基于HIF-1信号通路的朝药泽兰对缺氧诱导心肌细胞损伤的保护作用及其机制研究

Myocardial hypoxia injury （MHI）,one of the most common symptoms of ischemic heart disease,is characterized by Ca2+ overload and abnormal reactive oxygen species metabolism which caused myocardial apoptosis. Hypoxia inducible factor-1? (HIF-1) is an oxygen-dependent transcriptional factor, which plays a key role in oxygen homeostasis and therefore leads to cardioprotection via the upstream of MAPK or PI3K/AKT signaling pathway as well as downstream of expression of hypoxia related genes and regulation of mitochondrial oxidative stress signaling pathway. Lycopus lucidus Turcz (LLT), a perennial herb widely used as the 'Taiyin' Medicine of Korean Traditional Medicine (KTM), has the function of promoting blood circulation, removing blood stasis, accelerating 'Qi', and eliminating 'Zhuo'. According to KTM medication principle proposed that the drugs must be taken on the basis of 'Constitution', LLT is used to treat several kinds of ischemic heart diseases caused by 'Taiyin' physique characteristic of 'blood Zhuo and Qi retardarce'. The preliminary study documented that LLT elicits positive inotropic effects and its mechanism is related to activation of Ca2+ entry and Ca2+ release. Therefore, it would be provided three kinds of MHI models such as the isolated beating perfusion atrium model of acute hypoxia injury (in vitro), myocardiac cells model of hypoxia/reoxygenation (in vitro) and animal model of myocardial ischemia reperfusion injury (in vivo) in our projects. Therefore, the purpose of the present study was to investigate the mechanism by which LLT plays cardioprotection role in hypoxia induced injury and the results would be provided essential theoretical and experimental evidence to KTM research for anti-hypoxia induced myocardial injury.

心肌缺氧损伤是缺血性心脏病最常见的症状之一，可引起钙超载和氧自由基异常代谢，进而诱发心肌细胞凋亡。缺氧诱导因子-1（HIF-1）是一种依赖氧调控的转录因子，可通过上游的MAPK或PI3K/Akt信号通路及其下游的一系列缺氧相关基因的表达和对线粒体氧化应激途径的调控来发挥心肌保护作用。泽兰属朝医太阴人药，具有活血化瘀，行气化浊等功效，根据朝医"按象用药"的原则，用于治疗太阴人"血浊气涩"而引发的各种缺血性心脏疾病。前期研究发现，朝药泽兰可改善心肌正性肌力，其机制可能与钙离子调控相关。为阐明泽兰在缺氧状态下对心肌保护的作用机制，本课题组拟通过离体心脏灌流急性缺氧模型、心肌细胞缺氧/复氧损伤模型及心肌缺血再灌注动物模型，从整体和细胞分子水平上探讨朝药泽兰发挥保护缺氧心肌损伤的作用机制与HIF-1信号通路之间的相互联系。本研究成果将为朝药抗心肌缺氧损伤的基础研究提供必要的理论和实验依据。

心肌缺血再灌注或心肌缺氧/复氧损伤是目前临床医学在缺血性心脏疾病研究过程中所急需解决的问题。心肌缺氧可引起心肌细胞水肿、超微结构改变及心肌能量代谢变化等一系列不良影响。缺氧诱导因子-1（HIF-1）是细胞氧稳态调节的调控因子，缺血时HIF-1a作为功能亚基可引发各种缺氧应激蛋白表达的转录因子，在缺血适应性反应中起核心作用。因此，将HIF-1a作为缺血性心脏病的候选基因，通过发现更多新源药物更深入地研究抗心肌缺血再灌注或心肌缺氧/复氧损伤作用靶点是缺血性心肌病治疗的迫切需要。朝药泽兰通过调节HIF-1a信号通路干预心肌缺血再灌注损伤或心肌缺氧/复氧损伤。体外实验表明，心肌缺氧损伤时，泽兰能够激活PI3K/Akt信号通路，显著抑制HIF-1a及其下游因子活性。此外，泽兰能显著抑制缺氧诱导的心肌细胞凋亡。体内实验使用结扎冠状动脉左前降支的方法建立大鼠心肌缺血再灌注损伤模型。每天灌胃给予泽兰（200 mg/kg 或 500 mg/kg）。病理组织学结果显示泽兰能够干预心肌缺血再灌注损伤诱导的心肌细胞凋亡。同时，能够有效抑制HIF-1a的表达，增加PI3K/Akt信号通路活性及GSK-3beta的磷酸化作用，与体外结果一致。此外，泽兰的有效成分中迷迭香酸、桦木酸和齐墩果酸等单体物质也具有明显的抗心肌缺氧/复氧损伤作用。综上所述，朝药泽兰对拮抗心肌缺血再灌注或心肌缺氧/复氧损伤有显著的心肌保护作用，具有潜在的药理学治疗特性。但是作为新源药物仍需要进一步深入研究，确定其确切的心肌保护机制。