Nrf2参与水生动物氧化应激调控的研究进展*

严小军 祁鹏志 郭宝英 李继姬

Nrf2参与水生动物氧化应激调控的研究进展*

严小军1, 2祁鹏志1, 2①郭宝英1, 2李继姬1, 2

(1. 国家海洋设施养殖工程技术研究中心 舟山 316022; 2. 浙江海洋大学海洋科学与技术学院 舟山 316022)

环境变化会诱导机体活性氧(reactive oxygen species, ROS)水平升高, 从而产生氧化应激。氧化应激对所有生物的生存、生长、发育和进化都具有深远的影响。核因子E2相关因子2 (nuclear factor erythroid 2 related factor 2, Nrf2)被公认为细胞氧化应激调控的主导者, 与伴侣蛋白Kelch样环氧氯丙烷相关蛋白1 (kelch-like ECH-associated protein 1, Keap1)一起控制数百个解毒酶和抗氧化蛋白编码基因的表达。近年来, Nrf2在水生动物中逐渐获得重视, 并在一些模式鱼类如斑马鱼、鲤鱼及其他一些鱼类和水生无脊椎动物中得到研究。介绍了Nrf2的结构以及调控机制, 回顾了近年来水生动物Nrf2通路参与氧化应激调控所取得的进展。研究表明, Nrf2在水生动物中广泛存在, 在非生物(金属、有机污染物、无机盐、药物及微塑料等)、生物(细菌、病毒、有毒藻类)以及生境变化(冰融、盐胁迫)诱导的氧化应激调控中发挥重要作用。Nrf2一经激活入核, 在小Maf蛋白的协助下与抗氧化反应元件(antioxidant-response element, ARE)结合, 启动一系列ARE驱动基因的表达, 并和孕烷X受体(pregnane X receptor, Pxr)、丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)以及芳烃受体(aryl hydrocarbon receptor, AhR)等细胞通路协同作用参与一系列生理过程。Nrf2在水生动物响应环境变化过程中发挥重要的细胞保护机制, 有望发展成为抗逆育种潜在的基因靶点。

核因子E2相关因子2 (Nrf2); 氧化应激; 水生动物

细胞通过表达抗氧化蛋白和II相解毒酶来为自身提供保护, 这些蛋白在低水平氧化应激状态下即可被激活, 而这种激活是通过被称为抗氧化反应元件(antioxidant-response element, ARE)或亲电反应元件(electrophilic response element, EpRE)的顺式作用元件介导的(Kobayashi, 2005)。ARE最初发现于编码人和鼠两种主要解毒酶谷胱甘肽S转移酶Ya (GST-Ya)和NAD(P)H醌氧化还原酶1 (Nqo1)的启动子区(Jaiswal, 1994), 而后来的研究发现ARE广泛存在于抗氧化蛋白和II相解毒酶基因的5′启动子区, 从转录水平上调控细胞保护酶对氧化胁迫的诱导反应(Nguyen, 2003)。由ROS及亲电体水平升高和/或抗氧化能力降低而引起的细胞氧化还原状态的改变是触发ARE介导的转录反应的重要信号(Nguyen, 2009)。接下来的研究中, ARE的结合因子引起了科学家的兴趣。最终, 核因子E2相关因子2 (nuclear factor erythroid 2 related factor 2, Nrf2)成为人们关注的焦点, 被公认为细胞抗氧化防御的主导者(Vomund, 2017)。

Nrf2最早是被作为p45 NF-E2密切关联蛋白从K562细胞中克隆的(Moi, 1994)。p45 NF-E2是珠蛋白异源二聚体中较大的亚基, 具有NF-E2位点的结合活性, 是珠蛋白基因表达的关键顺式调节因子(Andrews, 1993)。p45 NF-E2相关蛋白中的4个成员p45 NF-E2、Nrf1、Nrf2和Nrf3已经在哺乳动物中分离出来, 被称为Cap'n'collar (CNC)型碱性亮氨酸拉链(bZIP)蛋白(Motohashi, 2002)。这个名称来源于与果蝇CNC蛋白的序列相似性。CNC蛋白首先在果蝇中被发现, 是唇和下颌发育所必需的(Mohler, 1995)。CNC蛋白其中一个异构体CNCC蛋白包含Nrf2经典的ETGE基序, 可能发挥和脊椎动物中Nrf2类似的功能(Kobayashi, 2005)。Nrf2在鼠、人、鸡和鱼中被鉴定出来, 并且被认为可能存在于所有其他脊椎动物中(Kobayashi, 2002), 而近年来的研究表明, Nrf2也广泛存在于一些水生无脊椎动物包括贝类、虾类等(Silvestre, 2020), 表明Nrf2介导的细胞防御功能在自然界中可能是保守的。Nrf2与ARE结合, 调节ARE介导的抗氧化基因表达从而参与响应环境变化的细胞反应(Dhakshinamoorthy, 2001; Jaiswal, 2004)。Nrf2的活性调节机制表明, Kelch样环氧氯丙烷相关蛋白1 (kelch-like ECH-associated protein 1, Keap1)是Nrf2的伴侣蛋白, 在Nrf2调控过程中发挥至关重要的作用。Keap1和Nrf2共同构成一个二元系统, 在人体中调控多达250个含ARE结构的基因。Keap1-Nrf2系统已发展成为机体对抗环境侵害的主要防御机制, 在维持机体稳态方面有着关键作用。

