HSF5/HSPB7和14-3-3η交互作用与心肌损伤保护

On the basis of previous studies, We hypothesized that the 14-3-3η/HSF5 expression is up-regulated and interacted，and imported into the nucleus to activate of HSPB7 in the downstream ,and to induce myocardium protection. The aim of this study is to investigate the role of 14-3-3η/HSF5/HSPS in the pathogenesis of acute myocardial injury in a new way. HSF5 protection was confirmed by gene transfection and RNAi. Co-IP, GST-pull down and yeast two-hybrid method were used to confirm the interaction between 14-3-3η/HSF5. The expression of 14-3-3η/HSF5 was detected by cytoplasmic/nuclear protein separation and Western blot analysis. Subcellular localization of 14-3-3η/HSF5 was detected by multi-fluorescent labeling/DAPI/Confocal assay. The effect of downstream effector HSPB7 was investigated by RNAi method. Subcellular localization in 14-3-3η/HSPB7 cells was preliminarily studied by multi-fluorescent labeling/Confocal method. Finally compare and comfirm the results through whole animal experiment. This project will help people to understand the myocardial ischemia/reperfusion phenomenon and its mechanism thoroughly and comprehensively, to further improve and enrich the relevant knowledge and theory, to elucidate possible targets for new drug effects, to lay a good theoretical and experimental basis for the development of myocardial protection drugs.

在总结前人与自身工作基础上，我们假设：心肌细胞遭受缺氧/复氧损伤刺激时，14-3-3η/HSF5表达同步上调且相互作用，入核启动下游HSPB7表达，发挥心肌保护作用。拟将14-3-3η/HSF5/HSPB7有机结合，用基因转染、RNAi技术确认HSF5保护作用，Co-IP、GST-pull down、酵母双杂交法证实14-3-3η/HSF5间相互作用；胞浆、胞核蛋白分离，Western blot检测14-3-3η/HSF5表达，多荧光标记/DAPI染色/Confocal测14-3-3η/HSF5细胞内之亚定位；用基因转染、RNAi法探讨下游蛋白HSPB7作用；用多荧光标记/Confocal法初步探讨14-3-3η/HSPB7细胞内之亚定位，最后在整体动物实验上比较论证。这将有助于深入了解心肌缺血/再灌注机制，丰富相关知识和理论，阐明新的药物作用靶点，为相应药物研发奠定良好的理论与实验基础。

本项目将14-3-3η/HSF5/HSPB7有机结合，探讨相关信号通路在急性心肌A/R损伤与保护中的作用。首先，确认了HSF5对大鼠乳鼠心肌细胞的保护作用：构建了相关腺病毒载体以及HSF5不同表达细胞；结果表明：HSF5高表达可增强心肌细胞抗急性A/R损伤能力，反之，则加重损伤。第二，确认了HSF5为14-3-3η配体蛋白：应用酵母双杂交、Co-IP及GST-pull down确认14-3-3η/HSF5间的交互作用。第三，探讨了14-3-3η不同表达对抗急性A/R损伤的影响：用已有HSF5高表达细胞，构建14-3-3η高/低表达细胞；结果表明：14-3-3η/HSF5高表达细胞，具有良好的抗急性A/R损伤能力，反之，则加重损伤。第四，探讨了14-3-3η/HSF5对下游HSPB7的影响：构建了相应腺病毒载体及HSPB7低表达细胞；结果表明：HSF5/14-3-3η高表达抗急性心肌A/R损伤能力，依赖于HSPB7表达。最后在整体大鼠心脏上进行了比较论证。本项目首次阐明HSF5在心肌急性A/R损伤时，为内源性心肌保护因子；确认HSF5为14-3-3η配体蛋白；HSPB7为14-3-3η/HSF5下游效应蛋白，三者协同作用，在心肌急性A/R损伤时发挥良好保护作用；这有助于人们深入了解心肌急性缺血/再灌注损伤机制及其14-3-3η/HSF5/HSPB7作用与贡献，进一步丰富相关知识和理论，阐明可能新的药物作用靶点，为心肌保护药物的研发奠定良好的理论与实验基础。我们还证实EGCG、辣椒素、川芎嗪、黄芪甲苷等中药成分显著上调/下调心肌细胞14-3-3/VDAC1/AMPK表达，激活适应性自噬、维护线粒体功能、减轻铁死亡，缓解急性A/R或阿霉素/LPS毒性损伤，为类似防治药物的研发奠定理论与实验基础。本课题全面完成研究计划，发表学术论文14篇（标注资助），并培养博士生1名，硕士生4名。