氧化应激对PI3K/Akt/eNOS通路的逆转在CAD痰浊壅塞证内皮祖细胞凋亡及中药干预中的作用机制

Mechnism underlying coronary artery disease tan-zhuo-yong-she syndrome is always the concernful scientific problem to be solved in the domains of the traditional Chinese medicine pharmacology and international cardiovascular pharmacology. After refered to considerable study evidence, we speculate key risk factors in the nosogenesis of this disease below: Metabolism dysfunction causes over oxidative stress in endothelial progenitor cells (EPCs) of patients, PI3K, eNOS lost their integrity, and the PI3K/Akt/eNOS survival signal pathway changes its role to triggering apoptosis. This change results in obviously apoptosis in EPCs. Consequently, the repair capacity of vascular injury is reduced. This project is designed to validate our hypothesis. After the function of PI3K,Akt,eNOS in EPCs is blocked or activated by their specific inhibitors or agonists, the cultivated EPCs will be exposured to the CAD tan-zhuo-yong-she syndrome model rats serum, ROS, antioxidants, qutan sanjie prescription medicated serum, respectively. Moreover, EPCs of the patients, rat models also will being discussed in vivo. The apoptosis ratio, T-ROS/T-AOC ratio,as well as the protein expression level, the integrity, and the activation of PI3K, Akt, eNOS in EPCs will be detected in vivo and in vitro. In this project, we will discuss systematically the mechnism by which oxidative stress induces apoptosis in EPCs via PI3K/Akt/eNOS signal pathway in CAD tan-zhuo-yong-she syndrome and its qutan sanjie prescription therapy. It is tried to find out that the key links of CAD tan-zhuo-yong-she syndrome development and targets of its qutan sanjie prescription treating, to reveal the modern scientific connotation of tan-zhuo-yong-she syndrome at the level of progenitor cell and functional protein, and provide scientific basis for exploring more effective treatment of this disease as well as new drug discovery.

冠心病(CAD)痰浊壅塞证及祛痰散结法干预的微观机制是中医基础领域的重要科学问题。经过系统梳理大量研究证据,结合前期发现(祛痰散结药可显著调节模型动物脂类代谢及血液的氧化应激成分,提高EPC缺血缺氧下的存活率),我们推测该病证的关键环节为:患者血液中异常的代谢成分诱发了EPC内的过度氧化应激,通过破坏信号蛋白的完整性逆转了PI3K/Akt/eNOS通路的调控方向,使细胞大量凋亡,血管修复能力低下。拟特异性地激活、阻断细胞内该通路的功能,培养液中分别添加氧化应激的诱导及抑制成分、瓜蒌薤白半夏汤含药血清培养EPC;结合利用l临床患者、模型大鼠进行体内研究。体内体外检测EPC的凋亡率、氧化应激状态,以及PI3K等mRNA及蛋白水平的表达量、激活程度;系统研究氧化应激对该通路和EPC凋亡的影响,及其在CAD痰浊壅塞证发病和祛痰散结中药干预中的作用及机制,祖细胞层次揭示"痰浊阻痹心脉"理论的科学内涵

冠心病(CAD)痰浊壅塞证及其祛痰散结治法的微观机制是中医基础领域的重要科学问题。我们前期发现瓜蒌薤白半夏汤(GXB)可显著降低模型大鼠血脂含量，纠正其血液异常的氧化应激状态，显著提高体外大鼠内皮祖细胞(EPCs)缺血缺氧条件下的存活率；因而推测：患者血液中异常的代谢成分诱发了EPCs内的过度氧化应激，使eNOS蛋白解偶联，PI3K/Akt/eNOS信号通路的功能从维护生存变为促进凋亡，进而使该细胞大量凋亡，机体血管修复能力低下。为验证该假说，本项目在临床、整体动物、体外细胞三个层次上进行了系列研究。首先检测了该病证患者及模型动物血浆中ox-LDL、AGEs等6种氧化诱导成分，以及总抗氧化能力(T-AOC)等5种抗氧化成分的含量或活性，计算了血液氧化成分/抗氧化成分的含量比值。其次，利用流式细胞仪技术检测了患者及模型动物外周血EPCs含量、凋亡率；以及其中PI3K、Akt、eNOS蛋白表达水平及激活的程度。第三，对培养的大鼠骨髓EPCs，先采用PI3K专一抑制剂阻断理细胞内PI3K/Akt/eNOS通路，再模拟患者体内环境，利用低氧气体+含ox-LDL的无血清培养液培养细胞，并在培养液中添加GXB水提物及三个有效组分，制造氧化应激损伤及祛痰散结中药保护细胞模型。同样检测细胞凋亡率以及胞内PI3K等蛋白的表达、激活水平。结果发现，① CAD痰浊壅塞证患者、模型大鼠血液中EPCs凋亡率显著升高，其中氧化诱导成分含量显著升高，抗氧化酶及其他抗氧化成分含量显著降低。② 体外氧化损伤的EPCs内PI3K、Akt、eNOS三种蛋白的表达及磷酸化水平显著降低。GXB或其有效组分的干预对体内、体外EPCs的凋亡率均有显著抑制作用，同时显著上调了PI3K等三种蛋白的表达及磷酸化。本项目结果证实，异常的血液氧化应激环境通过抑制PI3K/Akt/eNOS通路造成了EPCs凋亡，这是CAD痰浊壅塞证发病及GXB干预的重要环节。本项目的研究在祖细胞层次揭示中医“痰浊阻痹心脉”及中药“祛痰散结、开胸除痹”理论的科学内涵。