| Background:Atherosclerosis(AS) is the most common, the most important one in a vessel disease called arteriosclerosis. The common feature of atherosclerosis is the thickening of the oarterial wall hardens, loses its elasticity and lumen narrowing. Atherosclerosis is characterized by lesions of the affected artery from the endometrium, there have been a variety of diseases in combination, including the local accumulation of lipids and complex carbohydrates, fibrous tissue and calcification to plaque formation, and arterial media gradual degeneration.secondary lesions still including plaque hemorrhage, plaque rupture and local thrombosis (known as atherosclerosis-thrombosis.) formation. Modern cellular and molecular biology techniques indicated atherosclerotic lesions characteristics by macrophages wander, and smooth muscle cell proliferation; a large number of collagen fibers, elastic fibers and proteoglycans of connective tissue matrix formation; and cells inside and outside the lipid accumulation. Due to the appearance of the accumulation of lipid in the arterial intima yellow atherosclerotic, so called atherosclerosis.Nicotine is an unpleasant, bitter, colorless transparent oily liquid, highly volatile and easily oxidized in air into dark gray, and can rapidly dissolve in water and alcohol through the nose and mouth bronchial mucosa, the body can easily beabsorption. Glued to the surface of the skin nicotine can be absorbed into the body. When nicotine enters the body, will have many roles and become the main accomplice of many deseases, such as the limbs, peripheral vasoconstriction. tachycardia, increased blood pressure, faster breathing, mental status changes (for example, become the emotional stability or mental excitement), and to promote platelet aggregation.Nicotine is accessory cause for cardiovascular obstruction, hypertension, stroke and other cardiovascular diseases.Apolipoprotein E (ApoE) is a polymorphic protein, transformation and metabolic processes involved in the lipoproteins, their genes can regulate many biological functions, and the pathogenesis of many cardiovascular diseases.Research on ApoE and its gene polymorphism is one of the hot spots of medical research currently. To explore the intrinsic link between ApoE and its gene polymorphism has important clinical value to prevention, diagnosis and treatment of cardiovascular disease. Apolipoprotein E was found in CM and VLDL, IDL mainly, and part of in the HDL, normal human plasma ApoE concentration is0.03～0.05g/L. The concentration of Apo-E and plasma triglyceride content was positively correlated. The ApoE physiological functions including:(1) as the LDL receptor ligand, hepatocyte CM remnant receptor ligands, which are closely related to lipoprotein metabolism;②ApoE has polymorphism, the polymorphism is to determine the level of individual lipids and is closely related to the occurrence and development on atherosclerosis;③involved in the activation of lipolytic enzymes, immune regulation and regeneration of nerve tissue.Transforming growth factor-beta (TGF-β) are a group of newly discovered regulation of cell growth and differentiation of TGF superfamily. This family members including TGF-β,activins,inhibins,Mullerian inhibitor substance (MIS), and bone morpho-genetic proteins (BMPs). The naming of the TGF-β cytokines enable normal fibroblasts phenotypic conversion to occur in the simultaneous presence of epidermal growth factor (EGF), change the fibroblast towel wall growth characteristics obtained in the agar growth in capacity, and loss of inhibition of the growth of medium density-dependent. TGF-β and the previously reported growth inhibitory factor secreted from the epithelial cells of African green monkey kidney BSC-1is the same thing. At first, the research on biological function of TGF-β was in inflammation, tissue repair and embryonic development.while in recent years many scientist found that TGF-P has an important role in the regulation on cell growth, differentiation and immune function. The function of TGF-β1is similar with TGF-β2and TGF-β3function. Generally speaking. TGF-P stimulated the cells of mesenchymal origin, while inhibited epithelial or nerve cells of epiblast progenitors sources.The biological role of TGF-β include the following aspects:(1) inhibits the proliferation of immunocompetent cells;(2) the regulation of cell phenotype;(3) inhibition of lymphocyte differentiation;(4) inhibition of cytokine production:such as inhibition in PBMC IFN-y and TNF-alpha production.