Zhang Yan, Wang Shi-Jie, Han Zhen-Hua, Li Yong-Qin, Xue Jia-Hong, Gao Deng-Feng, Wu Xiao-San, Wang Cong-Xia
Department of Cardiology, The Second Hospital of Xi'an Jiaotong University Xi'an 710004, China.
Department of Physiology and Pathophysiology, Medical School of Xi'an Jiaotong University Xi'an 710061, China.
Int J Clin Exp Pathol. 2014 Oct 15;7(11):8112-7. eCollection 2014.
The aim of this study was to investigate the effect of PI3K/AKT signaling pathway in the activity of recombinant human angiotensin converting enzyme 2 (rhACE2) promoted the activity of endothelial nitric oxide synthase (eNOS). The human umbilical vein endothelial cells (HUVEC) were cultured in vitro. Then treated with Ang II (1×10(-6) mol/L) for 24 h. The rhACE2 (100 μmol/L) was added and incubated for 5, 10, 15, 30, 60 min respectively which was based on Ang II intervention. The effect of rhACE2 on phosphorylation eNOS level was also observed in the presence of LY294002 (10 μmol/L) (PI3K/AKT inhibitors). Griess reagent method was applied to measure NO contents in cell culture supernatant, RT-PCR to detect the expression of eNOSmRNA in HUVEC, and Western blot to detect the expression of eNOS and phosphorylated eNOS. In Ang II intervention group, NO contents were significantly lower than control group (P < 0.05). Through rhACE2 treatment, the NO contents in cell culture medium and the expression level of phosphorylated eNOS were significantly higher than in Ang II intervention group (P < 0.05), but eNOSmRNA and non-phosphorylated eNOS protein expression level showed no significant difference (P > 0.05). After HUVEC was intervened by PI3K/AKT pathway inhibitor LY294002, the expression level of phosphorylated eNOS was significantly lower than that in the rhACE2 30 min treatment group (P < 0.05). rhACE2 may reduce the activity of Ang II inhibited endothelial cell eNOS, which can be blocked by PI3K/AKT pathway inhibitor LY294002, suggesting PI3K/AKT signaling pathway plays an important role in rhACE2's promotion of the activity of endothelial cell eNOS.
本研究旨在探讨PI3K/AKT信号通路在重组人血管紧张素转换酶2（rhACE2）促进内皮型一氧化氮合酶（eNOS）活性中的作用。体外培养人脐静脉内皮细胞（HUVEC）。然后用血管紧张素II（1×10⁻⁶ mol/L）处理24小时。基于血管紧张素II干预，分别加入rhACE2（100 μmol/L）并孵育5、10、15、30、60分钟。在存在LY294002（10 μmol/L）（PI3K/AKT抑制剂）的情况下，也观察了rhACE2对磷酸化eNOS水平的影响。采用Griess试剂法测定细胞培养上清液中的NO含量，RT-PCR检测HUVEC中eNOSmRNA的表达，Western blot检测eNOS和磷酸化eNOS的表达。在血管紧张素II干预组中，NO含量显著低于对照组（P < 0.05）。通过rhACE2处理，细胞培养基中的NO含量和磷酸化eNOS的表达水平显著高于血管紧张素II干预组（P < 0.05），但eNOSmRNA和非磷酸化eNOS蛋白表达水平无显著差异（P > 0.05）。在用PI3K/AKT通路抑制剂LY294002干预HUVEC后，磷酸化eNOS的表达水平显著低于rhACE2 30分钟处理组（P < 0.05）。rhACE2可能降低血管紧张素II抑制的内皮细胞eNOS的活性，而PI3K/AKT通路抑制剂LY294002可阻断这种作用，提示PI3K/AKT信号通路在rhACE2促进内皮细胞eNOS活性中起重要作用。
