| 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. |
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