1 Nrf2、Keap1结构

1.1 Nrf2结构

Nrf2是由核因子NFE2L2 (erythroid-derived 2-like 2)编码的蛋白, 具有碱性亮氨酸拉链(basic region-leucine zipper, bZIP)结构, 隶属于CNC转录因子家族, 是该家族中活力最强的转录调控因子(Alam, 1999)。Nrf2广泛存在于从低等的昆虫到高等的哺乳动物中, 在持续暴露于外界环境的皮肤、肺、消化道以及解毒代谢器官如肝脏和肾脏中大量表达。

同源比对发现Nrf2包含7个高度保守的Neh (Nrf2-ECH homology)结构域(图1a)。Neh1位于Nrf2的C端, 包含bZIP基序, 能够与多种含有此同源结构的小Maf蛋白、c-Jun蛋白, c-Fos蛋白结合形成二聚体。细胞核内, Nrf2通过Neh1区与小Maf蛋白结合, 进而识别并结合ARE元件, 启动下游基因转录。此外, Neh1区还包含核转录因子普遍的核定位和输出信号, 能够与泛素连接酶UbcM2作用以调控Nrf2的核转位和降解(Jain, 2005)。Neh2位于Nrf2的最末N端, 含有DLG和ETGE基序的结合位点, 是负调控蛋白Keap1的作用靶点。该结构域包含丰富的赖氨酸残基, 可介导Nrf2泛素化及26S蛋白酶体的降解(McMahon, 2004)。与Keap1解耦联的Nrf2进入细胞核并以Nrf2-sMaf异二聚体形式与ARE结合后并不能立即启动下游基因的转录, 而是需要一类被称为转录共激活子的辅助蛋白参与。位于最末C端的Neh3和位于N端的Neh4、Neh5负责结合转录共激活子, 共同行使转录调控功能。其中, Neh3通过与解螺旋酶DNA结合蛋白6 (chromodomain helicase DNA binding protein 6, CHD6)的相互作用来协助Nrf2的顺式激活(Nioi, 2005), 而Neh4和Neh5结构域是通过与环磷酸腺苷反应元件结合蛋白(cyclic AMP response element-binding protein, CBP)的结合, 反式激活Nrf2的表达(Katoh, 2001)。对氧化还原不敏感的Neh6含有富含丝氨酸残基的DSGIS和DSAPGS基序, 可作为糖原合成酶激酶-3 (glycogen synthase kinase-3, GSK-3β)的磷酸化靶点, 启动Kepa1非依赖的Nrf2降解(Chowdhry, 2013)。Neh7是近年来新发现的Nrf2结构域, 其与视黄酸X受体α (retinoic X receptor α, RXRα)识别并结合后, 能够抑制Nrf2的转录(Wang, 2013a)。

图1 Nrf2 (a)和Keap1 (b)分子的结构域图示

注: Keap1-dependent degradation: Keap1-依赖型降解; Transactivation domain: 转录激活域; RXRα binding: RXRα结合域; DNA binding domain: DNA结合域; CUL3 association: CUL结合域; Self association: 自交联; Nrf2 association: Nrf2结合域

1.2 Keap1结构

Keap1为隶属于Kelch家族的多区域阻遏蛋白, 可作为氧化应激的传感器存在, 对Nrf2号通路起负调控作用(Kobayashi, 2006)。生理状态下, Keap1常以二聚体形式存在, 将Nrf2锚定在胞浆中, 抑制其活动。氨基酸序列及域功能分析发现Keap1包含5个不同的结构域: 氨基末端区(NTR)、羧基末端区(CTR)、Broad复合物即tramtrack和bric-a-brac结构域(BTB), 插入区(IVR), 六个Kelch/双甘氨酸重复序列(DGR) (图1b)。BTB和DGR区是主要的功能区(Kaspar, 2009)。Keap1通过BTB区交联形成同源二聚体, 同时BTB也是Cul3 (Cillion 3)依赖性E3泛素连接酶复合物的结合位点, 是介导Nrf2泛素化及蛋白降解的必要区域(Cullinan, 2004)。DGR区是Keap1与Nrf2的结合区, 该区中的6个Kelch能够形成β折叠结构与Nrf2的Neh2区结合(Adams, 2000)。IVR区含有大量的半胱氨酸(Cys, C)残基, 是整个蛋白的功能调节区。而Nrf2诱导剂也常通过修饰其中的Cys273和Cys288位点, 干扰Nrf2泛素化从而稳定Nrf2蛋白(Ogura, 2010)。

