戴甲培，石 悅

(1 中南民族大學(xué) 武漢神經(jīng)科學(xué)與神經(jīng)工程研究所，武漢 430074;2 中南民族大學(xué) 藥學(xué)院，武漢 430074；3 中南民族大學(xué) 生命科學(xué)學(xué)院，武漢 430074)

人體內(nèi)95%以上的ROS為氧自由基.ROS可通過(guò)脂質(zhì)過(guò)氧化反應(yīng)等途徑損傷組織細(xì)胞，導(dǎo)致細(xì)胞死亡、染色體突變和畸變，并可致癌.然而，越來(lái)越的多研究發(fā)現(xiàn)，活性氧也有好的一面.胞外信號(hào)分子(生長(zhǎng)因子、細(xì)胞因子、激素和神經(jīng)遞質(zhì)等)誘導(dǎo)的正常信號(hào)通路中，特異的質(zhì)膜氧化酶生成的ROS可作為第二信使參與啟動(dòng)多種生物效應(yīng).胞內(nèi)氧化酶產(chǎn)生的正常水平的ROS在信號(hào)轉(zhuǎn)導(dǎo)、生長(zhǎng)、Ca2+信號(hào)通路和調(diào)控氧化還原敏感的基因表達(dá)等生理活動(dòng)中發(fā)揮重要作用.由此可見(jiàn)，ROS對(duì)細(xì)胞的應(yīng)激功能具有雙向生物學(xué)效應(yīng).

1 細(xì)胞內(nèi)ROS的產(chǎn)生與調(diào)控

1.1 細(xì)胞內(nèi)ROS產(chǎn)生

多種正常的非吞噬細(xì)胞也能產(chǎn)生和釋放ROS(表1).有報(bào)道指出，很多非吞噬細(xì)胞表達(dá)具有NADPH/NADH氧化酶活性的酶和NADPH氧化酶亞基[1].在機(jī)體多器官平滑肌內(nèi)發(fā)現(xiàn)gp91phox的同源物Nox1，而且在小鼠NIH3T3細(xì)胞中過(guò)量表達(dá)Nox1導(dǎo)致ROS增多[2].而且，體外培養(yǎng)的多種正常類型的細(xì)胞在胞外刺激劑(細(xì)胞因子、神經(jīng)遞質(zhì)，多肽類生長(zhǎng)因子如轉(zhuǎn)移因子α和激素如胰島素)的作用下都產(chǎn)生H2O2[3].同時(shí)，細(xì)胞在胞外因子或生長(zhǎng)因子的刺激下自身能夠產(chǎn)生和釋放ROS，且腫瘤細(xì)胞能持續(xù)合成和釋放H2O2[4]，因此，推測(cè)ROS可能作為一種自分泌因子或胞內(nèi)和胞間的信號(hào)分子起作用[1,3].

總之，任何能進(jìn)行氧化還原反應(yīng)的蛋白質(zhì)或復(fù)合酶都能通過(guò)電子轉(zhuǎn)移反應(yīng)產(chǎn)生ROS[5].

1.2 細(xì)胞內(nèi)ROS的清除

同時(shí)，胞內(nèi)抗氧化巰基還原體系主要包括還原型谷胱甘肽(GSH)和硫氧還原蛋白(Trx)及其還原酶TrxR.Trx主要還原底物蛋白的二硫鍵為巰基.胞內(nèi)還原性GSH與氧化性GSSG形成動(dòng)態(tài)平衡，這兩者本身的含量或比值是衡量細(xì)胞抗氧化的重要指標(biāo)[16].

1.3 細(xì)胞的氧化還原平衡狀態(tài)

正常細(xì)胞內(nèi)ROS的產(chǎn)生與清除保持動(dòng)態(tài)平衡，如果有刺激(炎癥、病原入侵、紫外線等)引起ROS或抗氧化系統(tǒng)相對(duì)增強(qiáng)，改變細(xì)胞氧化還原狀態(tài)，通過(guò)相應(yīng)的信號(hào)通路改變對(duì)應(yīng)的基因表達(dá)，產(chǎn)生不同的生理效應(yīng).如ROS產(chǎn)生超出其清除，導(dǎo)致氧化應(yīng)激，損傷細(xì)胞或組織[16].

2 ROS與氧化應(yīng)激

有關(guān)ROS對(duì)機(jī)體和細(xì)胞危害的報(bào)道較多[17].ROS可通過(guò)過(guò)氧化細(xì)胞膜、線粒體膜的不飽和脂肪酸影響線粒體膜的通透性[18]，且其脂質(zhì)過(guò)氧化反應(yīng)的產(chǎn)物可分解為更多的自由基，導(dǎo)致連鎖反應(yīng)損傷組織和細(xì)胞.另外，ROS能與胞內(nèi)各種生物大分子反應(yīng)引起DNA斷裂和蛋白質(zhì)脂質(zhì)氧化[17]，其介導(dǎo)的損傷可導(dǎo)致細(xì)胞死亡、染色體突變和畸變，導(dǎo)致細(xì)胞癌化.ROS含量過(guò)多，引起氧化產(chǎn)物與細(xì)胞抗氧化能力失衡，導(dǎo)致氧化應(yīng)激與多種疾病的形成，如動(dòng)脈粥樣硬化、阿耳茨海默氏病[17]、巴金森病[17]、腦缺血[19]和衰老[20].

