基于Wnt/β-catenin通路探讨脑缺血后神经再生及丹龙醒脑方的促进机理研究

Using Chinese medicine targeting on the promotion of neural regeneration and brain repair for reconstructing new functional neurons are timly important topics in neuroscience research field. The studies are still in very early states. There are many unexplored questions. Among the cellular signal pathways, wnt/β-catenin pathway is the decisive pathway for proliferation, migration and differentiation of neural stem cells(NSCs). this project will base on our previous working basis that Danlong Xingnao Formula(DLXNF) can reduce the injury of the brain and neuron apoptosis induced by cerebral ischemia, promote NSCs proliferation, reduce nerve disfunction,improve learning and memory ability and the express of β-catenin,decrease the express of Gsk-3β to systematically study the effects of DLXNF on regulating the proliferation , migration and differentiation of NSCs in various in vitro and in vivo experimental models including cerebral ischemia rats models, hypoxic injured NSCs models. We will investigate the effects of the DLXNF on the upstream and downstream signals of Wnt/β-catenin pathway. and the activition of β-catenin(nucleulocation, DNA combination capability). To verify the Wnt/β-catenin pathway is the key targets of the DLXNF, Dickkopf-1 and siRNA will be used to block the Wnt/β-catenin pathway and we will investigate whether they can eliminate the effects of DLXNF or not.The study aims to explore the mechanisms of DLXNF in promoting neurogenisis. The study will provide new method and new thought for protective effect of traditional chinese medicine on cerebral ischemia injury.

中医药促进神经再生、恢复和重建神经功能已成为神经科学研究热点，但仍处初步阶段。Wnt/β-catenin信号通路是神经干细胞（NSCs）增殖、迁移、分化的决定性通路。本研究在前期丹龙醒脑方减轻脑缺血损伤、细胞凋亡、促进NSCs增殖、减轻神经功能缺失，提高学习记忆能力、升高β-catenin表达的基础上，研究丹龙醒脑方对NSCs增殖、迁移、分化的作用。以Wnt/β-catenin通路为切入点，研究丹龙醒脑方对该通路上、下游主要因子表达、β-catenin活化（核移位、DNA结合能力）、β-catenin磷酸化失活等的作用，以及Wnt拮抗剂Dickkopf-1、siRNA沉默β-catenin能否消除丹龙醒脑方的作用。旨在明确丹龙醒脑方的作用靶点和环节，揭示其促进神经发生的生物学机制，可为中医药防治脑缺血提供新方法、新思路。

（1）背景：中医药促进神经再生、恢复和重建神经功能已成为神经科学研究热点，但仍处初步阶段。Wnt/β-catenin信号通路是促进神经再生的决定性通路。前期研究表明，丹龙醒脑方能减轻神经功能缺失，减轻脑缺血损伤、细胞凋亡，促进NSCs增殖，提高学习记忆能力，其机理有待进一步明确。.（2）内容：从整体水平和细胞水平探讨丹龙醒脑方对NSCs增殖、迁移、分化及Wnt/β-catenin信号通路的影响，揭示丹龙醒脑方促进神经再生的机理。.（3）重要结果、关键数据：丹龙醒脑方能促进脑I/R大鼠模型和细胞模型NSCs增殖（丹中组60.8±12.6 vs 模型组12.2±2.1）、迁移（丹中组31.8±4.3 vs 模型组10.5±3.76）、分化（神经元：丹中组13.8±1.2 vs 模型组8.9±0.6；星形胶质细胞：丹中组32.8±6.1 vs 模型组16.9±2.4；NG2胶质细胞：丹中组20.8±2.6 vs 模型组8.9±2.4），升高脑I/R大鼠模型和细胞模型Wnt-1（整体实验丹中组4.32±0.24 vs 模型组1.50±0.05）、Wnt-3（丹中组3.06±0.04 vs 模型组1.45±0.08）、Frizzled（丹中组0.681±0.010 vs 模型组0.365±0.018）、Dsh（丹中组0.473±0.036 vs 模型组0.212±0.021）、β-catenin（丹中组0.648±0.024 vs 模型组0.306±0.018）、Cyclin D1（丹中组141±16.11 vs 模型组261±19.74）、c-myc（丹中组0.315±0.021 vs 模型组0.039±0.008，丹龙醒脑方组4.34±0.18 vs 缺氧组1.91±0.07）、c-jun表达（丹中组0.450±0.035 vs 模型组0.076±0.026，丹龙醒脑方组5.28±0.36 vs 缺氧组2.48±0.11），降低Gsk-3β表达（丹中组142.65±20.44 vs 模型组76.46±19.33）。.（4）科学意义：从Wnt/β-catenin信号通路揭示了丹龙醒脑方促进神经再生的作用及机理，为丹龙醒脑方抗脑缺血损伤临床应用提供了实验依据，为缺血性脑血管病中医治疗提供了新的治法和新的切入点。