2 Nrf2信号通路的调控机制

尽管Nrf2的激活入核能够诱导数百个具有细胞保护功能的基因的表达, 从而提高机体抗氧化应激的能力, 但Nrf2的无序激活也会对机体造成严重伤害。keap1缺陷型小鼠体内Nrf2无序表达, 小鼠在出生后3周内死亡(Wakabayashi, 2003)。因此, 控制Nrf2活动对维持细胞内环境稳态, 保障机体健康至关重要。现有研究表明, Nrf2信号通路的激活主要有两种调控方式: Keap1-依赖型调控(图2a)和Keap1-非依赖型调控(图2b)。

2.1 Keap1-依赖型调控

生理状态下, 两个Keap1蛋白以BTB区交联形成二聚体, 同时使用该区域与Cul3相互作用。二聚体Keap1的两个DGR分别与Nrf2的Neh2结构域的DLG和ETGE基序结合将Nrf2限制在胞浆中。Nrf2泛素化后被26S蛋白酶体降解以维持在较低的基础水平, 并以从头合成的方式更新(Stewart, 2003)。一小部分入核的Nrf2激活细胞保护基因的转录, 满足机体正常的抗氧化需求。

Keap1含有27个Cys残基, 因此常将该分子视作内源性以及环境氧化应激信号的传感器(Sihvola, 2017)。ROS氧化硫醇, 诱导大分子谷胱甘肽(GSH)化和烷基化, 因此具有修饰Keap1 Cys的能力(Holland, 2008)。Nrf2的Keap1-依赖型调控都是以Keap1的Cys修饰为基础的。一旦暴露于亲电体和ROS, Keap1的某些Cys残基(主要是C273和C288)被修饰, 导致Keap1构象改变, Keap1处于一个非功能性封闭状态。尽管Nrf2的DLG和ETGE基序能够与Keap1的DGR结合, 但不能够被泛素化蛋白酶体降解, 因此没有足够的处于游离状态的Keap1产生。导致新生产的Nrf2不能被Keap1捕获而发生入核激活(Baird, 2013)。另外还有一种“铰链和闩锁”模型。认为Nrf2的DLG基序对Keap1的亲和力比ETGE基序弱的多, 导致亲电体攻击Keap1的Cys时, DLG与Keap1 DGR区的结合会断开, 使Nrf2不能被泛素化降解, 从而发生入核转移(Jung, 2010)。Keap1抑制的另一个机制与它与Nrf2泛素化所需的CUL3复合物的相互作用有关。位于BTB结构域的C151影响Keap1与Cul3的结合。Nrf2激活剂巴多索隆(CDDO, RTA401)与Keap1在C151位点形成加合物, 从而破坏Keap1和Cul3之间的相互作用(Naidu, 2018), Keap1被阻塞在Nrf2结合构象中, 新合成的Nrf2逃脱泛素化从而产生入核激活(Robledinos-Antón, 2019)。入核后的Nrf2与小Maf蛋白(MafK, MafG, MafF)结合后识别ARE序列并启动下游基因转录(Yamamoto, 2018)。

图2 Nrf2信号通路的调控机制: (a) Keap1-依赖型调控和(b) Keap1-非依赖型调控

注: Keap1-dependent modulation: Keap1-依赖型调控; Keap1-independent modulation: Keap1-非依赖型调控; Basal state: 基态; Induced state:诱导态; Inducers: 诱导物; blocking: 阻断; proteasome 26S: 蛋白酶体26S; Nrf2 degradation: Nrf2降解; Nascent Nrf2: 新生Nrf2; Cytoprotective genes: 细胞保护基因; Cytoplasm: 细胞质; Nucleus: 细胞核

2.2 Keap1-非依赖性调控

糖原合酶激酶-3β (glycogen synthase kinase-3, GSK-3β)被认为参与Nrf2入核激活后的调控。GSK-3β蛋白激酶是一种多功能丝氨酸/苏氨酸激酶, 在多种信号通路中发挥重要作用(Kannoji, 2008)。GSK-3β能够磷酸化酪氨酸激酶Fyn的某个苏氨酸(Thr, T)残基, 介导Fyn的入核激活(Dai, 2017)。激活的Fyn磷酸化Nrf2的酪氨酸(Tyr, Y)568残基, 诱导Nrf2的核输出, 并被Keap1捕获后降解(Jain, 2006)。另外, 在胞浆中GSK-3β能够直接磷酸化Nrf2位于Neh6区的丝氨酸(Ser, S)335和338残基, 而后磷酸化的Nrf2易位到细胞核中, 并被E3泛素连接酶β-TrCP (β-transducin repeat containing E3 ubiquitin protein ligase, β-TrCP)直接识别, 诱导Nrf2的核泛素化和降解(Hayes, 2015)。除此之外, 一种竞争机制也会影响Nrf2对下游基因转录的激活。碱性亮氨酸拉链转录因子1 (basic leucine zipper transcrip-tion factor 1, Bach1)是机体内一种广泛存在转录抑制子(Sun, 2002), 和Nrf2有一定的亲缘关系(Kobayashi, 2006)。生理状态下, Bach1和小sMaf蛋白形成异二聚体并与ARE元件结合(Dhakshinamoorthy, 2005), 抑制基因表达。氧化应激时, Bach1从ARE中释放出来并被Nrf2取代。Bach1与Nrf2竞争与ARE的结合, 导致Nrf2下游基因的抑制(Jain, 2005)。