3 ROS與細(xì)胞正常生理活動(dòng)

1989年，基于理論背景以及對(duì)自由基在生物內(nèi)環(huán)境的體評(píng)價(jià)，Saran和Bors[21]假設(shè)ROS作為一種生物信號(hào)起作用，而不是介導(dǎo)細(xì)胞損傷.在生物環(huán)境中，ROS的半衰期為400×10-6ns，擴(kuò)散速度為55-3000nm[21].而且，有研究確立了ROS在調(diào)控造血干細(xì)胞命運(yùn)中所起的信號(hào)作用[22].有研究發(fā)現(xiàn)，ROS在缺血預(yù)處理(IPC)及缺血后過(guò)程中可改善心功能，降低凋亡細(xì)胞數(shù)目，保護(hù)心肌[23].且ROS在腦缺血再灌注過(guò)程中起神經(jīng)保護(hù)作用[24].ROS的雙向作用與NO很類似.有毒氣體NO可作為胞內(nèi)信號(hào)分子調(diào)節(jié)血管擴(kuò)張，有效地緩解心絞痛，也是免疫系統(tǒng)的有效武器，可消滅細(xì)菌、病毒等病原體.同樣，越來(lái)越多的研究表明亞微摩爾濃度的ROS作為一種新的胞內(nèi)或胞間的第二信使參與多種生長(zhǎng)因子和細(xì)胞因子誘導(dǎo)的正常信號(hào)轉(zhuǎn)導(dǎo)過(guò)程[25]進(jìn)而調(diào)節(jié)細(xì)胞生長(zhǎng)、增殖、分化和凋亡等生理過(guò)程.

4 ROS與細(xì)胞的增殖、分化和凋亡

4.1 ROS與細(xì)胞增殖

如150 μM的H2O2能作為激活信號(hào)活化T-淋巴細(xì)胞，但抗氧化劑則抑制T-細(xì)胞增殖[27].在倉(cāng)鼠和大鼠的成纖維細(xì)胞[10]以及HeLa細(xì)胞[28]的培養(yǎng)基中加入過(guò)氧化氫酶和過(guò)氧化物歧化酶不僅抑制細(xì)胞增殖，而且經(jīng)臺(tái)盼藍(lán)染色顯示死亡細(xì)胞數(shù)目增加，并且與培養(yǎng)皿分離的細(xì)胞數(shù)目也增加.同時(shí)，電子顯微鏡掃描發(fā)現(xiàn)這些“死亡”細(xì)胞顯示出凋亡特征：核膜內(nèi)膜附近染色體固縮，且DNA斷裂.

表2 ROS可引起生長(zhǎng)反應(yīng)的細(xì)胞類型

4.2 ROS與細(xì)胞分化和凋亡

可引起細(xì)胞氧化損傷的抗腫瘤藥物丁酸、阿霉素，在亞毒性濃度時(shí)改變胞內(nèi)ROS水平對(duì)于誘導(dǎo)人K562細(xì)胞分化為成熟紅細(xì)胞至關(guān)重要[40].小鼠PC12細(xì)胞在NGF(神經(jīng)生長(zhǎng)因子)的誘導(dǎo)下分化為神經(jīng)細(xì)胞，胚胎干細(xì)胞在IL-1(白細(xì)胞介素-1)的誘導(dǎo)下分化為心肌細(xì)胞的過(guò)程中都產(chǎn)生ROS[26].

細(xì)胞有兩種死亡方式：程序性死亡即凋亡(PCD)和壞死(necrosis).凋亡是由基因調(diào)控的有序的細(xì)胞自我死亡的正常生理過(guò)程.而壞死是由嚴(yán)重且持續(xù)的有害刺激所引起的，不受基因調(diào)控.大量研究證明多種類型的細(xì)胞凋亡與ROS有關(guān)[41].H2O2可活化凋亡誘導(dǎo)因子P53，提示ROS可增強(qiáng)P53誘導(dǎo)凋亡的活性[42].很多抗氧化劑(SOD、GSH)可抑制ROS引起的凋亡.

ROS較低濃度的時(shí)候可能通過(guò)啟動(dòng)一系列信號(hào)傳導(dǎo)，引起細(xì)胞凋亡，高濃度的時(shí)候?qū)е录?xì)胞壞死[43].所以，ROS通過(guò)多種信號(hào)通路調(diào)節(jié)細(xì)胞的凋亡和壞死，凋亡或者壞死取決于ROS的濃度，作用時(shí)間和細(xì)胞所處的環(huán)境[43].

5 ROS與信號(hào)轉(zhuǎn)導(dǎo)

盡管ROS可調(diào)節(jié)多種生理過(guò)程，但其靶信號(hào)分子仍不完全清楚.大量證據(jù)表明氧化還原作用可調(diào)節(jié)信號(hào)通路過(guò)程中從受體到細(xì)胞核的多個(gè)部分[5]，如ROS既可與某些受體直接作用或氧化信號(hào)轉(zhuǎn)導(dǎo)分子(蛋白激酶、蛋磷酸酶、轉(zhuǎn)錄因子或轉(zhuǎn)錄因子抑制劑)；也可能通過(guò)改變胞內(nèi)GSH和GSSG的比例而改變氧化還原狀態(tài)，間接影響信號(hào)轉(zhuǎn)導(dǎo)蛋白分子的活性，行使其信使分子的功能[5].ROS調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)的方式主要有兩種；氧化修飾蛋白質(zhì)和改變細(xì)胞內(nèi)氧化還原狀態(tài)，其可能的作用機(jī)制如下.

5.1 ROS與鈣離子

細(xì)胞膜、內(nèi)質(zhì)網(wǎng)和線粒體上的鈣泵和鈣通道的開(kāi)放程度決定了胞內(nèi)Ca2+濃度，通常情況下，細(xì)胞內(nèi)Ca2+含量穩(wěn)定在10-7mol/L，外來(lái)信號(hào)刺激下，Ca2+濃度可快速上升到10-6～ 10-5mol/L[44]，從而影響多種蛋白或Ca2+依賴性蛋白激酶的活性，實(shí)現(xiàn)對(duì)細(xì)胞功能的調(diào)節(jié).氧化還原作用可調(diào)控內(nèi)質(zhì)網(wǎng)上與Ca2+釋放相關(guān)的IP3(三磷酸肌醇)受體和ryanodine受體及Ca2+-Na+交換體[45].氧化劑通過(guò)Ca2+通路增加Ca2+內(nèi)流及抑制Ca2+泵，提高胞內(nèi)Ca2+濃度.

