NAP对幼年氟曝露小鼠的神经保护作用及机制研究

Chronic fluorosis is a global disease and China is one of the most serious endemic areas. Our previous studies have shown that developmental fluoride exposure of mice could cause central nervous system (CNS) disfunction, including cognition, emotion and nociception. NAP, an eight-amino-acid peptide, has been shown to have a brain bioavailability and neuroprotective effects in a wide variety of neurological disorders. The neuroprotective effects of NAP is closely related with microtubules. In the present study, we propose that the apoptosis of neuron is delayed by the interaction of NAP and end-binding proteins of microtubules. Meanwhile, NAP could maintain the fuction of CNS by promoting neurogenesis. Thus, experiment on neuron-like cells and mice will be applied in vivo and vitro, respectively. Using chimeric gene technique, we construct the recombinant AAV which could express neurotrophin 4 (NT4) and NAP in vivo. Then, nasal mucosa of mice suffered developmental fluoride exposure will be transfected by the NT4-NAP/AAV. Continuous secreted NT4-NAP fusion peptide will enter the brain through the nose-brain pathway. The neuroprotection and mechanism will be explored, including behavior tests, neural apoptosis, neurogenesis, Synaptic density, microtubules system, and so on. We are expecting this study will provide a reliable strategy for the prevention and therapy of neurotoxicity in chronic fluorosis.

幼年期曝露于高氟生长环境中可影响啮齿类动物的中枢神经系统（CNS）功能。NAP是一段由8个氨基酸构成的高效神经保护肽，可以抵抗多种外源性物质或环境的神经毒性，已有研究表明其作用机制与细胞微管系统密切相关。本项目在前期研究的基础上，提出“NAP通过与微管末端结合蛋白作用，使微管重排、延缓氟化物所致的细胞凋亡；同时NAP能够促进神经发生从而维持幼年期曝露小鼠CNS功能”的科学假说。首先拟在体外实验中，阐明NAP对染氟PC12细胞的神经保护作用；再利用基因嵌合技术构建可表达神经营养素4（NT4）和NAP的腺相关病毒载体（NT4-NAP/AAV）；将病毒转染幼年氟曝露小鼠的鼻黏膜，使持续分泌表达的融合肽NT4-NAP经鼻脑通路入脑，继而通过从行为学改变、神经凋亡、神经发生、树突棘形态和密度、突触密度、微管系统变化等多个方面阐明NAP对慢性氟曝露小鼠的神经保护作用及其机制。

氟元素普遍存在于自然界，人体主要通过饮水、饮茶、空气、食物等来摄取氟元素。长期过量摄入氟化物可以通过直接或者间接的方式对神经系统造成损伤。NAP是一段由 8 个氨基酸构成的高效神经保护肽，可以抵抗多种外源性物质或环境的神经毒性，已有研究表明其作用机制与细胞微管系统密切相关。本项目利用基因嵌合技术构建可稳定表达NAP的慢病毒载体，通过动物实验验证鼻-脑通路可持续有效的分泌目的蛋白；以类神经元细胞以及幼年曝露于长期髙氟饮水的SD大鼠为模型，通过细胞和动物实验，综合运用动物行为学、细胞生物学及分子生物学技术（CCK-8、流式细胞术、转录组测序、透射电镜、实时定量荧光PCR、免疫组织化学、Western blotting等），揭示NaF诱导的细胞凋亡是引起中枢神经系统损伤的机制之一；NAP通过增加EB1蛋白的表达，促进微管结构改建缓解NaF引起的细胞凋亡，从而发挥神经保护作用。以上研究结果为慢性氟中毒的预防和治疗提供思路，同时也为神经精神疾病的基因治疗提供依据。

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