3 Nrf2信号通路参与水生动物氧化应激调控

在长期的进化过程中, 水生动物发展了相对完善的细胞应激体系以应对复杂的生存环境。在哺乳动物中发现的一些典型细胞应激信号通路, 如丝裂原活化蛋白激酶(MAPK)通路, 核因子-κB (NF-κB)通路, 过氧化物酶体增殖物激活受体(PPAR)通路以及Nrf2通路等在水生动物中也被发现, 而Nrf2通路在其中发挥核心作用(Silvestre, 2020)。

3.1 Nrf2与环境污染相关性研究

金属作为水系统中最常见的污染物, 对其研究最早也最为深入。在水生动物中, Nrf2已被报道参与多种金属诱导的氧化应激反应(表1)。而斑马鱼作为一种水生模式生物, 在Nrf2抗氧化应激调控研究中发挥了引领作用。镉是一种嗅觉毒物, 诱导斑马鱼抗氧化基因谷胱甘肽硫转移酶pi (GSTpi)、谷氨酸半胱氨酸连接酶催化亚基(GCLC)、血红素氧化酶1 (HO-1)、过氧化物酶1 (Prdx1)表达, 但被吗啉代介导的Nrf2敲降阻断, 导致嗅觉驱动行为破坏、细胞死亡增加和嗅觉感觉神经元丢失。嗅觉神经元特异性基因在Nrf2吗啡啉突变体斑马鱼中表达下调。用Nrf2的激活剂萝卜硫素(SFN)预处理胚胎, 可减弱镉诱导的嗅觉组织损伤(Wang, 2013b)。环境相关浓度的铬(2 mg/L)胁迫下, 斑马鱼肝脏中Nrf2在mRNA及酶活水平上均显著上调, 免疫组化证实其发生了入核激活。Nrf2的激活诱导下游Nqo1和含铜和锌的超氧化物歧化酶(CuZnSOD)表达(Shaw, 2019)。紫草碱可以减轻铬诱导的斑马鱼肝细胞毒性, 其最终也是通过激活Nrf2-Keap1-ARE通路, 诱导细胞保护基因红细胞衍生核因子2样蛋白2 (Fe2l2)、Nqo1和热激蛋白70 (Hsp70)的表达, 提高细胞活力, 减少ROS产生来实现的(Shaw, 2020)。另外, Shaw等(2019) 研究还发现作为芳烃受体(AhR)通路中重要成分的细胞色素P4501亚族A多肽(CYP1A)在铬暴露后也显著表达上调, 作者认为这可能是通过Nrf2依赖的AhR通路间接诱导的, 表明细胞抗氧化机制的组成部分之间存在广泛的串话。类似的交互作用机制也在研究斑马鱼银和镉暴露时被发现(Hu, 2019)。在野生型斑马鱼胚胎中, 银和镉的积累和毒性受三磷酸腺苷结合盒(ABC)转运体的影响, 可以显著诱导ABC转运体的mRNA表达, 而孕烷X受体(Pxr)和Nrf2的突变降低了这些诱导效应, 但ABC转运蛋白基础表达的升高弥补了诱导性缺失。Pxr缺陷胚胎中金属离子的毒性未变, 然而, Nrf2的突变破坏了GSH的产生, 导致银和镉在斑马鱼胚胎中的毒性增强。此外, 在未进行攻毒的Pxr缺陷模型中, 其他转录因子如Ahr1b、Ppar-β、Nrf2表达均出现上调, 而Ahr1b、Ppar-β和Pxr的诱导增强仅在金属离子暴露的Nrf2缺陷胚胎中可见, 说明对转录因子缺失的不同补偿现象。