很多研究表明ROS可能通過(guò)直接改變胞內(nèi)Ca2+濃度激活下游基因或下調(diào)轉(zhuǎn)錄[46].Suzuki[47]等發(fā)現(xiàn)血管平滑肌細(xì)胞內(nèi)次黃嘌呤/黃嘌呤氧化酶體系生成的ROS可使IP3誘導(dǎo)內(nèi)質(zhì)網(wǎng)釋放Ca2+.Roveri[48]等發(fā)現(xiàn)300μmol/ L H2O2刺激血管平滑肌細(xì)胞，引起胞質(zhì)Ca2+濃度持續(xù)性上升,當(dāng)介質(zhì)中無(wú)Ca2+時(shí)，無(wú)此現(xiàn)象，表明H2O2可能影響細(xì)胞膜上的鈣離子通道或Ca2+與膜偶聯(lián)的轉(zhuǎn)運(yùn)機(jī)制.

H2O2誘導(dǎo)胞內(nèi)Ca2+增加程度與其濃度成正比，1mM H2O2處理體胎鼠皮層神經(jīng)細(xì)胞膜，H2O2可通過(guò)TRPM2鈣離子通道促進(jìn)胞外Ca2+內(nèi)流提高胞內(nèi)Ca2+濃度[49].同時(shí)，有研究表明，體外培養(yǎng)的胎鼠齒狀回顆粒細(xì)胞經(jīng)1～10μM H2O2灌流處理2h，膜片鉗檢測(cè)發(fā)現(xiàn)H2O2可通過(guò)L-型鈣離子通道促進(jìn)Ca2+內(nèi)流，導(dǎo)致胞內(nèi)Ca2+濃度增加[50].

5.2 ROS與受體

微摩爾濃度的外源性H2O2能誘導(dǎo)PDGF-a、PDGF-b和EGF等生長(zhǎng)因子受體的酪氨酸磷酸化和激活[51,52].溶血磷脂酸對(duì)誘導(dǎo)EGF受體的活化受ROS的調(diào)節(jié)，ROS不直接增強(qiáng)EGF受體自身的酪氨酸激酶活性，而是氧化PTPs(酪氨酸磷酸酶)活性中心的Cys來(lái)抑制其活性，進(jìn)而促進(jìn)EGF受體磷酸化而活化EGF受體[53].Knebel等也發(fā)現(xiàn)ROS可氧化抑制膜結(jié)合的PTPs的活性，進(jìn)而抑制RTKs(受體酪氨酸激酶)的去磷酸化促進(jìn)受體活化[54].也有相似研究報(bào)道[55]O2-可改變受體磷酸化-去磷酸化平衡，通過(guò)促進(jìn)受體磷酸化而激活PDGF受體.

5.3 ROS與酶

ROS和氧化還原作用都可能通過(guò)影響蛋白激酶和蛋白磷酸酶的活性改變蛋白質(zhì)酪氨酸殘基的磷酸化狀態(tài)，進(jìn)而有助于生長(zhǎng)因子介導(dǎo)的信號(hào)通路轉(zhuǎn)導(dǎo)[56].如，H2O2具有細(xì)胞膜通透性，在多肽類生長(zhǎng)因子刺激下，可在多種細(xì)胞中暫時(shí)積累，通過(guò)氧化PTPs和PTEN(脂類磷酸酶)參與受體介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)[25].而且，ROS可激活PKB(蛋白激酶B)[57]，也很可能通過(guò)改變酶的巰基/二硫鍵的平衡，來(lái)激活PKC(蛋白激酶C)[58].

ROS也可調(diào)節(jié)非受體酪氨酸激酶(PTKs)如JAKs(Janus激酶)家族和Src激酶家族的活性[59,60].Simon等證明PDGF誘導(dǎo)JAK-STAT通路的激活對(duì)氧化還原作用敏感，外源性氧化劑如H2O2可激活JAK-STAT通路[59].Bauskin[61]等發(fā)現(xiàn)H2O2等巰基氧化劑可通過(guò)氧化Ltk(61s，在淋巴細(xì)胞、白血病細(xì)胞和神經(jīng)元等細(xì)胞中大量表達(dá))形成通過(guò)二硫鍵連接的多聚體來(lái)使其激活.

氧化還原敏感的有絲分裂原激活蛋白激酶(MAPK)的信號(hào)通路也受ROS調(diào)節(jié)[62].MAPK信號(hào)轉(zhuǎn)導(dǎo)通路主要包括胞外信號(hào)調(diào)節(jié)激酶ERK1 (p44MAPK)/ERK2 (p42MAPK)，JNKs/SAPK(c-Jun N端激酶/應(yīng)激激活的蛋白激酶)和p38 MAPK，以及BMK1/ERK5(分裂原激活的大分子蛋白激酶).

有研究證明外源氧化物可激活ERK MAPK通路，但其具體作用機(jī)制和靶分子尚不清楚，有研究推測(cè)ROS可能氧化抑制PTPs和/或蛋白磷酸酶A來(lái)激活ERK MAPK通路[63].

十幾年前，已證明PDGF 刺激細(xì)胞產(chǎn)生的H2O2在BALB/3T3細(xì)胞生長(zhǎng)中發(fā)揮作用[64].Sundaresan等證明PDGF能暫時(shí)提高胞內(nèi)H2O2濃度，H2O2參與PDGF誘導(dǎo)的酪氨酸磷酸化，MAPK激活和DNA合成等生理活動(dòng)[65].在小鼠上皮細(xì)胞JB6，外源性H2O2可激活p70S6k和p90Rsk，并且磷酸化p42(MAPK)或p44(MAPK)，調(diào)節(jié)細(xì)胞增殖[66].

