Nrf2參與水生動物氧化應激調(diào)控的研究進展*

嚴小軍 祁鵬志 郭寶英 李繼姬

Nrf2參與水生動物氧化應激調(diào)控的研究進展*

嚴小軍1, 2祁鵬志1, 2①郭寶英1, 2李繼姬1, 2

(1. 國家海洋設施養(yǎng)殖工程技術研究中心 舟山 316022; 2. 浙江海洋大學海洋科學與技術學院 舟山 316022)

環(huán)境變化會誘導機體活性氧(reactive oxygen species, ROS)水平升高, 從而產(chǎn)生氧化應激。氧化應激對所有生物的生存、生長、發(fā)育和進化都具有深遠的影響。核因子E2相關因子2 (nuclear factor erythroid 2 related factor 2, Nrf2)被公認為細胞氧化應激調(diào)控的主導者, 與伴侶蛋白Kelch樣環(huán)氧氯丙烷相關蛋白1 (kelch-like ECH-associated protein 1, Keap1)一起控制數(shù)百個解毒酶和抗氧化蛋白編碼基因的表達。近年來, Nrf2在水生動物中逐漸獲得重視, 并在一些模式魚類如斑馬魚、鯉魚及其他一些魚類和水生無脊椎動物中得到研究。介紹了Nrf2的結構以及調(diào)控機制, 回顧了近年來水生動物Nrf2通路參與氧化應激調(diào)控所取得的進展。研究表明, Nrf2在水生動物中廣泛存在, 在非生物(金屬、有機污染物、無機鹽、藥物及微塑料等)、生物(細菌、病毒、有毒藻類)以及生境變化(冰融、鹽脅迫)誘導的氧化應激調(diào)控中發(fā)揮重要作用。Nrf2一經(jīng)激活入核, 在小Maf蛋白的協(xié)助下與抗氧化反應元件(antioxidant-response element, ARE)結合, 啟動一系列ARE驅動基因的表達, 并和孕烷X受體(pregnane X receptor, Pxr)、絲裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)以及芳烴受體(aryl hydrocarbon receptor, AhR)等細胞通路協(xié)同作用參與一系列生理過程。Nrf2在水生動物響應環(huán)境變化過程中發(fā)揮重要的細胞保護機制, 有望發(fā)展成為抗逆育種潛在的基因靶點。

核因子E2相關因子2 (Nrf2); 氧化應激; 水生動物

細胞通過表達抗氧化蛋白和II相解毒酶來為自身提供保護, 這些蛋白在低水平氧化應激狀態(tài)下即可被激活, 而這種激活是通過被稱為抗氧化反應元件(antioxidant-response element, ARE)或親電反應元件(electrophilic response element, EpRE)的順式作用元件介導的(Kobayashi, 2005)。ARE最初發(fā)現(xiàn)于編碼人和鼠兩種主要解毒酶谷胱甘肽S轉移酶Ya (GST-Ya)和NAD(P)H醌氧化還原酶1 (Nqo1)的啟動子區(qū)(Jaiswal, 1994), 而后來的研究發(fā)現(xiàn)ARE廣泛存在于抗氧化蛋白和II相解毒酶基因的5′啟動子區(qū), 從轉錄水平上調(diào)控細胞保護酶對氧化脅迫的誘導反應(Nguyen, 2003)。由ROS及親電體水平升高和/或抗氧化能力降低而引起的細胞氧化還原狀態(tài)的改變是觸發(fā)ARE介導的轉錄反應的重要信號(Nguyen, 2009)。接下來的研究中, ARE的結合因子引起了科學家的興趣。最終, 核因子E2相關因子2 (nuclear factor erythroid 2 related factor 2, Nrf2)成為人們關注的焦點, 被公認為細胞抗氧化防御的主導者(Vomund, 2017)。

Nrf2最早是被作為p45 NF-E2密切關聯(lián)蛋白從K562細胞中克隆的(Moi, 1994)。p45 NF-E2是珠蛋白異源二聚體中較大的亞基, 具有NF-E2位點的結合活性, 是珠蛋白基因表達的關鍵順式調(diào)節(jié)因子(Andrews, 1993)。p45 NF-E2相關蛋白中的4個成員p45 NF-E2、Nrf1、Nrf2和Nrf3已經(jīng)在哺乳動物中分離出來, 被稱為Cap'n'collar (CNC)型堿性亮氨酸拉鏈(bZIP)蛋白(Motohashi, 2002)。這個名稱來源于與果蠅CNC蛋白的序列相似性。CNC蛋白首先在果蠅中被發(fā)現(xiàn), 是唇和下頜發(fā)育所必需的(Mohler, 1995)。CNC蛋白其中一個異構體CNCC蛋白包含Nrf2經(jīng)典的ETGE基序, 可能發(fā)揮和脊椎動物中Nrf2類似的功能(Kobayashi, 2005)。Nrf2在鼠、人、雞和魚中被鑒定出來, 并且被認為可能存在于所有其他脊椎動物中(Kobayashi, 2002), 而近年來的研究表明, Nrf2也廣泛存在于一些水生無脊椎動物包括貝類、蝦類等(Silvestre, 2020), 表明Nrf2介導的細胞防御功能在自然界中可能是保守的。Nrf2與ARE結合, 調(diào)節(jié)ARE介導的抗氧化基因表達從而參與響應環(huán)境變化的細胞反應(Dhakshinamoorthy, 2001; Jaiswal, 2004)。Nrf2的活性調(diào)節(jié)機制表明, Kelch樣環(huán)氧氯丙烷相關蛋白1 (kelch-like ECH-associated protein 1, Keap1)是Nrf2的伴侶蛋白, 在Nrf2調(diào)控過程中發(fā)揮至關重要的作用。Keap1和Nrf2共同構成一個二元系統(tǒng), 在人體中調(diào)控多達250個含ARE結構的基因。Keap1-Nrf2系統(tǒng)已發(fā)展成為機體對抗環(huán)境侵害的主要防御機制, 在維持機體穩(wěn)態(tài)方面有著關鍵作用。