表1 Nrf2参与水生动物氧化应激调控

续表

续表

在斑马鱼中, Nrf2近年来也被报道参与除金属以外的其他多种环境污染物包括PAHs、POPs、无机盐、药物等的氧化应激调控过程(表1)。氟化钠(NaF)暴露时, 斑马鱼脑和肝脏中Nrf2表达上调, 而Keap1表达下调, 同时下游细胞氧化应激基因表达上调, 证实了Nrf2在NaF诱导的斑马鱼氧化应激中发挥重要作用, 且与哺乳动物中经典的Keap1负调控Nrf2的方式相吻合(Mukhopadhyay, 2015a, b)。在三氧化二砷(As2O3)暴露的斑马鱼中, Nrf2也以同样的方式在脑中激活, 诱导下游HO-1和Nqo1表达上调, 参与抗三氧化二砷诱导的氧化应激过程(Sarkar, 2014)。叔丁基过氧化氢(tBOOH)以及α-、β-萘黄酮(ANF, BNF)单独或者ANF+BNF联合暴露时, 斑马鱼胚胎中SOD、GSTpi、谷胱甘肽过氧化物酶(GPx)以及谷氨酰半胱氨酸连接酶(GCL)表达显著上调。当用吗啉代将Nrf2敲降后, 这些元件表达上调被明显抑制, 且使得tBOOH暴露后的斑马鱼胚胎死亡率增加, 并加剧ANF+BNF联合暴露导致的胚胎畸形(Timme-Laragy, 2009)。全氟辛烷磺酰基化合物(PFOS)暴露明显上调Nrf2和下游HO-1的表达。当与Nrf2的激活剂SFN共暴露时, ROS水平明显下降。当用吗啉代将Nrf2沉默后, PFOS诱导的HO-1表达明显下调(Shi, 2010)。高剂量的亚砷酸钠暴露下, Nrf2突变型Nrf2afh318(Nrf2 DNA结合区域发生突变)幼斑马鱼死亡率明显高于野生型。亚砷酸钠暴露诱导细胞应激保护因子GCLC、GSTpi、ABCC2以及Prdx1以Nrf2依赖的方式表达, 而SFN预处理显著提高亚砷酸盐暴露时的斑马鱼成活率, Nrf2在对抗急性亚砷酸钠毒性中发挥重要作用(Fuse, 2016)。

在除斑马鱼之外的其他鱼类中, 也有证据表明Nrf2参与细胞应激, 是触发抗氧化级联反应的关键事件之一。在建鲤中, 0.60 mg/L铜暴露4 d增加鱼脑Nrf2核积累, 诱导下游抗氧化元件CuZnSOD、GPx1a、GR表达上调。增强Nrf2与ARE结合的能力, 导致CuZnSOD表达水平升高。铜暴露还上调了Nrf2、MafG1和蛋白激酶C (PKC)的表达, 表明这些蛋白需要重新合成, 以延长对抗氧化基因的诱导时效(Jiang, 2014)。在建鲤肌肉中呈现了与之相反的结果。0.56 mg/L铜暴露4 d导致鱼肌肉中核Nrf2蛋白水平降低, ARE结合能力减弱, 半胱氨酸蛋白酶-3 (caspase-3)介导的DNA断裂增加, 诱导抗氧化元件CuZnSOD、GPx1a、GPx1b表达下调, 抗氧化酶活性降低从而引起肌肉的氧化损伤(Jiang, 2015)。这些研究表明即使同种污染对鱼体不同组织造成的伤害也不尽相同, 鱼体不同组织对同种污染诱导的氧化应激可能发展出了不同的应对机制。尽管如此, 这些研究仍证实Nrf2在鱼体应对急性Cu污染事件中发挥重要调控作用。在虹鳟中还发现, 头肾中Nrf2介导的抗氧化系统和PXR介导的解毒系统协同作用以对抗2,2',4,4'-四溴联苯醚(BDE-47)诱导的氧化压力(Liu, 2019)。此外, 在草鱼应对锌(Song, 2017)和姜黄素(Ming, 2020), 大黄鱼应对锌(Zheng, 2016)和汞(Zeng, 2016), 鰕虎鱼应对阿司匹林(Wang, 2020b)等事件中, 均能发现Nrf2信号通路的身影。

