| Objective:Many studies have now confirmed that endothelial cell senescence plays an important role in the vascular aging process. This experiment establish the endothelial cell senescence model by angiotensin-Ⅱinducing human umbilical vein endothelial cells (HUVECs) aging,and explore the influence of different concentrations of Ang-(1-7) on endothelial cell senescence,Sirtl mRNA expression and NO secretion induced by AngⅡto HUVECs.Methods:We culture HUVECs in vitro and the 3 to 5 generation cells were used in our study. Experiment was divided into:(1) control group:no intervention of factors; (2) Ang II group:add 10"6mol/L of Ang II;(3)Ang-(1-7) group:join the 10-6mol/L of Ang-(1-7);(4)Ang-(1-7)+AngⅡgroup:add 10"6mol/L of AngⅡon the basis of different concentrations of Ang-(1-7) (10-6～10-8mol/L). Cells and the culture medium were collected 72 hours after the intervention. Senescenceβ-gal staining and cell cycle analysis were used to identify cell aging status. RT-PCR observe the expression of Sirt1 mRNA in each group. And the cell culture medium were used to analyze the nitric oxide (NO) content by using NO detection kit.Resultsl.Flow cytometry results of cell cycle analysis:compared to the control group (Go/Gi phase and S phase), Go/G1 phase of AngⅡgroup increased and S phase decreased,and Ang-(1-7) group showed no significant difference(P>0.05), suggesting that individual to join Ang II on endothelial cells can induced Go/G1 phase arrest and S phase decreased, while the individual joined Ang-(1-7) had no effect on endothelial cell cycle; Ang-(1-7)+AngⅡgroup compared with AngⅡgroup(Go/G1 phase and S phase) showed significant difference(P<0.05), Ang-(1-7) inhibited Ang II on the endothelial cell cycle, making the reduction of Go/G1 phase and the increase of S phase; the different concentration of Ang-(1-7)+AngⅡgroups (P>0.05) showed not statistically significant and Ang-(1-7) was non-dose dependent inhibition of AngⅡon the endothelial cell cycle.2.Cell senescenceβ-galactosidase staining analysis showed that:compared with the control group, Ang II group P<0.05, Ang-(1-7) group P>0.05, suggesting that individual to join AngⅡon endothelial cells can enhance cell P-galactosidase activity, while the individual joined Ang-(1-7) had no effect on endothelial cells P-galactosidase activity; Ang-(1-7)+AngⅡgroup compared with Ang II group showed statistically difference(P<0.05), prompted Ang-(1-7) inhibited AngⅡrole in the enhancement of cell senescenceβ-galactosidase activity;the different concentration of Ang-(1-7)+AngⅡgroups(P>0.05) showed not statistically significant, prompted Ang-(1-7) in non-dose dependent inhibition of AngⅡon cell senescenceβ-galactosidase enzyme activity.3.Compared with Sirtl mRNA expression in the control group, Ang II group and Ang-(1-7) group (P＜0.05) were statistically significant,suggesting that AngⅡadded individually inhibited the expression of Sirtl mRNA on endothelial cells, on the contrary, individual to join Ang-(1-7) on endothelial cells promoted the expression of Sirtl mRNA; Ang-(1-7)+Ang II group compared with Ang II group showed statistically difference(P<0.05), prompted Ang-(1-7) inhibited Ang II role in the expression of Sirtl mRNA reduced on endothelial cells; the different concentration of Ang-(1-7)+AngⅡgroups(P<0.05) showed statistically difference, prompted Ang-(1-7) in a dose-dependent inhibition of the expression of Sirtl mRNA on endothelial cells induced by AngⅡ.4. The results of NO test showed that:compared with the control group, AngⅡgroup and Ang-(1-7) group were statistically significant, suggesting that individual to join AngⅡon endothelial cells can inhibit the secretion of NO, and Ang-(1-7) added alone can promote the secretion of NO on endothelial cells; other than Ang-(1-7)+AngⅡ(10-7mol/L) group, Ang-(1-7) +AngⅡgroup compared with AngⅡ(P＜0.05), suggesting that Ang-(1-7) inhibited AngⅡrole in reducing cell NO generation on endothelial cells; the different concentration of Ang-(1-7)+AngⅡgroups(P<0.05) showed statistically difference and did not show the trend of increasing or decreasing accompanying with the changes of concentration on each group, suggesting that Ang-(1-7) inhibited the role of AngⅡreducing endothelial cells NO generation in non-dose dependent.Conclusion:1. AngⅡcan induce human umbilical vein endothelial cells (HUVECs) senescence.2. Ang-(1-7) inhibit endothelial cell senescence in non-dose dependent induced by AngⅡ.3. The role of Ang-(1-7) in endothelial cell senescence induced by Ang II may be related to prompte the expression of Sirtl mRNA in endothelial cells and the secretion of NO.
|
PDF Full Text Request