活性氧:从毒性分子到信号分子--活性氧与细胞的增殖、分化和凋亡及其信号转导途径

活性氧(reactive oxygen species,ROS)是生物体有氧代谢产生的一类活性含氧化合物的总称,主要包括O2·-、H2O2、·OH等,机体细胞通过多种途径维持ROS产生与消解的动态平衡。近年的研究揭示ROS参与细胞正常的生理过程,与细胞的增殖、分化及凋亡密切相关。不同刺激诱导细胞产生的内源性ROS可作为第二信使,通过改变氧化还原状态调节增殖、分化和凋亡相关的信号转导通路中多种靶分子的活性,最终决定细胞的命运。     

叶绿体光合代谢中活性氧的产生与清除

有氧代谢不可避免产生活性氧(ROS),叶绿体的PSI和PSII反应中心均是ROS产生的主要位点。叶绿体产生的ROS主要有超氧阴离子(O2-)、过氧化氢(H2O2)、羟自由基(.OH)和单线氧(1O2),其中在PSI产生的O2-将进一步产生H2O2和.OH,而1O2产生在PSII。正常生理代谢条件下,叶绿体内抗氧化系统和光能吸收利用的调节保持活性氧产生和消灭的平衡,不会影响植物的正常生理功能。     

UVA诱发的1O2和O2-·产生与脂质过氧化水平关系的研究

紫外A(UVA,320 nm-400 nm)诱发的脂质过氧化反应是通过活性氧(ROS)介导的。在UVA照射之后,单线态氧(1O2)和超氧阴离子(O2-.)是细胞内最初产生的ROS,它们进一步生成过氧化氢(H2O2),羟自由基(.OH)等其它自由基。为了探讨UVA照射后最早生成的1O2和O2-.与细胞氧化损伤后果的关系,我们采用一种特异性检测1O2和O2-.的高灵敏度化学发光探针MCLA(2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimid-azo[1,2-α]pyrazin-3-one hydrochloride)检测人外周血淋巴细胞经UVA照射后的化学发光变化。发现不同剂量UVA照射后,细胞MCLA化学发光变化和MDA浓度变化一致。结果表明UVA照射后1O2和O2-.的水平与由此引发的脂质过氧化损伤存在正相关关系。因此,MCLA化学发光方法可望作为一种检测UVA诱发脂质过氧化水平的简单快速方法。     

铜锌超氧物歧化酶与过氧化氢反应中羟自由基的形成

应用脱氧核糖降解法研究了离体条件下Cu,Zn-SOD与H2O2反应产生·OH,并对其机理进行了探讨。H2O2可使Cu,Zn-SOD失活,在失活过程中有·OH产生,甲酸钠和苯甲酸钠均能不同程度地保护Cu,Zn-SOD和降低H2O2与Cu,Zn-SOD反应中·OH的产额;热失活SOD也可和H2O2反应生成·OH,且效能高于活性Cu,Zn-SOD;用螫合剂脱去Cu,Zn-SOD的金属辅基后,脱辅基的SOD蛋白不能和H2O2反应产生·OH;Cu2＋和H2O2反应产生·OH的效率很高,而Zn2＋产生·OH的效率很低。实验结果提示Cu,Zn－SOD与H2O2反应产生的·OH可能是SOD活性中心的Cu2＋与H2O2发生Fenton反应的结果．     

5''-磷酸腺苷对小鼠脾细胞氧化应激损伤的保护作用

目的研究5'-磷酸腺苷(5'-AMP)体外抗氧化和对体外氧化损伤脾细胞的损伤修复能力。方法用化学比色法测定5'-AMP体外清除二苯代苦味酰基自由基(DPPH自由基)的能力;建立过氧化氢(H2O2)氧化损伤体外培养小鼠脾细胞模型,用MTT法检测5'-AMP修复受损伤脾细胞的作用,并分析其对细胞抗氧化体系及抗氧化能力的影响。结果5'-AMP具有剂量依赖性的体外抗氧化和清除活性氧能力,添加0·5mmol/L、1mmol/L、5mmol/L和10mmol/L5'-AMP均能显著修复H2O2诱导的脾细胞氧化损伤(P<0·05),总抗氧化能力和抗氧化酶类活力(P<0·01),5'-AMP添加量大于1mmol/L时,可显著降低丙二醛(MDA)含量(P<0·01)。其细胞培养液的氧自由基(ROS)水平逐渐降低,5'-AMP添加量为10mmol/L时,ROS水平接近对照组水平。结论5'-磷酸腺苷能显著修复氧化损伤,具有显著的抗氧化作用。     

植物生理学实验教学中H_2O_2可视化检测方法的改进

活性氧（reactive oxygen species,ROS）,如超氧阴离子自由基（O2-·）、羟自由基（·OH）和过氧化氢（H2O2）均是植物尤其是高等植物在有氧代谢过程中产生的,     

