Research Of Sirt3 On Angiotensin Ⅱ-induced Human Umbilical Vein Endothelial Cells Senescence And Dysfunction

BackgroundAs the protective screen between vessel wall and blood, vascular endothelial cells have the abilities of anticoagulation, regulating angiotasis, participating in inflammation, etc. Endothelial dysfunction, mainly refer to an impairment of endothelium-dependent vasorelaxation caused by a loss of nitric oxide bioactivity in the vessel wall, is observed in the presence of major cardiovascular risk factors, including atherosclerosis, hypertension. The abilities of proliferation, migration, and diastolic are reduced in aging endothelial cells, which lead to receding of repair, angiogenesis and vasodilatation function, is one of reasons that cardiovascular disease increases with age. AngⅡ, a key substance of renin-angiotensin-aldosterone system can induce endothelial dysfunction and aging by increasing the production of ROS.Sirt3 is one of the seven mammalian sirtuins in the family of Sir2, which are a conserved family of proteins possessing NAD+-dependent deacetylase activity. Of the seven sirtuins, Sirt3, which mainly located in mitochondria and the nucleus, is the only sirtuin analogue whose increased expression was shown to be associated with longevity of humans. The distribution of Sirt3 in tissues and organs are very widely and high expression in metabolically active tissues such as muscle, liver, kidney and heart. Recent studies have reported that Sirt3 regulate the production and clearance of ROS through deacetylation of numerous mitochondrial enzymes. And Sirt3 can affect the occurrence of aging diseases such as cardiovascular diseases and cancers. So we suppose that Sirt3 may affect the progress of Ang Ⅱ-induced human umbilical vein endothelial cells(HUVECs) senescence and vascular endothelial cell function through reducing accumulation of ROS.Purpose1. To detect the role of Sirt3 in Ang Ⅱ-induced human umbilical vein endothelial cells senescence.2. To explore the influence of Sirt3 on the function of human umbilical vein endothelial cells stimulated by Ang Ⅱ.Methods1. Cell cultureHUVECs from ATCC were maintained in endothelial cell medium (ECM) in humidified incubator with 5% CO2 and 95% air at 37℃. HUVECs were divided into four groups:control, Sirt3-siRNA, AngⅡ, Sirt3-siRNA+AngⅡ.2. Sirt3-siRNAtransfectionThe Sirt3-siRNA used was synthesized by reagent company, and screened by Western blot. The sequence with the highest inhibition efficiency was used in the following experiment.3. Western blotTo extracted total protein of the cells, and to detect Sirt3, P16,P21, eNOS protein expression in each group via Western blot.4. Detection of cells aging statusSenescence β-gal staining was used to identify HUVECs aging status.5. eNOS activity assayeNOS activity was measured by use of a nitricoxide synthase assay kit.6. Measurement of NO concentrationThe NO levels in the cell culture supernatant were measured by using nitric oxide assay kit in nitrate reductase method.7. Assessment of intracellular ROS levelsIntracellular ROS was detected with 2’,7’-dichlorodihydro-fluorescein diacetate (DCFH-DA).8. Statistical analysisThe data are expressed as x±s. The difference between the experimental group and the control group use t test, the rest groups compared with each other was analyzed by a one-way analysis of variance. P<0.05 was considered statistically significant.Results1. The inhibition efficiency of sequence 1 is the highest in three sequences. Sequence 1 was screened for subsequent experiments.2. Treatment with 10-7,10-6,10-5 mol/L Angll for 24 h resulted in a significant increase of Sirt3 expression in HUVECs, with the peak at 10-6 mol/L. The dose of 10-6 mol/L was selected for the treatment condition in subsequent experiments.3. The protein expression of P16, P21 increased significantly in AngⅡ group and Sirt3-siRNA group. The protein expression of P16, P21 in Sirt3-siRNA+ AngⅡ group increased more significantly than that in Angll group.4. The cells positive rate of β-gal staining was significantly increased in Angll group. Compared with the Angll group, the cells positive rate of β-gal staining of Sirt3-siRNA+AngII group was higher.5. AngⅡ induced an obvious pathological phenotype of HUVECs, as shown by a reduced eNOS activity and reduced NO concentration, however, eNOS expression increased in response to Angll stimuli. When Sirt3 was knocked down using siRNA, the activity of eNOS in Sirt3-siRNA+AngⅡ group decreased more significantly than that in AngⅡ group. The concentration of NO further decreased when pretreated with Sirt3-siRNA. In addition, treatment with Sirt3 siRNA reduced eNOS expression under conditions with or without Angll.6. Compared with the NC group, ROS level of cells in AngⅡ group is higher. Sirt3-siRNA significantly increased ROS level of cells with or without intervention of AngⅡ.Conclusions1. Sirt3 can delay the progress of Ang Ⅱ-induced HUVECs senescence.2. Sirt3 play a protection role in Ang Ⅱ-induced HUVECs dysfunction.3. Sirt3 play the above roles through regulating ROS level of cells.