亚低温通过AMPK/Sirt1/FoXO1途径调控自噬保护心肺复苏后神经元细胞的机制研究

Enough evidences shown that moderate level of Autophagy has protection on neurons but the phenomenon of Autophagy was inhibited on the early stage after cardiopulmonary resuscitation in our prophase study. Further study we discovered that the activation of Autophagy could improve the neurologic function of rats after CPR, and the Autophagy was activated during the inducible hypothermia condition, so we speculated that the protections of hypothermia on neurons after CPR may be related to the activation of Autophagy.However, the mechanisms of it are still unclear. The AMPK/Sirt1/FoXO1 signaling pathway is a key way to regulate the Autophagy during ischemia and hypoxia, therefore, our projects are aim to explore the mechanisms of hypothermia on the AMPK/Sirt1/FoXO1 signaling pathway in regulating the Autophagy, and our hypothesis is that hypothermia activates the Autophagy via activation of AMPK, up-regulating the expression and activity of Sirt1 and then promoting the expressions of FoXO1、ULK 1、Rab7 and Atg5、Atg7、Atg8 or inactive the P53 or promoting the expression of anti-oxidant protein. Our projects are going to establish cardiopulmonary resuscitation animal model in transgenic mice and oxygen glucose deprivation (OGD) model in neurons to study the effects of hypothermia on the activation of AMPK, the expression and activity of Sirt1, the expressions of FoXO1、ULK 1、Rab7 、P53and Atg5、Atg7、Atg8, and the relations among them.Further study we will explore the molecular mechanisms among them through the technology of gene knock-out , Immunoprecipitation et al. At last, we will demonstrate the mechanisms of hypothermia regulation the Autophagy via the AMPK/Sirt1/FoXO1 signaling pathway, and find a new therapeutic target for cerebral resuscitation after CPR.

适度的自噬对神经元细胞具有保护作用。我们前期的研究表明心肺复苏（CPR）后早期神经元细胞自噬水平、p-AMPK降低，激活自噬,对CPR神经元细胞具有保护作用，亚低温治疗可提高自噬和p-AMPK水平。因此,本课题提出亚低温上调AMPK的活性，一方面上调ULK 1活性,抑制mTOR的活性；另一方面上调 SIRT1的活性，增加FoXO1、Rab7和自噬基因Atg5、Atg7、Atg8的表达及活性或灭活P53，促进自噬或增加抗氧化蛋白的表达，共同保护神经元的研究假说。本项目通过建立转基因小鼠CPR模型和神经元细胞OGD模型，研究亚低温对AMPK激活、Sirt1活性、FoXO1、ULK 1、Rab7、P53和Atg5、Atg7、Atg8表达的影响及它们之间的关系，进一步采用基因敲除、免疫沉淀等方法阐明亚低温调控自噬的机制，为CPR后脑复苏提供新的靶点。

本研究发现心肺复苏大鼠ROSC后2-4h大脑皮层自噬和p-AMPK水平降低。用自噬激活剂雷帕霉素（1mg/kg）明显激活自噬可减少ROSC 后72h海马和大脑皮层内的神经元细胞凋亡，表明适度激活自噬对ROSC脑损伤具有保护作用。ROSC后亚低温治疗使大脑皮层内p-AMPK和自噬水平增加，表明亚低温增加AMPK活性、促进自噬。. 体外实验我们建立神经元细胞OGD/R模型，发现OGD/R后6h、12h p-AMPK、FOXO1均较基础水平明显下降，与此同时自噬相关基因的表达也明显下降，p-AMPK、FOXO1的下降和自噬基因表达的下降成正相关。我们还发现OGD/R后Sirt1表达也显著下降，而亚低温可促进p-AMPK、FOXO1和Sirt1表达的增加，促进LC3Ⅱ/Ⅰ转换。我们进一步敲除Sirt1，发现敲除Sirt1的神经元细胞OGD/R后自噬减少、凋亡增加，亚低温处理也不能逆转这一现象，表明Sirt1是亚低温状态下调控自噬的关键分子。. 我们利用Sirt1基因敲除小鼠建立全脑缺血再灌注损伤模型（I/R）模拟ROSC后脑损伤过程，Sirt1基因敲除小鼠I/R后脑损伤明显加重，利用亚低温进行干预可减轻脑损伤，和野生型小鼠相比Sirt1-/-小鼠自噬减弱，Sirt1基因敲除并没有完全阻断自噬过程，说明亚低温状态下Sirt1并不是调控自噬的唯一途径，还有其它机制参与其中。. 我们利用全基因芯片筛选亚低温对大鼠脑组织差异性表达基因尤其关注AMPK pathway 和Autophagy pathway。结果表明与MAPK有关的炎症通路、P53凋亡通路和细胞炎症因子受体通路与心肺复苏后脑损伤有关。亚低温对这些通路中多个相关基因的表达有显著影响，通过影响CPR后炎症反应、细胞凋亡和细胞炎症因子受体介导的损伤通路，而发挥脑保护作用。针对Autophagy pathway的Q-PCR array结果表明亚低温治疗可以影响自噬通路中众多基因的改变，变化显著的基因有Caspase 8、CXCR4、Irgm和Tnfsf10等，这些基因分布比较广泛，有细胞表面受体、细胞质内效应器和穿梭于细胞质和细胞核之间的运载工具，表明亚低温可能在更高层次上，如蛋白质的的翻译、转录和转录后调控等，进行调控才能产生如此广泛的影响，这为今后的研究指明了方向。