藤茶抗氧化活性研究

本文建立体外二苯代苦味酰基自由基(DPPH·)体系,并采用连苯三酚法在碱性溶液中自氧化反应产生超氧阴离子自由基(O2-+)和邻二氮菲-Fe2+/H2 O2体系产生羟自由基(·OH)法,在酶标仪上检测藤茶总黄酮、二氢杨梅素和杨梅素对DPPH·、O2-+和·OH的清除作用.结果表明:藤茶各提取物均有不同程度的体外抗氧化效果,杨梅素对DPPH·的清除作用最强DPPH·IC50 (2.91±0.28) mg/L,二氢杨梅素对O2-+的清除作用最强IC50 (3.88±0.99) mg/L,对·OH的清除效果是杨梅素＞二氢杨梅素＞藤茶总黄酮.确定杨梅素和二氢杨梅素为藤茶的主要抗氧化相关活性成分.     

茶多糖的抗氧化活性及对细胞氧化损伤的保护机制

茶多糖是一种从茶叶中提取的酸性糖蛋白, 具有良好的抗氧化活性。以自由基清除率为指标, 分析皖西南地区夏秋茶多糖的抗氧化活性, 基于H₂O₂和EDTA-Fe²⁺建立的外源性羟基自由基(·OH)损伤细胞模型和PMA诱导内源性羟基自由基损伤模型, 进一步探讨茶多糖对自由基损伤的修复作用机制。结果表明, 茶多糖具有良好的体外抗氧化活性, 对DPPH·和·OH均具有较强的清除效果, EC₅₀值分别为209.5和535.2 µg∙mL^-1, 最大清除效率与Vc相当。细胞增殖实验表明, 外源性和内源性自由基氧化损伤模型中细胞存活率均随着茶多糖浓度的增加而升高, 在茶多糖浓度为800 µg∙mL^-1时细胞存活率分别高达87.41%和85.84%, 且显著高于模型组(47.67%和48.03%)。在修复机制上, 利用激光共聚焦显微镜显影细胞内活性氧(ROS)分布以及荧光强度, 分析结果显示, 与模型组相比, 茶多糖对于细胞模型中外源和内源性ROS均具有明显的清除效果, 与体外抗氧化实验结果一致。茶多糖在体外表现出良好的自由基清除效率, 可在细胞水平上改善自由基损伤。该研究在细胞水平上揭示了茶多糖清除自由基的抗氧化损伤机制, 为后续进一步阐明茶多糖抗衰老作用奠定了基础。     

血卟啉衍生物光敏引起NAD(P)H氧化作用研究

血卟啉衍生物(HPD)光敏引起NAD(P)H氧化为中介的luminol化学发光(CL)与多种因素有关,如pH、激光功率密度、照光时间以及luminol、HPD以及底物NAD(P)H浓度等的改变都可引起CL的变化.当NAD(P)H浓度远远大于HPD浓度时,CL强度与HPD浓度成正比关系,这表明化学发光测定可以作为检测HPD光敏反应的指标,而且HPD敏化的NAD(P)H光氧化过程中化学发光的产生是与活性氧物质(ROS)有关的.选择性应用O_2、H_2O_2、OH、~1O_2的专一性清除剂研究ROS及~1O_2在HPD敏化的NAD(P)H氧化过程中的作用,其主要结果如下:(1)NAD(P)H光氧化为中介的lumlnol化学发光在水溶液中受到铜锌超氧化物岐化酶(CuZn-SOD)、过氧化氢酶(CAT)和甲酸钠(F)的抑制,在D_2O中受到His和Met的抑制.1μg/ml的CuZn-SOD抑制可达84%,但再增加酶量,抑制程度不再增加;CAT的抑制作用也出现类似情况;F的抑制作用较弱,在14—42mM浓度范围内,抑制程度不超过60%.(2)CuZn-SOD对化学发光的抑制作用随照光时间延长而持续下降,而CAT和F的抑制作用却随照光时间延长而有上升的趋势.His和Met在D_2O中的作用比较复杂.(3)观察伴随NAD(P)H光氧化失活时OD_(340)值的变化,发现ROS清除剂在H_2O中对失活有保护作用,而~1O_2猝灭剂His和Met在D_2O中对失活有保护作用.上述清除剂和猝灭剂     

