ERK抑制剂经由线粒体动力学-自噬途径促进心肺复苏后神经细胞生存研究

Decreased cerebral injury is the key to improving the prognosis for patients suffering from cardiac arrest and cardiopulmonary resuscitation (CA/CPR). However, it is not clear which functional pathways lead to neurocyte survival tendency following CPR. The results in our previous studies showed the inhibitor of extracellular regulated protein kinases (ERK) could reduce neurocyte apoptosis, increase the expression of mitochondrial fusion protein 2, decrease the expression of the split protein p-Drp 1 as well as autophagy related proteins including beclin 1 and LC3 Ⅱ. Therefore, we speculate that inhibition of over activation of ERK may produce neuroprotective effect via modulating mitochondrial dynamics - autophagy. In the current study, we are going to investigate the relationship between ERK pathway activation and mitochondrial dynamics - autophagy by using rat CA/CPR model and neuronal hypoglycemic and hypoxic reperfusion model so as to understand the pathomechanism of cerebral injury following CPR. We want to identify how does ERK inhibitor affect the expression of autophagy associated protein? How does which interact mitochondrial fission/fusion protein, maintain mitochondrial morphology and functional homeostasis and produce neuroprotective effect after CPR. The current study, from the perspective of signal molecules and functional protein molecules, reveals the mechanism of the brain injury after CPR, and provides a new way of thinking for searching a possible target of cerebral protection.

降低心脏骤停（CA）/心肺复苏（CPR）后脑损伤是改善患者预后的关键，但哪些功能途径主导CPR后神经细胞生存倾向尚未清楚。我们前期研究发现：CPR后应用细胞外调节蛋白激酶(ERK)通路抑制剂能够减少脑细胞凋亡，上调线粒体融合蛋白Mfn2表达和下调分裂蛋白p-Drp1表达，使自噬相关蛋白Beclin1和LC3Ⅱ表达减少。据此我们推测：抑制ERK过度激活可能通过线粒体动力学-自噬途径产生CPR后神经保护作用。本项目拟采用CA/CPR大鼠模型及神经细胞缺糖缺氧再灌注模型，研究在CPR后脑损伤的病理机制中ERK通路与线粒体动力学-线粒体自噬的关系，阐明ERK抑制剂如何影响自噬相关蛋白的表达及其与线粒体分裂/融合蛋白的相互作用，维持线粒体形态和功能稳态，从而产生CPR后脑保护作用。本项目从信号分子和功能蛋白质分子的视角，揭示CPR后脑损伤的发生机制，为寻找可能的CPR后脑保护作用靶点提供新思路。

本项目探究了细胞外信号激酶（ERK）-线粒体动力学-自噬途径在心肺复苏后脑缺血再灌注损伤中的病理变化，揭示抑制ERK磷酸化激活产生维护线粒体功能以抑制过度自噬的脑保护作用及机制。首先在心跳骤停/心肺复苏大鼠模型上，证实ERK抑制剂PD98059提高复苏后24小时和48小时生存率和改善两个时间点的神经功能损伤；并对复苏后24小时和48小时的大脑皮质层线粒体功能（氧化磷酸化、ATP生成、氧化还原及膜通透性）和结构有保护作用。基于此，探讨其与线粒体动力学-自噬的联系，发现PD98059增加线粒体融合蛋白MFN2的含量以及减少线粒体分裂蛋白P-DRP1的含量，抑制线粒体分裂，并减少凋亡和自噬。其次在SH-SY5Y神经细胞OGD/R模型上，进一步证实ERK-Drp1/Mfn2-自噬途径涉及的缺血再灌注病理机制。结果发现，ERK、Drp1和自噬共同参与了OGD/R诱导的细胞损伤和死亡。单独使用PD98059、Drp1 si-RNA或自噬抑制剂3-MA后均表现出神经保护效应；而单独使用ERK激活剂TPA、过表达Drp1S616E或自噬激活剂雷帕霉素进一步加重细胞损伤和死亡。PD98059抑制Drp1的活化和自噬的激活；保护线粒体功能，逆转细胞损伤和死亡，与敲降Drp1效应一致，而这些作用则被过表达Drp1S616E所抵消。敲降Mfn2后的效应与敲降Drp1相反，促进线粒体分裂和细胞自噬，抵消PD98059的抗缺血再灌注作用。因此，ERK-Drp1/Mfn2-自噬信号通路可作为治疗神经细胞缺血再灌注损伤的潜在靶点，为改善脑缺血再灌注损伤患者的治疗效果提供新的思路。