大鼠l cam1和Vcam1的表达,但热量限制的时间持续13和31个月。本研究是在成年鼠后进行热量限制4个月进行检测,可能由于处理时间偏短,从而影响了结果。此外,热量限制还可以有效抑制Bgn(F=13.189, P=0.001), Acvrll(F=2.306, P=0.142)的表达,而上调HSPD1(F=3.191, P=0.075), Pexl9(2.474, P=0.126), Cyld(F=1.888, P=0.194)等基因的表达。高糖饮食则相反。
3.高糖高胆固醇诱导EA.hy926细胞凋亡中对HSPD1基因表达的影响
随胆固醇浓度的增加,EA.hy926细胞活性下降,呈浓度依赖性。0.8mM胆固醇+5mM葡萄糖共培养12h的细胞活性与正常对照组(5mM葡萄糖+0mM胆固醇)无显著差异(P>0.056),而0.8mM胆固醇+25mM葡萄糖共培养12h的细胞活性与正常对照组(25mM葡萄糖+0mM胆固醇)具显著性差异(F=579.023,P=0.001)。5mM或25mM葡萄糖+0.8mM胆固醇处理EA.hy926细胞不同时间后,细胞活性呈时间依赖性下调,48h后细胞大部分变圆,脱落(F=79.758,P<0.001)。流式检测表明此时,与低糖低脂处理的细胞比较,高糖高胆固醇处理的细胞大部分发生凋亡(F=127.068,P<0.001),HSPD1基因表达显著上调(F=6.959,P=0.006)。
通过上述三个部分的实验,我们得出以下结论：
1.随着年龄的增长,动脉硬化的易感基因表达上调,表明患动脉硬化的风险增加。
2.热量限制可以下调这些基因的表达,推测其可能预防或延缓动脉硬化的发生。
3.高糖高脂喂养的大鼠,长期的HC饮食可使血液中的OXLDL升高；血浆中TpP水平显著高于FC组；而CR组显著低于FC组；从动脉血管横切图看,HC组的血管内皮显著增厚,并不同程度的形成泡沫细胞。表明HC组大鼠动脉已有不同程度动脉硬化的病变,更有诱发动脉硬化的可能性。并且随年龄增长,长期高糖高脂饮食罹患动脉硬化的可能性更大,而热量限制饮食即可减低此风险。
4.热量限制可以上调成年大鼠动脉中Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gygl等参与糖代谢相关的基因,而高糖高脂的饮食则起到相反的作用。同时,热量限制还可以有效抑制Icaml, Tlnl, Bgn, Acvrll, Fgfrl, Bax的表达,而上调HSPD1, Pex19, Cyld等基因的表达。高糖饮食则相反。即并且随着年龄的增长,动脉硬化的易感基因表达上调,患动脉硬化的风险增加；而热量限制可以下调这些基因的表达,从而预防或延缓动脉硬化的发生。相反,高糖高脂饮食则起相反的作用,加快发病及病变的进程。
5.持续激活的HSP60通过激活人血管内皮细胞表达TNF-a,E-选择素,ICAM1,IL6等细胞因子,促进自身炎症发应从而诱发AS。无论是高糖,高糖高脂,或是低糖高脂处理EA.hy926内皮细胞,都可以诱导HSPD1的nRNA表达,但高糖高脂诱导表达的效果最显著。因此,适当控制饮食摄入糖与脂含量,可预防或延缓AS的发生
Cardiovascular disease is the predominant cause of death in developed countries population, mortality also showed a tendency to increase year by year in China. Atherosclerosis (AS) cardiovascular disease is called the number one killer in developed countries, and the incidence in developing countries is also high (Shimizu, T,2008). Coronary atherosclerotic heart disease referred to as coronary heart disease (CHD) is a common disease, has become a most common kinds of heart disease in the United States and Europe. In1990, The global population is5.3billion, and the death of50million, of which6.3million died of coronary heart disease, accounting for12.4%.1991American Heart Association announced that deaths of coronary heart disease account for45.3%to total deaths in1988, and about1/5of Americans die of cardiovascular disease before the age of65. As an Asian country, coronary heart disease morbidity and mortality of China is in the ranks of lower-prone countries, but with the changing life styles, it increased in recent years. In hospitalized patients with heart disease, the proportion of the disease are also increasing. The basic pathogenesis of coronary heart disease is atherosclerosis (As). The common mechanism are AS plaque instability, caused by local platelet aggregation and acute thrombosis, acute occlusion of the lumen, and acute coronary syndrome (ACS). With China's economic and social development and improved living standards and dietary level of habit change, and the consequent aging of the population, atherosclerosis sclerosis diseases gradually become China's leading causes of death (Mu Yi-ming,2001). The World Health Organization predicted that upto2020after worldwide infectious disease case fatality rate has been effectively controlled, Heart tube disease will become the world's first cause of death.
