天麻酚性成分介导反应性星形胶质细胞重编程促进脑缺血后神经修复的作用及机制研究

Endogenous neurorepair is activated following cerebral ischemia, which intents to restore neurofunction through axonal regeneration of impaired neurons, as well as migration and differentiation of neural progenitor cells. However, due to the very limited ability of the neurorepair in central nervous system and the glial scar formed by reactive astrocytes (RAs) after ischemia, the self-repairing mechanism is too far to promote the recovery of neurofunction. RAs have the characteristics of neural stem cells. Under the exogenous factors such as transcription factors and small molecule compounds，RAs can be reprogrammed into neurons. Reprogramming of RAs in the glial scar into neurons not only reduce the inhibitory effect on axonal regeneration, but also provide a new source of neurogenesis, which is an important way to promote endogenous repair mechanisms. In our previous study, we have demonstrated that phenolic compound 4# of Gastrodia elata. can improve the neurological function of rats undergo cerebral ischemic and increase the positive expression of Nestin, a marker of neuronal stem cells, and NeuN, a marker of neurons in the peri-infarction cortex. In vitro experiments show that compound 4# can reprogram RAs into neurons, suggesting that it may plays a pivotal role in promoting nerurorepair. Based on the previous work, this project aims to explore the effect of 4# on neurorepair via reprogramming RAs into neurons, and to elucidate the related mechanisms.

脑缺血激活的神经轴突再生、神经祖细胞(NPCs)增殖迁移及分化等内源性修复机制对卒中后神经功能的恢复至关重要，但由于中枢神经系统再生能力弱，加之缺血后反应性星形胶质细胞(RAs) 形成的胶质瘢痕(GS)会抑制轴突再生能力、限制梗死区周围皮质(PIC)的再生微环境，导致神经自我修复能力不足。RAs具有干细胞特性，在外因作用下可被重编程为神经元。促使GS内RAs重编程为神经元不仅可减轻对轴突再生的抑㓡、改善PIC再生微环境，还可协同NPCs补偿梗死区损失的神经元，是促进内源性神经修复机制的关键环节。课题组前期研究证实天麻4#酚性成分可改善脑缺血大鼠神经功能、增加PIC内Nestin+及NeuN+细胞数；体外实验显示其具有将RAs重编程为神经元的作用。本课题拟从介导GS内RAs重编程为神经元的角度，研究天麻4#酚性成分促进脑缺血后神经修复的作用，并阐明其作用机制。

脑缺血后,梗死区周围皮质(PIC)内反应性星形胶质细胞(RAs) 形成的胶质瘢痕(GS)抑制PIC再生微环境，削弱了神经自我修复能力。RAs具有干细胞特性，促使PIC内RAs重编程为神经元可能减轻对轴突再生的抑制、协同神经祖细胞（NPCs）补偿丢失的神经元，改善PIC再生微环境。本课题在前期研究基础上，从上述科学假说探讨天麻4#酚性成分（APCG-4）促进脑缺血后神经修复的作用及机制。.结果表明：1. APCG-4可改善暂时性大脑中动脉阻断（tMCAO）大鼠的运动及记忆功能，减轻卒中后遗症：①促进大鼠PIC内PAs重编程为兴奋性和抑制性神经元，分布到相应的大脑皮层分层，并建立运动功能相关的神经投射；②促进PIC内血管新生，并与RAs衍生的神经元伴行；③抑制PIC周围GS的形成，维持PIC内神经元与星形胶质细胞的耦合，保护神经血管单元（NVU）的结构；④上述作用机制与上调Wnt信号通路有关。2. 体外实验证明APCG-4可改善PIC再生微环境：①促进RAs重编程为成熟神经元，机制与抑制Notch1信号通路、调控转录因子NeuroD1及SOX2 的表达有关。②保护NVU结构、促进受损神经元的修复，机制增加营养因子NT3、BDNF等的分泌有关。③减轻RAs与成纤维细胞形成GS，促进受损神经元的轴突再生，并迁移进入GS内与星形胶质细胞建立突触连接，其机制为上调神经轴突再生因子Tenascin-C、Laminin、BDNF的表达，并下调抑制神经轴突再生因子CSPGs、Semaphorins的表达；④促进NPCs的迁移、增殖和存活，机制为增加RAs分泌EPO及PSA-NCAM促进神经祖细胞的迁移，升高神经祖细胞内BCL-2/BAX比值抑制其凋亡，上调RAs的Shh信号通路。.结论：APCG-4通过诱导PIC内RAs重编程为神经元、减轻GS形成，保护NVU结构，并促进NPCs迁移和增殖，从而改善PIC再生微环境，促进缺血后脑组织自我修复。APCG-4亮点：①只促进PIC内RAs重编程，不影响周围正常脑组织，不改变大脑结构；②只促进部分RAs重编程为神经元，将GS转换回富含神经元的组织，并非“融化”整个GS，保证脑缺血后亚急性期GS发挥有益作用。研究提示：诱导PIC内RAs重编程为神经元是促进内源性神经修复机制的潜在途径，p-HBA可能是药物干预的候选药物。