Lo等發(fā)現(xiàn)胞內(nèi)H2O2可能介導(dǎo)TNF-α和IL-1誘導(dǎo)的JNK的激活[69]，且ANG II[70]和EGF[71]與細(xì)胞相互作用時(shí)，也發(fā)現(xiàn)相似效應(yīng).Bae[72]等證明EGF刺激A431細(xì)胞(人表皮癌細(xì)胞)產(chǎn)生H2O2，暫時(shí)提高胞內(nèi)H2O2濃度，是EGF誘導(dǎo)的蛋白質(zhì)酪氨酸殘基磷酸化所必需的.用過(guò)氧化氫酶清除H2O2則抑制EGF誘導(dǎo)的多種蛋白質(zhì)酪氨酸磷酸化，如EGF受體和磷脂酶C-γ1.有研究表明，BMK1比ERK1/ERK2對(duì)H2O2更加敏感，BMK1可能是一種氧化還原敏感激酶[73].

PLA2(磷脂酶A2) 主要催化磷脂分解，產(chǎn)生前列腺素類物質(zhì)，白三烯、溶血磷脂、血小板激活因子等多種脂質(zhì)介質(zhì)，這些產(chǎn)物可作為第二信使分子,調(diào)節(jié)多種細(xì)胞功能如磷脂轉(zhuǎn)運(yùn)、膜修復(fù)、胞外水解及神經(jīng)元轉(zhuǎn)移因子的釋放等.EFG信號(hào)轉(zhuǎn)導(dǎo)過(guò)程中誘導(dǎo)的PLA2的激活和花生四稀酸的合成對(duì)抗氧化劑如NAC(N-乙酰半胱氨酸)、DTT(二硫蘇糖醇)和DPI(FAD依賴性氧化酶抑制劑)敏感，表明PLA2可能是ROS的靶分子[74].

H2O2和亞油酸羥基過(guò)氧化物可激活內(nèi)皮細(xì)胞PLD(磷脂酶D)[75]，促進(jìn)磷脂酰乙醇胺(PEt)和磷脂酸(PA)的合成，且H3標(biāo)記的脫氧葡萄糖放射性同位素示蹤法顯示氧化劑介導(dǎo)的PLD活化無(wú)細(xì)胞毒性.H2O2可誘導(dǎo)NIH-3T3成纖維細(xì)胞PLD活化，水解PEt，不水解磷脂酰膽堿[76].PKC的激活和蛋白質(zhì)酪氨酸殘基的磷酸化介導(dǎo)H2O2誘導(dǎo)內(nèi)皮細(xì)胞和成纖維細(xì)胞PLD的活化[77,78].Takekoshi等發(fā)現(xiàn)氧化型二酰甘油比其還原型能更有效地活化PKC[79].

5.4 ROS與轉(zhuǎn)錄因子

大量研究報(bào)道[80]核轉(zhuǎn)錄因子的活性與其氧化還原狀態(tài)密切相關(guān).氧化還原敏感的轉(zhuǎn)錄因子如NF-κB、AP-1、P53[81]、v-Ets[82]、v-Rel[83]、和v-Myb[84]等在DNA結(jié)合域都含有保守的cys殘基[85],推測(cè)此cys的氧化還原狀態(tài)可能直接調(diào)節(jié)基因的表達(dá)[81,84].

5.4.1 ROS和NF-κB轉(zhuǎn)錄因子

未激活的NF-κB位于胞漿，是由P65、P50和抑制亞基I-κB組成的三聚體.當(dāng)細(xì)胞受外來(lái)信號(hào)刺激后，NF-κB復(fù)合體活化將I-κB磷酸化，使其被遍在蛋白輟合酶降解，游離的NF-κB活化轉(zhuǎn)移入細(xì)胞核，與特異的DNA結(jié)合，調(diào)節(jié)基相應(yīng)的因表達(dá).

TNF-α刺激細(xì)胞時(shí)，線粒體產(chǎn)生的ROS可激活NF-κB[86].用100μM H2O2處理細(xì)胞(如T-細(xì)胞)發(fā)現(xiàn)H2O2促進(jìn)NF-κB與抑制亞基I-κB的分離，進(jìn)而激活NF-κB[87].加入20mM的抗氧化劑NAC可抑制H2O2對(duì)NF-κB的活化[87]，進(jìn)一步證明了氧化還原對(duì)于NF-κB的激活至關(guān)重要.但突變分析證明位于NF-κB亞基P50的DNA識(shí)別域的還原型Cys62是NF-κB與DNA結(jié)合的重要決定因素，表明NF-κB的還原狀態(tài)有助于其與DNA的結(jié)合[88].正如有研究[80]發(fā)現(xiàn)DTT預(yù)處理抑制TPA活化NF-κB，加入GR(谷胱甘肽還原酶抑制劑)后，部分抵消這一效應(yīng)，但在TPA處理1h后加入DTT，則增強(qiáng)NF-κB的活化.這提示胞內(nèi)ROS既能通過(guò)氧化作用激活NF-κB，促進(jìn)其釋放，又通過(guò)氧化NF-κB亞基的Cys抑制其與DNA的結(jié)合.

5.4.2 ROS和AP-1轉(zhuǎn)錄因子

同樣，ROS能引起轉(zhuǎn)錄因子AP-1的活化[89]，但抑制其與DNA的結(jié)合.AP-1是由c-Fos和c-Jun(原癌基因c-fos和c-jun的表達(dá)產(chǎn)物)組成的異二聚體蛋白，AP-1與DNA序列結(jié)合，誘導(dǎo)PKC活化主要促進(jìn)細(xì)胞增生和腫瘤形成、生長(zhǎng)和遷移，是目前腫瘤研究的焦點(diǎn).

