P66Shc氧化应激信号通路诱导的星形胶质细胞exosomes在脑缺血后神经功能恢复中的作用机制研究

Free radicals are harmful. However, antioxidant therapy is useless. This is a challenge for clinical treatment of ischemic brain injury with anti-oxidants, and its related mechanisms need to be clarified. Our previous and preliminary experiments found that in the late stage of oxygen-glucose deprivation/reperfusion (OGD/R), the p66Shc protein was phosphorylated and translocated into the mitochondria, and played an important role in mitochondrial ROS generation in astrocytes. OGD/R can induce release of exosomes from astrocytes, and this process can be reversed by application of antioxidant. These astrocyte-derived exosomes played a protective role in neurons and brain vascular endothelial cells injury induced by ischemia, and also promoted the brain vascular endothelial cell proliferation, indicating that the mitochondrial oxidative stress in the late stage of cerebral ischemia and reperfusion can stimulate release of exosomes from astrocytes and plays a constructive role in the remodeling process of neurovascular unit, but the detailed mechanism is unknown. Thus, this project designs to explore the effects and mechanisms of the p66Shc mediated mitochondrial oxidative stress signaling pathway in the release of protective exosomes from astrocytes and its promotive role in the recovery of cerebral nerve function through the ischemic models in vitro and in vivo. The expected results will reveal a novel mechanism which is related to the repairing of brain function after ischemia, and provide the experimental data for the rational use of antioxidants in clinical.

“自由基有害”而“抗氧化治疗无效”是临床治疗缺血性脑损伤的一大难题，其相关机制有待阐明。申请人前期实验和预实验发现氧糖剥夺再灌注后p66Shc蛋白磷酸化并线粒体易位，在星形胶质细胞线粒体ROS产生中扮演重要作用。氧糖剥夺再灌注可诱导星形胶质细胞释放exosomes, 并可被抗氧化剂所逆转；而该exosomes对神经元和脑血管内皮细胞的缺血性损伤具有保护作用，并促进脑血管内皮细胞的增殖，提示脑缺血再灌注后期线粒体氧化应激可刺激星形胶质细胞通过分泌exosomes调节脑神经血管单元的重构，但具体机制不详。据此，本课题拟通过整体及微流控芯片细胞缺血模型，观察缺血后期p66Shc线粒体氧化应激信号通路在星形胶质细胞分泌保护性exosomes及促进脑神经功能恢复中的作用机制，预期研究结果将揭示星形胶质细胞参与脑缺血再灌注损伤修复中的新机制，并为临床合理使用抗氧化剂提供必要的实验数据。

缺血性脑卒中是常见而严重的神经系统疾病，至今在临床上除了使用溶栓剂外仍缺乏有效且副作用较小的治疗药物。因此，寻找抗脑缺血药物作用的新靶点，开发新型的抗缺血性脑损伤及促进缺血后脑神经功能恢复的药物具有重大的意义。近年来研究发现外泌体在细胞之间的相互作用中发挥重要作用，且与多种疾病的发生发展密切相关。但是目前对外泌体在缺血性脑损伤中的作用及相关机制知之甚少。因此，本课题聚焦于缺血性损伤条件下星形胶质细胞源性外泌体对神经元及血管内皮细胞功能的调节作用及潜在分子机制。我们研究发现:（1）星形胶质细胞外泌体对神经元细胞的缺血性损伤具有保护作用，并能调节缺血后神经元的突触可塑性，上调synapsin I蛋白的表达；（2）星形胶质细胞外泌体对tMCAO导致的小鼠神经功能缺失及脑组织损伤具有明显的改善作用；（3）星形胶质细胞外泌体通过ERK1/2信号通路上调SH-SY5Y细胞synapsin I蛋白的表达；（4）星形胶质细胞外泌体能够影响血管内皮细胞的成管能力，但是与所处的缺血阶段密切相关；（5）星形胶质细胞外泌体参与调节血管内皮细胞VCAM-1和VEGF蛋白得到表达；（6）缺糖缺氧及再灌注影响星形胶质细胞外泌体miRNAs的表达谱；（7）缺糖缺氧再灌注激活星形胶质细胞 p66Shc信号通路，但是阻断该信号通路并没有显著影响星形胶质细胞外泌体对神经细胞缺血性损伤的保护作用和对突触可塑性相关蛋白synapsin I表达的调节作用。（8）星形胶质细胞的氧化应激状态影响其外泌体内一些内含物如synapsin I蛋白的释放。以上数据提示星形胶质细胞外泌体对缺血性脑损伤具有显著地神经保护作用。本课题的完成为今后星形胶质细胞外泌体或其模拟类似物运用于临床治疗缺血性脑损伤奠定了理论基础，同时也为研发脑缺血治疗的创新药物开辟新的方向。