Atherosclerosis (As) is an important pathological basis of cardiovascular and cerebrovascular diseases, prevention AS is a fundamental measure of prevention and treatment of cardiovascular diseases. Atherosclerosis (AS) is characterized by a lipid-rich plaques in the aorta wall gathering system disease, and is the major pathological basis of cardiovascular and cerebrovascular disease.it is a multi-factor chronic arterial disease, the concentrated expression of systemic vascular endothelial function obstacles (endothelialdysfunction,), intimal chronic inflammation, fibrosis, stenosis and thrombosis formation. The main clinical manifestations of myocardial infarction, stroke and peripheral vascular disease, often accompanied by hypertension, hypercholesterolemia or diabetes. Because of the complexity of the etiology and pathology, AS has not yet been fully elucidated, but it is certain that AS is related to a variety of genetic and environmental factors of complex diseases, the interaction between these genetic factors and environmental factors ultimately lead to plaque formation and the emergence of series of subsequent clinical events. Many AS vascular disease etiology, which is closely related to many lifestyles, including smoking, high fat diet, lack of physical activity habits. From the biological perspective, AS is a chronic inflammatory disease, and dyslipidemia is closely related to its development and it is a very complex process. The clinical events of AS disease are coronary heart disease and stroke.
Therefore, the AS mechanism have become an increasing concern, elevated blood lipids and disorder are the premise and basis of most of the AS. Endothelial dysfunction is the originating events of AS; and a variety of factors interact, result in the occurrence and development of AS. The main factors include:vascular endothelial cell dysfunction, vascular smooth muscle cells, platelets and blood coagulation state of exception, monocytes, macrophage infiltration, free radical damage, viral and bacterial infections, apoptosis, inflammation and immune response, calcium deposition, and genetic abnormalities. The pathogenesis is now generally recognized as "injury stress theory (Ross, R,1999). The process involves in AS containing the following steps:a variety of stimuli and injury of endothelial cells (vascular of endothelial cell, VEC); monocytes transmit through the lesion into the intima and tranform into macrophages; macrophages together with the role of T lymphocytes secrete cytokines transmit into macrophage-derived foam cells; medial vascular smooth muscle cells move into the intima, engulfed lipid to form foam cell then proliferation and migration to form a fibrous cap; fiber cap weakened by matrix metalloproteinases and y-dry detoxification secreted by macrophage and T lymphocyte, the formation of atherosclerosis physics and chemistry of the emboli.
