AMPK激酶失活在烧伤酸中毒心肌损伤中的重要作用及机制研究

Acidosis plays an important role in inducing cardiac injury in severe burn. However, the underlying mechanisms remain to be illustrated. Based on our previous findings and the latest advances, we hypothesize that under acidosis conditions in severe burns, the decreased activity of AMPK induces the consumption of acetyl CoA, which decreases the acetylation of histone H3 and H4, leading to cardiac injury. To confirm this hypothesis, in vivo rat models of severe burn and acidosis, in vitro heart Langendorff perfusion model and cardiomyocyte model treated with acidification medium are to be used. After modulating the protein expression with adenovirus vectors or interfering their activities by using activators/ inhibitors, the effects are to be determined by using various molecular biological and proteomics technologies. In this study, the key roles of AMPK inactivation in mediating cardiac injury under acidosis are to be explored in severe burn; and then the mechanisms and effects of the deficiency of acetyl CoA and decreased acetylation of histone H3 and H4 are to be investigated; in the end, the possible measures are to be applied in ameliorating the cardiac injury. It is hopeful that through this study, the key roles of the new pathway of “AMPK inactivation- acetyl CoA consumption - decreased acetylation of histone H3 and H4” in inducing cardiac injury are to be explored, which would provide new targets and theory for the clinical treatment of vital organ injuries induced by severe burn.

酸中毒是烧伤后引起心肌损伤、甚至死亡的重要原因，其机制未明。基于前期研究与最新进展，申请者率先提出“AMPK激酶失活引起乙酰CoA耗竭，进而降低组蛋白H3、H4乙酰化水平是严重烧伤酸中毒心肌损伤的关键分子机制”的科学假设。拟制备大鼠烧伤、酸中毒模型、离体心脏灌注模型与心肌细胞酸处理模型，从整体动物、离体心脏和心肌细胞三个层次，在基因工程调控、多种激活剂/抑制剂干预的基础上，采用分子生物学、蛋白质组学等技术，首先明确AMPK激酶失活在酸中毒心肌受损中的作用，再揭示AMPK激酶失活引起乙酰CoA耗竭、以及组蛋白H3、H4乙酰化水平降低的关键环节和作用机制，最后探讨调控各环节对烧伤心脏的保护效应。通过研究，可望证实“AMPK失活-乙酰辅酶A耗竭-组蛋白H3、H4乙酰化水平降低”在烧伤酸中毒心肌损伤中的关键作用，为烧伤后心肌损伤乃至全身脏器损伤的临床防治提供新靶点与新思路。

酸中毒是烧伤后引起心肌损伤、甚至死亡的重要原因，其机制未明。本项目制备SD大鼠烧伤模型、在体大鼠酸中毒模型、体外心肌细胞酸、碱处理模型，从在体动物与体外细胞水平研究酸中毒导致心肌受损的作用及其分子机制。证实AMPK激酶失活以及组蛋白去乙酰酶6活性升高等通过减少自噬介导酸中毒心肌细胞损害，揭示了其关键环节和作用机制，并探讨了调控相关环节对烧伤酸中毒心肌损伤的保护效应。.在心肌细胞中，发现细胞外pH改变会引起细胞内pH发生相应变化，并能调控细胞自噬水平：体外酸处理可降低心肌细胞自噬，碱处理可增加心肌细胞自噬水平。证实AMPK活性改变是环境pH变化调控心肌细胞自噬的关键分子，并发现在环境pH改变的条件下，AMPK调控细胞自噬不是通过调控mTOR信号，而是通过直接作用于自噬起始蛋白 ULK1发挥作用。.证明酸处理使心肌细胞内微管乙酰化最重要的活性酶之一——组蛋白去乙酰酶6（HDAC6）的活性升高，并介导微管乙酰化水平明显降低，是pH降低阻碍心肌细胞自噬体形成的分子机制。以Taxol上调微管乙酰化，或以HDAC6的选择性抑制剂Tubastatin A（简称TBSA）抑制HDAC6活性，均可使酸处理的心肌细胞微管乙酰化水平恢复至接近正常，心肌细胞自噬体生成增多、损伤减轻、活力改善。.本项目首次证实AMPK失活、HDAC6激活、微管乙酰化水平降低等是酸中毒导致心肌细胞损害的分子机制，激活AMPK、抑制HDAC6、上调微管乙酰化水平等可有效减轻细胞损伤、改善细胞活力。为酸中毒后心肌损害的防治与新药开发提供了有潜在临床应用价值的实验依据。.已发表标注论文3篇，其中SCI论著2篇；参编专著1部。部分实验结果在总结整理中，拟撰写论文与申请专利。

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