雷公藤甲素联合BDNF修饰的BMSC促进小胶质细胞介导的脑缺血后神经保护机制研究

Stroke remains one of the major diseases causing human morbidity and mortality. Secondary brain injury after cerebral ischemia is caused by strong inflammatory reactions, and the regulation of microglial activation is one key element to alleviate neuro-inflammations. Previous studies have confirmed that triptolide (TP) can conduct its protective effects on cerebral ischemia through inhibiting the inflammatory reactions. And bone marrow-derived mesenchymal stem cells (BMSCs) transplantation can also improve the neurological dysfunction after cerebral ischemic stroke, however the inflammatory reactions and nutritional deficiency can lead to cell apoptosis and cell death. Based on the early BDNF lentivirus construction, we are planning to approach transgenic mice model of cerebral ischemia with genetic manipulation of microglia, oxygen and glucose deprivation model, magnetic resonance imaging and RNAi methods, aiming to clarify the molecular activation mechanism and pathway of microglia by TP and how it affects the survival of BMSCs. Also we are expected to demonstrate the anti-inflammatory roles of TP combined with BDNF modified BMSCs transplantation by regulating microglial activity, autophagy function and TLR4/NLRP3 signal pathway. The main objectives of this study are inhibiting the cerebral ischemia inflammation reaction and function of autophagy, increasing nutrition support to improve BMSCs survival rate, then promote ischemic stroke repairs of brain function and to explore neural regeneration mechanism, thus providing theoretical foundations and new targets for ischemic brain injury treatments.

脑卒中是人类主要的致残和致死性疾病，脑缺血后强烈的炎症反应可导致脑组织继发性损伤，而调控小胶质细胞活化状态是减轻神经炎症反应的关键环节之一。前期研究证实雷公藤甲素(TP)能通过抑制炎症反应对脑缺血起到保护作用；骨髓间充质干细胞（BMSCs）移植能改善缺血性脑卒中后的神经功能障碍，但炎症反应和营养缺乏会导致植入细胞凋亡和死亡。在前期已构建BDNF慢病毒基础上，我们构建小胶质细胞转基因小鼠脑缺血模型、氧糖剥夺模型，采用磁共振成像和RNA干扰等方法，明确TP激活小胶质细胞的途径及影响BMSCs存活的分子机制，阐述TP抗炎联合BDNF修饰的BMSCs移植在调控小胶质细胞活性、自噬功能和TLR4/NLRP3信号通路机制，期望通过抑制脑缺血后炎症反应和自噬功能、增加营养支持从而提高BMSCs存活率，促进缺血性脑卒中后脑功能修复并探讨神经修复机制，为寻找缺血性脑损伤的新疗法和新靶点提供理论依据。

本研究基于氧糖剥夺复氧细胞体外模型和小鼠局灶性脑缺血在体模型，利用小胶质细胞自噬条件特异性敲除小鼠，聚焦探究Bdnf修饰的BMSCs细胞移植联合雷公藤甲素（TP）治疗脑缺血的神经保护作用。体外结果显示TP预处理能够提高OGD/R后PC12细胞活力，抑制细胞凋亡，下调iNOS。BMSC移植和TP预处理治疗脑缺血模型后，行为学显示小鼠运动能力显著改善,影像学证实了脑梗塞面积减少。同时，在体研究结果表明TP预处理能够影响脑缺血后小胶质细胞活性，降低Iba1和CSF1R表达，减少脑梗死面积，抑制脑缺血后炎症反应。进一步利用小胶质细胞特异性自噬缺陷小鼠构建脑缺血模型，通过结合TP治疗，观察到小胶质细胞活化显著增强，且追溯到其作用机制是通过上调细胞存活BDNF-Akt信号通路，继而改善脑缺血后突触蛋白表达，上调锥体神经元动作电位发放，提高神经元兴奋性，最终发挥保护作用。而TP神经保护通过脑缺血后激活Nrf2/Akt通路，发挥抗氧化功能来减轻脑损伤。本课题系统阐述了TP预处理和BMSC移植对脑卒中后的保护作用及机制，并全面验证TP与BMSC联合治疗调控小胶质细胞活化，自噬以及抗氧化等重要保护功能。