脑红蛋白对AMPK介导脑缺血损伤与修复的调控作用及机制研究

Ischemic stroke is the most common subtype of cerebrovascular disease which has been the first leading cause of death in China since 2010. Acute ischemic stroke treatment is quite challenging for the evidence-based therapies are so limited. AMP-activated protein kinase (AMPK), which acts as a key metabolic regulator, would be an important potential target for stroke treatment and prevention. However, the role of AMPK in ischemic brain injury remains controversial. Our previous study indicates that neuroglobin (Ngb) transducted by TAT into brain has a protective effect on cerebral ischemia, and the overexpression of Ngb could significantly inhibit AMPK activation in the brain tissue of the Ngb transgenic mice. We hypothesis that Ngb might regulate brain injury and repair by AMPK during cerebral ischemia. The focal cerebral ischemic model of AMPK-knockout mice and Ngb transgenic mice in vivo and oxygen glucose deprivation (OGD) model in vitro will be used in this study. The cell energy metabolism, autophagy, brain damage and repair, the expressions of AMPK pathway cascades will be detected too. The aim of this study is to clarify the effect and mechanism of AMPK in the ischemic cerebral injury or repair and elucidate the regulatory role and mechanism of Ngb on AMPK.

缺血性脑卒中是严重危害健康的常见病，AMP-活化蛋白激酶（AMPK）是调节细胞能量代谢的关键调控器，在脑缺血损伤与修复中发挥着重要作用，有望成为缺血性脑卒中治疗新靶点。申请人前期研究发现TAT转导的脑红蛋白（Ngb）在脑缺血中具有保护作用；而Ngb转基因小鼠脑组织AMPK活性明显减低，提示Ngb能在非缺血状态下抑制AMPK活性。那么，Ngb是否也能抑制脑缺血后的AMPK活性，进而调控脑缺血损伤与修复？明确这一重要科学问题将为缺血性脑卒中治疗研究提供新思路和新靶点。本项目立足上述前期研究，拟联合慢病毒转染、TAT蛋白质转导等技术，对AMPK基因敲除小鼠和Ngb转基因小鼠进行在体和离体研究，全面检测细胞代谢、细胞自噬、脑损伤与修复及AMPK信号通路等指标变化，明确AMPK在脑缺血损伤与修复中所发挥的作用及机制，阐明Ngb和TAT-Ngb融合蛋白对AMPK介导脑缺血损伤与修复的调控作用及机制。

缺血性脑卒中是严重危害健康的常见病，AMP-活化蛋白激酶（AMPK）是调节细胞能量代谢的关键调控器，在脑缺血损伤与修复中发挥着重要作用，有望成为缺血性脑卒中治疗新靶点。申请人前期研究发现脑红蛋白（Neuroglobin, Ngb）在脑缺血中具有保护作用；而Ngb转基因小鼠脑组织AMPK活性明显减低。本项目从在体与离体、野生型与转基因及基因敲除等不同层面，检测了细胞代谢、细胞自噬、脑损伤与修复及AMPK信号通路等指标变化，观察了AMPK在脑缺血损伤和修复中的作用及机制，探讨了Ngb对AMPK介导脑缺血损伤与修复的调控机制。体外研究结果表明抑制AMPK活性对谷氨酸诱导的HT22细胞损伤具有保护作用，可能通过减轻氧化应激损伤来实现的；而Ngb能调控AMPK活性并在HT22细胞中发挥抗谷氨酸损伤作用，可能与其减轻氧化应激水平、调控HIF-1α及细胞凋亡途径有关。体内研究结果表明二甲双胍激活小鼠AMPK活性具有抗慢性脑缺血损伤的作用；而敲除小鼠AMPKα基因功能则会加重慢性脑缺血损伤。本项目还发现血清Ngb水平增高与蛛网膜下腔出血的不良预后相关，表明Ngb可能成为预测预后的生物标志物；此外，脑胶质瘤患者脑瘤细胞胞浆Ngb表达量与肿瘤病理级别成正相关，表明 Ngb在胶质瘤的诊断、评价恶性程度中有重要意义。最后，本项目还充分利用临床资源优势，收集了107例基因确诊的CADASIL患者，建立了遗传流调队列，发现中国东南沿海地区CADASIL突变热点为NOTCH3基因R544C点突变，且发现R544C点突变患者容易伴发脑出血和脑微出血，但发病机制不明。因此，我们采用CRISPR/Cas9新技术，成功创建了该点突变的CADASIL小鼠，并发现高血压和慢性脑缺血损伤表型。总之，本项目为Ngb的临床转化提供了实验依据，为缺血性脑卒中等疾病的神经保护研究与药物研发提供了新思路和新靶点。