1 Nrf2、Keap1結構

1.1 Nrf2結構

Nrf2是由核因子NFE2L2 (erythroid-derived 2-like 2)編碼的蛋白, 具有堿性亮氨酸拉鏈(basic region-leucine zipper, bZIP)結構, 隸屬于CNC轉錄因子家族, 是該家族中活力最強的轉錄調(diào)控因子(Alam, 1999)。Nrf2廣泛存在于從低等的昆蟲到高等的哺乳動物中, 在持續(xù)暴露于外界環(huán)境的皮膚、肺、消化道以及解毒代謝器官如肝臟和腎臟中大量表達。

同源比對發(fā)現(xiàn)Nrf2包含7個高度保守的Neh (Nrf2-ECH homology)結構域(圖1a)。Neh1位于Nrf2的C端, 包含bZIP基序, 能夠與多種含有此同源結構的小Maf蛋白、c-Jun蛋白, c-Fos蛋白結合形成二聚體。細胞核內(nèi), Nrf2通過Neh1區(qū)與小Maf蛋白結合, 進而識別并結合ARE元件, 啟動下游基因轉錄。此外, Neh1區(qū)還包含核轉錄因子普遍的核定位和輸出信號, 能夠與泛素連接酶UbcM2作用以調(diào)控Nrf2的核轉位和降解(Jain, 2005)。Neh2位于Nrf2的最末N端, 含有DLG和ETGE基序的結合位點, 是負調(diào)控蛋白Keap1的作用靶點。該結構域包含豐富的賴氨酸殘基, 可介導Nrf2泛素化及26S蛋白酶體的降解(McMahon, 2004)。與Keap1解耦聯(lián)的Nrf2進入細胞核并以Nrf2-sMaf異二聚體形式與ARE結合后并不能立即啟動下游基因的轉錄, 而是需要一類被稱為轉錄共激活子的輔助蛋白參與。位于最末C端的Neh3和位于N端的Neh4、Neh5負責結合轉錄共激活子, 共同行使轉錄調(diào)控功能。其中, Neh3通過與解螺旋酶DNA結合蛋白6 (chromodomain helicase DNA binding protein 6, CHD6)的相互作用來協(xié)助Nrf2的順式激活(Nioi, 2005), 而Neh4和Neh5結構域是通過與環(huán)磷酸腺苷反應元件結合蛋白(cyclic AMP response element-binding protein, CBP)的結合, 反式激活Nrf2的表達(Katoh, 2001)。對氧化還原不敏感的Neh6含有富含絲氨酸殘基的DSGIS和DSAPGS基序, 可作為糖原合成酶激酶-3 (glycogen synthase kinase-3, GSK-3β)的磷酸化靶點, 啟動Kepa1非依賴的Nrf2降解(Chowdhry, 2013)。Neh7是近年來新發(fā)現(xiàn)的Nrf2結構域, 其與視黃酸X受體α (retinoic X receptor α, RXRα)識別并結合后, 能夠抑制Nrf2的轉錄(Wang, 2013a)。

圖1 Nrf2 (a)和Keap1 (b)分子的結構域圖示

注: Keap1-dependent degradation: Keap1-依賴型降解; Transactivation domain: 轉錄激活域; RXRα binding: RXRα結合域; DNA binding domain: DNA結合域; CUL3 association: CUL結合域; Self association: 自交聯(lián); Nrf2 association: Nrf2結合域

