多环芳烃通过NO介导的亚硝基化影响海马神经发育的机制研究

Polycyclic aromatic hydrocarbons (PAHs) exposure is proved to be associated with abnormal neurodevelopment and function of hippocampus, but the mechanisms are still elusive. Nitric oxide (NO) and NF-κB palys a critical role in neurodevelopment and function of hippocampus. Protein S-Nitrosylation is one of major molecular mechanisms of NO. Our study showed that PAHs might affect neurodevelopment via NO. In the present project, we choose benzo(a)pyrene (BaP) as a representive of PAHs. Firstly, environmental level BaP exposure to pregant C57BL/6 mice from gestation day 1 to weaning, to study the relationship between PAHs-induced neurodevelopmental defects and dysfunction of offsprings hippocampus and protein S-Nitrosylation, and focus on NF-κB related proteins using tools of ethology, proteomics, and molecular biology. Secondly, BaP exposure to embyronic hippocampus neurons which are pretreated with overexpression or knockdown nNOS, and activation or blocking nNOS-NO pathway, to explore the mechanisms of PAHs-induced hippocampus neurodevelopmental defects and dysfunction regulated by NF-κB through NO-meadiated S-Nitrosylation. The outcomes of the present project would be provide more precise scientifical evidences to evaluate the health risk of PAHs exposure to human and animals from gestation to weaning, and in favour of searching intervention strategies and targets for PAHs-induced neurodevelopmental defects and dysfunction.

多环芳烃(PAHs)暴露与海马神经发育和功能异常相关。一氧化氮(NO)及NF-κB在海马神经发育与功能中起关键作用，蛋白亚硝基化是NO作用的主要分子机制之一。申请人研究表明PAHs可能通过NO引起神经发育损伤，具体机制待阐明。本项目拟以代表性PAH[苯并(a)芘]，首先以C57BL/6小鼠为对象，环境水平从妊娠到哺乳期暴露，利用行为学、蛋白组学和分子生物学等技术，研究PAHs暴露损伤海马神经发育和功能与蛋白亚硝基化的相互关系，重点关注NF-κB等相关蛋白亚硝基化；其次以胎鼠海马神经元为对象，利用高表达和低表达nNOS模型以及激活剂和阻断剂干预nNOS-NO通路策略，重点探讨NO介导亚硝基化NF-κB调控PAHs海马神经发育和功能损伤的机制。本研究结果以期为从妊娠到哺乳期暴露PAHs的健康风险评估提供更详细的科学依据，有利于探寻PAHs损伤神经发育的预防策略和干预靶点。

PAHs作为一类广泛分布的持久性污染物，危害生态环境和人类健康。目前研究证明PAHs早期暴露能够影响神经发育，而暴露的效应和具体机制都不清楚。本项目围绕着代表性PAHs (BaP)神经发育毒性开展体内外试验和机制研究。本项目利用环境相关水平BaP哺乳期暴露没有影响母鼠和仔鼠学习记忆能力，但诱发母鼠和雄性仔鼠焦虑行为，导致神经生长、焦虑行为相关基因等差异表达。进一步发现，环境相关水平BaP哺乳期暴露主要引起仔鼠海马区小神经胶质细胞数目减少、自噬和内质网应激(ER stress)。在体外模型中，BaP可诱导小神经胶质细胞NO释放和ER stress和细胞自噬。为探究NO与ER stress调控关系，采用NO供体和NOS抑制剂干预研究ER stress水平，结果表明BaP诱导BV-2释放NO负调控ER stress，可能起到保护细胞作用。但自噬与ER stress之间调控关系尚不明确，我们通过构建诱导自噬和ER stress模型，发现二者同环氧化酶-2（COX-2）高表达相关。通过体外高低表达COX-2或者抑制剂干预，发现外源受试物可经COX-2诱导自噬。更进一步证明ER stress下游ATF4作为转录因子结合到COX-2基因启动子区促进其高表达，进而调控自噬发生，引起细胞毒性。综上，本项目发现了环境相关水平的PAHs通过哺乳暴露导致雄性仔鼠出现焦虑行为，小神经胶质细胞ER stress和自噬，为PAHs对人群健康风险评估提供新的科学依据；初步探明NO 调控ER stress 介导PAHs神经发育损伤的分子机制；阐释了ER stress经ATF4转录调控COX-2介导细胞自噬的分子机制，药物干预ER stress和COX-2可有效地降低外源物的毒性损伤作用，提示ER stress和COX-2是外源物毒性损伤潜在干预靶点，为人群健康提供了可能的预防策略。