As to the formation of AS, There are more and more attention paid to the immune and inflammatory theory in recent years. Studies suggest that AS is a protective inflammation-fiber proliferative response to the local damage of the vascular endothelial. If the injury sustained period of time, this response becomes excessive, and eventually become diseases that plaque formation. Lipid deposition in the plaque formation process is the most important factor in the damage response and is also one of the earliest performance. Along with lipid deposition, the formation of oxidized low-density lipoprotein cholesterol (by oxLDL-C), circulating leukocytes and monocytes are activated and migrate to the lesion, which become activated by oxLDL-C under macrophages through their scavenger receptor uptake by oxLDL-C into a foam cell generation and accumulation of foam cells lead to the formation of lipid stripes. The release of the deposition of cholesterol cool plasma lipoproteins from the foam cell death constitute the plaque lipid core. Inflammatory response to the continued development of T cell activation, triggered fiber proliferative response ultimately the formation of the fibrous cap. Early lipid core plaque formation in small, fibrous cap thickness, plaque steady state, along with the continued death of foam cells and the deposition of plasma lipids, lipid core plaque growing on the other hand a large number of infiltration of macrophages to release large amounts of hydrolytic enzymes, especially metalloproteinases series, gradually thinning the fibrous cap by degrading the fibrous cap, as well as inhibit the formation of collagen fibers, so that stable plaques transmit into unstable plaques, and the latter influenced by the inner and outside factors, and the final breakdown lead to acute coronary events. The pathologist also found a large number of macrophage infiltration, and the increased expression of inflammatory markers in the plaque rupture site. Thus, the continuous progress of the plaque and the extent of the inflammatory response are closely related. The rupture of the plaque is closely related to the intensity of inflammation and macrophage infiltration in plaque. The formation of the fatty streak or plaque within the arterial wall is the characteristic lesions of AS. However, Macrophages as well as central and membrane migration to the intima of the arterial intima smooth muscle cells via the scavenger receptor (scavenger receptor, SR) uptaking of oxidized low-density lipoprotein (oxidizedLDL, oxLDL), and thus the formation of macrophage/smooth muscle cells derived foam cells, is a central event in the development of AS process.
Over the past century, people have done some research on aging and longevity but so far, no news has been announced. Although aging itself is not a disease but many diseases associated with aging such as cancer, Alzheimer's (Alzheimer's disease), atherosclerosis, metabolic disorders (metabolic disorders). Heart disease and stroke mortality with age exponential increase in mortality at age65-74more than40percent, nearly60%of the mortality rate of85years. Growing body of research found that, even in the absence of other risk factors as hypertension, diabetes and smoking, cardiovascular system increases with age have become more prone to disease and damage, this may homeostasis and aging vascular cells related (Ungvari et al,2010). In experimental animals and human body have confirmed that the oxidative damage generated by reactive oxygen species reactive oxygen species (ROS) in activation of NAD (P) H oxidase and lowered the activity of NO, leading to endothelial cell dysfunction of the aging process, causing the elderly coronary artery disease and stroke.
Calorie restriction (calorie restiction, CR) is to provide the organism full of nutrients such as essential amino acids, vitamins, etc., to ensure that organisms do not occur malnutrition, limiting the daily intake of total calories. General CR diet is reduced by30%-40%of the animal free food intake. The function of caloric restriction in a passive way to reduce oxidation and other damage, leading to prolonged life cycle, can regulate the life cycle from yeast to mammals and other organisms. Since1935McCay, first reported in the CR to prolong rats life, a large number of studies have shown that CR is the most powerful anti-aging methods In addition to genetic manipulation. Calorie restriction not only to maintain the physiological state of many a young man, but also delay and prevent the age-related diseases such as cardiovascular disease, diabetes, tumor occurrence and development. Calorie restriction (caloric restriction, CR) has been shown to have anti-aging and extend the life of the role (Russell JC2008; Fontana, L,2009), including calorie intake limit and reduce the consumption of the number of two ways. Multi-effect protective effect of CR on the cardiovascular system, such as protection of blood vessels (to prevent atherosclerosis process) to improve myocardial tolerance and delay cardiac aging (Shinmura K,2011). In the aging process, the activation of IL-1, IL-6and TNF-a and other cytokine by the transcription factor of NF-kB, mediated body chronic inflammation, which lead to the key of a series of aging-related diseases. Therefore, it was suggested by antioxidant drugs (such as curcumin (curcumin)) to suppress the inflammatory response dependent on NF-kB signaling and aging (Sikora, the Eab et al.,2010). It is shown that the CR can reduce the inflammatory cytokine IL-6and leptin expression (Reed, JL,2010).