1.2 Keap1結構

Keap1為隸屬于Kelch家族的多區(qū)域阻遏蛋白, 可作為氧化應激的傳感器存在, 對Nrf2號通路起負調(diào)控作用(Kobayashi, 2006)。生理狀態(tài)下, Keap1常以二聚體形式存在, 將Nrf2錨定在胞漿中, 抑制其活動。氨基酸序列及域功能分析發(fā)現(xiàn)Keap1包含5個不同的結構域: 氨基末端區(qū)(NTR)、羧基末端區(qū)(CTR)、Broad復合物即tramtrack和bric-a-brac結構域(BTB), 插入?yún)^(qū)(IVR), 六個Kelch/雙甘氨酸重復序列(DGR) (圖1b)。BTB和DGR區(qū)是主要的功能區(qū)(Kaspar, 2009)。Keap1通過BTB區(qū)交聯(lián)形成同源二聚體, 同時BTB也是Cul3 (Cillion 3)依賴性E3泛素連接酶復合物的結合位點, 是介導Nrf2泛素化及蛋白降解的必要區(qū)域(Cullinan, 2004)。DGR區(qū)是Keap1與Nrf2的結合區(qū), 該區(qū)中的6個Kelch能夠形成β折疊結構與Nrf2的Neh2區(qū)結合(Adams, 2000)。IVR區(qū)含有大量的半胱氨酸(Cys, C)殘基, 是整個蛋白的功能調(diào)節(jié)區(qū)。而Nrf2誘導劑也常通過修飾其中的Cys273和Cys288位點, 干擾Nrf2泛素化從而穩(wěn)定Nrf2蛋白(Ogura, 2010)。

2 Nrf2信號通路的調(diào)控機制

盡管Nrf2的激活入核能夠誘導數(shù)百個具有細胞保護功能的基因的表達, 從而提高機體抗氧化應激的能力, 但Nrf2的無序激活也會對機體造成嚴重傷害。keap1缺陷型小鼠體內(nèi)Nrf2無序表達, 小鼠在出生后3周內(nèi)死亡(Wakabayashi, 2003)。因此, 控制Nrf2活動對維持細胞內(nèi)環(huán)境穩(wěn)態(tài), 保障機體健康至關重要?，F(xiàn)有研究表明, Nrf2信號通路的激活主要有兩種調(diào)控方式: Keap1-依賴型調(diào)控(圖2a)和Keap1-非依賴型調(diào)控(圖2b)。

2.1 Keap1-依賴型調(diào)控

生理狀態(tài)下, 兩個Keap1蛋白以BTB區(qū)交聯(lián)形成二聚體, 同時使用該區(qū)域與Cul3相互作用。二聚體Keap1的兩個DGR分別與Nrf2的Neh2結構域的DLG和ETGE基序結合將Nrf2限制在胞漿中。Nrf2泛素化后被26S蛋白酶體降解以維持在較低的基礎水平, 并以從頭合成的方式更新(Stewart, 2003)。一小部分入核的Nrf2激活細胞保護基因的轉錄, 滿足機體正常的抗氧化需求。

Keap1含有27個Cys殘基, 因此常將該分子視作內(nèi)源性以及環(huán)境氧化應激信號的傳感器(Sihvola, 2017)。ROS氧化硫醇, 誘導大分子谷胱甘肽(GSH)化和烷基化, 因此具有修飾Keap1 Cys的能力(Holland, 2008)。Nrf2的Keap1-依賴型調(diào)控都是以Keap1的Cys修飾為基礎的。一旦暴露于親電體和ROS, Keap1的某些Cys殘基(主要是C273和C288)被修飾, 導致Keap1構象改變, Keap1處于一個非功能性封閉狀態(tài)。盡管Nrf2的DLG和ETGE基序能夠與Keap1的DGR結合, 但不能夠被泛素化蛋白酶體降解, 因此沒有足夠的處于游離狀態(tài)的Keap1產(chǎn)生。導致新生產(chǎn)的Nrf2不能被Keap1捕獲而發(fā)生入核激活(Baird, 2013)。另外還有一種“鉸鏈和閂鎖”模型。認為Nrf2的DLG基序對Keap1的親和力比ETGE基序弱的多, 導致親電體攻擊Keap1的Cys時, DLG與Keap1 DGR區(qū)的結合會斷開, 使Nrf2不能被泛素化降解, 從而發(fā)生入核轉移(Jung, 2010)。Keap1抑制的另一個機制與它與Nrf2泛素化所需的CUL3復合物的相互作用有關。位于BTB結構域的C151影響Keap1與Cul3的結合。Nrf2激活劑巴多索隆(CDDO, RTA401)與Keap1在C151位點形成加合物, 從而破壞Keap1和Cul3之間的相互作用(Naidu, 2018), Keap1被阻塞在Nrf2結合構象中, 新合成的Nrf2逃脫泛素化從而產(chǎn)生入核激活(Robledinos-Antón, 2019)。入核后的Nrf2與小Maf蛋白(MafK, MafG, MafF)結合后識別ARE序列并啟動下游基因轉錄(Yamamoto, 2018)。

圖2 Nrf2信號通路的調(diào)控機制: (a) Keap1-依賴型調(diào)控和(b) Keap1-非依賴型調(diào)控

注: Keap1-dependent modulation: Keap1-依賴型調(diào)控; Keap1-independent modulation: Keap1-非依賴型調(diào)控; Basal state: 基態(tài); Induced state:誘導態(tài); Inducers: 誘導物; blocking: 阻斷; proteasome 26S: 蛋白酶體26S; Nrf2 degradation: Nrf2降解; Nascent Nrf2: 新生Nrf2; Cytoprotective genes: 細胞保護基因; Cytoplasm: 細胞質; Nucleus: 細胞核

