Research Of SIRT3on Proliferation Of VSMC Induced By AngⅡ

BackgroundCoronary atherosclerotic heart disease (CHD) is a common disease which is a seriously hazard to human health. Each year about20million people died because of the acute cardiovascular events around the world, more than half them were caused by CHD. In recent years, with the improvement of people’s living standards, change in diet, and the increases of living pressure, CHD outstanding performance characteristics of younger, high incidence, high morbidity and high mortality. Therefore, the work to prevent, early diagnose of CHD is imminent, have to cause a common concern of the whole world. Familiar with the pathogenesis of CHD is the basis of our work. Vascular smooth muscle cells (VSMCs) is the main cells in the medial layer of the vessel wall, studies have shown that VSMCs will change quickly from the contractile phenotype to proliferative phenotype in pathological conditions, cell function will also change at the same time from the adjustment of vascular tone to proliferation and migration capacity, this phenotype transition has an important role in the onset and development of CHD and in-stent restenosis.The Sirtuin family is genes discovered in the study of yeast transcriptional silencing widely present in various species, can adjust the acetylation state of histone which must need NAD+participation. And Sirtuin plays an important role in gene silencing, chromatin stability, repair of faulted DNA as well as the extension of cell cycle. There are7Sirt homologous genes in mammalian and named respectively from Sirtl to Sirt7. The silencing signal adjustment factor3(Sirt3) is a NAD-dependent deacetylase family member which was found positioned in mammalian cells mitochondrial firstly. Activation of Sirt3gene has been linked to prolong human life span. Sirt3is widely present in mitochondrial matrix and nucleus, the distribution in tissues and organs are very widely:the brain, liver, spleen, testes, skeletal muscle, kidney, heart, thymus, lungs, bone marrow, uterus and ovaries and in some other organs also can detected to its mRNA expression, and high expression in metabolically active tissues such as muscle, liver, kidney and heart. Recent studies have reported that Sirt3overexpression can protect myocardial cells from multiple stimuli. Sirt3-LKB1-AMPK pathway activated by exogenous NAD has a protective effect in myocardial remodeling induced by agonist. Whether Sirt3expression in vascular tissue and whether there will be a similar protective effect in the vessels have not been reported.Purpose(1) To detect whether Sirt3expressed in VSMCs of mice.(2) To explore the role of Sirt3in VSMCs.Methods1. Cell cultureVSMCs of C57mice was kindly provided by key laboratory of Cardiovascular Remodeling and Function Research,Qilu Hospital of Shandong University. The culture medium was compounded by DMEM,10%fetal bovine serum and1%double antibiotics. Cells were cultured at37℃,5%CO2incubator. Using concentration gradient Ang Ⅱ (10-7mol/L,10-6mol/L,10-5mol/L) stimulate cells, to produce cell proliferation model.2. Real-time quantitative PCRThrough the following steps:total RNA extracted, reverse transcription and RT-PCR to detect the expression of Sirt3at mRNA level in different groups.3. Western blotVia total protein extracted, electrophoresis, transfer, blocking, mark primary antibody and secondary antibody and coloration to detect Sirt3protein expression in each group.4. Sirt3-SiRNA and plasmid transfectionSirt3-SiRNA was synthesised by Reagent Company, and screened by PCR, Sirt3-plasmid was extracted in E. coli. Sirt3-SiRNA and plasmid were transfected by Lipo2000to silencing or overexpression the expression of Sirt3, and then detect the role of Sirt3in the proliferation of VSMCs.5. Edu kit to detect cell proliferationUsing5-ethynyl-2’deoxyuridine (Edu) kit to detect the level of cell proliferation. 6. Statistical analysisEach group of experimental data was presented by mean±standard deviation (x±s), the comparison between the experimental group and the control group use t test, the rest three experimental groups compared with each other use One-way ANOVA analysis, and the pairwise comparisons were used by SNK method. We consider P<0.05as statistically significant standard.Results1. In preliminary experiments we used Western blot to detect the expression of Sirt3in VSMCs in mice and confirmed Sirt3is stably expressed in VSMCs.2. Real-time quantitative PCR data through calculate and statistical analysis display, the Sirt3mRNA expression in three AngⅡ groups was significantly higher than those in the control group (P<0.01), and the Sirt3mRNA expression in AngⅡ10-6mol/L group was more than that of AngⅡ10-7mol/L and AngⅡ10-5mol/L group (P<0.05), but the comparison between AngⅡ10-7mol/L and AngⅡ10-5mol/L group showed no significantly difference.3. Western blot showed that Sirt3protein expression in three the AngⅡ groups were significantly higher than that in the control group (P<0.01), and similarly with the RT-PCR results:the Sirt3protein expression level of AngⅡ10-6mol/L group was higher than the other AngⅡ groups (P<0.05), and the comparison between AngⅡ10-7mol/L and AngⅡ10-5mol/L group showed no significantly difference.4. After SiRNA and plasmid transfection the expression of Sirt3was detected by western blot, the results exhibited that the expression of Sirt3protein was significantly reduced in siRNA transfection group (P<0.01) and significantly increased in sirt3-plasmid transfection group (P<0.01).5. Using Edu kit to detecte cell proliferation rate in each group, the results showed that the rate in the Angll group is significantly increased compared with the control group (P<0.05), that rate in the AngⅡ+Sirt3-SiRNA group is significantly rise compared with the Angll group compared with the Angll group (P<0.01),the rate in the AngⅡ+Sirt3-plasmid group is significantly decreased compared with the Angll group (P<0.01).Conclusions1) Sirt3is stably expressed in VSMCs.2) The stimulation of AngⅡ can increase the expression of Sirt3both at mRNA level and protein level.3) Angll can replicable cell proliferation model on VSMC, silence Sirt3could reduce cell tolerance to the pathological stimuli.4) High expressed Sirt3can inhibit cell proliferation in VSMCs induced by Ang Ⅱ.