补阳还五汤促进缺血性脑中风后康复“生新”的药效物质基础及其作用机制研究

Buyanghuanwu decoction (BYHWD) has significant clinical curative effect on cerebral ischemic stroke treatment. The former research discovered that BYHWD has the function of “benefiting qi and promoting circulation of blood to promote regeneration” in the thesis group. But its target and the effective component is not clear. This project intends to use the microdialysis technique in real time collecting drug-containing cerebrospinal fluid in rats as samples of cell membrane chromatography for neuronic and vascular endothelial cell membrane chromatography filtering the effective substance basis and targets of “Regeneration”. To enrich activity components by preparative high performance liquid chromatography, that is applied for a variety of cell models verifying the pharmacological effects of active ingredients, and getting the molecular structure of the active substance through LC/MS and NMR technology; Cooperating on speckle imaging techniques to monitor brain blood flow, microvascular morphology, and blood rheology after administration in animal experiments. Make use of immunohistochemical methods to determine the microvascular density in ischemic lesions, VEGF, RGMa protein expression and the length of neuron axon regeneration. Use microvascular and axonal regeneration to clarify the pharmacodynamic mechanism of “Regeneration” of BYHWD. Combining cell experiments with the animal experiments to expore hypothetical pathway “BYHWD → RGMa expression around the focal ischemia → axonal regeneration →Combined with new cerebrovascular → Repair the stroke damage”, thus clearly state the pharmacodynamic Substance basis and specific target points of BYHWD, laying the foundation for stable effective Traditional Chinese Medicine preparations and its clinical application.

补阳还五汤促进缺血性脑中风后康复具有显著的临床疗效，研究表明能“益气行血”进而“生新”，但其物质基础与作用机制尚不明确。本项目运用微透析技术采集大鼠含药脑脊液为受试对象，采用神经元及血管内皮细胞膜固相色谱法，筛选“生新”有效组分及作用靶点；以制备型HPLC富集活性成分，靶细胞模型验证其药理效应，并应用LC/MS、NMR技术在线检测，获取活性物质的分子结构信息；配合激光散斑成像技术监测给药后大鼠脑部血流、微血管形态、及血液流变学指标；并运用免疫组织化学等方法测定缺血病灶区的微血管密度、VEGF、RGMa蛋白表达、神经轴突再生长度，从微血管及神经元轴突再生的角度探讨补阳还五汤“生新”的作用机制；结合体外细胞实验及动物实验探究“补阳还五汤→新生脑微血管→缺血灶周围RGMa的表达→轴突再生→修复脑中风损伤”通路，从而阐明补阳还五汤促进脑中风后康复的药效物质基础及分子作用机制，指导该方临床合理应用。

本课题从补阳还五汤“益气行血”进而“生新”的角度，通过补阳还五汤→新生脑微血管→缺血灶周围RGMa的表达→轴突再生→修复脑中风损伤的途径对其治疗缺血性脑中风进行机制研究，运用神经元及血管内皮细胞膜固相色谱法，筛选“生新”作用靶点及有效组分，富集活性成分，进行靶细胞模型验证其药理学效应，研究发现从脑微血管内皮细胞胞膜固相色谱法筛选出6个活性成分，这6个活性成分均有不同程度的促进脑微血管内皮细胞的作用，且具有浓度依赖性；从神经元样PC12细胞膜固相色谱法筛选出6个活性成分，6个活性成分对OGD/R模型的原代皮层、海马神经元均具有保护作用；从海马神经元细胞膜固相色谱法筛选出2个活性成分，2个活性成分可以缓解OGD/R模型对细胞造成的损伤。同时活性成分（毛蕊异黄酮苷和芒柄花苷）可以上调氧糖剥夺模型细胞的MLC、VEGF、GAP43 和 BDNF 的表达水平，下调p-MLC蛋白水平；从微血管及神经元轴突再生的角度探讨补阳还五汤“生新”的作用机制研究，结果表明补阳还五汤可能通过影响脑血流量，抑制脑损伤后RGMa 的表达，促进轴突延伸，调节轴突再生和重塑来促进MCAO损伤大鼠的恢复。