补阳还五汤调节中风后神经再生及脑内炎性微环境与信号通路作用

In the adult mammalian brain, neurogenesis from neural stem/progenitor cells continues in two regions: the dentate gyrus zone (DG) and the subventricular zone (SVZ) lining the lateral ventricles. The generated neuroblasts migrate to their appropriate location and differentiate to mature granule cells and olfactory bulb interneurons...The ischemia-induced neurogenesis gives rise to new neurons not only in areas where new neurons are normally formed but also in areas which are nonneurogenic in the intact brain..These findings have raised the possibility that stimulation of neuronal replacement by neurons produced by endogenous neurogenesis could become of value for restoring function after stroke conditions leading to neuronal loss. A major problem with endogenous neurogenesis as a potential mechanism for cell replacement is that only a fraction of the new neurons will survive long-term. About 80% of the stroke-generated neurons die during the first 2 weeks after their formation. ..Stroke may be characterized by pro-inflammatory cytokine changes and increased numbers of activated microglia. Inflammation is one key pathological change observed during stroke. The poor survival is most likely to a large extent due to the inflammatory microenvironment encountering the newborn neurons. Therefore, neuro-inflammation may in turn be a valuable therapeutic target in the treatment of stroke...JAK/STAT3 cellular signal activation intensively influence the neural stem cell proliferation and differentiation which can be modulated by many cytokines...Buyang huanwu decoction (BHD) is a traditional formula in chinese medcine which is used to treat the patients with stroke for hundreds of years...Our preliminary results showed that BHD enhanced proliferation and neuronal differentiation of NSCs. Also, It induced the activation of JAK/STAT3 pathway. In the meantime, our data indicated that BHD down-regulated many cytokines such as IL18, IL20, IL4 etc. Therefore, we hypothesize that BHD can promote neurogenesis for brain repair by regulating the inflammatory microenvironment and activating JAK/STAT3 signaling and/or other signaling pathways in ischemic stroke condition. To verify this hypothesis, this proposal will address four specific questions: (1) Whether BHD positively modulates neurogenesis in adult NSCs under normoxia and hypoxia in vitro. (2) Whether BHD play a role in promotion of neurogenesis in post-ischemic brains in vivo. (3) What’s the mechanism behind the neurogenesis-promoting effects of BHD. (4) what’s the relationship between inflammatory microenvironment and neurogenesis. We will use primary cultured neural stem/progenitor cells, middle cerebral artery occlusion animal model to detect the proliferation and differentiation of neural stem/progenitor cells. After verifying the hypothesis, we can develop the new concept that BHD regulate adult neurogenesis by modulating the inflammatory microenvironment and related cellular signal pathway. The study will extend the view on interpreting the mechanism on BHD modulated neurogenesis and advance current knowledge about the molecular mechanisms of inflamation mediated neurogenesis. which may have implications for developing therapeutic approaches to promoting brain repair in stroke.

神经再生在特定脑区域里一直发生，包括侧脑室下区和海马齿状回区，脑缺血能引起神经再生，新生神经元能迁徙至病灶代替死亡的神经元发挥功能。中风后脑内有持续的炎症反应,产生大量的炎症和抗炎因子。这些脑部炎症因子及其信号通路对神经再生有显著的抑制或促进的双重调节作用，因此成为干预中风后神经再生的重要靶点。中药补阳还五汤历来用于临床治疗中风后遗症并有显著疗效。前人研究发现补阳还五汤对周围免疫反应有重要的调控作用。我们初期数据发现在大鼠大脑中动脉梗塞模型中，补阳还五汤可以促进中风后神经干细胞增殖、分化并调控炎症因子如IL18, IL20, 等的表达、激活JAK-STAT3信号通路。然而，补阳还五汤对中风后神经再生的确切作用及其是否通过调控脑部炎症因子从而起到疗效尚待研究。本项目拟用大鼠大脑中动脉梗塞模型研究缺血再灌注损伤后补阳还五汤对脑神经再生的动态作用及机制并研究其所调控脑部炎症因子和神经再生的关系.

补阳还五汤（BHD）是清代名医王清任开创的治疗脑卒中的传统方剂，至今在临床使用了200余年。促进神经再生是治疗脑中风后遗症以及神经退行性病变的一个新方向。而神经再生是涉及神经损伤后神经干细胞生存、增殖及分化多领域协同的课题。在本研究中，我们观察了BHD在实验性大鼠卒中模型中对神经元凋亡，神经干细胞增殖和分化，神经突形成以及神经功能恢复的作用。我们发现BHD治疗14天后可以显着减少大鼠缺血性脑梗死的面积并减少细胞凋亡。BHD在体内和体外呈剂量依赖性地促进缺血大鼠脑中神经干/祖细胞的增殖。此外，BHD可以促进原代培养的神经干细胞中神经元和星形胶质细胞的分化。水迷宫实验表明BHD还有利于大鼠学习功能的恢复。二维凝胶电泳显示BHD通过作用多信号系统发挥其调控作用，包括PI3K / Akt / Bad和Jak2 / Stat3/cyclin D1等。.我们在研究BHD复方的同时，对其中的活性成分也做了相应的研究，我们筛选了其中50多个活性成分，通过实验发现BHD君药中的黄芪内黄芪皂苷VI具有强烈的脑缺血再灌注损伤后神经保护及促神经再生的功能。.我们的实验结果表明缺血1.5小时再灌注7天的大鼠，每天通过静脉注射黄芪皂苷VI（2ug / kg）7天。通过使用不同的生物标志物，我们发现黄芪皂苷VI可促进大鼠脑中齿状回区，脑室下区和皮质的神经再生。同时在体外可以促进干细胞的自我更新和增殖。蛋白质印迹分析表明，黄芪皂苷VI上调了Nestin，p-EGFR和p-MAPK在体内外的表达。表皮生长因子（EGF）介导的EGFR / MAPK信号通路在促进神经再生和脑修复中起关键作用。结果显示黄芪皂苷VI可以激活EGF介导的EGFR / MAPK信号级联反应，通过联合使用EGF受体抑制剂吉非替尼和ERK抑制剂PD98059能消除其促神经再生作用。行为学检测表明，黄芪皂苷VI可以促进MCAO大鼠的空间学习和记忆能力恢复，并改善其运动功能。总之，补阳还五汤及其活性物质黄芪皂苷VI可以有效地促进短暂性缺血性脑卒中大鼠神经再生，并改善因缺血导致的神经功能损伤的修复。尤其是黄芪皂苷VI，我们相信很有潜力开发成为治疗中风的新药物。