超声STE评估CG诱导IL-10调控JAK2/STAT3、PI3K/Akt减轻鼠心肌I/R的基础研究

Myocardial Ischemia reperfusion injury is closely related with apoptosis. Calycosin-7-O-β-D-glucoside (CG), able to reduce neuron damage caused by I/R, is one of the main flavonoid ingredients of astragalus membranaceus. Early experiments indicated that CG could inhibit myocardial cell apoptosis through the activation of PI3K/Akt signaling pathway and eventually relieve myocardial Ischemia reperfusion injury. Related studies have shown that IL-10, JAK2/STAT3 and PI3K/Akt signaling pathway plays an important role in the protective mechanism of myocardial ischemia-reperfusion injury. Th1 inflammatory cytokines (IFN - gamma and IL - 2) and Th2 inflammatory cytokines (IL-4 and IL-10) were down-regulated and up-regulated, respectively, by astragalus membranaceus during its heart protection in autoimmune myocarditis. Several studies have shown that IL-10 can promote the activation of JAK2/STAT3 and PI3K/Akt signal pathway. To sum up, a hypothesis that myocardial ischemia-reperfusion injury alleviation of CG is developed by inducing IL-10 mediated activation of JAK2/STAT3 and PI3K/Akt signaling pathway was accordingly claimed. Based on the previous work, the present study built the model of myocardial ischemia-reperfusion injury of Wistar rat through coronary artery ligation, to explore the role of CG in its myocardial ischemia-reperfusion injury. Also, STE technology was used to evaluate effects of CG on myocardial function. Besides, anti-IL-10 antibody as well as JAK2 / STAT3, PI3K/Akt signaling pathway specific inhibitors was used to verify that CG realizes its important function, inhibiting myocardial apoptosis, by inducing IL-10 mediated activation of JAK2/STAT3 and PI3K/Akt signal pathway and thus protect myocardia from reperfusion injury. The present study will firstly report the protective mechanism of CG, which will be of important theoretical significance and application value and provide new thought, new targets and new technology for prevention and control of acute myocardial infarction (AMI).

心肌缺血再灌注损伤（I/R）与细胞凋亡密切相关。毛蕊异黄酮葡萄糖苷（CG）是黄芪的主要黄酮类成分之一，在大脑I/R过程中能够减轻I/R引起的神经元损伤。预实验表明，CG能够通过激活PI3K/Akt信号通路抑制心肌细胞凋亡，减轻心肌I/R。相关研究表明IL-10、JAK2/STAT3和PI3K/Akt信号通路在I/R的保护机制中发挥重要作用。因此，推测“CG是通过诱导IL-10调控JAK2/STAT3和PI3K/Akt信号通路减轻心肌I/R”。 本课题将在原有工作基础上构建Wistar大鼠I/R模型，应用STE评价CG对心肌功能的影响；探讨CG通过诱导IL-10调控JAK2/STAT3和PI3K/Akt信号通路，发挥抑制心肌细胞凋亡，减轻心肌I/R的作用和机制。目前，国内外未见相关报道。本课题的完成，将为急性心肌梗死的防治提供一种新思路、新靶点和新的技术平台，具有重要的理论意义和应用价值。

急性心肌梗死严重影响人类的生活质量，而再灌注治疗后心脏出现不可逆性结构伤害，即心肌I/R损伤。本课题组前期证实黄芪有效活性成分毛蕊异黄酮葡萄糖苷（Calycosin-7-O-β-d-glucoside，CG）具有保护心肌免遭I/R损伤的功能，因此本项目展开对其相关分子机制的研究。本项目从细胞与动物水平对相关分子机制充分证实，首先利用缺氧复氧处理大鼠心肌细胞H9C2，构建心肌I/R损伤细胞模型，并使用CG进行干预处理，经分子生物学实验检测发现，I/R诱导H9C2细胞发生凋亡、细胞中IL-10的表达水平显著降低、JAK2/STAT3信号通路灭活，而CG可有效逆转这一现象。其次采用结扎小鼠LAD法，构建心肌I/R损伤小鼠模型，并使用CG进行干预处理，经分子生物学实验检测发现，I/R诱导心肌组织梗死面积增大、血清与组织中IL-10的表达水平显著降低、JAK2/STAT3信号通路灭活，而CG对这种异常现象可有效逆转。此部分研究结果初步证实IL-10与JAK2/STAT3信号通路参与CG保护心肌免遭I/R损伤的过程。随后展开进一步证实，构建心肌I/R损伤小鼠模型，并使用CG干预的基础上，同时给予IL-10RA抗体阻断IL-10的表达，经分子生物学实验检测发现，IL-10RA抗体破坏了CG对I/R诱导损害的缓解作用，包括血流动力学参数、心肌损伤面积、JAK2/STAT3信号通路以及心肌细胞凋亡等。此结果充分证明CG可通过调节IL-10激活JAK2/STAT3信号通路发挥保护心肌免遭I/R损伤的功能。以上研究结果可为毛蕊异黄酮葡萄糖在临床治疗心肌I/R损伤中的应用提供理论依据。