低温预适应通过AMPK激活线粒体自噬改善心肺复苏后心功能的机制研究

Studies have shown that hypothermic preconditioning (HPC) can significantly reduce the heart and brain ischemia-reperfusion injury. Our previous studies have found that HPC significantly improved ventricular fibrillation Wistar rats ROSC rate, reduced the number of defibrillation times and the dosage of adrenaline used during CPR, improved heart function. HPC increased ATP content and p-AMPK protein expression levels in myocardial after ROSC. Therefore, our hypothesis is that HPC increase glucose and fatty acid metabolism, promote the production of ATP by activating AMPK; On the other hand activate mitochondrial autophagy by AMPK-ULK1-mTOR pathway to selectively remove off damaged mitochondria, at the same time, the AMPK can phosphorylate PGC-1α directly or increase the expression of PGC-1α, promote the biosynthesis of mitochondria, jointly improve cardiac function. This project through the establishment of Wistar rats CPR model and myocardial cell OGD model to research the activation of AMPK on expression of mTOR, HPC, ULK1, GLUT1, GLUT4, ACC and PINK1 / Parkin, p62, AMBRA1, NIX, BNIP, PARIS, and PGC-1α, and to analyze the relationships among them, further, the AMPK agonist and blocking agent, gene knockout, immune precipitation methods are used to clarify the mechanism that HPC regulate mitochondria autophagy, which will offer a new target for prevention and cure of heart failure after CPR.

我们前期的研究发现低温预适应（HPC）可提高室颤Wistar大鼠的ROSC率，减少电除颤次数和肾上腺素的用量，改善心功能。HPC增加ROSC后心肌内ATP的含量和p-AMPK水平。本课题提出HPC激活心肌内的AMPK，通过AMPK→ULK1← mTOR激活线粒体自噬，选择性清除受损的线粒体，改善心功能。本项目通过建立Wistar大鼠CPR模型和心肌细胞OGD模型，研究HPC对AMPK激活、mTOR、ULK1、PINK1/Parkin、p62、AMBRA1、NIX、BNIP、PARIS表达的影响，进一步采用AMPK激活剂和阻断剂、基因敲除、免疫沉淀等方法阐明HPC调控线粒体自噬的机制，为CPR后心力衰竭的防治提供新的靶点

本研究发现低温预适应（HPC）可提高室颤Wistar大鼠的ROSC率，减少电除颤次数和肾上腺素的用量，改善心功能，提高ROSC后大鼠的存活率，存活率和常规亚低温治疗组之间没有显著差异。我们发现HPC增加ROSC后大鼠心肌内ATP的含量，稳定线粒体膜电位和减轻线粒体组织学损伤，表明HPC可以减轻ROSC后线粒体损伤。进一步研究（体内和体外）发现HPC增加心肌内P-AMPKα和P-ULK1水平，同时线粒体自噬相关基因PINK1、Parkin、p62、AMBRA1和BNIP表达增加，表明HPC通过AMPK-ULK途径激活线粒体自噬优化ROSC后心肌能量代谢，从而改善心功能。本研究的意义在于发现短暂的低温预适应可以获得等于或优于传统亚低温治疗的效果，这将极大简化临床亚低温治疗的措施，具有一定的临床应用前景。