2.2 Keap1-非依賴性調(diào)控

糖原合酶激酶-3β (glycogen synthase kinase-3, GSK-3β)被認為參與Nrf2入核激活后的調(diào)控。GSK-3β蛋白激酶是一種多功能絲氨酸/蘇氨酸激酶, 在多種信號通路中發(fā)揮重要作用(Kannoji, 2008)。GSK-3β能夠磷酸化酪氨酸激酶Fyn的某個蘇氨酸(Thr, T)殘基, 介導Fyn的入核激活(Dai, 2017)。激活的Fyn磷酸化Nrf2的酪氨酸(Tyr, Y)568殘基, 誘導Nrf2的核輸出, 并被Keap1捕獲后降解(Jain, 2006)。另外, 在胞漿中GSK-3β能夠直接磷酸化Nrf2位于Neh6區(qū)的絲氨酸(Ser, S)335和338殘基, 而后磷酸化的Nrf2易位到細胞核中, 并被E3泛素連接酶β-TrCP (β-transducin repeat containing E3 ubiquitin protein ligase, β-TrCP)直接識別, 誘導Nrf2的核泛素化和降解(Hayes, 2015)。除此之外, 一種競爭機制也會影響Nrf2對下游基因轉錄的激活。堿性亮氨酸拉鏈轉錄因子1 (basic leucine zipper transcrip-tion factor 1, Bach1)是機體內(nèi)一種廣泛存在轉錄抑制子(Sun, 2002), 和Nrf2有一定的親緣關系(Kobayashi, 2006)。生理狀態(tài)下, Bach1和小sMaf蛋白形成異二聚體并與ARE元件結合(Dhakshinamoorthy, 2005), 抑制基因表達。氧化應激時, Bach1從ARE中釋放出來并被Nrf2取代。Bach1與Nrf2競爭與ARE的結合, 導致Nrf2下游基因的抑制(Jain, 2005)。

3 Nrf2信號通路參與水生動物氧化應激調(diào)控

在長期的進化過程中, 水生動物發(fā)展了相對完善的細胞應激體系以應對復雜的生存環(huán)境。在哺乳動物中發(fā)現(xiàn)的一些典型細胞應激信號通路, 如絲裂原活化蛋白激酶(MAPK)通路, 核因子-κB (NF-κB)通路, 過氧化物酶體增殖物激活受體(PPAR)通路以及Nrf2通路等在水生動物中也被發(fā)現(xiàn), 而Nrf2通路在其中發(fā)揮核心作用(Silvestre, 2020)。

3.1 Nrf2與環(huán)境污染相關性研究

金屬作為水系統(tǒng)中最常見的污染物, 對其研究最早也最為深入。在水生動物中, Nrf2已被報道參與多種金屬誘導的氧化應激反應(表1)。而斑馬魚作為一種水生模式生物, 在Nrf2抗氧化應激調(diào)控研究中發(fā)揮了引領作用。鎘是一種嗅覺毒物, 誘導斑馬魚抗氧化基因谷胱甘肽硫轉移酶pi (GSTpi)、谷氨酸半胱氨酸連接酶催化亞基(GCLC)、血紅素氧化酶1 (HO-1)、過氧化物酶1 (Prdx1)表達, 但被嗎啉代介導的Nrf2敲降阻斷, 導致嗅覺驅動行為破壞、細胞死亡增加和嗅覺感覺神經(jīng)元丟失。嗅覺神經(jīng)元特異性基因在Nrf2嗎啡啉突變體斑馬魚中表達下調(diào)。用Nrf2的激活劑蘿卜硫素(SFN)預處理胚胎, 可減弱鎘誘導的嗅覺組織損傷(Wang, 2013b)。環(huán)境相關濃度的鉻(2 mg/L)脅迫下, 斑馬魚肝臟中Nrf2在mRNA及酶活水平上均顯著上調(diào), 免疫組化證實其發(fā)生了入核激活。Nrf2的激活誘導下游Nqo1和含銅和鋅的超氧化物歧化酶(CuZnSOD)表達(Shaw, 2019)。紫草堿可以減輕鉻誘導的斑馬魚肝細胞毒性, 其最終也是通過激活Nrf2-Keap1-ARE通路, 誘導細胞保護基因紅細胞衍生核因子2樣蛋白2 (Fe2l2)、Nqo1和熱激蛋白70 (Hsp70)的表達, 提高細胞活力, 減少ROS產(chǎn)生來實現(xiàn)的(Shaw, 2020)。另外, Shaw等(2019) 研究還發(fā)現(xiàn)作為芳烴受體(AhR)通路中重要成分的細胞色素P4501亞族A多肽(CYP1A)在鉻暴露后也顯著表達上調(diào), 作者認為這可能是通過Nrf2依賴的AhR通路間接誘導的, 表明細胞抗氧化機制的組成部分之間存在廣泛的串話。類似的交互作用機制也在研究斑馬魚銀和鎘暴露時被發(fā)現(xiàn)(Hu, 2019)。在野生型斑馬魚胚胎中, 銀和鎘的積累和毒性受三磷酸腺苷結合盒(ABC)轉運體的影響, 可以顯著誘導ABC轉運體的mRNA表達, 而孕烷X受體(Pxr)和Nrf2的突變降低了這些誘導效應, 但ABC轉運蛋白基礎表達的升高彌補了誘導性缺失。Pxr缺陷胚胎中金屬離子的毒性未變, 然而, Nrf2的突變破壞了GSH的產(chǎn)生, 導致銀和鎘在斑馬魚胚胎中的毒性增強。此外, 在未進行攻毒的Pxr缺陷模型中, 其他轉錄因子如Ahr1b、Ppar-β、Nrf2表達均出現(xiàn)上調(diào), 而Ahr1b、Ppar-β和Pxr的誘導增強僅在金屬離子暴露的Nrf2缺陷胚胎中可見, 說明對轉錄因子缺失的不同補償現(xiàn)象。

