心肌缺血再灌注时Notch-1与自噬间关系及其机制研究

Although rapid restoration of coronary blood flow is indispensable after acute myocardial infarction (AMI), this reperfusion can paradoxically cause cardiac tissue injury, referred to as myocardial ischemia-reperfusion (I-R) injury. However, the molecular mechanisms underlying myocardial I-R injury are unknown. . The cellular homeostasis can be maintained by autophagy via eliminating excessive or injuried organelles. Under physiological conditions, cardiac autophagy is at a low level. However, sustained myocardial ischemia can cause an increased autophagy level, and during the period of reperfusion, the level of autophagy will further increase, termed as excessive autophagy. Recent studies reveal that a moderate level of autophagy is beneficial for alleviating ischemic injury, whereas an excessive autophagy will be detrimental for the heart. However, during myocardial I-R, the upstream signaling molecules regulating autophagy are poorly understood.. Notch-1 signaling plays a critical role in cell fate determination. Notch-1 signaling was reported to be an important regulator in cardiac development, cardiac progenitor cell fate, and proliferation of immature cardiomyocytes. A recent study demonstrated that Notch-1 is activated in adult mouse infarcted myocardium and the up-regulated Notch-1 signaling acts as a cardioprotective regulator. Recently,our study found that Notch-1 was down-regulated during reperfusion following ischemia, and cardiomyocyte-specific over-expression of Notch-1 intracellular domain (N1ICD) blunted myocardial injury by promoting cell survival. . The present study is designed to determine whether Notch-1 functions as a critical up-stream regulator on autophagy during myocardial I-R. To achieve this aim, the models of myocardial I-R injury in mice and hypoxia-reoxygenation injury in neonatal rat cardiomyocyte were established. The activation of Notch-1 was achieved by delivery of recombinant adeno-associated virus serotype-8-N1ICD (rAAV8-N1ICD) into the heart of wild-type adult mice and neonatal rat ventricular cardiomyocytes. And the down-regulation of Notch-1 was achieved by Notch-1-siRNA in vivo and in vitro. If our expected results were achieved, inhibition of autophagy by Notch-1 activation may represent a novel and effective strategy for alleviation of the reperfusion injury after AMI. .

自噬是机体重要的防御机制。新近研究表明：心肌缺血时，适度自噬可减轻心肌损伤，而再灌注期，过度自噬则加重心肌损伤。但心肌缺血再灌注过程中，调控自噬的上游信号分子尚未明确。Notch-1信号通路与细胞命运休戚相关。我们前期研究发现：Notch-1在心肌缺血时表达上调，再灌注后显著降低，且Notch-1过表达可显著减轻心肌缺血再灌注损伤。据此，我们提出假说：Notch-1可能作为调控自噬的上游信号分子，在心肌缺血时被激活后，通过维持适度自噬减轻心肌损伤，而再灌注时，其表达下调则导致心肌过度自噬，从而加重心肌损伤。该项目拟通过建立小鼠心肌缺血再灌注损伤模型及新生大鼠心肌细胞缺氧复氧模型，利用腺相关病毒及siRNA技术分别实现Notch-1过表达及下调，从而研究心肌缺血及再灌注这两种不同病理状态下，Notch-1与自噬间的关系及其机制，为心梗患者心肌再灌注损伤的防治提供新的思路和策略。

本项目通过构建小鼠心肌缺血(myocardial ischemia, MI)及心肌缺血再灌注损伤(myocardial ischemia/reperfusion injury, MIRI)模型和SD乳鼠心肌细胞缺氧及缺氧复氧模型，对 Notch-1 调控自噬在心肌缺血及心肌缺血再灌注过程中的作用进行研究。首先，通过western blot、透射电镜、免疫荧光，反映在心肌缺血期及再灌注期自噬水平的差异，从而导致心肌的损伤程度不同；进一步通过western blot、免疫组化证实Notch1在心肌缺血期表达显著增加及再灌注期表达显著减少；进而采用腺相关病毒载体(AAV8-N1ICD)实现Notch-1过表达，以研究心肌缺血再灌注期Notch-1表达增加与减少对心肌细胞自噬水平及组织损伤的影响。结果显示：Notch1过表达，缺血再灌注期心肌自噬水平降低、损伤减轻。综上所述，本项目揭示了Notch-1 调控自噬在心肌缺血及心肌缺血再灌注过程中的作用，为缺血性心肌病和心肌缺血再灌注损伤的临床防治提供新的思路。项目资助发表SCI 论文 10 篇（其中项目负责人王先宝为第一/通讯作者4篇），核心论文2 篇，待发表 SCI 论文 1 篇（已投稿）。培养博士生2 名，硕士生 5 名，其中 1 名博士已经取得博士学位， 1 名在读， 3 名硕士已经取得硕士学位，2 名在读。项目投入经费23万元，支出约19.5万元，各项支出基本与预算相符。