活性氧双向生物学效应和机制

戴甲培，石 悦

(1 中南民族大学 武汉神经科学与神经工程研究所，武汉 430074;2 中南民族大学 药学院，武汉 430074；3 中南民族大学 生命科学学院，武汉 430074)

人体内95%以上的ROS为氧自由基.ROS可通过脂质过氧化反应等途径损伤组织细胞，导致细胞死亡、染色体突变和畸变，并可致癌.然而，越来越的多研究发现，活性氧也有好的一面.胞外信号分子(生长因子、细胞因子、激素和神经递质等)诱导的正常信号通路中，特异的质膜氧化酶生成的ROS可作为第二信使参与启动多种生物效应.胞内氧化酶产生的正常水平的ROS在信号转导、生长、Ca2+信号通路和调控氧化还原敏感的基因表达等生理活动中发挥重要作用.由此可见，ROS对细胞的应激功能具有双向生物学效应.

1 细胞内ROS的产生与调控

1.1 细胞内ROS产生

多种正常的非吞噬细胞也能产生和释放ROS(表1).有报道指出，很多非吞噬细胞表达具有NADPH/NADH氧化酶活性的酶和NADPH氧化酶亚基[1].在机体多器官平滑肌内发现gp91phox的同源物Nox1，而且在小鼠NIH3T3细胞中过量表达Nox1导致ROS增多[2].而且，体外培养的多种正常类型的细胞在胞外刺激剂(细胞因子、神经递质，多肽类生长因子如转移因子α和激素如胰岛素)的作用下都产生H2O2[3].同时，细胞在胞外因子或生长因子的刺激下自身能够产生和释放ROS，且肿瘤细胞能持续合成和释放H2O2[4]，因此，推测ROS可能作为一种自分泌因子或胞内和胞间的信号分子起作用[1,3].

总之，任何能进行氧化还原反应的蛋白质或复合酶都能通过电子转移反应产生ROS[5].

1.2 细胞内ROS的清除

同时，胞内抗氧化巯基还原体系主要包括还原型谷胱甘肽(GSH)和硫氧还原蛋白(Trx)及其还原酶TrxR.Trx主要还原底物蛋白的二硫键为巯基.胞内还原性GSH与氧化性GSSG形成动态平衡，这两者本身的含量或比值是衡量细胞抗氧化的重要指标[16].

1.3 细胞的氧化还原平衡状态

正常细胞内ROS的产生与清除保持动态平衡，如果有刺激(炎症、病原入侵、紫外线等)引起ROS或抗氧化系统相对增强，改变细胞氧化还原状态，通过相应的信号通路改变对应的基因表达，产生不同的生理效应.如ROS产生超出其清除，导致氧化应激，损伤细胞或组织[16].

2 ROS与氧化应激

有关ROS对机体和细胞危害的报道较多[17].ROS可通过过氧化细胞膜、线粒体膜的不饱和脂肪酸影响线粒体膜的通透性[18]，且其脂质过氧化反应的产物可分解为更多的自由基，导致连锁反应损伤组织和细胞.另外，ROS能与胞内各种生物大分子反应引起DNA断裂和蛋白质脂质氧化[17]，其介导的损伤可导致细胞死亡、染色体突变和畸变，导致细胞癌化.ROS含量过多，引起氧化产物与细胞抗氧化能力失衡，导致氧化应激与多种疾病的形成，如动脉粥样硬化、阿耳茨海默氏病[17]、巴金森病[17]、脑缺血[19]和衰老[20].

