基于Wnt/β-catenin信号通路探讨电针对脑缺血再灌注大鼠模型神经血管单元的保护机制

Wnt/β-catenin signaling pathway plays an important role in regulating neurovascular unit (NVU). On the basis of previous studies, Electro-acupuncture(EA) can improve middle cerebral artery occlusion and reperfusion (MCAO/R) of rats’motor function by protecting NVU, but its mechanism is still ambiguous. In this project, Sprague-Dawley (SD) rats are used to establish MCAO/R and the methods of Real-time PCR, Western-blot, immunohistochemistry and double-labelling technique of immunofluorescence are employed to observe the expression of protein and gene in Wnt-1, β-catenin, GSK-3β, VEGF, NeuN, GFAP. Besides, 9.4T/400mm mammals magnetic resonance is applied to observe the influence from electric acupuncture on the structure and function of neurovascular unit (NVU). Moreover, DKK-1 is adopted to block Wnt/β-catenin signaling pathway and methods of cell co-culture technique is used to establish in vitro model of neurovascular unit (NVU) in order to observe the effects of Electro-acupuncture(EA) on Wnt/β-catenin signaling pathway and NVU’s important component as well as its function. At the level of molecules, cells, protein and the overall structure and function, EA provides protective effect on NVU by canonical Wnt/β-catenin signaling pathway, which reveals the mechanism of EA in the treatment of ischemic cerebrovascular disease（ICVD）.

Wnt/β-catenin信号通路在调控神经血管单元（NVU）的过程中发挥重要作用。课题组前期研究发现，电针能通过保护NVU发挥其改善大脑中动脉缺血再灌注模型（MCAO/R）大鼠运动功能的作用，但其机制不明。本项目拟以SD大鼠构建MCAO/R模型，运用Real-time PCR，Western-blot，免疫组化，免疫荧光双标记等方法观察电针后Wnt-1，β-catenin，GSK-3β，VEGF，NeuN，GFAP等在蛋白、基因水平的表达；通过9.4T 磁共振活体观察NVU结构及功能；并利用DKK-1阻断Wnt/β-catenin信号通路和采用细胞共培养技术构建“脑神经血管单元”体外模型，观察电针对Wnt/β-catenin信号通路、NVU重要组分及其功能的影响，在分子、细胞、蛋白水平探讨电针通过经典Wnt/β-catenin信号通路对NVU的保护效应，揭示电针治疗缺血性脑血管病的机制。

本研究探讨了电针通过调控神经血管单元（NVU）对缺血性脑卒中（ICS）发挥保护作用及相关机制。主要研究内容与结果如下：1）通过观察大脑中动脉闭塞模型大鼠脑组织形态和神经功能的变化，发现电针刺激能显著改善缺血引起的神经功能损伤。2）利用磁共振影像系统检测脑梗死体积，免疫组化、荧光定量PCR技术、Western blotting 法检测NVU主要标志蛋白——血管内皮生长因子（VEGF）、胶质纤维酸性蛋白（GFAP）、神经元核抗原（NeuN），Wnt/β-catenin信号通路关键成分Wnt3、β-连环蛋白（β-catenin）、糖原合成酶激酶-3β（GSK-3β）、支架蛋白（Axin2）蛋白的表达，证实了电针可激活Wnt/β-catenin信号通路，有效保护脑缺血大鼠神经血管单元，减少脑梗死体积，改善MCAO大鼠神经功能缺损症状。3）电针血清作用于体外NVU模型，可减少糖氧剥夺/复糖复氧（OGD/R）诱导的神经元凋亡，降低氧化应激水平并改善血脑屏障（BBB）损伤，且该作用与Wnt/β-catenin信号通路激活相关。将在体研究与离体验证相结合，从宏观和微观层面验证了预定设想，完成预期计划。