(5) other regulation. TGF-β is not only a powerful activator of connective tissue, but also the most powerful value-added smooth muscle inhibitor by far. From a sense that the majority of cells able to form a TGF-β. and the formation mainly by platelets and active macrophages. Most cells secrete TGF-beta was lurking, the only activity was in the PH value lower or hydrolyzed protein split. Smooth muscle cell proliferation inhibitors such as TGF-β and irritants such as PDGF balance was very important to smooth muscle cell proliferative responses and led to the formation of atherosclerotic lesions. Therefore, by macrophage-derived foam cells, including subcutaneous interstitial appropriate active can significantly affect the secretion of growth factors, which chemokines attract smooth muscle cells from the interstitial transferred to the intima. and the formation of intimal.muscle fibers, proliferation lesions. TGF-β have potential applications to the treatment of wound healing, and promote the repair of cartilage and bone, and by the immunosuppressive treatment of autoimmune diseases and transplant rejection.Regulatory T cells (Treg) is a kind of negative regulatory role of T cells subsets,included in the thymus gland differentiation within a naturally regulatory T cells (such as CD4+CD25+T cells) and in the thymus outside the adaptability of hyperparasites regulatory T cells (such as Tregl cells and Th3cells). Regulatory T cells is different from Thl and Th2with the control function of the T cells group, because of its immune suppression effect, in a variety of autoimmune disease characterized by plays an important role in the regulation, recently under people’s extensive concern. TGF-β is one of the main medium which related atherosclerosis of Treg cell function.Since people found TGF-β defects mice causes a variety of inflammatory disease, the function of TGF-beta in the immune system caused scientist’s attention. These function was related to strengthen T cells proliferation, activate and T cells differentiation to Th1and Th2. In this case the T cells activate due to TGF-β has the function of inhibit proliferation, activation, and T cells differentiation to Th1and Th2. In addition, TGF-β1of peripheral tissue like a collaborative stimulating factor to maintain Treg cells express Foxp3. The double effect of effect T cells and Treg cells may contribute to the TGF beta regulation of peripheral T cell tolerance.Apoptosis is a factors from inside and outside of body trigger intracellular stored death program leads to cell death process, also known as programmed cell death. Apoptosis as a physiological process in ensuring normal development, growth, maintain inner environment plays an important role, and play an active defence function. Nicotine can regulated apoptosis, this regulation can be through a variety of receptors such as the nAChR nicotinic acetylcholine receptors, dopamine receptors, the effects of glutamate receptor expression to the regulation of apoptosis, or nicotine through multiple signaling pathways regulating apoptosis, it has been found associated with nicotine related signaling pathways of Ca2+,3MAPK pathway (ERK, p38and JNK/SAPK), AKT pathways and mitochondrial pathways. People have studied nicotine regulated cell apoptotic phenomena extensively, the molecular mechanism of the apoptosis of nicotine regulation also has a relatively systematic understanding, and biological significance a deeper understanding between nicotine and apoptosis. Objective:1. To investigate under the different concentration of nicotine stimulation, atherosclerotic plaque formation on Apo-/-E mice and the changes of peripheral blood regulatory T cells and TGF-β1value.Methods:Through2different concentrations of [2.0,0.5mg/(kg, d)] nicotine and saline on Apo-E-/-mice peritoneal injection after12weeks (18mice were divided into3groups randomly:high-dose nicotine group,low-dose nicotine, blank control group, with6mice in each group), detected peripheral blood regulatory T cell with flow cytometry, detected peripheral blood TGF-β1value by ELISA.Apo-E-/-mice aortic root were carried out and maken pathological sections and follow by HE staining.Statistical analysis was performed using the statistical software of SPSS13, measurement data with mean±SD (x±s), using repeated measures analysis of variance on the mouse body quality, regulatory T cells/CD4, TGF-β1values to analyzed its difference, data which in the groups before and after treatment were used the paired t test to analysis of its differences, using One-way ANOVA analysis differences between groups; One-way ANOVA was used to analysis mice lipids and plaque area difference among3groups; using Pearson correlation coefficient on the treatment of regulatory T cells/CD4and plaque area, TGF-β1value after the treatment and plaque area. Multiple comparison of homogeneity of variance of each group by LSD test, variances in Tamhane inspection. Probability values of P<0.05were considered statistically significant.Results:1. The time factor is the impact of body mass in mice with significant difference (time main effect F=40.193, P<0.01), grouping factors on body mass in mice without significant difference (group main effect F=0.079, P>0.05), the interaction effect is not significant (F=0.728, P=0.499).After12weeks treatment, the mouse body mass greater than before the treatment, effects of nicotine on the mouse body mass without significant effect.2. Nicotine have the trend to lower the level of total cholesterol, high density lipoprotein cholesterol and elevated low-density lipoprotein cholesterol, but in the lipid four indicators, each indicator between groups showed no significant differences (P>0.05).3. Effect of nicotine to the mice aortic root plaque histological features:12weeks after the treatment, mice aorta was sliced in cross-section and HE staining, three groups of