AP-1的DNA結(jié)合域含有高度保守的cys殘基，用還原劑保證此cys殘基的還原狀態(tài)有助于提高AP-1與DNA序列的結(jié)合能力，氧化此cys殘基則抑制AP-1與DNA的結(jié)合[87].

同樣，其他轉(zhuǎn)錄因子如P53[81]、v-Ets[82]、v-Rel[83]、和v-Myb[84]的DNA結(jié)合域的活性也受氧化還原調(diào)節(jié)，與特殊位點(diǎn)的cys殘基有關(guān)[81,84]，cys殘基的還原狀態(tài)都有助于轉(zhuǎn)錄因子與DNA結(jié)合.

在兔肺血管平滑肌細(xì)胞中，PLA2和花生四烯酸能促進(jìn)H2O2誘導(dǎo)c-fos和c -jun基因的表達(dá)，且PLA2抑制劑可以阻斷H2O2誘導(dǎo)c-fos和c-jun基因表達(dá)，下調(diào)PKC僅部分地抑制H2O2或花生四烯酸對(duì)c-fos和c-jun的誘導(dǎo)，提示此過(guò)程中PKC依賴性和非依賴性調(diào)節(jié)同時(shí)存在[90].

6 ROS調(diào)節(jié)細(xì)胞的氧化還原狀態(tài)

ROS可通過(guò)改變GSSG(氧化型谷胱苷肽)與GSH(還原型谷胱苷肽)的比例調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)因子的氧化還原狀態(tài)來(lái)影響其活性.在T-細(xì)胞(Molt-4)中，體內(nèi)NF-κB的活化和其與DNA結(jié)合的抑制均可由GSSG調(diào)節(jié)，且胞內(nèi)GSH的水平下降被認(rèn)為可能作為信號(hào)通路的一個(gè)組成部分調(diào)節(jié)NF-κB的活性[91].在體外，GSSG抑制NF-κB的DNA結(jié)合活性比抑制AP-1更有效，氧化的Trx(硫氧還蛋白)則能更有效地抑制AP-1的DNA結(jié)合活性[91].如上所述，其他轉(zhuǎn)錄因子如Ets、Rel、Myb、Fos/Jun(AP-1)、p53和NF-κB也受到類似的調(diào)節(jié)，GSH/GSSG比例或胞內(nèi)GSSG增高引起轉(zhuǎn)錄因子活化，抑制其DNA結(jié)合活性[3].

有證據(jù)證明GSH與GSSG的比例對(duì)某些信號(hào)轉(zhuǎn)導(dǎo)蛋白如PKC活性的有重要調(diào)節(jié)作用[92].在體外，低濃度的GSSG可活化從兔腦中提取的部分純化的PKC[93].200μmol/L tBOOH(叔丁基過(guò)氧化氫)可誘導(dǎo)肝細(xì)胞中GSSG 的堆積增加Ca2+濃度,而不是通過(guò)提高Ca2+內(nèi)流和Ca2+泵抑制來(lái)升高Ca2+水平[94].Henschke也證明胎牛肺動(dòng)脈內(nèi)皮細(xì)胞內(nèi)GSSG積累可影響內(nèi)質(zhì)網(wǎng)IP3受體(鈣釋放通道)導(dǎo)致IP3依賴性Ca2+增加[95].上述結(jié)果表明,胞內(nèi)GSH/GSSG比例與氧化介導(dǎo)的Ca2+濃度變化有一定關(guān)系.

綜上所述，高濃度ROS對(duì)細(xì)胞有損傷作用，低濃度的ROS作為第二信使調(diào)節(jié)蛋白激酶、蛋白磷酸酶、NF-κB和AP-1的活性，而且還影響Ca2+通道、K+通道、Na+通道[96]和Na+-Ca2+交換[97]等多種離子通道的活性，或通過(guò)改變GSH的水平影響細(xì)胞的氧化還原狀態(tài)，在信號(hào)通路過(guò)程中的多個(gè)位點(diǎn)調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)的效率(如圖1)，參與信號(hào)轉(zhuǎn)導(dǎo)、生長(zhǎng)、Ca2+信號(hào)通路和調(diào)控氧化還原敏感的基因表達(dá)各種細(xì)胞的正常生理活動(dòng)，如引起細(xì)胞適應(yīng)性反應(yīng)和誘導(dǎo)防御基因的表達(dá).但其作用機(jī)制仍有待研究，且對(duì)于不同細(xì)胞其作用機(jī)制可能不同.

圖1 ROS通過(guò)與多層次的細(xì)胞靶分子相互作用 調(diào)節(jié)細(xì)胞的信號(hào)通路過(guò)程

[1] Dickinson BC,Chang CJ.Chemistry and biology of reactive oxygen species in signaling or stress responses[J].Nat Chem Biol,2011,7:504-511.

[2] Arnold RS,Shi J,Murad E,et al.Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1[J].Proc Natl Acad Sci USA,2001,98:5550-5555.

[3] Bae YS,Oh H,Rhee SG,et al.Regulation of reactive oxygen species generation in cell signaling[J].Mol Cells,2011,32:491-509.

[4] Vurusaner B,Poli G,Basaga H.Tumor suppressor genes and ROS: complex networks of interactions[J].Free Radic Biol Med,2012,52:7-18.

[5] Matsuyama T,Ziff M.Increased superoxide anion release from endothelial cells in response to cytokines[J].J Hnmunol,1986,137:3295-3304.

[6] Meier B,Radeke HH,Selle S,et al.Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha[J].Biochem J,1989,263:539-545.

[7] Heinecke JW,Rosen H,Suzuki LA,et al.The role of sulfur-containing amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells[J].J Biol Chem,1987,262:10098-10103.

