天麻活性成分通过重建神经血管单元稳态抗脑缺血再灌注损伤的作用及机制研究

Cerebral ischemia reperfusion injury (CIRI) is considered to be the most important reason which leads to the deterioration of ischemic cerebrovascular disease (ICVD). As a dynamic process, the pathological change of CIRI involves complex cascade of time and space which contribute to the disorders of the brain. Therefore, little effect are observed so far when applying therapeutic drugs which targeting a single pathological segment of ICVD. Accordingly, global cerebral protection strategy was proposed in 2001 by National Institute of Neurological Disorders and Stroke (NINDS) to maintain the homeostasis of the cerebral neurovascular unit (NVU). Our previous studies have proved that the ethyl acetate extract of Gastrodia elata Blume. (EEG) as well as three phenolic compounds separated from EEG protected the brain aganist CIRI. Further studies revealed that these three compounds passed through the brain blood barrier (BBB), and each compound showed certain selectivity on different pathological segment during the process of the destruction of NVU homeostasis. However, the efficacy of each compound on CIRI was significantly lower than that of EEG or the combination of two or three compounds. Our preliminary data indicates that multiple active compounds extracted from G. elata might penetrate the BBB and exert the anti-CIRI effect to reconstructe the homeostasis of NVU. The present project is designed to clarify the effects and underlying mechanisms of G. elata on the reconstruction of NVU homeostasis in CIRI. Furthermore, this project will investigate effects of the three compounds extracted from G. elata exerting on NVU homeostasis by targeting the pathological changes of the NVU destruction at different time points in CIRI in vitro and in vivo.

脑缺血再灌注损伤（CIRI）是导致缺血性脑血管病恶化的重要原因，其病理改变涉及复杂的时间、空间级联反应所导致的脑稳态失调，故针对CIRI单一病理环节进行的药物治疗收效甚微，2001年NINDS提出维护神经血管单元（Neurovascular Unit,NVU)稳态的策略。我们前期发现天麻乙酸乙酯提取物（EEG）具有显著抗CIRI作用，并证实EEG中所含的3个活性成分能通过血脑屏障，且对NVU稳态破坏的不同病理环节具有选择性作用，但这3个成分无论对整体还是离体CIRI模型保护作用都较EEG弱，联合使用则药效增强，提示天麻是通过入脑的多个活性成分共同重建NVU稳态而发挥抗CIRI作用。本课题拟以天麻3个入脑成分为研究对象，采用CIRI动物模型及细胞共培养技术，从NVU稳态破坏不同时间点的病理变化关键事件出发，对3个成分重建NVU稳态的作用机制、时间窗进行研究，以期阐明天麻抗CIRI的科学内涵。

鉴于缺血性脑卒中(IS)发生发展过程中，神经血管单元(NVU)稳态的破坏存在时间、空间的复杂联系，早期以神经炎症、细胞凋亡的损伤为主；后期则以神经再生、血管新生的修复为主。本研究在国家自然科学基金项目“ 云南昭通天麻抗脑缺血药效物质基础的研究”(编号：30760305)和“基于脑内分布的天麻酚性成分对缺血性脑损伤神经保护作用及机制研究”(编号：81560664)的基础上，对前期研究活性最明显的天麻三个入脑成分，在IS发生的不同时间点，对NVU稳态破坏事件的影响进行系统研究，观察三个入脑成分对NVU稳态破坏的重建作用及作用机制。结果表明，在时间上天麻三个入脑成分可减轻NVU稳态破坏早期BBB的损伤、对抗细胞凋亡、对抗小胶质细胞的活化；可减轻NVU稳态破坏晚期的神经炎症，促进NVU稳态破坏晚期的神经修复和血管新生。在空间上可减轻NVU组成细胞结构的损伤，使血脑屏障（BBB)超微结构破坏减轻、星型胶质细胞足突水肿减轻；神经元损伤减轻。作用机制涉及：（1）减轻BBB损伤：上调Occludin和Claudin-5 的表达水平；成分 C 还可激活 PI3K/AKt 信号通路；成分D还能启动Ang-1/Tie-2信号通路；(2) 对抗神经细胞凋亡：成分C介导JNK信号通路；成分D、H可调控Bcl-2/Bax信号通路；（3）减轻小胶质细胞的活化：调控TLR4介导的MyD88依赖性通路下游的MAPK和NF-κB信号通路。（4）对抗神经炎症的发生：成分C作用于COX-2途径，成分D作用于5-LOX途径，成分H调节小胶质细胞M1/M2型表型转化；（5）促进神经修复：促进内皮细胞释放神经营养因子VEGF-A和BDNF；成分H还能启动BDNF/Trkb信号通路；（6）促进血管新生：上调促血管新生因子VEGF-A、Ang-1的mRNA表达；成分C还可增加脑内VEGFR-2、α-SMA、Smad-3及降低Ang-2的表达，从而促进血管的成熟；成分D、H还能上调促血管新生因子Ang-2的mRNA的表达。提示，天麻三个入脑成分重建NVU稳态的作用涉及了神经元、胶质细胞、微血管等多个层面，在NVU稳态破坏过程中，于不同的时间点、作用于不同的病理环节，从不同的时间、空间角度共同重建NVU稳态，从而发挥抗IS的作用。