This topic is divided into four parts, Firstly, based on published microarray data results, we do bioinformatics analysis and data mining, as the following:derived from the GSE7281microarray data sets submitted by the United States Miller, the SJ, Fischer344rats of natural aging, aged3,6,15,28months, the artery mRNA was extracted from rat to do whole genome microarray analysis. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7281, Submitted from the United States Becker, KG the GSE11845chip data set, select the data set in normal controls (free diet), calorie restriction (discontinuous diet) and fat (hydrogenated coconut oil, heat higher than60%of the normal control group) of three groups of samplesdata, heart, liver and muscle-related gene analysis. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11845.
Mining of atherosclerosis and aging-related genes. Then set up Wistar rat model, Using the RT-PCR, ELISA methods, we do serum oxidized low-density lipoprotein (of OXLDL), C-reactive protein (C-of reactive protein, CRP), thrombus precursor protein (TpP) targets detection and pathological analysis of the observed vascular morphology, and further experimental validation of the results of the microarray data mining. High carbohydrate diet and calorie restriction in a rat model based on the real-time fluorescence quantitative PCR (Real-time quantitative Polymerase Chain Reaction and Real Time PCR), to verify the results of bioinformatics and filter31 while responding to caloric restriction gene expression and high-fat high-sugar diet. EA.hy926cell apoptosis through the establishment of the HC induced cell model of atherosclerosis (cell line of EA. Hy926), high glucose and high cholesterol induced a preliminary study of a molecular chaperone family of HSP60protein expression. The results are as follows:
1. Bioinformatics mining of atherosclerosis and aging-related genes.
The total of166probes (155genes) were obtained from the Age-related rat arterial microarray, of which36genes respond to calorie restriction and high-fat high-sugar diet. The Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, etc. involved in glucose metabolism, a downward trend in the aging process in rats; Gyg1involved in glycogen synthesis, increased with age and continued upward. HK2, PFKP, Dlat, Pdp2, Acyl, etc. are a key regulatory enzyme of glycolysis and the citric acid cycle, Tkt, TECR, and ACS15are the key enzyme of the NADPH pathway. Along with the aging process in rats, the glucose metabolism enzyme expression and NADPH formation routes blocked, suggesting that individual mature, the body may reduce the energy demand of NADPH, the decline may lead to decreased activity of SIRT1, It is reported that SIRT1has a protective effect to blood vessels and atherosclerosis, and it play a very important role in mediating the body the life extension by calorie restriction. Therefore, caloric restriction may increase the expression of these genes, the body's "fuel" burning, reduce fat accumulation, and to extend the lifespans.
Bioinformatics analysis showed that the increase with age, rat arterial vascular cell adhesion molecule Vcam1, Icaml, Postn, the Fn1, Sdcl, Mfge8, Sppl, Jup, Tln1upregulated, indicating that the decline of endothelial cells increased with agelikely to cause atherosclerosis. Agt, Collal, Gja5which related to the vascular development, will decrease, and Bgn, Acvrll, Fgfrl, Bax which are involved in vascular regulation and remodeling will rose. These genes may be involved in the occurrence of hypertension, suggesting that caloric restriction may inhibit the expression of Bgn, Acvrl1. In addition, the HSPD1involved in the immune and antioxidant genes such as Pex19, Cyld declined with aging, calorie restriction significantly increased their expression. With aging, atherosclerosis susceptibility genes were up-regulated, indicating that the increase in the risk of suffering from arteriosclerosis; calorie restriction lowered the expression of these genes, suggesting that it may prevent or delay atherosclerosis.
2. High glucose and fat induced atherosclerosis Rat model and experimental verification of bioinformatics result.
44, six months and40, eleven months SPF male Wistar rats were randomized to FC (free diet group) to give basic feed and count runoff feeding; CR (calorie restriction group) time every day to give the basic feed100～150g/3mice, according to the mouse day had just eaten the feed weight; HC (high-sugar high-fat group), feed composition:basic feed+15%lard and3%cholesterol,5%refined sugar. Rats in each group after2months and4months feeding, cardiac blood was collected by the serum OXLDL and the plasma TpP content testing, and blood CRP mRNA detection showed that this experiment in rats fed by high glucose and fat more the possibility of induced atherosclerosis. And with aging, they are more likely to suffer from arteriosclerosis in long-term high-fat diet, however, calorie restriction diet can reduce this risk.
