调控HIF信号通路恢复老龄心肌缺血后处理心肌保护作用的分子机制研究

Cardiac complications caused by myocardial ischemia / reperfusion (I/R) injury are common and serious. Studies have shown that ischemic postconditioning (IPostC) myocardial protection is weakened by aging, the mechanism is unclear. Applicant has found that IPostC cardioprotective mechanisms is associated with activation of hypoxia-inducible factors (HIF) signaling pathways, thereby up-regulating its downstream key target genes. But HIF pathway regulation disorder in the aging state, suggesting that the mechanism of weakened IPostC protection in aging myocardial may be related to abnormal HIF signaling pathway. We propose that activation of aging myocardial HIF signaling pathway, so that up-regulate its downstream key target genes may be the key node to restore IPostC myocardial protection. Based on previous research, this project is intended to: (1) elucidate the molecular mechanism of HIF signaling pathway regulation abnormal in aging patients leads to weaken the myocardial protective effect of IPostC; (2) explore the new strategy to restore the protective effect of IPostC in aging myocardial with gene intervention through the activation of HIF signal pathway and up-regulate its downstream key target gene. The solution of the above problem not only helps to clarify the mechanism of weakening the protective effect of IPostC in aging myocardial, but also provides a more systematic scientific basis for the myocardial protection of aging patients.

心肌缺血/再灌注(I/R)损伤导致的心脏并发症常见且后果严重，研究表明：衰老弱化缺血后处理(IPostC)的心肌保护作用，其机制尚不清楚。申请人研究发现：IPostC发挥心肌保护作用的机制与激活缺氧诱导因子(HIF)信号通路并上调其下游的关键靶基因有关，但老龄状态下HIF通路调控失常，提示老龄弱化IPostC心肌保护作用的机制可能与HIF信号通路失常有关。我们提出：激活老龄心肌的HIF信号通路，上调其下游关键靶基因可能是恢复IPostC心肌保护的关键节点。本项目拟在前期研究基础上：(1) 阐明老龄心肌HIF信号通路调控异常致IPostC心肌保护作用弱化的分子机制；(2) 探索通过基因干预手段激活HIF信号通路及上调其下游的关键靶基因，恢复老龄心肌IPostC保护作用的新策略。上述问题的解决不仅有助于阐明老龄心肌IPostC保护作用弱化的机制，更将为老龄患者的心肌保护提供系统的科学依据。

急性心肌梗死（AMI）是目前严重危害人类健康的重要心血管疾病，而再灌注治疗的同时，会引起致命性的心肌缺血再灌注（I/R）损伤，申请人研究发现：缺血后处理（IPostC）发挥心肌保护作用的机制与激活缺氧诱导因子（HIF）信号通路并上调其下游的关键靶基因有关，但老龄状态下HIF通路调控失常，提示老龄弱化IPostC心肌保护作用的机制可能与HIF信号通路失常有关。我们提出假设：激活老龄心肌的HIF信号通路，上调其下游关键靶基因可能是恢复IPostC心肌保护的关键节点。因此本研究从细胞和器官水平，运用腺病毒转染技术，乳鼠心肌细胞培养技术，定量RT-PCR、western blot、免疫组化等，明确IPostC心肌保护作用与HIF信号通路和该通路下游低氧敏感基因（AMPK、VEGF、GLUT及PDK等）的内在联系，并成功构建携带HIF-1α基因的重组9型腺相关病毒（AAV-9）载体靶向转导老龄心肌，通过上调 HIF-1α水平并诱导下游关键靶基因的表达，阐明靶向转导 HIF-1α对IPostC抗老龄心肌I/R损伤的心肌保护作用。并完成了AAV9-HIF靶向调控HIF下游基因己糖激酶Ⅱ（HKⅡ）与3'磷酸肌醇依赖的激酶1（PDK1）的实验研究，同时加入药物后处理（七氟醚后处理），观察其对心肌HIF-1信号通路以及巨噬细胞迁移抑制因子（MIF）通路的调控作用，并观察调控MIF通路后对下游AMPK通路的影响。以上内容的完成，更有利于明确AAV9-HIF对老龄心肌HIF通路以及下游靶蛋白的调控机理，为临床围术期保护老龄患者心肌功能提供新的资料。