3 ROS与细胞正常生理活动

1989年，基于理论背景以及对自由基在生物内环境的体评价，Saran和Bors[21]假设ROS作为一种生物信号起作用，而不是介导细胞损伤.在生物环境中，ROS的半衰期为400×10-6ns，扩散速度为55-3000nm[21].而且，有研究确立了ROS在调控造血干细胞命运中所起的信号作用[22].有研究发现，ROS在缺血预处理(IPC)及缺血后过程中可改善心功能，降低凋亡细胞数目，保护心肌[23].且ROS在脑缺血再灌注过程中起神经保护作用[24].ROS的双向作用与NO很类似.有毒气体NO可作为胞内信号分子调节血管扩张，有效地缓解心绞痛，也是免疫系统的有效武器，可消灭细菌、病毒等病原体.同样，越来越多的研究表明亚微摩尔浓度的ROS作为一种新的胞内或胞间的第二信使参与多种生长因子和细胞因子诱导的正常信号转导过程[25]进而调节细胞生长、增殖、分化和凋亡等生理过程.

4 ROS与细胞的增殖、分化和凋亡

4.1 ROS与细胞增殖

如150 μM的H2O2能作为激活信号活化T-淋巴细胞，但抗氧化剂则抑制T-细胞增殖[27].在仓鼠和大鼠的成纤维细胞[10]以及HeLa细胞[28]的培养基中加入过氧化氢酶和过氧化物歧化酶不仅抑制细胞增殖，而且经台盼蓝染色显示死亡细胞数目增加，并且与培养皿分离的细胞数目也增加.同时，电子显微镜扫描发现这些“死亡”细胞显示出凋亡特征：核膜内膜附近染色体固缩，且DNA断裂.

表2 ROS可引起生长反应的细胞类型

4.2 ROS与细胞分化和凋亡

可引起细胞氧化损伤的抗肿瘤药物丁酸、阿霉素，在亚毒性浓度时改变胞内ROS水平对于诱导人K562细胞分化为成熟红细胞至关重要[40].小鼠PC12细胞在NGF(神经生长因子)的诱导下分化为神经细胞，胚胎干细胞在IL-1(白细胞介素-1)的诱导下分化为心肌细胞的过程中都产生ROS[26].

细胞有两种死亡方式：程序性死亡即凋亡(PCD)和坏死(necrosis).凋亡是由基因调控的有序的细胞自我死亡的正常生理过程.而坏死是由严重且持续的有害刺激所引起的，不受基因调控.大量研究证明多种类型的细胞凋亡与ROS有关[41].H2O2可活化凋亡诱导因子P53，提示ROS可增强P53诱导凋亡的活性[42].很多抗氧化剂(SOD、GSH)可抑制ROS引起的凋亡.

ROS较低浓度的时候可能通过启动一系列信号传导，引起细胞凋亡，高浓度的时候导致细胞坏死[43].所以，ROS通过多种信号通路调节细胞的凋亡和坏死，凋亡或者坏死取决于ROS的浓度，作用时间和细胞所处的环境[43].

5 ROS与信号转导

尽管ROS可调节多种生理过程，但其靶信号分子仍不完全清楚.大量证据表明氧化还原作用可调节信号通路过程中从受体到细胞核的多个部分[5]，如ROS既可与某些受体直接作用或氧化信号转导分子(蛋白激酶、蛋磷酸酶、转录因子或转录因子抑制剂)；也可能通过改变胞内GSH和GSSG的比例而改变氧化还原状态，间接影响信号转导蛋白分子的活性，行使其信使分子的功能[5].ROS调节信号转导的方式主要有两种；氧化修饰蛋白质和改变细胞内氧化还原状态，其可能的作用机制如下.

5.1 ROS与钙离子

细胞膜、内质网和线粒体上的钙泵和钙通道的开放程度决定了胞内Ca2+浓度，通常情况下，细胞内Ca2+含量稳定在10-7mol/L，外来信号刺激下，Ca2+浓度可快速上升到10-6～ 10-5mol/L[44]，从而影响多种蛋白或Ca2+依赖性蛋白激酶的活性，实现对细胞功能的调节.氧化还原作用可调控内质网上与Ca2+释放相关的IP3(三磷酸肌醇)受体和ryanodine受体及Ca2+-Na+交换体[45].氧化剂通过Ca2+通路增加Ca2+内流及抑制Ca2+泵，提高胞内Ca2+浓度.

