Botch经抑制Notch通路实现缺血性脑卒中模型中神经保护作用及机制研究

Stroke is the leading cause of adult death and disability in the world. It is well recognized that transient mild ischemic preconditioning in neurons or animals could increase their tolerance to later lethal ischemic insult. According to the literature and our pilot study, Botch expression is elevated after ischemic preconditioning in neurons. And Botch inhibition polishes the neuroprotection provided by such preconditioning. From perspective of mechanism, Botch promotes neurogenesis by blocking S1 furin-like cleavage of Notch1. Meanwhile it is reported that Notch pathway is activated in the ischemic hemisphere, and inhibiting Notch protein maturation by DAPT could protect the neurons from death and decrease the ischemic infarction volume. Hereby we assume that Botch has great potential in neuroprotection. In basic study, oxygen and glucose (OGD) insult in neurons is frequently used as in vitro stroke model. And middle cerebral artery occlusion (MCAO) is most widely used as in vivo stroke model. To make the translation from bench to bedside easier, before promising achievement in rodents could be applied in clinic trials, basic research on human species is recommended. With the development of human pluripotent stem cell technology, human embryonic stem cells (hESCs) and their differentiated human neurons has become perfect source of human disease models and brought curing hope for multiple neurological diseases. Applying OGD injury to human neurons derived from hESCs, we could establish an in vitro stroke model for Botch neuroprotection study. And combining the human neural progenitor transplantation and MCAO injury in mouse we could produce a powerful in vivo model for human stroke with which we could study the Botch function in living human neurons. Using lentivirus system to up-regulate the Botch expression or shRNA to silent Botch expression, we could verify the relationship between Botch expression and tolerance to ischemia in human neurons and explore whether Botch exert neuroprotection via inhibiting Notch signaling activation. Our study investigates neuroprotection of Botch both in mouse and in human, both in vitro and in vivo. If our hypothesis proves true, it will add to the treatment and pathological mechanism of stroke.

脑卒中是中国人群第一位致死和致残因素，寻找溶栓以外的治疗方法非常必要。多项神经保护剂临床试验的失败提示神经保护靶点的基础研究必须基于客观依据，且在多个种属水平得到验证。文献和我们的前期工作表明：Botch是发挥缺血预适应神经保护作用的关键分子。Botch能抑制Notch蛋白的剪切和激活，而Notch激活导致缺血损伤恶化、抑制Notch信号能缓解卒中损伤。据此我们推测Botch可能通过抑制Notch信号发挥神经保护作用。这一假设在我们前期小鼠神经元体外研究已经得到初步验证。本项目将引入Botch敲除的模式动物，利用人类神经元OGD损伤作为人类神经元体外卒中模型，结合人类神经前体细胞移植和MCAO技术构建人类神经元在体卒中模型；利用慢病毒载体上调或者shRNA沉默Botch基因表达。从小鼠和人类两个种属，从体外和体内两个层次研究Botch表达对脑卒中结局的影响和机制，具有较好的临床转化价值。

脑卒中是中国人群第一位的致死和致残病因，给社会经济造成沉重负担。卒中后的神经保护治疗一直是卒中后治疗的热点。文献及我们的前期工作基础提示，Botch是发挥缺血预适应神经保护作用的关键分子。Botch能抑制Notch1蛋白的剪切和激活，是内源性的Notch通路抑制剂。为验证Botch在脑卒中的神经保护作用，本项目利用体内大脑中动脉栓塞（middle cerebral artery occlusion and reperfusion，MCAO/R）为体内卒中模型、神经元氧糖剥夺模型（oxygen and glucose deprivation，OGD）为体外卒中模型，利用腺病毒载体上调或者shRNA沉默Botch基因表达，从体内和体外两个层次研究Botch表达对脑梗死结局的影响和可能的机制。在缺血性卒中以外，另外将Botch的神经保护作用在出血性卒中进一步验证。本研究证实，缺血缺氧损伤诱导神经元和小胶质细胞内Botch分子的动态表达。Botch通过减小梗死体积、神经元死亡和小胶质细胞介导的神经炎症反应起到对缺血性脑卒中的神经保护作用。Botch抑制缺血缺氧损伤诱发的NICD表达上升及其线粒体转移，是Botch抑制小胶质细胞炎症反应的可能途径。对于出血性卒中而言， Botch可以减少脑出血后的神经细胞死亡、神经功能损伤和神经炎症反应。体内及体外研究提示Botch通过抑制Notch1剪切发挥神经保护作用。体外机制研究提示，115位点是Botch与Notch1结合，并抑制其成熟剪切的关键位点。本研究验证了Botch对于脑血管病的神经功能保护作用及可能的机制，具有临床的转化及应用价值。