蛋白激酶Akt介导血管硝酸甘油耐受形成的机制研究

Continuous application of nitroglycerin for a period of time will make its effects that on the hemodynamics of peripheral vessels and the dilatation of coronary arteries weaken or even disappear, which is termed as nitroglycerin tolerance. Our previous work showed that: the decreased protein level of cGMP-dependent protein kinase ( PKG ) induced by continuous application of nitroglycerin palys an important role in the development of tolerance, but the mechanism of which is unclear. Based on our previous work and premilinary experiments, we put forward the hypothesis: After being activated by continuous application of nitroglycerin, Akt inhibits the activity of FoxO1 by phosphorylation, then inhibits the transcription of PKG, thereby leads to the decrease of PKG protein and tolerance. According to this hypothesis, we intend: ①to determine whether or not Akt activation casued by continuous application of nitroglycerin is mediated by PI3K or PTEN, and to determine the role of PKG and ROS in it; ②to identify whether or not the regulation of PKG transcription is inhibited by phosphorylation of FoxO1 caused by activated Akt; ③to examine whether or not modulation of Akt signaling pathway improves the development of tolerance. By employing vessel tension recording, biochemical and molecular biological techniques the proposed study is expected to reveal the new mechanisms of Akt mediated in the development of nitroglycerin tolerance.

硝酸甘油连续应用一段时间后会导致其扩张外周血管改善血流动力学效应和扩张冠状动脉改善心肌缺血作用减弱甚至消失，即产生耐受。我们以往工作表明：硝酸甘油通过降低环鸟苷酸依赖的蛋白激酶（PKG）的蛋白水平导致耐受形成，但PKG蛋白水平降低的机制尚未阐明。基于以往工作和预实验结果，我们提出假说：Akt被硝酸甘油持续应用激活后，通过FoxO1途径抑制了PKG的转录，从而降低PKG的蛋白水平。针对该假说，本项目拟研究：①硝酸甘油持续作用是否通过调节PI3K与PTEN的活性从而激活Akt；②活化的Akt是否通过磷酸化FoxO1抑制了其对PKG的转录调控；③通过调控Akt信号通路能否改善耐受现象。我们拟利用离体血管，培养细胞和动物模型，结合功能测定、生化与分子生物学分析进行研究，期待为硝酸甘油耐受的形成机制提供新的认识。

硝酸甘油是临床上治疗心血管疾病的一种常用药物，但其长期使用所产生的耐受严重影响了其使用和疗效。本项目紧密围绕硝酸甘油舒张血管的作用机理以及其耐受形成的机制为中心展开研究。在本计划的资助下，我们发现持续缺氧超过30分钟可以通过激活磷脂酰肌醇3激酶/蛋白激酶Akt信号通路，进而激活其下游的Rho激酶，引起猪冠状动脉收缩，这可能是缺氧诱导心绞痛和心肌梗死发生机制之一；我们还发现硝酸甘油和一氧化氮等硝基类舒血管药物通过激活sGC和PKG，进而活化蛋白磷酸酶1，使猪冠状动脉和肺动脉蛋白激酶Akt的磷酸化水平降低，导致其活性抑制；在此基础上，我们一方面在猪肺动脉上发现脂酰肌醇3激酶/蛋白激酶Akt信号通路受抑后可以抑制5型磷酸二酯酶降解cGMP的作用，从而形成正反馈循环促进一氧化氮的舒张血管作用；另一方面，我们还发现预先给予猪冠状动脉孵育硝酸甘油，可以减轻缺氧诱导的冠状动脉收缩。在执行本研究计划中我们共发表标注本基金的SCI论文3篇。