Effect of ghrelin on angiotensin II induced human umbilicus vein endothelial cell oxidative stress and endothelial cell injury.

OBJECTIVE: To determine the effect of ghrelin on protecting the human umbilical vein endothelial cells (HUVEC) from injury by angiotensin II (Ang II) in vitro.
METHODS: (1) HUVEC was incubated for 24 h with AngII whose final concentration in the medium varied from 10⁻⁹ to 10⁻⁶ mol/L or pretreated with 10⁻⁹ to 10⁻⁶ mol/L ghrelin for 2 h before incubation for 24 h with Ang II whose final concentration in the medium was 10⁻⁶ mol/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 10⁻⁶ mol/L Ang II, respectively. Before HUVECs were incubated with 10⁻⁶ mol/L Ang II for 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 10⁻⁶ mol/L Ang II 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 10⁻⁶ mol/L Ang II and ghrelin, respectively,and then were incubated with 10⁻⁶ mol/L of Ang II for 3, 6, 12, or 24 h. Furthermore, HUVECs were pretreated with 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/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 10⁻⁶ mol/L Ang II for 24 h, or pretreated with ghrelin plus PD98059, wortmannin, and [D-Lys³]GHRP-6 before incubation with Ang II for 24 h. NO was measured in the endothelial cell supernatants by Griess method. (4) HUVECs were cultivated with blank or Ang II with 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: Ang II injuried the endothelial cell vitality,increased the cell apoptosis rates in HUVECs, and decreased NO production in HUVEC supernatants,whereas ghrelin protected HUVECs from Ang II injury. Ghrelin decreased the reactive oxygen species in HUVECs induced by Ang II. The effect could be attenuated by [D-Lys³]GHRP-6 pretreatment; PD98059 alleviated Ang II 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. Ang II reduced 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 Ang II induced injury, which is related to decreasing oxidative stress, increasing the protein expression of eNOS, and activating PI3K/Akt signal pathway through GHSR1a receptor.