[8] Kather H,Kreiger-Brauer HI.A stimulus sensitive NADPH-oxidase present in human fat cell plasma membrane defines a novel pathway of signal transduction[J].Biol Chem Hoppe-Seyler,1992,373:746.

[9] Henrotin Y,Deby-Dupont G,Deby C,et al.Production of active oxygen species by isolated human chondrocytes[J].Br J Rheumatol,1993,32:562-567.

[10] Immenschuh S,Baumgart-Vogt E.Peroxiredoxins,oxidative stress,and cell proliferation[J].Antioxid Redox Signal,2005,7:768-777.

[11] Craven PA,Pfanstiel J,De Robertis FR.Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation[J].Clin Invest,1986,77:850-859.

[12] Shibanuma M,Kuroki T,Nose K.Stimulation by hydrogen peroxide of DNA synthesis competence family gene expression and phosphyorylation of a specific protein in quiescent Balb/3T3 cells[J].Oncogene,1990,3:27-32.

[13] Takasu N,Komatsu M,Aizawa T,et al.Hydrogen peroxide generation in whole rat pancreatic islets,synergistic regulation by cytoplasmic free calcium and protein kinase-C[J].Biochem Biophys Res Commun,1988,155:569-575.

[14] Robertson FM,Beavis AJ,Oberyszyn TM,et al.Production of hydrogen peroxide by murine epidermal keratinocytes following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate[J].Cancer Res,1990,50:6062-6067.

[15] Tiku ML,Liesch JB,Robertson FM.Production of hydrogen peroxide by rabbit articular chondrocytes: Enhancement by cytokines[J].J Lmmunol,1990,145:690-697.

[16] Thannickal VJ,Fanburg BL.Reactive oxygen species in cell signaling[J].Am J Physiol Lung Cell Mol Physiol,2000,279:1005-1028.

[17] Correia S C,Santos R X,Perry G,et al.Mitochondrial importance in Alzheimer's,Huntington's and Parkinson's diseases[J].Adv Exp Med Biol,2012,724:205-21.

[18] Cui K,Luo X,Xu K,et al.Role of oxidative stress in neurodegeneration: recent developments in assay methods for oxidative stress and nutraceutical antioxidants[J].Progr NeuroPsychopharmocol Biol Psychiat,2004,28:771-799.

[19] Banasiak KJ,Xia Y,Haddad GG.Mechanisms underlying hypoxia-induced neuronal apoptosis[J].Prog Neurobiol,2000,62:215-249.

[20] Halliwell B,Gutteridge JM,Cross CE.Free radicals,antioxidants,and human disease: where are we now[J].J Lab Clin Med,1992,119:598-620.

[21] Saran M,Bors W.Oxygen radicals acting as chemical messengers: a hypothesis[J].Free Radic Res Commun,1989,7:213-20.

[22] Owusu-Ansah E,Baneriee U.Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation[J].Nature,2009,461:537-541.

[23] Cai Z,Zhong H,Bosch-Marce M,et al．Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partial deficiency of HIF-1 alpha[J].Cardiovasc Res,2008,77:463-470．

[24] Wang Q,Sun AY,Simonyi A,et a1.Ethanol preconditioning protects against ischemia reperfusion．induced brain damage：role of NADPH oxidase-derived ROS[J]．Free Radic Biol Med,2007,43:1048-1060.

[25] Rhee SG,Kang SW,Jeong W,st al.Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins[J].Curr Opin Cell Biol,2005,17:183-189.

[26] Sauer H,Wartenberg M,Hescheler J.Reactive oxygen species as intracellular messengers during cell growth and differentiation[J].Cell Physiol Biochem,2001,11:173-186.

[27] Wasylyk C,Wasylyk B.Oncogenic conversion of Ets affects redox regulation in-vivo and in-vitro[J].Nucleic Acids Res,1993,21:523-529.

[28] Burdon RH,Gill V.Cellularly generated active oxygen species and HeLa cell proliferation[J].Free Radic Res Comm,1993,19:203-213.

[29] Burdon RH,Rice-Evans C.Free radicals and the regulation of mammalian cell proliferation[J].Free Radic Res Comm,1989,6:345-358.

[30] Murrell GAC,Francis MJO,Bromley L.Modulation of fibroblast proliferation by oxygen free radicals[J].Biochem J,1990,265:659-665.

[31] Shibanuma M,Kuroki T,Nose K.Superoxide as a signal for increase in intracellular pH[J].Cell Physiol,1988,136:379-383.

[32] Ikebuchi Y,Masumoto K,Tasaka K,et al.Superoxide anion increases intracellular pH,intracellular free calcium and arachidonate release in human amnion ceils[J].Biol Chem,1991,266:13233-13237.

[33] Stirpe F,Higgins T,Tazzori P L,et al.Stimulation by xanthine oxidase of 3T3 Swiss fibroblasts and human lymphocytes[J].Exp Cell Res,1991,192:635-638.

[34] Crawford D,Zbinden I,Amstad P,et al.Oxidant stress induces the protooncogenes c-fos and c-myc in mouse epidermal cells[J].Oncogene,1989,3:27-32.

[35] Craven P A,Pfanstiel J,De Robertis F R.Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation[J].Clin Invest,1986,77:850-859.

[36] Nose K,Shibanuma M,Kuroki T.Involvement of hydrogen peroxide in the actions of TGF/31[J].In: Pasquier C,Olivier R Y,Auclair C,et al.Oxidative stress,cell activation and viral infection[M].Basel: Birkhauser Verlag,1994,21-23.

[37] Shibanuma M,Kuroki T,Nose K.Stimulation by hydrogen peroxide of DNA synthesis competence family gene expression and phosphyorylation of a specific protein in quiescent Balb/3T3 cells[J].Oncogene,1990,3:27-32.

