过氧亚硝基调节脑缺氧缺血后神经再生及其信号通路的研究

Neural stem/progenitor cells (NSCs) have limited capacities of growth and differentiation, offering possible regeneration therapy for stroke and neurodegenerative diseases. Hypoxic/ischemic stimulation mediates the growth and differentiation of NSCs into mature neurons. However, the mechanisms of hypoxia/ischemia-mediated neurogenesis are largely unknown. In hypoxic/ischemic brain, nitric oxide and superoxide are simultaneously produced and they rapidly react to form peroxynitrite. Recent progress indicates that peroxynitrite could promote endothelial cell growth for angiogenesis and hypoxia-induced muscle cell proliferation. However, whether peroxynitrite contributes to adult neurogenesis in hypoxic/ischemic brains is still unknown. The cell growth-promoting effects or cytotoxic effects of peroxynitrite could be dependent on its concentration. Because peroxynitrite has short lifetime, lack of technical methods for accurately monitoring peroxynitrite is a major obstacle. The development of sensitive and specific peroxynitrite probes makes it possible to address whether peroxynitrite can promote adult neurogenesis under hypoxia/ischemia. Thus, we recently developed new peroxynitrite-specific probes and conducted pilot studies. Our preliminary results showed that hypoxia increased endogenous peroxynitrite formation, and the increased peroxynitrite was correlated with the up-regulation of vascular endothelial growth factors (VEGF) and the enhanced proliferation and neuronal differentiation of NSCs. Also, low dose of peroxynitrite promoted NSCs proliferation. Therefore, we hypothesize that peroxynitrite can act as cellular signaling molecule to promote neurogenesis for brain repair by activating VEGF signaling and/or other signaling pathways in ischemic/hypoxic neural stem/progenitor cells. To verify this hypothesis, this proposal will address three specific questions: (1) Whether peroxynitrite positively modulates neurogenesis in adult NSCs under normoxia and hypoxia in vitro. (2) Whether endogenous peroxynitrite play a role in promotion of neurogenesis in post-ischemic brains in vivo. (3) What's the mechanism behind the neurogenesis-promoting effects of peroxynitrite. We will use primary cultured neural stem/progenitor cells, middle cerebral artery occlusion and hypoxic animal model to detect peroxynitrite production, the proliferation and differentiation of neural stem/progenitor cells. After verifying the hypothesis, we can develop the new concept that peroxynitrite functions as a cellular signaling to regulate adult neurogenesis in hypoxic/ischemic neural stem/progenitor cells. The study will advance current knowledge about the molecular mechanisms of hypoxia/ischemia-mediated neurogenesis. Using this knowledge and our new peroxynitrite probe, we can seek for drugs targeting on peroxynitrite signaling, which may have implications for developing therapeutic approaches to promoting brain repair in stroke and neurodegenerative diseases.

研究发现脑缺氧缺血刺激内源性神经干细胞增殖并定向分化，为治疗中风及神经退行性病变带来了新希望,但由于神经再生的机制不明确，使发展药物治疗策略大为滞后。缺血再灌注产生一氧化氮及超氧阴离子，生成过氧亚硝基阴离子（ONOO-）。但由于ONOO-半衰期短，且缺少精确检测方法，有关生理功能尤其在神经再生中机制不明。为此我们研发了一系列高灵敏度及选择性的荧光探针。在这些探针帮助下，我们发现缺氧产生ONOO-并伴随血管内皮细胞生长因子表达增加及干细胞向神经元方向分化；少量ONOO-促进了神经干细胞增殖；且ONOO-的解构剂FeTPPS和FeTMPyP能抑制神经干细胞的神经元分化。因此ONOO-可能是调节缺血后神经再生的重要信号分子。本项目拟通过神经干细胞培养，小鼠缺氧及脑缺血模型，结合ONOO-专属探针，系统研究ONOO- 在神经再生中的作用。

成人神经发生的发现为治疗脑卒中及神经退行性病变带来了新的希望。神经干细胞是脑卒中后脑结构与功能的修复和重建的重要治疗靶细胞。目前很多的报导显示缺血缺氧能激活人或动物大脑的神经干细胞，能促进神经干细胞的增殖以及定向分化，但是因为其背后的机制研究不甚清楚，使发展药物治疗策略大为滞后。缺血再灌注产生一氧化氮及超氧阴离子，生成过氧亚硝基阴离子（ONOO-）。一直以来，ONOO-的形成被认为是造成大脑缺血再灌注损伤的重要病例过程。我们的假说认为ONOO-也可以作为氧化还原的主要信号分子促进神经干细胞在缺氧、缺血过程中的增长。我们的研究发现在脑卒中的临床病人及动物模型中均有明显的ONOO-的形成。在动物缺氧缺血模型中，神经干细胞的增殖伴随着ONOO-的形成，且两者有明显正相关的关系。通过采用我们自主研发的ONOO-的荧光探针进行神经干细胞的分选实验我们发现，內源的ONOO-含量相对较高的神经干细胞拥有更强的干细胞增殖能力。缺氧刺激了內源ONOO-的生成并且促进了神经干细胞自我更新，增殖和神经元的分化。采用ONOO-的分解剂FeTMPyP和FeTPPs可以明显抑制神经干细胞的自我更新，增殖和分化。外源性加入合成的ONOO-或者其供体均可以促进神经干细胞的自我更新，增殖和向神经元方向的分化。进一步的机制研究发现ONOO-促进神经再生的机制部分通过激活HIF-1α以及相关的Wnt/β-catenin信号通路。此外，我们的研究也发现，ONOO-的浓度是它在缺血缺氧过程中发挥不同功能的关键因素。相对低浓度的ONOO-可以显著促进神经再生，而高浓度的ONOO-则促进了细胞的死亡。综上所述，ONOO-可以发挥其信号分子的作用在缺氧缺血的情况下促进神经干细胞的自我更新，增殖和向神经元方向分化。