| Cigarette smoking is an important risk factor for cardiovascular diseases, including myocardial infarction, coronary heart disease, cerebrovascular diseases, aortic aneurysm, peripheral vascular diseases, et al. It was reported that about 1.6 millions of cardiovascular deaths were caused by cigarette smoking worldwide in 2000. Smoking was shown to increase inflammation and oxidative stress, leading to endothelial dysfunction, and contributing to thrombus formation, and smoking cessation was associated reduced risk of above mentioned disorders.Nicotine is the most abundant component of cigarette, which is considered to play the major role of cigarette smoking However, the mechanism of nicotine-induced endothelial dysfunction is not fully elucidated.Previous studies focused on nicotinic acetylcholine receptor (nicotinic acetylcholine receptor, nACHR). Studies in vitro found nACHR is involved in nicotine mediated endothelial cell proliferation, migration and angiogenesis and those in vivo demonstrated that nicotine contributed to angiogenesis, and this angiogenic effects could lead to plaque instability and further development of atherosclerosis, tumor growth and other smoking-related diseases. It was also reported that renin - angiotensin - aldosterone system was over-activated in smokers, angiotension converting enzyme inhibitors (ACEI), by alleviating endothelial dysfunction and inhibiting oxidative stress, could regulate the expression of eNOS in endothelial cells, and reduce endothelial cells damage caused by nicotine. Therefore, we hypothesize that nicotine induce endothelial dysfunction through AT1 receptor.Objective:1. To study whether nicotine have influence on expression of AT1R on HUVEC.2. To study whether nicotine influence the expression of eNOS and ICAM-1.3. To study whether the change of AT1R effect the change of eNOS and ICAM-1on nicotine.Methods:HUVEC was co-cultured with 10-6M nicotine at different time poin(t0h,2h,6h,12h,18h,24h)to observe the change of AT1R on membrane , eNOS and ICAM-1 in cytoplasm; HUVEC incubated with 0,10-8,10-7,10-6M nicotine for 24 hours when the change of ATIR was most apparent and 12 hours when the change of eNOS was most apparent and the ICAM-1 have significance simutaneously. We obtained the notable time and concentration point about the change of AT1R,eNOS and ICAM-1 from this experiment and then randomly divided experiment into control group, nicotine treatment group, losartan + nicotine treated group, AT1R siRNA interference + nicotine intervention group, the change of eNOS and ICAM-1 detected by western blot. Using SPSS18.0 to analysis, data are presented as means±standard, one-way ANOVA was used to compare more than two means, significance was determined by P<0.05.Results:1. HUVEC incubated with 0,10-8,10-7,10-6M nicotine for 24h ,the AT1R increased gradually as the concentration of nicotine increased. Compared with control group, there was no significantly statistic difference on concentration of 10-8 or 10-7M nicotine intervention ,but 10-6M nicotine intervention have(p﹤0.05). HUVEC incubated with 10-6M nicotine 2h induced upregulation of AT1R, and the expression of AT1R gradually increased as the intervention time increased.2. Incubatation with 10-6M nicotine upregulated the eNOS on HUVEC at 6 h, with its peak at 12h and decline at 24h, ICAM-1 upregulationed at 2h, peaked at 6h and reduced after 12h. Incubated with 0,10-8,10-7,10-6M nicotine for 12h, eNOS and ICAM-1 increased followed with the increase of nicotine concentration.3. Giving losartan 1h in advance or silenced AT1R reduced the expression of eNOS, but had no effect on ICAM-1.Conclusions:AT1 recptor activation plays a role in the nicotine-induced endothelial dysfunction.
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