[38] Burdon RH,Gill V,Rice-Evans C.Oxidative stress and tumour cell proliferation[J].Free Radic Res Comm,1990,11:65-76.

[39] Gallagher KE,Betteridge LJ,Patel MK,et al.Effects of oxidants on vascular smooth muscle proliferation[J].Biochem Soc Trans,1993,21:98S.

[40] Chénais B,Andriollo M,Guiraud P,et al.Oxidative stress involvement in chemically induced differentiation of K562 cells[J].Free Radic Biol Med,2000,28:18-27.

[41] Carmody RJ,Cotter TG.Signalling apoptosis: a radical approach[J].Redox Rep,2001,6:77-90.

[42] Hockenbery DM,Oltvai ZN,Yin XM,et al.Bcl-2 functions in an antioxidant pathway to prevent apoptosis[J].Cell,1993,75:241-251.

[43] Jabs T.Reactive oxygen intermediates as a mediators of programmed cell death plant and animals[J].Biochem Pharmacol,1999,57:231-245.

[44] Gómez TM,Snow DM,Letourneau PC.Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration[J].Neuron,1995,14:1233-1246.

[45] Gordeeva AV,Zvyagilskaya RA,Labas YA.Cross-talk between reactive oxygen species and calcium in living cells[J].Biochemistry (Mosc),2003,68:1077-1080.

[46] Wartenberg M,Diedershagen H,Hescheler J,et al.Growth stimulation versus induction of cell quiescence by hydrogen peroxide in prostate tumor spheroids is encoded by the duration of the Ca2+response[J].Biol Chem,1999,274:27759-27767.

[47] Suzuki YJ,Ford GD.Superoxide stimulates IP3-induced Ca2+release from vascular smooth muscle sarcoplasmic reticulum[J].Am J Physiol,1992,262:114-116.

[48] Roveri A,Coassin M,Maiorino M,et al.Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells[J].Arch Biochem Biophys,1992,297:265-270.

[49] Kaneko S,Kawakami S,Hara Y,et al.A critical role of TRPM2 in neuronal cell death by hydrogen peroxide[J].J Pharmacol Sci,2006,101:66-76.

[50] Akaishi T,Nakazawa K,Sato K,et al.Hydrogen peroxide modulates whole cell Ca2+currents through L-type channels in cultured rat dentate granule cells[J].Neurosci Lett,2004,356:25-28.

[51] Goldkorn T,Balaban N,Matsukuma K,et al.EGF receptor phosphorylation and signaling are targeted by H2O2redox stress[J].Am J Respir Cell Mol Biol,1998,19:786-798.

[52] Gonzalez-Rubio M,Voit S,Rodriguez-Puyol D,et al.Oxidative stress induces tyrosine phosphorylation of PDGF-a and -b receptors and pp60c-src in mesangial cells[J].Kidney Int,1996,50:164-173.

[53] Cunnick JM,Dorsey JF,Standley T,et al.Role of tyrosine kinase activity of epidermal growth factor receptor in the lysophosphatidic acid-stimulated mitogen-activated protein kinase path-way[J].J Biol Chem,1998,273:14468-14475.

[54] Knebel A,Rahmsdorf HJ,Ullrich A,et al.Dephosphory-

lation of receptor tyrosine kinases as target of regulation by radiation,oxidants or alkylating agents[J].EMBO J,1996,15: 5314-5325.

[55] Cai J,Jones DP.Superoxide in apoptosis.Mitochondrial generation triggered by cytochrome c loss[J].J Biol Chem,1998,273:11401-11404.

[56] Stern A.Oxidative stress and growth factor-mediated signal transduction.In Pasquier C,Olivier RY,Auclair C,et al.Oxidative stress,cell activation and viral infection[M].Basel: Birkhauser Verlag,1994,35-42.

[57] Ushio-Fukai M,Alexander RW,Akers M,et al.Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells[J].J Biol Chem.1999,274:22699-22704.

[58] Lee HB,Yu MR,Song JS,et al.Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by human peritoneal mesothelial cells[J].Kidney Int,2004,65:1170-1179.

[59] Simon AR,Rai U,Fanburg BL,Cochran BH.Activation of the JAK-STAT pathway by reactive oxygen species[J].Am J Physiol Cell Physiol,1998,275:1640-1652.

[60] Aikawa R,Komuro I,Yamazaki T,et al.Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats[J].J Clin Invest,1997,100:1813-1821.

[61] Yoshida Y,Maruyama M,Fujita T,et al.Reactive oxygen intermediates stimulate interleukin-6 production in human bronchial epithelial cells[J].Am J Physiol Lung Cell Mol Physiol,1999,276: 900-908.

[62] Han MJ,Kim BY,Yoon SO,et al.Cell proliferation induced by reactive oxygen species is mediated via mitogen-activated protein kinase in Chinese hamster lung fibroblast (V79) cells[J].Mol Cells,2003,15:94-101.

[63] Whisler RL,Goyette MA,Grants IS,et al.Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in Jurkat T cells[J].Arch Biochem Biophys,1995,319:23-35.

[64] Shibanuma M,Kuroki T,Nose K.Stimulation by hydrogen peroxide of DNA synthesis,competence family gene expression and phosphorylation of a specific protein in quiescent Balb/3T3 cells[J].Oncogene,1990,5:1025-1032.

[65] Sundaresan M,Yu ZX,Ferrans VJ,et al.Requirement for generation of H2O2for platelet-derived growth factor signal transduction[J].Science,1995,270:296-299.

[66] Bae GU,Seo DW,Kwon HK,et al.Hydrogen peroxide activates p70S6ksignaling pathway[J].J Biol Chem,1999,274:32596-32602.

[67] Lee SL,Wang WW,Finlay GA,et al.Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion[J].Am J Physiol Lung Cell Mol Physiol,1999,277:282-291.

