NF-κB信号通路在老龄心肌缺血再灌注损伤中的作用机制及其靶向干预研究

Aging is considered as a major risk factor for acute myocardial infraction. Recently, we found that aging increased the sensitivity of myocardial ischemia-reperfusion injury in a myocardial ischemia-reperfusion injury(I/R)mouse model. Compared with the control group, the cardiac NF-κB pathway activity significantly increased, but the mechanism is not clear. IκBα gene can specifically inhibit NF-κB signaling pathway to protect against myocardial ischemic injury. Accordingly, to explore the susceptible mechanism of NF-κB pathway in aging related myocardial ischemia, this study intends to use the myocardial ischemia-reperfusion injury model in aged wild-type and transgenic mice that can express the mutant IκBα in a cardiac-specific manner. By constructing the double-stranded recombinant AAV9 vector which can express IκBα gene rapidly in vivo and in vitro transfection experiments , the role of IκBα in regulating critical target genes of NF-κB pathway at the cellular, tissue and animal level could be clarified, and then the molecular mechanism that NF-κB pathway overactivation promotes myocardial ischemic injury could be revealed. Based on the intervention experiments of exogenous IκBα importation in aged mice, the effect and molecular mechanism of targeted inhibition of NF-κB signaling pathway could be identified in the prevention of myocardial ischemia-reperfusion injury from aging. The utility of exogenous IκBα as a novel therapeutic target for aged myocardial ischemic injury would be confirmed. The study would also provides new ideas and basis to alleviate and improve the prognosis of ischemia-reperfusion injury in patients with coronary heart disease.

老龄是急性心肌梗死(AMI)发病的高危因素。我们最近利用心肌缺血再灌注损伤（I/R）模型，发现老龄加重心肌缺血再灌注损伤的敏感性，且心肌NF-κB通路活性较对照显著激活，但机制不清楚。IκBα基因能特异性抑制NF-κB信号通路，抵抗心肌缺血损伤。据此，本课题拟采用老龄及心肌特异性IκBα转基因小鼠的I/R损伤模型，探讨NF-κB通路在老龄心肌缺血损伤易感性中的作用机制。通过构建快速表达IκBα基因的双链重组AAV9载体进行体内外转染实验，在细胞、组织及动物整体水平上，明确IκBα对NF-κB通路关键靶基因的调控，揭示NF-κB通路激活加重心肌缺血损伤的分子机制。基于老龄鼠体内外源导入IκBα干预实验，阐明靶向抑制心肌NF-κB信号通路防治老龄心肌缺血再灌注损伤的效果及分子机制，确定外源IκBα防治老龄心肌缺血损伤的效果，为减轻冠心病患者缺血再灌注损伤提供新的思路和依据。

本项目结合老龄是急性心肌梗死(AMI)发病的高危因素的临床特色，以前期研究结果为基础，建立心肌缺血再灌注损伤（Ischemia/reperfusion，I/R）模型，发现老龄心肌缺血再灌注易损性增加，且心肌核转录因子（Nuclear factor kappa B，NF-κB通路活性较对照显著激活，提示NF-κB通路是老龄心肌缺血损伤易感性增加的关键靶点，但具体机制尚不清楚。据悉，IκBα（inhibitor of nuclear factor kappa B alpha，IκBα）基因能特异性抑制NF-κB信号通路。本研究通过构建快速表达双链重组AAV9载体携带IκBα突变基因的进行体内外转染实验，在细胞、组织及动物整体水平上，同时应用心肌特异性过表达IκBα基因小鼠，观察通过抑制NF-κB信号通路，调控心肌细胞焦亡、自噬、凋亡以及线粒体功能等方面的作用，从而改善I/R损伤，明确IκBα对NF-κB通路关键靶基因的调控，揭示NF-κB通路激活加重心肌缺血损伤的分子机制及其靶向干预效果。结果发现：（1）老龄小鼠I/R后内源性心肌损伤加重，自噬效应、细胞凋亡、细胞焦亡效应增加，而NF-κB信号通路是导致急性心肌缺血再灌注损伤的年龄异质性的关键靶点。（2）细胞试验，外源性转导IκBα基因，通过抑制NF-κB信号通路减轻H/R诱导的氧化损伤和炎症反应，降低自噬水平，减少因心肌细胞凋亡和细胞焦亡，稳定线粒体功能，最终提高心肌细胞活力，达到保护衰老心肌细胞的作用。（3）动物实验，靶向转导IκBα或心肌特异性过表达IκBα可有效抑制NF-κB信号通路的过度激活、减轻炎症反应和氧化损伤、降低细胞程序性死亡、缩小心肌梗死面积、改善心功能等多种效应，达到防治老龄心肌I/R损伤的目的。因此，外源性IκBα基因靶向转导有望为老龄心肌缺血/再灌注损伤提供新型有效的治疗策略。