灯盏花乙素靶向调控NOX2-ROS-Cx43通路影响脑缺血边缘区及远隔海马神经元损伤的机制研究

Reactive oxygen species caused by peroxidation is an important mechanism of ischemic brain injury. Inhibition of NADPH oxidase (NOX2) or knockout NOX2 subtypes improved cerebral ischemia-reperfusion injury. In clinical,we investigated that lamps Shengmai capsule can effectively improve the degree of disability of stroke patients, without increasing cerebral hemorrhage and other bleeding risk. Previous studies have shown that Scutellarin,as the main ingredient as lamps Shengmai capsule, improved neurological function and reduced infarction size and brain edema after cerebral ischemia-reperfudion, but the mechanism remains unclear. Gap junction （Cx43）is vital to the regulation of the level of second messengers such as reactive oxygen after cerebral ischemia-reperfusion injury. So we proposed the following hypothesis: "The protection of Scutellarin on neuron cell death in perilesional zones and hippocampal after cerebral ischemia regulated by Nox2-ROS-Cx43 signal pathway": Scutellarin-the radical-Nox2-ROS-Cx43-gap junction communication- apoptosis-related factors - treatment of nerve injury, which can help us further investigate the protective effect of scutellarin on nerve injury after ischemia-reperfusion injury and its mechanism, on the one hand, there is conducive to the modernization of Chinese medicine and serve the clinical better, the other hand, which can provide important reference for other flavonoids drug on the brain protective and provide the techniques and methods for drug screening research.

活性氧引起的过氧化反应是缺血性脑损伤发生的重要机制，抑制NADPH氧化酶2（NOX2）或基因敲除NOX2亚型减少了再灌注损伤。临床实践发现灯盏生脉胶囊能有效改善卒中患者的残障程度，且不增加脑出血及其他出血性风险，基础研究显示作为灯盏生脉胶囊主要成分的灯盏花乙素（Scutellarin）能够改善脑缺血再灌注后神经功能评分，减少脑梗死面积和脑水肿，但其作用机制尚不清楚。缝隙连接（Cx43）调控脑缺血再灌注损伤第二信使如活性氧水平至关重要。本项目提出"灯盏花乙素靶向NOX2-ROS-Cx43通路影响脑缺血边缘区及远隔海马神经元损伤的假说"即：灯盏花乙素-清除自由基-Nox2下调-ROS减少-Cx43增加-缝隙连接通讯的改变-凋亡相关因子改变-治疗神经损伤，明确其作用机制，有利于中药现代化研究，更好地服务于临床，为其他黄酮类化合物在脑保护作用研究方面提供重要参考，也为相关新药筛选研究提供技术和方法

活性氧（ROS）引起的过氧化反应是缺血性脑损伤发生的重要机制，抑制NADPH氧化酶2（NOX2）能减少再灌注损伤。临床实践发现灯盏生脉胶囊能有效改善卒中患者的残障程度，且不增加脑出血及其他出血性风险，基础研究显示作为灯盏生脉胶囊主要成分的灯盏花乙素（Scutellarin，Scu）能够改善脑缺血再灌注后神经功能评分，减少脑梗死面积，但其作用机制尚不清楚。本项目首先构建体外培养的星型胶质细胞缺氧/复氧模型，结果显示Scu干预使得缺糖缺氧损伤的星形胶质细胞活力增加，细胞内ROS含量减少，NOX2表达下调，并与NOX2抑制剂夹竹桃麻素干预效果比较，结果显示Scu能发挥NOX2抑制剂作用。同时应用AutoDock软件对Scu和NOX2的3D分子结构对接的结果显示二者的活性口袋形状吻合，3D分子结构对接良好。我们进一步构建在体大鼠MCAO/再灌注模型，验证Scu调控NOX2对脑缺血再灌注损伤作用机制，结果显示应用Scu后，MCAO大鼠神经功能评分降低，脑梗死面积减少，过氧化损伤减轻，NOX2和Cx43表达下调，并与NOX2抑制剂夹竹桃麻素干预效果比较，结果表明Scu可以通过靶向NOX2减轻脑缺血再灌注损伤，发挥神经保护作用。本项目研究结果一方面明确了Scu对脑缺血损伤的保护作用，另一方面明确了Scu对脑缺血损伤的保护作用是通过靶向NOX2，抑制过氧化反应减轻脑缺血再灌注损伤，该研究成果有利于中药现代化研究更好地服务于临床，为其他黄酮类化合物在脑保护作用研究提供重要参考，也为相关新药筛选提供技术和方法