急性心肌梗死中NF-κB/Drp1调控线粒体动力学在心肌细胞凋亡的分子机制研究

The molecular mechanism of mitochondrial dynamics in mitochondrial dysfunction and apoptosis has become a new research hotspot in the treatment of acute myocardial infarction (AMI). Our previous study found: the NF-κB signaling pathway was activated; the expression of mitochondrial fission protein Drp1 was significantly increased and mitochondrial division was increased during AMI but the mechanisms are not clear. The project will establish the hypoxia ischemia model of cell and animal to explore the mechanisms among NF-κB/Drp1 and mitochondrial dysfunction and dynamics during AMI by knocking out the Drp1 gene, over-expressing and silencing the key molecule IκBα in the NF-κB signaling pathway. This study has two parts: ① Using acute ischemia hypoxia model of wild type and Drp1-/- knockout myocardial cells and mice to explore the molecular between regulatory mechanism NF-κB and Drp1 by detecting the expressions of key molecular of NF-κB pathway and Drp1 and expression way and their combination binding locus. ② To explain the molecular of NF-κB/Drp1 mediates the mitochondria dynamic and its functions by detecting the key molecules of NF-κB/Drp1 pathway, mitochondrial dynamic related proteins, mitochondrial morphology, inflammatory factors and apoptosis in vivo and in vitro levels during AMI .

线粒体动力学在急性心肌梗死（AMI）治疗中已成为新的研究热点。课题组发现：小鼠AMI模型中心肌细胞NF-κB信号通路被显著激活、心肌细胞线粒体分裂蛋白Drp1表达显著上调、线粒体分裂增加，但NF-κB是如何调控心肌细胞缺血后线粒体分裂的机制尚不清楚。本研究采用缺血缺氧模型，通过敲除Drp1基因，过表达和沉默NF-κB信号通路中关键分子IκBα，揭示急性心肌梗死中NF-κB/Drp1调控线粒体动力学在心肌细胞凋亡的分子机制。包括两个研究内容：①采用野生型、Drp1-/-基因敲除心肌细胞和小鼠急性缺血缺氧模型，通过检测NF-κB和Drp1表达、结合方式和位点，探索NF-κB、Drp1之间的分子调控机制。②通过细胞和在体两个水平，通过检测NF-κB/Drp1通路中关键分子、线粒体动力学相关蛋白、线粒体形态、炎症相关及细胞凋亡等指标，明确AMI中NF-κB/Drp1对线粒体分裂及其功能的调节机制。

线粒体动力学在急性心肌梗死（AMI）治疗中已成为新的研究热点。本课题：① 改良并建立简便高效的小鼠原代乳鼠心肌细胞分离培养方法，通过缺血缺氧诱导心肌细胞缺血缺氧模型，检测NF-кB通路中关键分子p65、p50和线粒体融合-分裂相关蛋白，线粒体形态与功能，细胞凋亡。研究发现缺血缺氧导致心肌细胞NF-κB通路激活，其通过促进线粒体分裂蛋白Drp1的表达，增加心肌细胞线粒体过度分裂，从而导致心肌细胞线粒体功能障碍和凋亡显著增加。采用NF-κB通路抑制剂PDTC和过表达NF-κB通路中关键的NFKB1基因，则明显降低Drp1表达，减轻线粒体分裂程度和心肌细胞凋亡。② 通过结扎野生型和NFKB1-/-小鼠冠状动脉前降支建立小鼠急性心肌梗死模型，检测NF-кB通路关键分子p65、p50和线粒体融合-分裂相关蛋白表达、线粒体超微结构、血浆炎症相关因子、心肌酶标志物水平、心肌梗死面积等。研究发现，与野生型小鼠相比，术前注射NF-κB通路抑制剂PDTC的小鼠，心肌梗死面积显著降低，线粒体分裂程度显著降低，炎症因子IL-6、IL-1β、TNF-α水平显著降低。而NFKB1-/-小鼠心肌梗死面积显著增加，心肌细胞线粒体分裂程度增加，循环中炎症因子水平明显增加。本研究从体外细胞和载体小鼠水平，阐明了NF-κB通路介导Drp1蛋白的表达对心肌细胞线粒体分裂和功能的调控中的细胞学机制，证实靶向干预NF-κB/Drp1通路的心肌保护效应及可行性。