无脊椎动物缺乏脊椎动物那样获得性的细胞应激机制, 机体防御反应仅依靠非特异的固有调控机制, 其应对外部刺激的机体反应逐渐得到重视。近年来, Nrf2在一些无脊椎动物中逐渐被发现, 并报道在机体抗外界刺激诱导的氧化应激中发挥重要作用。低浓度镉暴露会引起背角无齿蚌肾脏中Nrf2表达上调同时Keap1表达下调, 而高浓度镉暴露下, Nrf2表达水平无明显变化, Keap1表达明显下调(井维鑫, 2019)。在克氏原螯虾中, 镉暴露显著降低四氢大麻酚(THC)和酚氧化酶原(proPO)水平, 而ROS水平以时间和剂量依赖的方式升高。同时镉暴露明显提升p38丝裂原活化蛋白激酶(p38MAPK)和Nrf2的表达和激活, 且p38MAPK和Nrf2表达与proPO活性密切相关, 肝胰脏可能通过ROS介导的MAPK/Nrf2途径参与氧化还原活动(Wei, 2020)。叔丁基对苯二酚(tBHQ)作为Nrf2的激活剂, 处理太平洋牡蛎后, GR mRNA和蛋白水平上调, 证实其诱导作用可能是通过Nrf2途径产生。太平洋牡蛎Keap1和Nrf2蛋白的保守结构域以及经典Nrf2诱导剂tBHQ对相关抗氧化防御的明确诱导, 支持了双壳类Nrf2/Keap1通路与哺乳动物中功能相一致的观点(Danielli, 2017a)。苯并芘(Bap)处理菲律宾蛤仔后, 在第1天和第6天, Nrf2的转录水平显著提高, 且与Keap1呈现负相关, 与抗氧化元件GST、SOD、GPx和CAT的转录表达呈现正相关。RNAi将Nrf2敲低后, 抗氧化元件表达呈现与Nrf2一致的变化。与对照组相比, 脂质过氧化水平明显升高。结果表明, Keap1能够感知氧化应激, 与Nrf2组成经典的二元系统在双壳贝类对Bap应激调控事件中发挥作用(Wang, 2018a)。这一观点在作者对另外一种双壳贝类栉孔扇贝的研究中得以强化。另外, 在苯并芘处理栉孔扇贝后, 除抗氧化元件外, PKC、c-JNK和p38MAPK也呈现与Nrf2一致的表达变化, PKC、MAPKs以及Nrf2通路在双壳动物抗Bap氧化防御中可能发挥协同的作用机制(Wang, 2019a)。本团队在厚壳贻贝中也证实Nrf2参与Bap的氧化应激调控过程。Bap暴露显著上调Nrf2转录表达, 同时下游抗氧化元件SOD、CAT、GPx和GR转录及蛋白表达上调, Nrf2与抗氧化基因转录表达呈现正相关。Nrf2原核表达后注射厚壳贻贝, 导致Bap诱导的ROS和脂质过氧化水平比对照组显著降低(Qi, 2020)。在矮小拟镖水蚤内, 还发现纳米和微米级苯丙乙烯微球能够诱导细胞内ROS的升高, 并激活Nrf2信号通路, 上调GPx、GR、SOD和GST酶活水平, 减轻细胞氧化应激反应(Jeong, 2017)。

3.2 Nrf2与微生物胁迫相关性研究

在哺乳动物中的大量研究已经证实, 作为应激状态下最重要的细胞防御通路, Nrf2在包括嗜血杆菌(Lugade, 2011)、肺炎链球菌(Gomez, 2016)、结核分歧杆菌(Rothchild, 2019), 以及肝炎病毒(Ivanov, 2011; Schaedler, 2010)、甲型流感病毒(Kosmider, 2012)、水疱性口炎病毒(Olagnier, 2017)在内的多种病原微生物的感染中发挥着重要作用。微生物感染可诱导机体产生大量的ROS, 改变正常的氧化应激状态。在此情况下, Nrf2信号通路被激活, 一方面诱导下游抗氧化基因表达以增强对ROS的清除, 维持细胞内环境稳态; 另一方面与免疫信号通路如TNF-α、NF-κB等协同作用, 通过诱导抗炎, 抗细胞凋亡基因的表达增强整体的免疫耐受能力。Nrf2单独参与水产动物抗菌应激的研究尚未见报道, 现有研究多涉及Nrf2参与其他物质诱导的抗菌调控过程。Jiang等(2016)报道, 嗜水气单胞菌能够诱导建鲤氧化应激。与肌醇摄入处于最优水平的建鲤相比, 肌醇摄入过少或过多的鱼体头肾和脾脏中Nrf2及下游抗氧化元件包括CuZnSOD、MnSOD、CAT、GPx1a和GR的转录表达均受到抑制。适宜剂量的肌醇摄入会通过激活Nrf2和E2F转录因子4 (E2F4)介导的通路上调抗氧化基因的表达, 促进损伤细胞更新来应对嗜水气单胞菌诱导的氧化应激, 促进机体健康。在草鱼中同样发现适宜水平的肌醇补充能在草鱼受到嗜水气单胞菌胁迫时显著下调Keap1的表达, 同时上调Nrf2的表达, 从而激活Nrf2信号通路, 降低ROS水平, 提高抗氧化酶活性, 增强其抗氧化能力(胡凯等, 2019)。这些研究证明Nrf2在肌醇补充诱导的抗细菌感染能力提升过程中发挥重要作用, 此外, 一些益生菌类细菌也可通过Nrf2通路发挥作用。Yu等(2018)报道了异育银鲫饮食中凝结芽孢杆菌的适量补充可以激活Nrf2-Keap1通路, 上调抗氧化元件如NADPH氧化酶2 (Nox2)和过氧化物酶2 (Prx2)表达水平, 增强抗氧化反应, 提高生长性能。

