Epac-Rap1通路在牡荆素介导的抗心肌缺血/再灌注损伤中的作用及机制

Ischemic heart disease is the leading cause of death worldwide, which develops as a consequence of a number of etiological risk factors. This disease always coexists with such disease states as systemic arterial hypertension and related left ventricular hypertrophy, hyperlipidemia, and atherosclerosis, diabetes and insulin resistance, heart failure, as well as aging. Myocardial ischemia reperfusion (MI/R) injury is known to occur on the restoration of coronary blood flow after a period of myocardial infarction (MI) during using such treatment strategies as thrombolysis, percutaneous transluminal coronary angioplasty, and coronary bypass surgery. MI/R injury is associated with the increased reactive oxygen species (ROS), calcium overloading, and the loss of membrane phospholipids especially during the reperfusion. We have previously found that vitexin, a natural product, can alleviate the myocardial IRI damage, but the underlying mechanisms remains unknown. Exchange protein directly activated by cAMP (epac) is a multidomain cAMP mediator, which regulates diverse biological functions. These novel cAMP mediators including Epac1 (cAMP-GEF-I) and Epac2 (cAMP-GEF-II) are known as guanine nucleotide exchange factors for Ras-like small GTPases. They are activated by cAMP, leading to the activation of Rap1. They also regulate calcium homeostasis, ion transportation and myocardial metabolism in the cardiomyocytes. Our preliminary study showed that myocardial MI/R injury up-regulated Epac and Rap1 protein expression, which was attenuated by vitexin. We thus hypothesize that Epac-Rap1 signaling pathway participates in vitexin-mediated protection of MI/R injury. To test this hypothesis, we will examine the impact of MR/I injury on Epac and Rap1 expression and the protective role of vitexin in our established MI/R rat model. Furthermore, cell biological, pharmacological and molecular approaches will also be used to determine the cellular and molecular mechanisms in MI/R injury. This study will provide valuable insights into developing therapeutic approaches targeting myocardial ischemic reperfusion injury.

心肌缺血再灌注损伤(IRI)严重危害人类健康，本课题组前期研究发现牡荆素对心肌IRI有保护作用，其机制尚未阐明。环腺苷酸结合蛋白(Epac)是一种新型cAMP通路的效应分子，在心肌细胞内，Epac调节兴奋-收缩偶联和钙离子稳态。最新预实验发现大鼠心肌IRI后心肌Epac蛋白表达增加与心肌损伤程度成正比；牡荆素后处理大鼠心肌IRI可抑制心肌Epac上调和下游Rap1激活，解除Epac对缺氧复氧心肌细胞ATP敏感钾通道的抑制作用。据此我们假定牡荆素可通过调控Epac-Rap1通路发挥心肌IRI的保护作用。为验证该假说，本项目通过建立在体大鼠心肌IRI模型和原代心肌细胞缺氧复氧模型，研究心肌IRI过程中Epac及其下游信号分子的动态变化，并探明牡荆素通过Epac-Rap1途径对心肌IRI保护作用机制。本研究结果将揭示牡荆素治疗心肌IRI的潜在分子机制，为临床治疗心肌IRI提供新靶标和实验依据。

心脏缺血再灌注损伤严重危害人类健康，Epac-Rap1信号通路在心肌IRI是否发挥重要作用，项目将探讨牡荆素（VT）介导Epac-Rap1信号通路抗大鼠心肌缺血再灌注损伤的分子机制。项目在整体动物模型和体外细胞两个层面考察Epac-Rap1信号通路参与心肌IRI病理生理机制。研究结果显示，大鼠MIRI后，心肌细胞内信号分子Epac1激活，并导致其下游的Rap1激活，促进心肌损伤； 8-CPT可进一步促进心肌细胞Epac1过表达和下游Rap1激活，加重心肌损伤； ESI-09可抑制Epac1和下游Rap1的激活，并抑制Epac1mRNA及Rap1mRNA基因表达，抑制CaMKⅡ蛋白的表达和ERK的磷酸化；VT可显著抑制MIRI大鼠心肌损伤，抑制心肌细胞Epac1的激活和下游Rap1活化，抑制CaMKⅡ蛋白的表达和ERK磷酸化，抑制Epac1mRNA及Rap1mRNA基因表达，ESI-09与VT具有协同作用。在心肌细胞OGD模型上， 细胞活力降低，LDH释放增加，心肌细胞内Epac1激活，下游Rap1的表达增加； 8-CPT增强心肌细胞Epac1过表达，增加下游Rap1的表达，并导致胞内钙超载，加重心肌细胞损伤； ESI-09可抑制心肌细胞Epac1的激活与过表达，抑制下游Rap1的表达与CaMKⅡ蛋白的表达，并增加ERK的磷酸化，抑制钙超载；VT（10μmol/L）可显著抑制OGD心肌细胞Epac1的激活及过表达，抑制其下游Rap1的表达，抑制CaMKⅡ蛋白表达，增加ERK磷酸化，减轻心肌细胞损伤。Epac1基因沉默后，心肌细胞Epac1及其下游相关蛋白、胞内钙离子浓度下降，并抑制ERK磷酸化；Epac2基因沉默后，心肌细胞Epac2表达下调，Epac1及其下游相关蛋白、胞内钙离子浓度无明显变化， ERK磷酸化减少。VT预处理后，慢病毒稳转Epac1或Epac2进细胞， Epac1或Epac2基因沉默后， ERK磷酸化均抑制；Epac1沉默后，胞内钙离子浓度降低；但Epac2沉默后，胞内钙离子浓度无明显变化。免疫共沉淀实验中，下拉Epac1后可检测到其下游Rap1的蛋白表达，证实细胞内Epac1 与Rap1蛋白间存在相互作用。项目初步探明了牡荆素调控心肌Epac-Rap1信号通路参与保护缺血心肌的分子机制，为牡荆素应用于临床治疗心肌IRI提供实验基础和理论依据。