表1 Nrf2參與水生動物氧化應激調(diào)控

續(xù)表

續(xù)表

在斑馬魚中, Nrf2近年來也被報道參與除金屬以外的其他多種環(huán)境污染物包括PAHs、POPs、無機鹽、藥物等的氧化應激調(diào)控過程(表1)。氟化鈉(NaF)暴露時, 斑馬魚腦和肝臟中Nrf2表達上調(diào), 而Keap1表達下調(diào), 同時下游細胞氧化應激基因表達上調(diào), 證實了Nrf2在NaF誘導的斑馬魚氧化應激中發(fā)揮重要作用, 且與哺乳動物中經(jīng)典的Keap1負調(diào)控Nrf2的方式相吻合(Mukhopadhyay, 2015a, b)。在三氧化二砷(As2O3)暴露的斑馬魚中, Nrf2也以同樣的方式在腦中激活, 誘導下游HO-1和Nqo1表達上調(diào), 參與抗三氧化二砷誘導的氧化應激過程(Sarkar, 2014)。叔丁基過氧化氫(tBOOH)以及α-、β-萘黃酮(ANF, BNF)單獨或者ANF+BNF聯(lián)合暴露時, 斑馬魚胚胎中SOD、GSTpi、谷胱甘肽過氧化物酶(GPx)以及谷氨酰半胱氨酸連接酶(GCL)表達顯著上調(diào)。當用嗎啉代將Nrf2敲降后, 這些元件表達上調(diào)被明顯抑制, 且使得tBOOH暴露后的斑馬魚胚胎死亡率增加, 并加劇ANF+BNF聯(lián)合暴露導致的胚胎畸形(Timme-Laragy, 2009)。全氟辛烷磺?；衔?PFOS)暴露明顯上調(diào)Nrf2和下游HO-1的表達。當與Nrf2的激活劑SFN共暴露時, ROS水平明顯下降。當用嗎啉代將Nrf2沉默后, PFOS誘導的HO-1表達明顯下調(diào)(Shi, 2010)。高劑量的亞砷酸鈉暴露下, Nrf2突變型Nrf2afh318(Nrf2 DNA結合區(qū)域發(fā)生突變)幼斑馬魚死亡率明顯高于野生型。亞砷酸鈉暴露誘導細胞應激保護因子GCLC、GSTpi、ABCC2以及Prdx1以Nrf2依賴的方式表達, 而SFN預處理顯著提高亞砷酸鹽暴露時的斑馬魚成活率, Nrf2在對抗急性亞砷酸鈉毒性中發(fā)揮重要作用(Fuse, 2016)。

在除斑馬魚之外的其他魚類中, 也有證據(jù)表明Nrf2參與細胞應激, 是觸發(fā)抗氧化級聯(lián)反應的關鍵事件之一。在建鯉中, 0.60 mg/L銅暴露4 d增加魚腦Nrf2核積累, 誘導下游抗氧化元件CuZnSOD、GPx1a、GR表達上調(diào)。增強Nrf2與ARE結合的能力, 導致CuZnSOD表達水平升高。銅暴露還上調(diào)了Nrf2、MafG1和蛋白激酶C (PKC)的表達, 表明這些蛋白需要重新合成, 以延長對抗氧化基因的誘導時效(Jiang, 2014)。在建鯉肌肉中呈現(xiàn)了與之相反的結果。0.56 mg/L銅暴露4 d導致魚肌肉中核Nrf2蛋白水平降低, ARE結合能力減弱, 半胱氨酸蛋白酶-3 (caspase-3)介導的DNA斷裂增加, 誘導抗氧化元件CuZnSOD、GPx1a、GPx1b表達下調(diào), 抗氧化酶活性降低從而引起肌肉的氧化損傷(Jiang, 2015)。這些研究表明即使同種污染對魚體不同組織造成的傷害也不盡相同, 魚體不同組織對同種污染誘導的氧化應激可能發(fā)展出了不同的應對機制。盡管如此, 這些研究仍證實Nrf2在魚體應對急性Cu污染事件中發(fā)揮重要調(diào)控作用。在虹鱒中還發(fā)現(xiàn), 頭腎中Nrf2介導的抗氧化系統(tǒng)和PXR介導的解毒系統(tǒng)協(xié)同作用以對抗2,2',4,4'-四溴聯(lián)苯醚(BDE-47)誘導的氧化壓力(Liu, 2019)。此外, 在草魚應對鋅(Song, 2017)和姜黃素(Ming, 2020), 大黃魚應對鋅(Zheng, 2016)和汞(Zeng, 2016), 鰕虎魚應對阿司匹林(Wang, 2020b)等事件中, 均能發(fā)現(xiàn)Nrf2信號通路的身影。

