药物预处理对心肌细胞缺血再灌注损伤的保护机制研究- - -异甜菊醇对KATP通道的作用

Brief periods of repetitive ischemia can protect cardiomyocytes against injury in a subsequent long period of ischemia. This phenomenon is called "ischemic preconditioning" (IPC) or "ischemic adaption". KATP channel as a final effector of the signal transduction pathway plays an important role in the process of ischemic preconditioning. Isosteviol is a derivative of stevioside, which is extracted from natural plant Stevia rebaudiana, and has been suggested to play a protective role in the ischemia-reperfusion process by activating the mitochondrial KATP channel. By experimental methods combining patch clamp with molecular biology and cell biology, this project will deeply investigate the protective effects of isosteviol on ischemia-reperfusion damage.Through simultaneously measuring the transmembrane current of sarcKATP and the fluorescence intensity of flavoprotein,we are expected to clarify which channel,mitoKATP or sarcKATP,palys an more important protective role aganist the I-R injury.Though directly clamping the mitochondria membrane we will identifying the interaction of isoteviol with mitoKATP.In additon,through examining the interaction of isosteviol with other types of channels(such as BKCa and Cav1.2),we will investigate if isosteviol can paly its protective role through other pathways. The implementation of this project will be helpful for understanding the mechanism of ischemic preconditioning and drug preconditioning against myocardial ischemia reperfusion injury on cell and molecular level.

长时间缺血前几次短暂的缺血会提高心肌细胞对后面长时间缺血再灌注损伤的耐受程度，对心肌细胞起一定的保护作用，称为 "缺血预适应"。KATP通道作为细胞内信号转导途径中的一种"终末效应因子"在缺血预适应过程中起重要作用。异甜菊醇是天然植物甜叶菊的提取产物，研究表明它有可能通过激活线粒体KATP通道在缺血再灌注过程中发挥保护作用。本课题拟通过膜片钳、分子生物学和细胞生物学相结合的实验手段，深入研究异甜菊醇对心肌细胞缺血再灌注损伤的保护机制。通过同时检测心肌细胞膜电流和线粒体呼吸链黄素蛋白的荧光强度,来阐明异甜菊醇究竟是通过线粒体KATP通道还是心肌细胞膜KATP通道来发挥保护作用的；通过检测异甜菊醇对其它通道（如BKCa、Cav1.2）的作用，研究异甜菊醇是否还能通过其它途径介导保护作用。本课题的实施将有助于在细胞、分子水平理解缺血预处理、药物预处理对心肌细胞缺血再灌注损伤的保护机制。

随着生活水平的提高，缺血性心脏病已成为威胁人类特别是中老年人身体健康的主要疾病。 KATP通道作为细胞内信号转导通路中的“终末效应因子”在缺血预适应过程中起重要的保护作用。异甜菊醇（STV）是天然植物甜叶菊的提取产物。研究表明异甜菊醇能够心肌减少缺血再灌注损伤的梗死面积，这一保护作用可被KATP通道的阻断剂阻断，提示心肌细胞KATP通道参与了异甜菊醇对缺血再灌注损伤的保护机制。本项目通过膜片钳和激光共聚焦相结合的实验技术研究了STV对心肌细胞膜（sarc-）和线粒体膜（mito-）KATP通道的作用，此外还研究了STV对心肌细胞动作电位、hERG通道电流以及L型钙离子通道电流的影响。得到如下实验结果：心肌细胞膜KATP通道电流能够被通道开放剂Pinacidil诱发并能被阻断剂Glibenclamide阻断；STV能够提高同样浓度Pinacidil诱导的sarcKATP通道的电流密度（52.5 ± 7.8 pA/pF for 10μM STV group and 30.0 ± 6.0 pA/pF for control group），增强sarcKATP通道对Pinacidil的敏感性；STV对sarcKATP 通道敏感性的增加有时间依赖性和浓度依赖性；STV对hERG通道电流和L型钙通道电流没有显著影响；STV对心肌细胞动作电位的时程和静息电位没有显著影响；STV能够提高Diazoxide诱导的黄素荧光蛋白的荧光强度(35.6 ± 8.0 % for 10μM STV group and 13.4 ± 2.4 % for control group)，从而间接提高线粒体膜KATP（mitoKATP）的敏感性；活性氧的清除剂NAC能够阻断STV对sarcKATP通道和mitoKATP通道的影响。这些结果表明STV能够提高sarcKATP通道和mitoKATP通道对开放剂的敏感性，并且STV对KATP的增敏作用是通过ROS介导的。以上实验结果有两个重要意义：首先其发现了异甜菊醇发挥缺血保护作用的分子机理，为其它文献报道的在异甜菊醇动物水平上的缺血保护作用提供理论依据，其次，证实了sarc- 和mitoKATP通道在缺血预适应过程中发挥协同作用来减轻心肌缺血再灌注损伤。这些实验结果为将来异甜菊醇纳入临床研究和应用奠定了理论基础。