很多研究表明ROS可能通过直接改变胞内Ca2+浓度激活下游基因或下调转录[46].Suzuki[47]等发现血管平滑肌细胞内次黄嘌呤/黄嘌呤氧化酶体系生成的ROS可使IP3诱导内质网释放Ca2+.Roveri[48]等发现300μmol/ L H2O2刺激血管平滑肌细胞，引起胞质Ca2+浓度持续性上升,当介质中无Ca2+时，无此现象，表明H2O2可能影响细胞膜上的钙离子通道或Ca2+与膜偶联的转运机制.

H2O2诱导胞内Ca2+增加程度与其浓度成正比，1mM H2O2处理体胎鼠皮层神经细胞膜，H2O2可通过TRPM2钙离子通道促进胞外Ca2+内流提高胞内Ca2+浓度[49].同时，有研究表明，体外培养的胎鼠齿状回颗粒细胞经1～10μM H2O2灌流处理2h，膜片钳检测发现H2O2可通过L-型钙离子通道促进Ca2+内流，导致胞内Ca2+浓度增加[50].

5.2 ROS与受体

微摩尔浓度的外源性H2O2能诱导PDGF-a、PDGF-b和EGF等生长因子受体的酪氨酸磷酸化和激活[51,52].溶血磷脂酸对诱导EGF受体的活化受ROS的调节，ROS不直接增强EGF受体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来抑制其活性，进而促进EGF受体磷酸化而活化EGF受体[53].Knebel等也发现ROS可氧化抑制膜结合的PTPs的活性，进而抑制RTKs(受体酪氨酸激酶)的去磷酸化促进受体活化[54].也有相似研究报道[55]O2-可改变受体磷酸化-去磷酸化平衡，通过促进受体磷酸化而激活PDGF受体.

5.3 ROS与酶

ROS和氧化还原作用都可能通过影响蛋白激酶和蛋白磷酸酶的活性改变蛋白质酪氨酸残基的磷酸化状态，进而有助于生长因子介导的信号通路转导[56].如，H2O2具有细胞膜通透性，在多肽类生长因子刺激下，可在多种细胞中暂时积累，通过氧化PTPs和PTEN(脂类磷酸酶)参与受体介导的信号转导[25].而且，ROS可激活PKB(蛋白激酶B)[57]，也很可能通过改变酶的巯基/二硫键的平衡，来激活PKC(蛋白激酶C)[58].

ROS也可调节非受体酪氨酸激酶(PTKs)如JAKs(Janus激酶)家族和Src激酶家族的活性[59,60].Simon等证明PDGF诱导JAK-STAT通路的激活对氧化还原作用敏感，外源性氧化剂如H2O2可激活JAK-STAT通路[59].Bauskin[61]等发现H2O2等巯基氧化剂可通过氧化Ltk(61s，在淋巴细胞、白血病细胞和神经元等细胞中大量表达)形成通过二硫键连接的多聚体来使其激活.

氧化还原敏感的有丝分裂原激活蛋白激酶(MAPK)的信号通路也受ROS调节[62].MAPK信号转导通路主要包括胞外信号调节激酶ERK1 (p44MAPK)/ERK2 (p42MAPK)，JNKs/SAPK(c-Jun N端激酶/应激激活的蛋白激酶)和p38 MAPK，以及BMK1/ERK5(分裂原激活的大分子蛋白激酶).

有研究证明外源氧化物可激活ERK MAPK通路，但其具体作用机制和靶分子尚不清楚，有研究推测ROS可能氧化抑制PTPs和/或蛋白磷酸酶A来激活ERK MAPK通路[63].

