Tie-2介导Ang-1、Ang-2调节大鼠失血性休克血管反应性双相变化的作用研究

Tie-2 Receptor Mediates the Regulation of Ang-1 and Ang-2 on the Biphasic Change of Vascular Reactivity after Hemorrhagic Shock in Rats

关键词:  失血性休克  血管反应性  双相变化  血管生成素-1  血管生成素-2  Tie-2

Key words:  hemorrhagic shock  vascular reactivity  biphasic change  angiopoietin-1  angiopoietin-2  Tie-2

摘要:

目的 观察Tie-2在血管生成素-1(angiopoietin-1，Ang-1)、血管生成素-2(angiopoietin-2，Ang-2)调节失血性休克血管反应性双相变化中的作用。方法 采用离体微血管环张力测定技术和Western blot技术，观察失血性休克后不同时间点肠系膜上动脉(superior mesenteric artery，SMA)中Tie-2蛋白表达和磷酸化变化、抑制Tie-2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，以及给予Ang-1和Ang-2后缺氧的血管内皮细胞(vascular endothelial cell, VEC)和血管平滑肌细胞(vascular smooth muscle cell，VSMC)混合细胞中Tie-2蛋白表达和磷酸化变化，并观察抑制Tie-2对缺氧早期和晚期的混合细胞NO含量的影响。结果 ①肠系膜上动脉Tie-2蛋白表达和酪氨酸磷酸化在正常时很低，失血性休克后逐渐增高，在休克早期(休克10 min)，Tie-2蛋白表达变化不大，但酪氨酸磷酸化已明显增高(P<0.01)；随着休克时间延长，Tie-2蛋白表达和酪氨酸磷酸化均进一步显著增高(P<0.01)。②Tie-2抑制剂可降低缺氧10 min的血管高反应性(NE的Emax由13.479 mN降低至10.122 mN，P<0.05)，并显著抑制Ang-1对缺氧10 min血管反应性的维持作用(Emax由15.283 mN降低至10.253 mN，P<0.01)；Tie-2抑制剂可改善缺氧4 h的血管低反应性(NE的Emax由5.875mN增高至8.003 mN，P<0.05)，并显著拮抗Ang-2进一步降低缺氧4 h血管反应性的作用(Emax由3.444 mN增高至7.643 mN，P<0.01)。③缺氧10 min时，降低血管高反应性的Ang-2可降低Tie-2磷酸化，使其由0.040 3降低至0.012 3(P<0.01)；缺氧4 h时，恢复血管低反应性的Ang-1可降低Tie-2蛋白表达，使其由0.227 6降低至0.085 1(P<0.01)，也可以降低Tie-2磷酸化，使其由0.143 7降低至0.035 9(P<0.01)。④NO含量在缺氧早期显著增高，Ang-2和Tie-2抑制剂显著抑制其增高(P<0.01)；缺氧晚期NO含量较正常对照组增高得更为显著，Ang-1和Tie-2抑制剂可抑制其增高(P<0.01)。结论 Ang-1、Ang-2通过Tie-2受体调节大鼠失血性休克血管反应性双相变化。

Abstract:

OBJECTIVE To observe the role of Tie-2 in the regulation of angiopoietin-1(Ang-1) and angiopoietin-2(Ang-2) on the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHODS The protein expression and tyrosine phosphorylation of Tie-2 in the superior mesenteric artery(SMA) after hemorrhagic shock were measured via Western blot technique, the effect of Tie-2 inhibitor on the vascular reactivity of SMA in the early (hyperreactivity) and late (hyporeactivity) period of hypoxia treated with Ang-1 and Ang-2 were observed via the isolated organ perfusion system, and the protein expression and tyrosine phosphorylation of Tie-2 in the hypoxia mixture of vascular endothelial cell(VEC) and vascular smooth muscle cell(VSMC) treated with Ang-1 and Ang-2 were measured via Western blot technique. Effect of Tie-2 on the NO concentration of VSMC and VEC mixture in the early and late hypoxia was detected by the NO kit. RESULTS ①The protein expression and tyrosine phosphorylation of Tie-2 in the SMA were low in normal control group, and were increased after hemorrhagic shock, the protein expression of Tie-2 was increased after 30 min shock (P<0.01), and the tyrosine phosphorylation of Tie-2 was increased after 10 min shock (P<0.01), both protein expression and tyrosine phosphorylation of Tie-2 were further increased as shock went on (P<0.01). ②Tie-2 inhibitor could decrease the vascular hyperreactivity in 10 min hypoxia (the Emax of NE was decreased from 13.479 mN to 10.122 mN, P<0.05), and repress the maintenance effect of Ang-1 on vascular reactivity in 10 min hypoxia (the Emax of NE was decreased from 15.283 mN to 10.253 mN, P<0.01); Tie-2 inhibitor could improve the vascular hyporeactivity in 4 h hypoxia (the Emax of NE was increased from 5.875 mN to 8.003 mN, P<0.05), and inhibit the decrease effect of Ang-2 on vascular reactivity in 4 h hypoxia (the Emax of NE was increased from 3.444 mN to 7.643 mN, P<0.01). ③At 10 min hypoxia, Ang-2 could decrease the tyrosine phosphorylation of Tie-2 from 0.040 3 to 0.012 3(P<0.01), and at 4 h hypoxia, Ang-1 could decrease the protein expression of Tie-2 from 0.227 6 to 0.085 1(P<0.01), and decrease the tyrosine phosphorylation of Tie-2 from 0.143 7 to 0.035 9(P<0.01). ④The NO concentration in the cells mixture was significantly increased in the early hypoxia, and the increase could be inhibited by Ang-2 and Tie-2 inhibitor(P<0.01); in the late hypoxia, the NO concentration was further increased, and could be inhibited by Ang-1 and Tie-2 inhibitor(P<0.01). CONCLUSION Ang-1 and Ang-2 regulate the biphasic change of vascular reactivity after hemorrhagic shock in rats through Tie-2 receptor.