基于神经血管单元稳态重建研究风药佐助补虚药抗脑缺血损伤的分子机制

Deficinecy-nourishing medicine has the function of reinforcing foundation in treatment of stroke. Wind -dispelling medicine combination with deficiency-nourishing medicine can improve the curative effect of stroke. Our initial research showed wind-dispelling medicine combination with deficiency-nourishing medicine could protect neurovascular unit after cerebral ischemia based on factorial analysis of wind -dispelling medicine and deficiency-nourishing medicine of houshiheisan. Neurotrophic factor and nerve growth guidance cues play an important role in the reconstruction of neurovascular unit homeostasis after cerebral ischemia. It is the scientific problem of this study that how wind-dispelling medicine and deficiency-nourishing medicine affect neurotrophic factor and nerve growth guidance cues signaling pathways to promote reconstruction of homeostasis of neurovascular unit. Therefore, we will explore the integration, synergy and diversity of wind-dispelling medicine combination with deficiency-nourishing medicine promoting reconstruction of homeostasis of neurovascular unit after cerebral ischemia through replication of middle cerebral artery occlusion based on hyperlipemia.The study will lay emphasis on modulating signal of neurotrophic factor and nerve growth guidance cues. Moreover, The molecular targeted sites of wind-dispelling medicine combination with deficiency-nourishing medicine promoting reconstruction of homeostasis of neurovascular unit will be confirmed by oxygen- glucose deprivation of SY5Y、EA.hy 926、U87 co-culture in vivo model through using signal pathway blockers. The significance of this research is to enrich the scientific connotation on synergistic effect of wind-dispelling medicine and extend the doctor's thoughts of stroke treatment with Traditional Chinese Medicine.

补虚药在脑中风治疗中具有固本作用。风药能佐助补虚药提高脑中风的临床疗效。我们前期对侯氏黑散方中"风药"、"补虚药"进行析因分析，结果表明风药佐助补虚药对脑缺血后神经血管单元进行整体保护。神经营养因子和神经导向因子的信号传递在脑缺血后神经血管单元稳态重建中起着重要作用。风药、补虚药如何调控其信号通路，促进脑缺血后神经血管单元稳态重建是本课题拟解决的关键科学问题。我们采用高脂血症基础上的MCAO脑缺血大鼠模型，以神经营养因子和神经导向因子的信号调控为研究重点，探讨风药佐助补虚药促进脑缺血后神经血管单元稳态重建的协同、整合作用及其差异性。并采用SY5Y、EA.hy 926、U87三种人类细胞株共培养构建体外神经血管单元模型，通过氧糖剥夺模拟体内缺血，利用信号通路阻断剂，确定风药佐助补虚药促进脑缺血后神经血管单元稳态重建的分子靶位。研究意义在于充实"风药增效"的科学内涵，拓展中医治疗脑中风的思路。

补虚药在脑中风治疗中具有固本作用。合理配伍风药是提高脑中风临床疗效的有效方法。风药、补虚药如何促进脑缺血后神经血管单元稳态重建，是本项目主要研究内容。采用整体动物实验与离体细胞实验相结合的研究方法，从神经营养因子、神经抑制因子和神经导向因子相关信号通路探究风药佐助补虚药保护神经血管单元的分子机制。通过神经功能评分、横木行走实验、旷场实验等多种方法对风药、补虚药以及风药补虚药联用抗脑缺血损伤的疗效进行综合评定，结果显示风药与补虚药联用改善神经功能的作用明显优于风药及补虚药单独使用。病理学检测结果风药佐助补虚药可明显抑制脑缺血后神经元的脱失，减轻神经胶质细胞及血管的水肿并且可以促进缺血脑组织微血管的新生。风药佐助补虚药可明显上调缺血脑组织神经营养因子BDNF蛋白和基因的表达，促进TrkB基因的转录；并通过提高AKT蛋白的磷酸化水平，增加核转录因子CREB的基因表达；风药佐助补虚药下调轴突生长抑制蛋白Nogo-A/NgR及其下游信号分子RhoA/ Rock2基因、蛋白的表达，抑制神经再生抑制信号抑，促进神经元再生、轴突生长和突触重塑；风药佐助补虚药通过对Netrin-1/DCC和分子开关Rac1/Cdc42/RhoA的调节促进脑缺血后神经功能的修复。风药与补虚药的作用靶点不同，实验结果提示风药侧重于对轴突抑制蛋白和神经导向因子的调节而补虚药侧重于对神经营养因子及其信号通路的调节。