十几年前，已证明PDGF 刺激细胞产生的H2O2在BALB/3T3细胞生长中发挥作用[64].Sundaresan等证明PDGF能暂时提高胞内H2O2浓度，H2O2参与PDGF诱导的酪氨酸磷酸化，MAPK激活和DNA合成等生理活动[65].在小鼠上皮细胞JB6，外源性H2O2可激活p70S6k和p90Rsk，并且磷酸化p42(MAPK)或p44(MAPK)，调节细胞增殖[66].

Lo等发现胞内H2O2可能介导TNF-α和IL-1诱导的JNK的激活[69]，且ANG II[70]和EGF[71]与细胞相互作用时，也发现相似效应.Bae[72]等证明EGF刺激A431细胞(人表皮癌细胞)产生H2O2，暂时提高胞内H2O2浓度，是EGF诱导的蛋白质酪氨酸残基磷酸化所必需的.用过氧化氢酶清除H2O2则抑制EGF诱导的多种蛋白质酪氨酸磷酸化，如EGF受体和磷脂酶C-γ1.有研究表明，BMK1比ERK1/ERK2对H2O2更加敏感，BMK1可能是一种氧化还原敏感激酶[73].

PLA2(磷脂酶A2) 主要催化磷脂分解，产生前列腺素类物质，白三烯、溶血磷脂、血小板激活因子等多种脂质介质，这些产物可作为第二信使分子,调节多种细胞功能如磷脂转运、膜修复、胞外水解及神经元转移因子的释放等.EFG信号转导过程中诱导的PLA2的激活和花生四稀酸的合成对抗氧化剂如NAC(N-乙酰半胱氨酸)、DTT(二硫苏糖醇)和DPI(FAD依赖性氧化酶抑制剂)敏感，表明PLA2可能是ROS的靶分子[74].

H2O2和亚油酸羟基过氧化物可激活内皮细胞PLD(磷脂酶D)[75]，促进磷脂酰乙醇胺(PEt)和磷脂酸(PA)的合成，且H3标记的脱氧葡萄糖放射性同位素示踪法显示氧化剂介导的PLD活化无细胞毒性.H2O2可诱导NIH-3T3成纤维细胞PLD活化，水解PEt，不水解磷脂酰胆碱[76].PKC的激活和蛋白质酪氨酸残基的磷酸化介导H2O2诱导内皮细胞和成纤维细胞PLD的活化[77,78].Takekoshi等发现氧化型二酰甘油比其还原型能更有效地活化PKC[79].

5.4 ROS与转录因子

大量研究报道[80]核转录因子的活性与其氧化还原状态密切相关.氧化还原敏感的转录因子如NF-κB、AP-1、P53[81]、v-Ets[82]、v-Rel[83]、和v-Myb[84]等在DNA结合域都含有保守的cys残基[85],推测此cys的氧化还原状态可能直接调节基因的表达[81,84].

5.4.1 ROS和NF-κB转录因子

未激活的NF-κB位于胞浆，是由P65、P50和抑制亚基I-κB组成的三聚体.当细胞受外来信号刺激后，NF-κB复合体活化将I-κB磷酸化，使其被遍在蛋白辍合酶降解，游离的NF-κB活化转移入细胞核，与特异的DNA结合，调节基相应的因表达.

TNF-α刺激细胞时，线粒体产生的ROS可激活NF-κB[86].用100μM H2O2处理细胞(如T-细胞)发现H2O2促进NF-κB与抑制亚基I-κB的分离，进而激活NF-κB[87].加入20mM的抗氧化剂NAC可抑制H2O2对NF-κB的活化[87]，进一步证明了氧化还原对于NF-κB的激活至关重要.但突变分析证明位于NF-κB亚基P50的DNA识别域的还原型Cys62是NF-κB与DNA结合的重要决定因素，表明NF-κB的还原状态有助于其与DNA的结合[88].正如有研究[80]发现DTT预处理抑制TPA活化NF-κB，加入GR(谷胱甘肽还原酶抑制剂)后，部分抵消这一效应，但在TPA处理1h后加入DTT，则增强NF-κB的活化.这提示胞内ROS既能通过氧化作用激活NF-κB，促进其释放，又通过氧化NF-κB亚基的Cys抑制其与DNA的结合.

