磷酸肌醇3－激酶调控缺氧诱导因子1α对大鼠缺氧性肺动脉高压的作用

目的:观察缺氧性肺动脉高压（HPH）大鼠肺组织中蛋白激酶B（AKT））和缺氧诱导因子1α(HIF-1α)以及血管内皮生长因子(VEGF)的表达，探讨磷酸肌醇3-激酶（PI3K）通路与HIF-1α和VEGF的关系及其在HPH发病中的可能机制。 方法:40只成年雄性Wistar大鼠随机分成对照组、低氧3、7、14和21 d组，每组8只，测各组大鼠平均肺动脉压（mPAP）、右室肥大指数（RVHI）、血管形态学指标Western印迹检测磷酸化AKT(P-AKT)；原位杂交和免疫组化检测HIF-1α的表达。免疫组化检测P-AKT和VEGF水平。 结果:① 低氧7 d起大鼠mPAP、管壁厚度与血管外径比值及管壁面积与血管面积比值分别为(23.53 ±1.78)mmHg, (45.5±3.1)%和(54.7±3.2)%,与对照组［(16.15±1.97)mmHg、(36.8±2.5)%、(63.2±2.5)%］比较差异显著(P＜0.05),低氧14 d起稳定于高水平;低氧14 d RVHI为(26.5±2.9)%,与对照组［(22.9±2.2)%］比较差异也显著(P＜0.05)。②p-AKT蛋白在对照组表达不明显，缺氧3 d后表达上升，与对照组比较差异显著(P＜0.05)，且在缺氧3 d、7 d、14 d、21 d组肺小动脉内膜、中膜表达均为阳性。③HIF-1α蛋白对照组表达不明显，缺氧3 d、7 d、14 d、21 d组肺血管内膜均为阳性，肺血管中膜，缺氧3 d组表达开始升高（0.209±0.009），与对照组比较差异显著(P＜0.05),缺氧7 d达高峰（0.232±0.008,P＜0.05），14 d和21 d下降；HIF-1α mRNA在对照组肺动脉血管壁内表达弱阳性，缺氧3 d和7 d肺血管中表达无明显变化，与对照组比较差异无显著(P＞0.05)。缺氧14 d后表达增高(0.305±0.104, P＜0.05),并持续维持于高水平。VEGF蛋白水平在低氧7 d显著高于对照组(0.188±0.018, P<0.05),14 d达高峰(0.238±0.017, P<0.05)。相关分析表明mPAP与肺血管重塑呈正相关(r=0.983, P<0.01)。在肺小血管内膜:P-AKT与 HIF-1α蛋白呈正相关(r=0.883, P<0.01)， HIF-1α与VEGF蛋白呈正相关(r=0.897, P<0.01)。 结论:磷酸化AKT与HIF-1α和VEGF均在大鼠HPH的发病机制中发挥作用。磷酸化AKT可能通过使HIF-1α蛋白表达增加的方式上调HIF-1α，进而上调下游目标基因VEGF，导致HPH的发生和发展。

Phosphatidylinositol 3-kinase regulates hypoxia-inducible factor 1α in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

AIM:To investigate relationship among phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in lung of rats with hypoxia-inducible pulmonary hypertension. METHODS:Forty male adult Wistar rats were randomly divided into five groups (eight rats in each group):control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H3, H7, H14 and H21 group). Mean pulmonary arterial pressure (mPAP), right ventric hypertrophy index (RVHI) and vessel morphometry were measured. The levels of HIF-1α mRNA expression in lung tissue was measured by in siteu hybridization (ISH). The protein expression of HIF-1α，VEGF and phosphorylated protein kinase β (P-AKT) were observed by immunohistochemistry or Western blotting. RESULTS:mPAP increased significantly 7 days after hypoxia ［(23.53±1.78) mmHg］, peaked 14 days after hypoxia, then remained on the high level. Pulmonary artery remodeling index (extern diameter 100 μm) and RVIH became evident 14 days after hypoxia. Expression of P-AKT protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima and tunica media in all hypoxia rats. HIF-1α mRNA staining was poorly positive in control，hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly 14 days after hypoxia (0.305±0.104, P<0.05), then remained stable. Expression of HIF-1α protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima in all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein was markedly up-regulated after 3 days (0.029±0.019, P<0.05 ), reached its peak 7 days after hypoxia (0.232±0.008, P<0.05), then tended to decline 14 days and 21 days after hypoxia. Expression of VEGF protein began to increase 7 days after hypoxia (0.188±0.018, P<0.05), reached its peak 14 days after hypoxia (0.238±0.017, P<0.05), then remained on the high level in pulmonary arterial tunica intima. Linear correlation analysis showed that P-AKT, HIF-1α mRNA, VEGF and mPAP were correlated with vessel the morphometry and RVHI (P<0.01). P-AKT was positively correlated with HIF-1α and VEGF (tunica intima). CONCLUSION:P-AKT, HIF-1α and VEGF are all involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.