尽管Nrf2已被广泛证实参与哺乳动物病毒感染过程, 但关于其在水生动物病毒感染中的作用却鲜有报道。尽管如此, 近年来的一些研究已确证实Nrf2在水生动物病毒感染过程中发挥关键性作用。鲤春病毒血症病毒(SVCV)感染黑头软口鲦上皮瘤细胞(EPC)细胞后, Nrf2的核蛋白和总蛋白含量以及转录水平均提升明显, 表明SVCV感染能激活Nrf2, 增加其基因转录和蛋白表达, 导致在细胞核内的累积。Nrf2的激活剂2-氰基-3,12-二氧代齐墩果-1,9(11)-二烯-28-羧酸(CDDO-Me)对EPC中Nrf2的激活效应有限, 而莱菔硫烷(SFN)能显著增加Nrf2的核转运, 上调下游效应基因的表达, 提高的总抗氧化能力。但介导的激活对SVCV的复制无显著性影响(杨毅, 2014)。SFN和CDDO-Me刺激胖头鲤肌肉细胞系(FHM)可以激活Nrf2-ARE 信号通路, 提高细胞总抗氧核能力。当FHM受到SVCV感染时, SFN和CDDO-Me预处理可以极显著降低SVCV-G的转录水平, 降低病毒滴度, 抑制 SVCV的复制。当Nrf2被敲降后, Nrf2蛋白表达和转录水平受到明显抑制, 且废除两种激活剂对Nrf2-ARE信号通路的激活作用(邵军辉, 2016)。这些研究表明Nrf2的激活有利于机体降低病毒诱导的氧化应激, 维持细胞稳态, 对病毒的感染发挥抵抗性作用。但在白斑综合征病毒(WSSV)感染日本囊对虾过程中, Nrf2发挥的作用与之相反。WSSV感染引起对虾血细胞内ROS水平升高, 导致的Nrf2的mRNA表达以及细胞核中的蛋白水平表达上升。将Nrf2敲低后, 对虾体内病毒蛋白的复制受到抑制, 同时存活率明显上升。注射SFN后, Nrf2表达量上升的同时病毒蛋白的表达量也上升。进一步研宄发现, WSSV结构蛋白VP41B前端具有ARE元件, 可与Nrf2结合。敲低Nrf2可以抑制VP41B的表达, 而SFN处理则增强其表达, 而RNA干扰实验证实VP41B与WSSV复制相关。这些结果说明WSSV可以利用对虾的Nrf2-ARE系统启动自身含有ARE元件的基因的表达, 进而促进病毒复制(陈敬, 2018)。

在软体动物如翡翠贻贝、褶纹冠蚌中还发现Nrf2参与有害藻类及藻毒素诱导的氧化应激反应。利马原甲藻短期暴露导致翡翠贻贝鳃中Keap1、CAT以及ABC转运蛋白转录表达上调, 而GPx1和Nqo1表达下调, 鳃明显损伤。随着暴露时间延长, Nrf2表达显著上调, 而KEAP1下调, 同时Nqo1、SOD、GST-ω和ABCB1上调, 96h后鳃损伤恢复。作者认为利马原甲藻可能导致鳃的氧化损伤。然而, 长时间高密度暴露可激活Nrf2信号通路, 从而降低毒素对贻贝鳃组织的影响(He, 2019)。Nrf2在褶纹冠蚌的外套膜、闭壳肌、腮、血淋巴、肝胰脏各组织中都均有表达, 且在肝胰脏中表达最高。微囊藻毒素刺激后, 肝胰腺和血淋巴中Nrf2表达上调, 显示Nrf2通路被激活(王晓波, 2018)。Wu等(2020)也发现微囊藻毒素会诱导褶纹冠蚌肝胰腺中Nrf2转录水平升高, 而下游抗氧化元件MnSOD、CuZnSOD、SeGPx和GST等转录及蛋白表达均上调, 认为Nrf2通路对保护软体动物免受微囊藻毒素侵害至关重要。

3.3 Nrf2与生境变化相关性研究

近年来研究表明, Nrf2也与一些鱼类特殊的生境适应相关联。侧纹南极鱼胚胎发育的最后阶段正值海冰融化和辐射强度增加, 微藻群落的释放和光合作用过程的激活提高了氧浓度, 而大量的溶解有机物和无机营养盐发生光解反应, 生成羟基自由基和过氧化氢, 此时侧纹南极鱼不可避免地遭受氧化压力的威胁(Regoli, 2014)。Giuliani等(2017)研究发现与温带物种Nrf2相比, 侧纹南极鱼Nrf2蛋白序列显示出对催化功能所必需的氨基酸的高度保守性, 但在非必需区域出现了一些特定取代, 这可能代表了一种分子适应性, 以提高在低温下活性位点的灵活性和可变性。另外, 在孵化前期Nrf2表达与胚胎发育初期相比明显上调, 其调控的抗氧化元件如CAT、GST、SeGPx也出现转录及翻译水平的上调, 证实了Nrf2在南极洲早期生命阶段抗冰融化应激保护过程中的重要性。Nrf2也被证实在抗盐胁迫中发挥重要角色, 例如在凤鲚中发现了一个Nrf2介导的盐应激调控网络(Wang, 2019b)。当凤鲚遭受盐胁迫时, Nrf2在鳃、脑、肠和肾四种被认为主要的渗透调节器官(Laverty, 2012)中被激活, 与水通道蛋白1 (AQP1)协同作用参与渗透调节过程。此时, 下游抗氧化酶SOD、GPx被激活以应对盐胁迫诱导的氧化压力。另外, Nrf2还通过刺激溶菌酶活性和提高白细胞计数来触发免疫增强作用以应对盐胁迫可能带来的免疫水平下降。