無脊椎動物缺乏脊椎動物那樣獲得性的細胞應激機制, 機體防御反應僅依靠非特異的固有調(diào)控機制, 其應對外部刺激的機體反應逐漸得到重視。近年來, Nrf2在一些無脊椎動物中逐漸被發(fā)現(xiàn), 并報道在機體抗外界刺激誘導的氧化應激中發(fā)揮重要作用。低濃度鎘暴露會引起背角無齒蚌腎臟中Nrf2表達上調(diào)同時Keap1表達下調(diào), 而高濃度鎘暴露下, Nrf2表達水平無明顯變化, Keap1表達明顯下調(diào)(井維鑫, 2019)。在克氏原螯蝦中, 鎘暴露顯著降低四氫大麻酚(THC)和酚氧化酶原(proPO)水平, 而ROS水平以時間和劑量依賴的方式升高。同時鎘暴露明顯提升p38絲裂原活化蛋白激酶(p38MAPK)和Nrf2的表達和激活, 且p38MAPK和Nrf2表達與proPO活性密切相關, 肝胰臟可能通過ROS介導的MAPK/Nrf2途徑參與氧化還原活動(Wei, 2020)。叔丁基對苯二酚(tBHQ)作為Nrf2的激活劑, 處理太平洋牡蠣后, GR mRNA和蛋白水平上調(diào), 證實其誘導作用可能是通過Nrf2途徑產(chǎn)生。太平洋牡蠣Keap1和Nrf2蛋白的保守結構域以及經(jīng)典Nrf2誘導劑tBHQ對相關抗氧化防御的明確誘導, 支持了雙殼類Nrf2/Keap1通路與哺乳動物中功能相一致的觀點(Danielli, 2017a)。苯并芘(Bap)處理菲律賓蛤仔后, 在第1天和第6天, Nrf2的轉錄水平顯著提高, 且與Keap1呈現(xiàn)負相關, 與抗氧化元件GST、SOD、GPx和CAT的轉錄表達呈現(xiàn)正相關。RNAi將Nrf2敲低后, 抗氧化元件表達呈現(xiàn)與Nrf2一致的變化。與對照組相比, 脂質過氧化水平明顯升高。結果表明, Keap1能夠感知氧化應激, 與Nrf2組成經(jīng)典的二元系統(tǒng)在雙殼貝類對Bap應激調(diào)控事件中發(fā)揮作用(Wang, 2018a)。這一觀點在作者對另外一種雙殼貝類櫛孔扇貝的研究中得以強化。另外, 在苯并芘處理櫛孔扇貝后, 除抗氧化元件外, PKC、c-JNK和p38MAPK也呈現(xiàn)與Nrf2一致的表達變化, PKC、MAPKs以及Nrf2通路在雙殼動物抗Bap氧化防御中可能發(fā)揮協(xié)同的作用機制(Wang, 2019a)。本團隊在厚殼貽貝中也證實Nrf2參與Bap的氧化應激調(diào)控過程。Bap暴露顯著上調(diào)Nrf2轉錄表達, 同時下游抗氧化元件SOD、CAT、GPx和GR轉錄及蛋白表達上調(diào), Nrf2與抗氧化基因轉錄表達呈現(xiàn)正相關。Nrf2原核表達后注射厚殼貽貝, 導致Bap誘導的ROS和脂質過氧化水平比對照組顯著降低(Qi, 2020)。在矮小擬鏢水蚤內(nèi), 還發(fā)現(xiàn)納米和微米級苯丙乙烯微球能夠誘導細胞內(nèi)ROS的升高, 并激活Nrf2信號通路, 上調(diào)GPx、GR、SOD和GST酶活水平, 減輕細胞氧化應激反應(Jeong, 2017)。

