苯并(a)芘暴露产活性氧介导海洋鱼类抗菌肽的表达机制及其在核转录因子信号通路中的作用

The immunotoxic effect caused by benzo[a]pyrene (BaP) exposure is commonly believed to be harmful to fish health, but study in mechanism on interactions between this effect and the immune response of fish is little known. Our previous study found that the hepcidin gene from marine cultured fish is significantly induced either by LPS challenge or upon BaP exposure. As we know, LPS-induced hepcidin expression is involved in the immune function. So what is the biological function for high expression of hepcidin induced upon exposure to BaP? Which factor is involved in regulating the hepcidin expression during BaP exposure? These questions are worth elucidating. It has been reported that ROS generated during BaP exposure mediates the activation of the transcription factor NF-κB which is crucial in a series of cellular processes, thus possibly influencing NF-κB signalling pathway. To answer these questions, marine medaka and marine cultured black porgy will be used to carry out this study. The study includes: (1) To compare the hepcidin gene expression patterns regulated by ROS with the one via NF-κB/STAT; (2) To understand the correlation between CYP1A1 mediated reactions via AhR and the responses stimulated by inflammatory factors; (3) To clarify the relationships among the generation of ROS by BaP exposure, the NF-κB activation and the expression of hepcidin mRNA through inhibition of NF-κB/STAT genes using RNAi technique. Through the studies above the question why the hepcidin gene is significantly expressed upon exposure to BaP and the role of ROS would be answered. Furthermore, the biological function implicated for the high expression of hepcidin induced upon exposure to BaP will be testified using RNAi technique combined with bacterial challenge experiments in comparison with the immune response induced with LPS challenge. The study, by revealing the interactions between the immunotoxic effects caused by BaP and the immune response of fish,will provide new insightful information and benefit the field of aquatic toxicology.

传统认为苯并(a)芘（BaP）的免疫毒性效应可影响鱼类健康，但从机制上研究其毒性效应与免疫反应之间的关系少有报道。我们发现，细菌LPS刺激和BaP暴露都显著诱导鱼类抗菌肽hepcidin（Hepc）表达，LPS诱导表达与免疫功能相关，而BaP诱导Hepc高表达的生物学意义及表达调控的通路为何？值得深入研究。已报道污染物产生的活性氧（ROS）与核转录因子NF-κB参与的信号通路相关。本研究用模式生物海水青鱂和黑鲷，比较研究BaP暴露产生的ROS和NF-κB/STAT信号调节下Hepc的表达模式，及诱导的P450代谢反应与炎症因子的关系；以RNAi技术研究BaP暴露产生的ROS与NF-κB及Hepc mRNA表达的关系；从而阐明ROS诱导Hepc高表达的机制与信号通路。并研究了BaP暴露诱导Hepc高表达的生物学功能。该研究将从机制上揭示污染物毒性效应与免疫应答的相互关系，具有重要的科学意义。

传统认为典型环境污染物苯并(a)芘（BaP）的免疫毒性效应可影响鱼类健康，但从机制上研究其毒性效应与免疫反应之间的关系少有报道。该项研究基于LPS 3小时刺激和BaP暴露3天（BaP-3d）后均可显著诱导hepcidin表达的结果，采集海水青鳉肝脏进行转录组测序，建立了青鳉转录组数据库，其中信号转导相关基因数量较多；转录组数据分析发现，LPS刺激与BaP暴露对多条免疫通路尤其是典型NF-κB通路的调控存在差异，LPS既能调控先天性免疫又能调控适应性免疫，BaP处理2d时主要调控先天性免疫基因，而处理3d时则主要调控适应性免疫基因；定量分析发现，LPS刺激与BaP暴露对一些适应性免疫基因表达存在交互作用；转录组数据分析发现，相对于BaP-2d组，BaP-3d组能够调控更多氧化还原相关基因，而氧化还原酶SOD与CAT的酶活在3d时被显著诱导，说明两组对hepcidin基因应答的差异很可能与ROS有关。BaP对青鳉肝脏ROS的调控与暴露时间的相关，以及与BaP对免疫调控的时间点的接近，为揭示ROS和鱼类免疫相关因子的关系提供了依据。通过改良构建的luminol瞬时检测系统发现，BaP能够持续诱导产生生理学浓度的ROS，区别于通常直接添加大剂量瞬时ROS的方法。在DIT-29细胞上证实了BaP对NF-κB通路的调控与H2O2持续表达系统产生的ROS（生理学浓度）对该通路的调控相一致，均为通过抑制P65的磷酸化与入核结合到启动子位点而抑制了该通路的激活，而常规方法中的直接添加高浓度过氧化氢则能够通过增加结合量而激活NF-κB通路，该结果为研究ROS对NF-κB通路调控的作用提供了新的思路；基于上述有关BaP可通过ROS抑制NF-κB通路的试验结果，用抑制剂抑制青鳉的典型NF-κB通路，mRNA定量分析发现hepcidin基因被显著诱导，同时通过转录组数据分析，发现许多其它免疫基因以及免疫通路上调，青鳉鱼体对致病菌感染的抵抗能力增强，该结果提示免疫通路间存在Cross-talk。该研究结果表明BaP诱导hepcidin表达的机制与ROS和NF-κB通路相关，这为进一步阐明污染物与致病菌共存的复杂海洋环境中鱼类如何健康生存具有重要的指导意义。