地黄梓醇协控缺血脑区血管-神经新生偶联机制

Recent key point on drug strategies to enhance angiogenesis and neurogenesis may provide promising opportunities to improve clinical outcomes during brain functional recovery after stroke. NSFC81073084 project suggests catalpol has potentials to provide benign neurovascular niche through remodeling angiogenesis. However, the cellular and molecular mechanisms underlying the effect of catalpol on angiogenesis and neurogenesis coupling after stroke remain elusive. This project will explore the effect of catalpol treatment on angiogenesis and neurogenesis coupling as follows：using GFP(green)-transgenic rat, BrdU injection, Confocal laser scanning microscopy and double-label immunofluorescence assays, siRNA, Signal pathway inhibitor, RT-PCR and western blot, to establish focal cerebral cortex ischemia model and to study the effect of catalpol on neurogenesis, including NSCs (neural stem cell, NSCs) stages of fate specification, migration, synaptic integration, maturation and survival; to study the effect of catalpol on growth factor secretion from neurovascular niche to provide friendly microenvironment for NSCs. Using co-culture system of cerebral microvascular endothelial cells and NSCs from SVZ （sub-ventricular zone, SVZ） to study mechanisms of effect of catalpol on angiogenesis and neurogenesis coupling. This project explores the roles of catalpol in regulating angiogenesis and neurogenesis coupling in vivo and in vitro via SDF1/CXCR4,VEGF/KDR,Ang1,2/Tie,PI3k/Akt and apoptosis cross-talking.

中风后缺血脑区循环重建和神经新生策略是研发中风药物关注的焦点。前期（NSFC81073084）研究提示梓醇促进缺血脑区血管新生成熟重塑，为新生神经元提供了良好的神经血管小生境，但梓醇协调缺血脑区血管新生-神经新生偶联机制尚不明确。本项目建立永久性局灶性脑缺血模型，采用GFP转基因大鼠、BrdU注射、免疫双标激光共聚焦、siRNA、膜片钳电生理、信号抑制剂、ELISA、RT-PCR、western blot技术，研究梓醇对脑缺血后神经干细胞增殖、迁移、分化和存活的影响；血管新生和神经新生的解剖学偶联关系；梓醇调控缺血脑区相关因子分泌改善神经血管小生境；离体共培养模型研究梓醇协控血管新生和神经新生的偶联机制，从整体、离体水平，细胞-蛋白-基因层面阐明梓醇通过SDF1/CXCR4、VEGF/KDR、Ang1,2/Tie、PI3k/Akt和凋亡信号串话来调控局灶脑缺血后神经-血管新生偶联机制。

中风后缺血脑区循环重建和神经新生策略是研发中风药物关注的焦点。前期研究提示梓醇促进缺血脑区血管新生成熟重塑，为新生神经元提供了良好的神经血管小生境，但梓醇协调缺血脑区血管新生-神经新生偶联机制尚不明确。本项目建立永久性局灶性脑缺血模型，采用GFP转基因病毒、BrdU注射、免疫双标激光共聚焦、siRNA、膜片钳电生理、信号抑制剂、ELISA、RT-PCR、western blot技术，研究了梓醇调控局灶脑缺血后神经-血管新生偶联机制。结果：延迟给药梓醇对局灶性脑缺血组织有保护和修复作用，促进中风大鼠神经功能恢复；明确了梓醇的神经新生、血管新生及其偶联作用；梓醇促进脑缺血大鼠损伤侧SVZ区神经干细胞增殖，促进SVZ区来源的神经干细胞迁移到缺血周围皮质区，并分化为神经元和胶质细胞，促进缺血区域附近神经元存活，减少星形胶质细胞过度增殖；梓醇通过VEGF/KDR信号通路促进局灶性脑缺血后缺血脑区血管新生；梓醇激活了SDF-1/CXCR4及其下游PI3K/AKT/ERK信号通路，改善神经血管小生境促进脑缺血大鼠缺血周围皮质神经-血管新生。离体结果进一步证实：梓醇介导SDF-1/CXCR4信号通路促进原代神经干细胞增殖、迁移、分化，这种作用可以被CXCR4信号阻断剂AMD3100部分阻断，提示梓醇促进内皮细胞增殖，通过介导SDF-1α旁分泌信号为神经新生迁移提供较好的微环境，促进原代神经干细胞迁移；有趣的是梓醇对原代培养的海马神经干细胞向神经元和胶质细胞分化的作用有剂量效应，10 μM梓醇促进NSCs向神经元方向分化，0.1 μM促进向星型胶质细胞方向分化，和整体实验结果相互验证，提示梓醇可以抑制星形胶质细胞过度增生，减少胶质瘢痕的形成，抑或为神经干细胞分化和迁移提供了有利环境和路径，其背后的机制后续进一步深究。.上述实验结果提示梓醇通过SDF1/CXCR4、VEGF/KDR信号协控脑缺血后血管新生、神经新生偶联，为梓醇防治缺血性脑血管疾病提供了新机制阐释。目前梓醇已获得临床I期实验治疗糖尿病；未来梓醇防治缺血性中风等脑血管疾病，促进神经血管修复，具有良好临床转化前景。