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

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

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(H IF-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H IF-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W istar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(RVH I)、血管形态学指标W estern印迹检测磷酸化AKT(P-AKT);原位杂交和免疫组化检测H IF-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 RVH I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。③H IF-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;H IF-1αmRNA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H IF-1α蛋白呈正相关(r=0.883,P<0.01),H IF-1α与VEGF蛋白呈正相关(r=0.897,P<0.01)。结论:磷酸化AKT与H IF-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H IF-1α蛋白表达增加的方式上调H IF-1α,进而上调下游目标基因VEGF,导致HPH的发生和发展。

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 （H3, H7 , H14 and H21 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.