针对缺血性脑中风的线粒体靶向纳米抗氧化治疗

The project is proposed to demonstrate the effects and the underlying mechanism of mitochondrial targeted nano-antioxidant therapy against the ischemic cerebral stroke insults. Based on this purpose, three types of nano-antioxidants which include the self-assembled nano-antioxidants, the release controlled nano-antioxidants and the mitochondrial targeted combinational type are designed and synthesized, respectively. After that, changes and controls of key genes and proteins will be investigated in the presence of these antioxidants using OGD (oxygen–glucose deprivation) model for cultured hippocampus neurons. The processes of apoptosis inhibition and regeneration of neurons after injury of hypoxia may mainly include the mitochondrial dependent apoptosis, PI3K/Akt and Nrf2/HO-1 signalling pathways. Meanwhile, the protections of nano-antioxidants on the apoptosis of neurons in hypoxic C. elegans will be studied according to analyses of the protein expressions of AKT-1 and SKN-1 and gene expressions of ced-3 or ced-9. Referring to the above results, the kinetic effects of the duration of hypoxic injury on the expressions of key gene or protein will be revealed in the presence of nano-antioxidants, and consequently, a so-called ”antioxidative kinetic singnalling pathway” is probably being discovered..Finally, the effects and the related mechanism of these nano-antioxidants will be elucidated in the rat middle cerebral artery occlusion and reperfusion model. Namely, in combination with the current stroke clinical interventions, we hope that an optimized kinetic therapy of nano-antioxidants can be presented for treating the hypoxic injury of neurons.

本课题从设计表面自组装型、缓释型与线粒体靶向复合型纳米抗氧化剂出发，以神经细胞氧糖剥夺损伤实验为模型研究纳米抗氧化剂对神经细胞损伤修复（或细胞凋亡抑制）过程中调控的关键蛋白与基因。检测的相关细胞损伤修复（细胞凋亡抑制）通路主要包括线粒体凋亡信号通路、Nrf2/HO-1 与Akt/PI3K 信号通路。活体实验采用秀丽隐杆线虫缺氧模型，拟检验纳米抗氧化剂对缺氧线虫AKT-1、SKN-1 表达及凋亡相关的ced-3 与ced-9 基因的调控作用。在上述研究基础上，进一步通过改变缺氧损伤时间长度检验纳米抗氧化剂对所获抗氧化剂调控关键蛋白与基因的动力学调控效果。即：细胞内“抗氧化动力学信号通路”。最后，经大鼠大脑中动脉阻塞/再灌注（MCAO/R）模型验证线粒体靶向纳米抗氧化药物的治疗效果与对所述关键蛋白或基因的时间相关调控规律，并总结出优化纳米抗氧化剂对缺血性脑中风的动力学治疗方案。

缺血性脑卒中（俗称中风）是危害中老年人生命与健康的一个凶险杀手。但由于中风复杂的致病机制，临床上急需针对缺血损伤具有高效治疗的药物。基于此，本课题经过前期采用神经细胞SH-SY5Y氧糖剥夺/复氧（OGD/R）与秀丽隐杆线虫缺氧损伤保护模型实验分析筛选出以改性寡聚壳聚糖为自组装载体的丹参酮IIA高效抗中风制剂。经研究发现，相关神经细胞损伤的保护通路主要包括Bcl2/Bax线粒体凋亡信号通路、Nrf2/HO-1抗氧化应激信号通路及mTOR介导的自噬信号通路。最后经大鼠大脑中动脉阻塞/再灌注（MCAO/R）中风模型验证该丹参酮IIA纳米制剂不但具备高效血脑屏障通透性能与脑靶向抗氧化神经保护功能，而且还可通过诱导大脑中动脉阻塞大鼠损伤后的神经干细胞的增值、迁移与分化来实现对神经功能损伤的再生行修复。近期深入研究发现：此再生修复机制可能关联蛋白酶体（proteasome）介导的Nrf2/ARE与MAPK/ p-CREB/BDNF信号通路之间的信号交互。由于大多抗氧化神经保护剂都可有效激活Nrf2/ARE抗氧化信号通路，这为缺血性中风损伤（甚至其他神经退行性疾病损伤）的再生性修复提供一条治疗的新策略。