Effect of ghrelin on angiotensin Ⅱ induced human umbilicus vein endothelial cell oxidative stress and endothelial cell injury
10.3969/j.issn.1672-7347.2010.10.003
- VernacularTitle:Ghrelin对血管紧张素Ⅱ诱导的脐静脉内皮细胞氧化应激和内皮功能损伤的影响
- Author:
Bin DENG
;
Li FANG
;
Xiaobin CHEN
;
Meifang CHEN
;
Xiumei XIE
- Publication Type:Journal Article
- Keywords:
ghrelin;
angiotensin Ⅱ;
reactive oxygen species;
nitric oxide;
phosphoinosi-tide-3-kinase/serine threonine kinase;
endothelial nitric oxide synthase
- From:
Journal of Central South University(Medical Sciences)
2010;35(10):1037-1047
- CountryChina
- Language:Chinese
-
Abstract:
Objective To determine the effect of ghrelin on protecting the human umbilical vein endothelial cells (HUVEC) from injury by angiotesin Ⅱ(AngⅡ) in vitro. Methods (1)HUVEC was incubated for 24 h with AngⅡ whose final concentration in the medium varied from 10-9 to 10-6mol/L or pretreated with 10-9to 10-6mol/L ghrelin for 2 h before incubation for 24 h with AngⅡwhose final concentration in the medium was 10-6mol/L. HUVECs were harvested to measure the cell vitality and cell apoptosis. The cell vitality was determined by MTT and cell apoptosis rates were measured by Annexin V-FITC apoptosis detection kit. (2)HUVECs were incubated for 3,6,12,or 24 h with 10-9,10-8,10-7,or 10-6mol/L AngⅡ, respectively. Before HUVECs were incubated with 10-6 mol/L AngⅡfor 24 h, ghrelin (10-9,10-8,10-7, and 10-6 mol/L) was used to pretreat the cells for 2 h. Growth hormone secregogue receptor 1a blocker [D-Lys3]GHRP-6 was added to the cells which were incubated for 24 h with 10-6mol/L AngⅡ and pretreated with 10-6 mol/L ghrelin for 2 h. Cell reactive oxygen species were measured by dichlorofluorescin (DCF) fluorescence probe method. (3)HUVECs were incubated for 24 h with 10-9,10-8,10-7, or 10-6mol/L AngⅡ and ghrelin, respectively,and then were incubated with 10-6mol/L of AngⅡ for 3,6,12,or 24 h. Furthermore,HUVECs were pretreated with 10-9,10-8,10-7, or 10-6mol/L ghrelin for 30 min,1 h,or 2 h, and then were incubated with the inhibitor of mitogen-activated protein kinase /extracellular regulated kinase (MAPK/ERK1/2),PD98059, the inhibitor of phosphoinositide-3-kinase/serine threonine kinase( PI3K/Akt)wortmannin, and [D-Lys3]GHRP-6 for 24 h. NO production was compared among groups. HUVECs were pretreated with ghrelin,PD98059, wortmannin, and [D-Lys3]GHRP-6 for 2 h and co-cultured with 10-6mol/L AngⅡfor 24 h,or pretreated with ghrelin plus PD98059, wortmannin, and [D-Lys3]GHRP-6 before incubation with AngⅡfor 24 h. NO was measured in the endothelial cell supernatants by Griess method. (4)HUVECs were cultivated with blank or AngⅡwith or without pretreatment with ghrelin or both ghrelin and wortmannin. The protein expression of eNOS and phospho-protein expression of Akt were measured by Western blot analysis. Results AngⅡinjuried the endothelial cell vitality,increased the cell apoptosis rates in HUVECs, and decreased NO production in HUVEC supernatants,whereas ghrelin protected HUVECs from AngⅡ injury. Ghrelin decreased the reactive oxygen species in HUVECs induced by AngⅡ. The effect could be attenuated by [D-Lys3]GHRP-6 pretreatment; PD98059 alleviated AngⅡ inhibition of NO production in HUVEC supernatants. Wortmannin and [D-Lys3]GHRP-6 could abolish protection of ghrelin from reducing NO production in HUVEC supernatants. AngⅡreduced the expression of eNOS,but ghrelin increased eNOS expression. Wortmannin could cancel this effect of ghrelin. Ghrelin increased p-Akt expression and reached the peak in 10 and 20 min. Conclusion Ghrelin may protect HUVECs from AngⅡinduced injury, which is related to decreasing oxidative stress, increasing the protein expression of eNOS, and activating PI3K/Akt signal pathway through GHSR1a receptor.