ApoE knock out mice are visible in various arterialatherosclerotic lesions, and can be seen in the plaques of different stages of development of fibrosis and atherosclerotic lesions, causing the blood vessels of different degree of stenosis. After12weeks’ treatment, with the blank control group, plaque areas of low-dose and high-dose nicotine group were increased, the higher the concentration of nicotine, the greater the plaque area. Significant difference between the groups, pairwise comparisons (Tamhane method) showed a significant difference (F=89.213. P=0.000).4. With repeated measures analysis of variance, the interaction effects of time factor and the grouping factor is significantly (F=12.521, P=0.001). Regulatory T cells/CD4in the different groups before it is processed difference not statistically significant (F=0.406. P=0.674);While after the treatment, regulatory T cells/CD4between different groups was statistically significant (F=11.934, P=0.004); regulatory T cells/CD4value in the control group before and after treatment was no significant difference (t=-0.035, P=0.973), low-dose nicotine group before and after treatment regulatory T cells/CD4value of the difference was statistically significance (t=8.933, P=0.000), high-dose nicotine group before and after treatment regulatory T cells/CD4difference was statistically significant (t=4.887. P=0.005). Regulatory T cells/CD4value of treated was less than that before treatment, different concentrations of nicotine can reduce the regulatory T cells/CD4value.5. Analysised with repeated measures ANOVA, the interaction effects of time factor and the grouping factor is significantly (F=15.302,P=0.000). No significant differences in TGF-β1values between the different groups before treatment (F=0.053, P=0.949). TGF-β1difference after treatment between different groups was statistically significant (F=35.640, P=0.000); Difference in control group TGF-β1value before and after treatment was no statistically significant (t=-0.247, P=0.815), low-dose nicotine group before and after treatment of TGF-β1value difference was statistically significant (t3.748, P=0.013), high-dose nicotine group before and after treatment of TGF-[31value of the difference was statistically significant (t=7.374. P=0.001). TGF-β1values before treatment more than that after treatment, the two dose of concentrations of nicotine could reduced the TGF-β1values both.6. Using Pearson product-moment correlation coefficient to analysis correlation between atherosclerotic plaque area and TGF-β1value, results show that the atherosclerotic plaque area and TGF-β1value is negative correlation significantly (r=-0.892, P=0.000).7. Using Pearson product-moment correlation coefficient to analysis correlation between atherosclerotic plaque area and regulatory T cells/CD4, results showed that the atherosclerotic plaque area and regulatory T cells/CD4were negatively correlated (r=-0.739, P=0.000). Conclusion:1.Nicotine could induced the formation of atherosclerotic plaque, may be related to nicotine reduces regulatory T cell numbers and TGF-beta1value.2.Atherosclerotic plaque area and TGF-beta1value was negatively correlated.3.Atherosclerotic plaque area and regulatory T cells/CD4value was negatively correlated. Objective:To investigated the effect of different concentrations of nicotine on apoptosis and necrosis in human umbilical vein endothelial cells (HUVECs).Methods:Using CD34by immunohistochemical method for identification of HUVECs; By3different concentrations of nicotine (3,30,300ng/ml) stimulation of HUVECs24h, using flow cytometry measured its apoptosis and necrosis rate.SPSS13.0software was used to analysis the experimental results, experimental data using the mean±SD (x±s) to express, among multiple groups compared with single factor analysis of variance, if the variance is equal used LSD test, while the unequal variances is used Tamhane test, correlation analysis using Pearson product-moment correlation coefficient, Probability values of P<0.05were considered statistically significant.Results:1. In apoptosis rate, the different among all groups was statistically significant (F=12.955, P<0.05); low concentrations(3ng/ml) of nicotine to promote apoptosis strongest, with the remaining three groups were statistically significant (P <0.05);However, no significant differences between control group and the group of high concentrations(300ng/ml)(P>0.05)or moderate concentrations(30ng/ml)of nicotine concentration and high concentrations of nicotine group (P>0.05).2. In cell necrosis, different between the four groups was statistical significance (F=83.728, P=0.000), along with nicotine concentration increased, cell necrosis rate also increasingly grow in quantity, high concentrations of nicotine promotes cell death was strongest, any two groups among four groups comparisons were statistically significant difference (P<0.05).3.Cell necrosis rate and nicotine concentrations were positively correlated (r=0.866, P=0.000).Conclusion:Effects of nicotine on the apoptosis of HUVECs was concentration dependent, cell necrosis rate was positively correlated with nicotine concentrations. |
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