5.4.2 ROS和AP-1转录因子

同样，ROS能引起转录因子AP-1的活化[89]，但抑制其与DNA的结合.AP-1是由c-Fos和c-Jun(原癌基因c-fos和c-jun的表达产物)组成的异二聚体蛋白，AP-1与DNA序列结合，诱导PKC活化主要促进细胞增生和肿瘤形成、生长和迁移，是目前肿瘤研究的焦点.

AP-1的DNA结合域含有高度保守的cys残基，用还原剂保证此cys残基的还原状态有助于提高AP-1与DNA序列的结合能力，氧化此cys残基则抑制AP-1与DNA的结合[87].

同样，其他转录因子如P53[81]、v-Ets[82]、v-Rel[83]、和v-Myb[84]的DNA结合域的活性也受氧化还原调节，与特殊位点的cys残基有关[81,84]，cys残基的还原状态都有助于转录因子与DNA结合.

在兔肺血管平滑肌细胞中，PLA2和花生四烯酸能促进H2O2诱导c-fos和c -jun基因的表达，且PLA2抑制剂可以阻断H2O2诱导c-fos和c-jun基因表达，下调PKC仅部分地抑制H2O2或花生四烯酸对c-fos和c-jun的诱导，提示此过程中PKC依赖性和非依赖性调节同时存在[90].

6 ROS调节细胞的氧化还原状态

ROS可通过改变GSSG(氧化型谷胱苷肽)与GSH(还原型谷胱苷肽)的比例调节信号转导因子的氧化还原状态来影响其活性.在T-细胞(Molt-4)中，体内NF-κB的活化和其与DNA结合的抑制均可由GSSG调节，且胞内GSH的水平下降被认为可能作为信号通路的一个组成部分调节NF-κB的活性[91].在体外，GSSG抑制NF-κB的DNA结合活性比抑制AP-1更有效，氧化的Trx(硫氧还蛋白)则能更有效地抑制AP-1的DNA结合活性[91].如上所述，其他转录因子如Ets、Rel、Myb、Fos/Jun(AP-1)、p53和NF-κB也受到类似的调节，GSH/GSSG比例或胞内GSSG增高引起转录因子活化，抑制其DNA结合活性[3].

有证据证明GSH与GSSG的比例对某些信号转导蛋白如PKC活性的有重要调节作用[92].在体外，低浓度的GSSG可活化从兔脑中提取的部分纯化的PKC[93].200μmol/L tBOOH(叔丁基过氧化氢)可诱导肝细胞中GSSG 的堆积增加Ca2+浓度,而不是通过提高Ca2+内流和Ca2+泵抑制来升高Ca2+水平[94].Henschke也证明胎牛肺动脉内皮细胞内GSSG积累可影响内质网IP3受体(钙释放通道)导致IP3依赖性Ca2+增加[95].上述结果表明,胞内GSH/GSSG比例与氧化介导的Ca2+浓度变化有一定关系.

综上所述，高浓度ROS对细胞有损伤作用，低浓度的ROS作为第二信使调节蛋白激酶、蛋白磷酸酶、NF-κB和AP-1的活性，而且还影响Ca2+通道、K+通道、Na+通道[96]和Na+-Ca2+交换[97]等多种离子通道的活性，或通过改变GSH的水平影响细胞的氧化还原状态，在信号通路过程中的多个位点调节信号转导的效率(如图1)，参与信号转导、生长、Ca2+信号通路和调控氧化还原敏感的基因表达各种细胞的正常生理活动，如引起细胞适应性反应和诱导防御基因的表达.但其作用机制仍有待研究，且对于不同细胞其作用机制可能不同.

图1 ROS通过与多层次的细胞靶分子相互作用 调节细胞的信号通路过程

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