Akt-eNOS-NO信号途径介导了Ang-1和Ang-2对大鼠失血性休克早期血管高反应性的调节作用

Akt-eNOS-NO signal pathway mediates regulatory effect of Ang-1 and Ang-2 on vascular hyperreactivity in early hemorrhagic shock rats

目的:观察内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)在血管生成素1(angiopoi-etin-1,Ang-1)和血管生成素2(angiopoietin-2,Ang-2)调节失血性休克大鼠血管反应性双相变化中的作用及其机制。方法:采用Western blotting技术观察失血性休克后不同时点肠系膜上动脉(superior mesenteric artery,SMA)中eNOS蛋白表达变化,采用离体微血管环张力测定技术观察eNOS抑制剂对Ang-1和Ang-2调节缺氧早期和晚期血管反应性作用的影响,并观察给予Ang-1、Ang-2以及Tie-2、Akt、p38 MAPK、ERK抑制剂后缺氧血管内皮细胞(vascular endothelial cells,VECs)和血管平滑肌细胞(vascular smooth muscle cells,VSMCs)混合培养物中eNOS蛋白表达和培养上清一氧化氮(nitric oxide,NO)含量的影响。结果:(1)eNOS在正常SMA中表达很低,失血性休克后逐渐增高,休克10 min、30 min、1 h、2 h和4 h时分别增高至正常对照的1.95、2.10、3.01、3.42和3.57倍(P<0.01)。(2)eNOS抑制剂可显著抑制缺氧10 min的血管高反应性,去甲肾上腺素(NE)的Emax由13.479mN降低至9.043 mN(P<0.01),也可以显著抑制Ang-1对缺氧10 min血管高反应性的维持作用,NE的Emax由15.283 mN降低至11.219 mN(P<0.01),但不改变Ang-2降低缺氧10 min血管高反应性作用,也不改变缺氧4 h的血管低反应性和Ang-1、Ang-2对缺氧4 h血管反应性的调节作用。(3)缺氧10 min的eNOS表达较正常对照增高,Ang-2和Tie-2、Akt抑制剂可抑制其增高(P<0.01),p38 MAPK、ERK抑制剂对其无显著影响。(4)NO含量在缺氧10 min显著增高,Ang-2和Tie-2、Akt、eNOS抑制剂可抑制其增高(P<0.01),p38 MAPK、ERK抑制剂对其无显著影响。结论:失血性休克早期,Ang-1和Ang-2通过Akt-eNOS-NO途径来调节血管高反应性。

AIM： To observe the role of endothelial nitric oxide synthase （eNOS） in the regulatory effect of angiopoietin - l （ Ang - 1 ） and angiopeietin - 2 （ Ang - 2） on the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHODS： The protein expression of eNOS was measured in the superior mesenteric artery （SMA） after hemorrhagic shock by Western blotting. The effect of eNOS inhibitor on the vascular reactivity of SMA treated with Ang - 1 and Ang - 2 in the early （hyperreactivity） and late （hyporeactivity） periods of hypoxia were observed via an isolated organ perfusion system. The protein levels of eNOS in the hypoxic mixture of vascular endothelial ceils （VECs） and vascular smooth muscle cells （ VSMCs）, and the concentration of nitric oxide （NO） in the medium supernatant of the mixture cells treated with Ang- 1, Ang- 2 and the inhibitors of Tie -2, Akt, p38 MAPK and ERK were measured. RESULTS： The protein expression of eNOS in SMA was low in normal control group, and increased significantly after hemorrhagic shock, which was 1.84, 3.55, 4.75, 5.96 and 6.33 folds of the normal control level in shock 10 rain, 30 rain, 1 h, 2 h and4 h groups, respectively （P 〈 O. O1 ）. Inhibitor of eNOS decreased the vascular hyperreactivity in hypoxia 10 min group, in which the Emax of norepinephrine （NE） was decreased from 13. 479 mN to 9. 043 mN （P 〈 0：05 ）. It also repressed the maintenance effect of Ang - 1 on vascular reactivity in hypoxia 10 min group, in wihieh the Emax of NE was decreased from 15. 283 mN to 11. 219 mN （P 〈0. 01 ）. The effect of Ang -2 on the vascular hyperreaetivity in hypoxia 10 min group, the vascular hyporeaetivity in hypoxia 4 h group, or the effect of Ang - 1 or Ang - 2 on the vascular reactivity in hypoxia 4 h group did not change. The protein expression of eNOS was increased 10 min after hypoxia as compared with the normal con- trol, which was decreased by Ang - 2 and the inhibitors of Tie - 2 and Akt （ P 〈 0. 01 ）, but was not decreased by p38 MAPK and ERK inhibitors. The concentration of NO in the medium supernatant was increased 10 min after hypoxia, and was significantly decreased by Ang - 2 and the inhibitors of Tie - 2, Akt and eNOS, while the inhibitors of p38 MAPK and ERK had no influence on it. CONCLUSION ： Ang - 1 and Ang - 2 regulate the vascular hyperreactivity in the early hemorrhagie shock rats through Akt -eNOS- NO pathway.