[68] Grewal JS,Mukhin YV,Garnovskaya MN,et al.Serotonin 5-HT2A receptor induces TGF-β1 expression in mesangial cells via ERK: proliferative and brotic signals[J].Am J Physiol Renal Physiol,1999,276:922-930.

[69] Lo YYC,Wong JMS,Cruz F.Reactive oxygen species mediate cytokine activation of c-Jun NH2-terminal kinases[J].J Biol Chem,1996,271:15703-15707.

[70] Wen Y,Scott S,Liu Y,et al.Evidence that angiotensin II and lipoxygenase products activate c-Jun NH2-terminal kinase[J].Circ Res,1997,81:651-655.

[71] Assefa Z,Garmyn M,Bouillon R,et al.Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes[J].J Invest Dermatol,1997,108:886-891.

[72] Bae YS,Kang SW,Seo MS,et al.Epidermal growth factor (EGF)-induced generation of hydrogen peroxide.Role in EGF receptor-mediated tyrosine phosphorylation[J].J Biol Chem,1997,272:217-221.

[73] Abe J,Kusuhara M,Ulevitch RJ,et al.Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase[J].J Biol Chem,1996,271:16586-16590.

[74] Goldman R,Moshonov S,Chen X,et al.Crosstalk between elevation of [Ca2+]i,reactive oxygen species generation and phospholipase A2 stimulation in a human keratinocyte cell line[J].Adv Exp Med Biol,1997,433:41-45.

[75] Natarajan V,Taher MM,Roehm B,et al.Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide[J].J Biol Chem,1993,268:930-937.

[76] Kiss Z,Anderson WH.Hydrogen peroxide regulates phospholipase D-mediated hydrolysis of phosphatidylethanolamine and phosphatidylcholine by different mechanisms in NIH 3T3 fibroblasts[J].Arch Biochem Biophys,1994,311:430-436.

[77] Min DS,Kim EG,and Exton JH.Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2in Swiss 3T3 broblasts[J].J Biol Chem,1998,273:29986-29994.

[78] Natarajan V,Vepa S,Verma RS,et al.Role of protein tyrosine phosphorylation in H2O2-induced activation of endothelial cell phospholipase D[J].Am J Physiol Lung Cell Mol Physiol,1996,271:400-408.

[79] Takekoshi S,Kambayashi Y,Nagata H,et al.Activation of protein kinase C by oxidized diacylglycerols[J].Biochem Biophys Res Commun,1995,217:654-660.

[80] Haddad JJ.Antioxidant and prooxidant mechanisms in the regulation of redox(y)-sensitive transcription factors[J].Cell Signal,2002,14:879-897.

[81] Hainaut P,Milner J.Redox modulation of p53 conformation and sequence-specific DNA binding in vitro[J].Cancer Res,1993,53:4469-4473.

[82] Wasylyk C,Wasylyk B.Oncogenic conversion of Ets affects redox regulation in vivo and in vitro[J].Nucleic Acids Res,1993,21:523-529.

[83] Kumar S,Rabson AB,Gélinas C.The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein[J].Mol Cell Biol,1992,12:3094-106.

[84] Guehmann S,Vorbrueggen G,Kalkbrenner F,et al.Reduction of a conserved Cys is essential for Myb DNA-binding[J].Nucleic Acids Res,1992,20(9):2279-2286.

[85] Abate C,Patel L,Rauscher FJ,et al.Redox regulation of fos and jun DNA-binding activity in vitro[J].Science,1990,249:1157-1161.

[86] Li YP,Schwartz RJ,Waddell ID,et al.Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in response to tumor necrosis factor alpha[J].FASEB J,1998,12:871-880.

[87] Schreck R,Rieber P,Baeuerle PA.Reactive oxygen intermediates as apparently widely used messengers in the activation of NF-kappa B transcription factor and HIV-1[J].EMBO Journal,1991,10:2247-2258.

[88] Matthews JR,Kaszubska W,Turcatti G,et al.Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B[J].Nucleic Acids Res,1993,21:1727-1734.

[89] Puri PL,Avantaggiati ML,Burgio VL,et al.Reactive oxygen intermediates mediate angiotensin II-induced c-Jun.c-Fos heterodimer DNA binding activity and proliferative hypertrophic responses in myogenic cells[J].J Biol Chem,1995,270:22129-22134..

[90] Chakraborti S,Chakraborti T.Down-regulation of protein kinase C attenuates the oxidant hydrogen peroxide-mediated activation of phospholipase A2 in pulmonary vascular smooth muscle cells[J].Cell Signal,1995,7:75-83.

[91] Galter D,Mihm S,Dr?ge W.Distinct effects of glutathione disulphide on the nuclear transcription factor kappa B and the activator protein-1[J].Eur J Biochem,1994,221:639-648.

[92] Kass GE,DuddySK,Orrenius S.Activation of hepatocyte protein kinase C by redox-cycling quinones[J].Biochem J,1989,260:499-507.

[93] Keyse SM,Emslie EA.Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase[J].Nature,1992,359:644-647.

[94] Livingston FR,Lui EM,LoebGA,et al.Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages[J].Arch Biochem Biophys,1992,299:83-91.

[95] Henschke P N,Elliott S J.Oxidized glutathione decreases luminal Ca2+content of the endothelial cell ins(1,4,5)P3-sensitive Ca2+store[J].Biochem J,1995,312:485-489.

[96] Yao JA，Jiang M，Tseng GN.Mechanism of enhancement of slow delayed rectifier current by extracellular sulfhydryl modification[J].Am J Physiol,1997,273:208-219.

[97] Chiamvimonvat N,O'Rourke B,Kamp T J,et al.Functional consequences of sulfhydryl modification in the pore-forming subunits of cardiovascular Ca2+and Na+channels[J].Circ Res,1995,76:325-334.