4 水生动物Nrf2研究存在的问题及展望

ROS的持续产生是水生动物应对外源胁迫时一个非常普遍的效应。非生物的水污染以及生物的细菌、病毒都会诱导机体ROS的过多累积, 从而导致氧化应激。尽管在水生动物中已经深入研究了参与抗氧化反应的主要酶, 但对其关键调控途径的理解还远未完成。Nrf2通路被认为是细胞氧化应激最主要的防御机制, 无论是在成体还是在胚胎发生过程中, 各种外源刺激都可以破坏细胞的氧化通道并触发Nrf2途径。当前Nrf2通路在水生动物中的研究大多聚焦于模式鱼类, 如斑马鱼和鲤鱼, 在其他鱼类和水生非脊椎动物中所涉不多。仅有的一些研究也多集中于Nrf2的鉴定, 以及对其能够参与抗氧化应激调控这样粗浅的认识。对Nrf2通路各种成分之间高水平的相互作用, 及其与其他抗氧化机制联合激活一个复杂的细胞应激调控网络还远未涉及。接下来, 应加深水生动物Nrf2调控异源物暴露详细路径及与其他通路如MAPK、AhR等协同作用的研究, 这不仅可以提供有关污染物作用方式(mode of action, MOA)的新线索, 而且还有助于开发高通量方法来评估生态毒理风险。另外, 这些问题的深入研究也可增进对水生动物响应外界胁迫应激调控机制的理解, 为制定水生动物资源可持续开发利用对策提供新思路。

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THE ADVANCES IN RESEARCH OF Nrf2 PATHWAY INVOLVED IN OXIDATIVE STRESS REGULATION IN AQUATIC ANIMALS

YAN Xiao-Jun1, 2, QI Peng-Zhi1, 2, GUO Bao-Ying1, 2, LI Ji-Ji1, 2

(1. National Engineering Research Center of Marine Facilities Aquaculture, Zhoushan 316022, China; 2. School of Ocean Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China)

Environmental changes can induce the increase of reactive oxygen species (ROS) level, which leads to oxidative stress. Oxidative stress has a profound impact on the survival, growth, development, and evolution of all organisms. The nuclear factor erythroid 2 related factor 2 (Nrf2) has been recognized as a dominant regulator of oxidative stress. Together with Kelch-like ECH associated protein 1 (Keap1), Nrf2 controls the expression of hundreds of detoxification enzymes and antioxidant protein coding genes. In recent years, Nrf2 has been gained with more and more attention in aquatic animal study, and has been studied in some model fish such as zebrafish, carp, other fish, and aquatic invertebrates. In this paper, the structure and regulatory mechanism of Nrf2 is introduced, and the progress of Nrf2 pathway involved in the regulation of oxidative stress in aquatic animals in recent years is reviewed. Studies have shown that Nrf2 exists widely in aquatic animals, and plays an important role in the regulation of oxidative stress induced by abiotic (metals, organic pollutants, inorganic salts, drugs and micro plastics), biological (bacteria, viruses, toxic algae), and habitat changes (ice melting, salt stress). Once Nrf2 is activated into the nucleus, it binds with anti-oxidant response element (ARE) with the help of small Maf protein, and starts the expression of a series of are driven genes, and interacts with pregnane X receptor (Pxr), mitogenactivated protein kinase (MAPK), and aromatic hydrocarbon receptor (AhR) and other cellular pathways are involved in a series of physiological processes. Nrf2 plays an important role in cell protection of aquatic animals in response to environmental changes, and is expected to become a potential gene target for stress resistance breeding.

nuclear factor erythroid 2 related factor 2; oxidative stress; aquatic animals

* 国家自然科学基金项目, 42020104009号, 41976111号, 42076119号; 舟山市科技计划项目, 2020C21119号, 2019F12004号。严小军, 博士生导师, 教授, E-mail: yanxiaojun2019@sina.com, yanxj@zjou.edu.cn

祁鹏志, 博士, 副研究员, E-mail: qpz2004@vip.sina.com, qipengzhi@zjou.edu.cn

2020-11-22,

2020-12-29

Q599; Q789; X17

10.11693/hyhz20201100316