田蓟苷抗心肌缺血再灌注损伤心肌保护作用的线粒体机理的研究

Myocardial ischemia reperfusion injury is a common cause of sudden death in clinical heart diseases patients. Mitochondria as the "power plant" plays a very important role in MIRI because it has the most direct connection with cell energy metabolism. Tilianin is a kind of natural flavonoids from Dracocephalum Moldavica with a clear against myocardial ischemia effect and its mechanism against MIRI may be related to mitochondria role according to our before research. Based on preliminary findings and the models of neonate rats' myocardial and rats' myocardial ischemia reperfusion injury established, the targets and mechanism of tilianin against MIRI with mitochondrial protection as breakthrough point will be investigated in this study by adopting the modern Chinese medicine pharmacology research methods and means. New targets and mechanism of tilianin against MIRI from the level of overall, cells, protein and molecular will be clarified to explore a new targets and thourghts for developing and researching new grug from Dracocephalum Moldavica and to establish a theoretical basis for further exploitation and utilization of this traditional Uighur genuine medicine.This research not only will lay a certain foundation for drug discovery research on active ingredients from xinjiang property plant medicine, at the same time, it will have important directive significance and modeling effect on research xinjiang characteristic advantage resources.

心肌缺血再灌注损伤（MIRI）是临床心脏病患者猝死的常见原因。线粒体作为机体的"动力工厂"与细胞的能量代谢有着最直接的联系，在MIRI中扮演了极为重要的角色。田蓟苷是新疆优势特色资源香青兰中主要的黄酮类成分。前期研究显示，田蓟苷抗心肌缺血作用确切，线粒体可能在田蓟苷抗 MIRI 的作用中扮演着重要的角色。本研究依据前期研究结果，采用乳鼠心肌细胞及大鼠心肌缺血再灌注损伤的模型，以线粒体保护作用机制为切入点，采用现代中药药理学的研究方法和手段,从整体水平、细胞水平和分子水平探讨田蓟苷抗心肌缺血再灌注损伤的线粒体作用的靶点和作用机制，为维吾尔药香青兰创新药的研发提供新的靶点与思路，为进一步发掘利用这一传统的维吾尔道地药材奠定基础。课题的顺利实施不仅对新疆地产植物药活性成分创新药研究奠定了一定的基础，同时，对于新疆特色植物药新药研发水平的促进和提高也具有一定的示范意义和引领作用。

采用结扎冠状动脉左前降支方法建立在体大鼠心肌缺血再灌注损伤模型，使用三气培养箱模拟缺氧3 h，复氧2 h的方法复制心肌细胞缺氧/复氧损伤模型。应用香青兰总黄酮、田蓟苷、环孢素A、普萘洛尔等进行干预，监测大鼠Ⅱ导联心电图确定模型的成功建立；Evan’s Blue-TTC及TTC 法测定心肌梗死面积；TUNEL 检测凋亡程度；测定心肌ATP、ROS、Ca2+的含量；观察线粒体微观结构、线粒体活力及mPTP开放程度。细胞方面，采用MTT法选择香青兰总黄酮及田蓟苷的高、中、低给药浓度；台盼蓝染色法检测细胞损伤程度；流式细胞仪测定mPTP的开放程度、细胞内ROS及Ca2+含量；差速离心法提取心肌组织中的线粒体，检测线粒体活力；Western Blot检测胞浆中AIF的表达。结果，香青兰总黄酮及田蓟苷显著降低心肌梗塞面积，提高ATP含量，抑制线粒体活力的降低，抑制mPTP的开放及线粒体Ca2+浓度的升高，降低ROS含量，改善线粒体微观结构。细胞方面，分别选取香青兰总黄酮的50 μg·mL-1、100 μg·mL-1、200 μg·mL-1和田蓟苷的5 μg·mL-1、10 μg·mL-1、20 μg·mL-1作为低、中、高剂量，香青兰总黄酮及田蓟苷均可以显著提高心肌细胞活力，减弱mPTP的开放，抑制ROS、Ca2+浓度的升高，提高线粒体活力，降低AIF的表达。研究结果表明香青兰总黄酮及田蓟苷可以通过保护线粒体起到抗缺血/再灌注（缺氧/复氧）损伤的作用，其机制可能是通过抑制线粒体mPTP开放、减轻Ca2+超载及ROS爆发，以维持线粒体的正常结构和功能而实现