基于神经血管单元探讨“三焦次第治疗”对脑缺血损伤大鼠Wnt/β-catenin信号通路的调控机制

The overall protection against nerves and blood vessels has become the focus of stroke research in recent years. The Wnt signaling pathway plays an important role in the protection of neurovascular units in ischemic stroke, however, its related molecular regulation mechanism remains to be revealed. Sequential treatment of triple energizer is a significant therapeutic rule on the basis of yang deficiency in cerebral ischemia/ reperfusion injury, and its mechanism remains to be further elucidated. In this study, genes expression of Wnt3a, β-catenin, Frizzled-1, LRP6, TCF3, VEGF in Wnt/β-catenin pathway and inflammatory suppressor MMP-9 in ischemic penumbra were detected by qRT-PCR and Western Blot after preparation of rat MCAO model. And neuronal apoptosis will be determined with Tunel assay, blood brain barrier permeability by Evans blue, co-localization of β-catenin/TCF3 by laser confocal microscopy. Then Yanghua Decoction and Fuyang Decoction were used to observe the regulation of Wnt-mediated signaling pathway to further explore the mechanism of neurovascular unit protection and to provide technical support for the prevention and treatment of cerebral I/R damage.

针对神经和血管的整体保护成为近年来脑卒中研究的焦点。Wnt信号通路在缺血性中风神经血管单元保护中发挥重要作用，但其相关分子调控机制有待揭示。基于阳虚为本的“三焦次第治疗”是对脑缺血再灌注（I/R）损伤有显著疗效的治则，其作用机理有待于进一步阐明。本研究通过制备大鼠MCAO模型，采用qRT-PCR及Western Blot等技术检测I/R损伤后不同时间点缺血半暗带Wnt/β-catenin通路Wnt3a、β-catenin、Frizzled-1、LRP6、TCF3和VEGF等基因以及该通路的炎性抑制因子MMP-9等的表达变化；TUNEL法检测神经元凋亡；EB法检测血脑屏障通透性；激光共聚焦显微镜检测β-catenin/TCF3的共定位。并以阳化汤和扶阳方做干预，观察其对Wnt介导的信号通路的调控作用，以进一步探讨其神经血管单元保护机制。以期为脑I/R损伤的防治提供技术支撑。

项目基于扶阳理论指导，通过神经血管单元研究，探讨了“三焦次第治疗”对脑缺血损伤大鼠Wnt/β-catenin信号通路的调控机制，采用了大鼠大脑中动脉梗阻（MCAO）模型，温阳法“三焦次第治疗”干预后，应用TTC染色检测脑梗死体积、TUNEL法检测神经元凋亡和透射电镜检测梗死灶周围脑组织超微结构，发现次第治疗组急性期和恢复期脑缺血再灌注损伤模型大鼠的脑梗死面积显著缩小，缺血半暗带区细胞减少了凋亡，同时促进该区神经细胞的存活，在结构和功能上改善血脑屏障通透性。观察“次第治疗”对NVU的保护作用。结果显示阳化汤对急性期脑缺血再灌注损伤有一定保护作用，高剂量（24g/kg/d）为治疗脑缺血再灌注损伤的最佳疗效剂量。“次第治疗”能显著下调MMP-3、MMP-9;并上调VEGF、BDNF蛋白及基因的表达，同时可激活Wnt/β-catenin信号通，研究表明“次第治疗”对急性期和恢复期脑缺血再灌注损伤模型的神经血管单元具有保护作用，该保护作用与下调MMP-3与MMP-9，上调VEGF与BDNF的表达，及Wnt/β-catenin通路的活化相关。