3.2 Nrf2與微生物脅迫相關性研究

在哺乳動物中的大量研究已經(jīng)證實, 作為應激狀態(tài)下最重要的細胞防御通路, Nrf2在包括嗜血桿菌(Lugade, 2011)、肺炎鏈球菌(Gomez, 2016)、結核分歧桿菌(Rothchild, 2019), 以及肝炎病毒(Ivanov, 2011; Schaedler, 2010)、甲型流感病毒(Kosmider, 2012)、水皰性口炎病毒(Olagnier, 2017)在內(nèi)的多種病原微生物的感染中發(fā)揮著重要作用。微生物感染可誘導機體產(chǎn)生大量的ROS, 改變正常的氧化應激狀態(tài)。在此情況下, Nrf2信號通路被激活, 一方面誘導下游抗氧化基因表達以增強對ROS的清除, 維持細胞內(nèi)環(huán)境穩(wěn)態(tài); 另一方面與免疫信號通路如TNF-α、NF-κB等協(xié)同作用, 通過誘導抗炎, 抗細胞凋亡基因的表達增強整體的免疫耐受能力。Nrf2單獨參與水產(chǎn)動物抗菌應激的研究尚未見報道, 現(xiàn)有研究多涉及Nrf2參與其他物質誘導的抗菌調(diào)控過程。Jiang等(2016)報道, 嗜水氣單胞菌能夠誘導建鯉氧化應激。與肌醇攝入處于最優(yōu)水平的建鯉相比, 肌醇攝入過少或過多的魚體頭腎和脾臟中Nrf2及下游抗氧化元件包括CuZnSOD、MnSOD、CAT、GPx1a和GR的轉錄表達均受到抑制。適宜劑量的肌醇攝入會通過激活Nrf2和E2F轉錄因子4 (E2F4)介導的通路上調(diào)抗氧化基因的表達, 促進損傷細胞更新來應對嗜水氣單胞菌誘導的氧化應激, 促進機體健康。在草魚中同樣發(fā)現(xiàn)適宜水平的肌醇補充能在草魚受到嗜水氣單胞菌脅迫時顯著下調(diào)Keap1的表達, 同時上調(diào)Nrf2的表達, 從而激活Nrf2信號通路, 降低ROS水平, 提高抗氧化酶活性, 增強其抗氧化能力(胡凱等, 2019)。這些研究證明Nrf2在肌醇補充誘導的抗細菌感染能力提升過程中發(fā)揮重要作用, 此外, 一些益生菌類細菌也可通過Nrf2通路發(fā)揮作用。Yu等(2018)報道了異育銀鯽飲食中凝結芽孢桿菌的適量補充可以激活Nrf2-Keap1通路, 上調(diào)抗氧化元件如NADPH氧化酶2 (Nox2)和過氧化物酶2 (Prx2)表達水平, 增強抗氧化反應, 提高生長性能。

盡管Nrf2已被廣泛證實參與哺乳動物病毒感染過程, 但關于其在水生動物病毒感染中的作用卻鮮有報道。盡管如此, 近年來的一些研究已確證實Nrf2在水生動物病毒感染過程中發(fā)揮關鍵性作用。鯉春病毒血癥病毒(SVCV)感染黑頭軟口鰷上皮瘤細胞(EPC)細胞后, Nrf2的核蛋白和總蛋白含量以及轉錄水平均提升明顯, 表明SVCV感染能激活Nrf2, 增加其基因轉錄和蛋白表達, 導致在細胞核內(nèi)的累積。Nrf2的激活劑2-氰基-3,12-二氧代齊墩果-1,9(11)-二烯-28-羧酸(CDDO-Me)對EPC中Nrf2的激活效應有限, 而萊菔硫烷(SFN)能顯著增加Nrf2的核轉運, 上調(diào)下游效應基因的表達, 提高的總抗氧化能力。但介導的激活對SVCV的復制無顯著性影響(楊毅, 2014)。SFN和CDDO-Me刺激胖頭鯉肌肉細胞系(FHM)可以激活Nrf2-ARE 信號通路, 提高細胞總抗氧核能力。當FHM受到SVCV感染時, SFN和CDDO-Me預處理可以極顯著降低SVCV-G的轉錄水平, 降低病毒滴度, 抑制 SVCV的復制。當Nrf2被敲降后, Nrf2蛋白表達和轉錄水平受到明顯抑制, 且廢除兩種激活劑對Nrf2-ARE信號通路的激活作用(邵軍輝, 2016)。這些研究表明Nrf2的激活有利于機體降低病毒誘導的氧化應激, 維持細胞穩(wěn)態(tài), 對病毒的感染發(fā)揮抵抗性作用。但在白斑綜合征病毒(WSSV)感染日本囊對蝦過程中, Nrf2發(fā)揮的作用與之相反。WSSV感染引起對蝦血細胞內(nèi)ROS水平升高, 導致的Nrf2的mRNA表達以及細胞核中的蛋白水平表達上升。將Nrf2敲低后, 對蝦體內(nèi)病毒蛋白的復制受到抑制, 同時存活率明顯上升。注射SFN后, Nrf2表達量上升的同時病毒蛋白的表達量也上升。進一步研宄發(fā)現(xiàn), WSSV結構蛋白VP41B前端具有ARE元件, 可與Nrf2結合。敲低Nrf2可以抑制VP41B的表達, 而SFN處理則增強其表達, 而RNA干擾實驗證實VP41B與WSSV復制相關。這些結果說明WSSV可以利用對蝦的Nrf2-ARE系統(tǒng)啟動自身含有ARE元件的基因的表達, 進而促進病毒復制(陳敬, 2018)。