Q-PCR was done to verificated the bioinformatics results, Based on the high-sugar diet and calorie restriction rat model, screened31gene expression in response to calorie restriction and high-fat high-sugar diet. The results show that calorie restriction can increase the adult rat arterial Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gygl and other genes involved in glucose metabolism, high-sugar high-fat diet have the opposite effect. The same time, calorie restriction can effectively inhibit the expression of Icam1and Tln1, but not obvious effect on Vcaml and Fnl gene. Zou, Y reported that caloric restriction can effectively inhibit the expression of different ages the rat Icam1and Vcam1, but the heat restrictions are thirteen months and thirty-one months. In this study, the adult mice were calorie restriction for four months for testing, maybe due to the shorter processing time, thus affecting the results. In addition, calorie restriction can effectively inhibit the expression of Bgn, Acvrl1, Fgfr1, Bax, and activate HSPD1, Pex19, Cyld gene expression. The high carbohydrate diet is the opposite.
3. High glucose and high cholesterol induce EA.hy926cell apoptosis and have effect on HSPD1gene expression.
With increasing cholesterol concentration of EA. Hy926cell activity dropped to a concentration dependent manner. But the cell activity has no significantly different between the0.8mM cholesterol+5mM and glucose co-cultured for12h and normal control group (5mM glucose+0mM cholesterol)(P>0.05); There is significant difference between the0.8mM cholesterol+25mM and glucose co-cultured12h and normal control group (25mM glucose+0mM and cholesterol)(P<0.01).5mM or25mM glucose+0.8mM cholesterol dealing with EA.hy926cells at different times, cell activity was time-dependent decrease, most of the cells rounded off after48h. Flow cytometry showed that the majority ofcells treated with high sugar and high cholesterol treated cells undergo apoptosis (50%) contrast to low-sugar low-fat treatment, and HSPD1gene expression was significantly upregulated (F=6.959, P=0.006).
Through above three-Part test, we draw the following conclusions:
1. With aging, atherosclerosis susceptibility genes upregulated, indicating that the risk of an increased risk of atherosclerosis.
2. Calorie restriction can downregulate the expression of those genes, suggesting that it may prevent or delay atherosclerosis occurrence.
3. High sugar and high fat fed rats, the long-term HC diet can increase blood OXLDL; the plasma TpP level significantly higher than the FC group; CR group were significantly lower than the FC group; from the arterial cross-section diagram, HC group of vascular endothelial significantly is thicker, and varying degrees of formation of foam cells. So that, the HC rats artery has varying degrees of atherosclerosis lesions, the possibility of more-induced atherosclerosis. And with aging, It is more likely to suffer from arteriosclerosis in long-term high-fat diet, however, calorie restriction diet can reduce this risk.
4. Caloric restriction can increase the Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gyg1and other genes involved in glucose metabolism in the adult rat arteries, high-sugar and high-fat diet have the opposite effect. The same time, calorie restriction can effectively inhibit the expression of Icam1, Tlnl, Bgn, Acvrll, Fgfr1, Bax, and to upregulate HSPD1, Pex19, Cyld gene expression. The high carbohydrate diet is the opposite. And with aging, atherosclerosis susceptibility gene expression increased, which raised risk of atherosclerosis; calorie restriction lowered the expression of these genes, thus preventing or delaying the occurrence of atherosclerosis. In contrast, high-fat diet is counterproductive to speed up the process of onset and lesions.
5. The sustained activation of HSP60activate the expression of TNF-a, E-selectin factors, ICAM1, IL6, cytokines by the human vascular endothelial cells to promote the inflammation and induce the AS. Although high-sugar, high sugar and fat or low sugar and high fat treatment on lipid EA.hy926endothelial cells can induce HSPD1mRNA expression, but high glucose and fat-induced expression of the most significant effect. Therefore, an appropriate control of dietary intake of sugar and fat content, can prevent or delay the occurrence of AS.
引文
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