在軟體動物如翡翠貽貝、褶紋冠蚌中還發(fā)現(xiàn)Nrf2參與有害藻類及藻毒素誘導的氧化應激反應。利馬原甲藻短期暴露導致翡翠貽貝鰓中Keap1、CAT以及ABC轉運蛋白轉錄表達上調(diào), 而GPx1和Nqo1表達下調(diào), 鰓明顯損傷。隨著暴露時間延長, Nrf2表達顯著上調(diào), 而KEAP1下調(diào), 同時Nqo1、SOD、GST-ω和ABCB1上調(diào), 96h后鰓損傷恢復。作者認為利馬原甲藻可能導致鰓的氧化損傷。然而, 長時間高密度暴露可激活Nrf2信號通路, 從而降低毒素對貽貝鰓組織的影響(He, 2019)。Nrf2在褶紋冠蚌的外套膜、閉殼肌、腮、血淋巴、肝胰臟各組織中都均有表達, 且在肝胰臟中表達最高。微囊藻毒素刺激后, 肝胰腺和血淋巴中Nrf2表達上調(diào), 顯示Nrf2通路被激活(王曉波, 2018)。Wu等(2020)也發(fā)現(xiàn)微囊藻毒素會誘導褶紋冠蚌肝胰腺中Nrf2轉錄水平升高, 而下游抗氧化元件MnSOD、CuZnSOD、SeGPx和GST等轉錄及蛋白表達均上調(diào), 認為Nrf2通路對保護軟體動物免受微囊藻毒素侵害至關重要。

3.3 Nrf2與生境變化相關性研究

近年來研究表明, Nrf2也與一些魚類特殊的生境適應相關聯(lián)。側紋南極魚胚胎發(fā)育的最后階段正值海冰融化和輻射強度增加, 微藻群落的釋放和光合作用過程的激活提高了氧濃度, 而大量的溶解有機物和無機營養(yǎng)鹽發(fā)生光解反應, 生成羥基自由基和過氧化氫, 此時側紋南極魚不可避免地遭受氧化壓力的威脅(Regoli, 2014)。Giuliani等(2017)研究發(fā)現(xiàn)與溫帶物種Nrf2相比, 側紋南極魚Nrf2蛋白序列顯示出對催化功能所必需的氨基酸的高度保守性, 但在非必需區(qū)域出現(xiàn)了一些特定取代, 這可能代表了一種分子適應性, 以提高在低溫下活性位點的靈活性和可變性。另外, 在孵化前期Nrf2表達與胚胎發(fā)育初期相比明顯上調(diào), 其調(diào)控的抗氧化元件如CAT、GST、SeGPx也出現(xiàn)轉錄及翻譯水平的上調(diào), 證實了Nrf2在南極洲早期生命階段抗冰融化應激保護過程中的重要性。Nrf2也被證實在抗鹽脅迫中發(fā)揮重要角色, 例如在鳳鱭中發(fā)現(xiàn)了一個Nrf2介導的鹽應激調(diào)控網(wǎng)絡(Wang, 2019b)。當鳳鱭遭受鹽脅迫時, Nrf2在鰓、腦、腸和腎四種被認為主要的滲透調(diào)節(jié)器官(Laverty, 2012)中被激活, 與水通道蛋白1 (AQP1)協(xié)同作用參與滲透調(diào)節(jié)過程。此時, 下游抗氧化酶SOD、GPx被激活以應對鹽脅迫誘導的氧化壓力。另外, Nrf2還通過刺激溶菌酶活性和提高白細胞計數(shù)來觸發(fā)免疫增強作用以應對鹽脅迫可能帶來的免疫水平下降。

4 水生動物Nrf2研究存在的問題及展望

ROS的持續(xù)產(chǎn)生是水生動物應對外源脅迫時一個非常普遍的效應。非生物的水污染以及生物的細菌、病毒都會誘導機體ROS的過多累積, 從而導致氧化應激。盡管在水生動物中已經(jīng)深入研究了參與抗氧化反應的主要酶, 但對其關鍵調(diào)控途徑的理解還遠未完成。Nrf2通路被認為是細胞氧化應激最主要的防御機制, 無論是在成體還是在胚胎發(fā)生過程中, 各種外源刺激都可以破壞細胞的氧化通道并觸發(fā)Nrf2途徑。當前Nrf2通路在水生動物中的研究大多聚焦于模式魚類, 如斑馬魚和鯉魚, 在其他魚類和水生非脊椎動物中所涉不多。僅有的一些研究也多集中于Nrf2的鑒定, 以及對其能夠參與抗氧化應激調(diào)控這樣粗淺的認識。對Nrf2通路各種成分之間高水平的相互作用, 及其與其他抗氧化機制聯(lián)合激活一個復雜的細胞應激調(diào)控網(wǎng)絡還遠未涉及。接下來, 應加深水生動物Nrf2調(diào)控異源物暴露詳細路徑及與其他通路如MAPK、AhR等協(xié)同作用的研究, 這不僅可以提供有關污染物作用方式(mode of action, MOA)的新線索, 而且還有助于開發(fā)高通量方法來評估生態(tài)毒理風險。另外, 這些問題的深入研究也可增進對水生動物響應外界脅迫應激調(diào)控機制的理解, 為制定水生動物資源可持續(xù)開發(fā)利用對策提供新思路。

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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

* 國家自然科學基金項目, 42020104009號, 41976111號, 42076119號; 舟山市科技計劃項目, 2020C21119號, 2019F12004號。嚴小軍, 博士生導師, 教授, E-mail: yanxiaojun2019@sina.com, yanxj@zjou.edu.cn

祁鵬志, 博士, 副研究員, E-mail: qpz2004@vip.sina.com, qipengzhi@zjou.edu.cn

2020-11-22,

2020-12-29

Q599; Q789; X17

10.11693/hyhz20201100316