库克诺你果汁提取物体外清除自由基及抗氧化活性研究

本文对诺你果汁多糖、乙醇溶出物和乙酸乙酯萃取物体外对超氧阴离子(O2·)、羟自由基(·OH)、DPPH和脂质过氧化(LPO)的抑制作用进行了研究。超氧阴离子(O2·)由邻苯三酚自氧化产生;羟自由基(·OH)由Fenton反应产生;利用Fe2 诱发卵黄脂蛋白产生丙二醛(MDA),TBA法测定。所有测定均为分光光度法。结果表明,与已知抗氧化剂L抗坏血酸相比,乙醇溶出物和乙酸乙酯萃取物均有明显的捕捉自由基和抗氧化能力,而多糖捕捉自由基和抗氧化能力很低,且对O2·没有抑制作用,反而会增加其生成速度。     

A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

NNK诱发BEP2D细胞产生活性氧及其对DNA的损伤

通过测定细胞内和细胞上清中活性氧（ｒｅａｃｔｉｖｅ ｏｘｙｇｅｎ ｓｐｅｃｉｅｓ,ＲＯＳ）水平,以及ＤＮＡ 加合物——８－羟基脱氧鸟嘌呤核苷（８－ｈｙｄｒｏｘｙｄｅｏｘｙｇｕａｎｏｓｉｎｅ,ＯＨ８ｄＧ）含量,对烟草特异亚硝胺类化合物４－甲基亚硝胺－１（３－吡啶基）－１－丁酮（４－（ｍ ｅｔｈｙｌｎｉｔｒｏｓａｍ ｉｎｏ）－１－（３－ｐｙｒｉｄｙｌ）－１－ｂｕｔａｎｏｎｅ,ＮＮＫ）诱发人乳头状病毒永生化的人支气管上皮细胞（ｈｕｍ ａｎ ｐａｐｉｌｌｏｍ ａｖｉｒｕｓ－ｉｍ ｍ ｏｒｔａｌｉｚｅｄ ｈｕｍ ａｎｂｒｏｎｃｈｉａｌｅｐｉｔｈｅｌｉａｌｃｅｌｌｌｉｎｅ,ＢＥＰ２Ｄ）产生的ＲＯＳ及其对ＤＮＡ 的氧化损伤进行研究,并观察纳米硒的保护作用．结果表明,ＢＥＰ２Ｄ 细胞经不同浓度的ＮＮＫ 作用后,细胞内和细胞上清中ＲＯＳ以及ＯＨ８ｄＧ含量均显著增加,并有较好的剂量效应关系．１ μｍ ｏｌ·Ｌ－ １纳米硒（ｎａｎｏｓｅｌｅｎｕｉｍ ,ＮＳ）能明显抑制ＮＮＫ 诱发ＢＥＰ２Ｄ细胞产生的ＲＯＳ及ＯＨ８ｄＧ 水平．揭示ＮＮＫ 能造成细胞的氧化损伤,而ＮＳ对ＮＮＫ 所致细胞的氧化损伤有保护作用．     

Validation of different chemilumigenic substrates for detecting extracellular generation of reactive oxygen species by phagocytes and endothelial cells

Chemiluminescence is a widely used tool to detect extracellular generation of reactive oxygen species (ROS). In the present study we tested four different chemilumigenic substrates (CLS)—luminol, isoluminol, lucigenin and pholasin—to detect extracellular CL in different cell types: polymorphonuclear leukocytes (PMN); DMSO‐differentiated HL‐60 cells; murine macrophages (RAW 264.7); and TNFα‐stimulated human endothelial cells (HUVEC). Extracellular ROS production was calculated by subtracting intracellular CL response in the presence of superoxide dismutase and catalase from the overall CL response in the absence of enzymes. CL varied considerably in dependence on the CLS and the stimulus used to evoke ROS generation. Luminol (oxidized LDL and zymosan stimulation) and isoluminol (FMLP and PMA stimulation) were the most effective CLS for PMN. Using 5 µmol/L lucigenin as CLS, small but consistent CL responses could be obtained in macrophages stimulated with PMA, zymosan or oxidized LDL. FMLP‐stimulated extracellular CL in H‐60 cells, HUVEC and macrophages was detected with the greatest sensitivity by pholasin. Our results demonstrate that none of the investigated CLS consistently yielded the highest CL quantum, either in different cell types with one stimulating agent or by different stimulating agents in one cell type. To get the highest CL quantum in experimental studies, we recommend optimizing the CLS depending on the cell type and the ROS‐generating stimulus used. Copyright © 2003 John Wiley & Sons, Ltd.     

Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.     

Lack of effect of pertussis toxin on TNF-alpha-induced formation of reactive oxygen intermediates by human neutrophils

When human PMN were plated on fetal calf serum-coated polystyrene surfaces, addition of TNF-alpha, FMLP or PMA elicited adhesion and H2O2 formation. These effects of TNF-alpha and FMLP, but not of PMA, were impaired by removal of extracellular Ca2+. In addition, H2O2 formation induced by FMLP but not by TNF-alpha or PMA was inhibited by prior treatment with pertussis toxin (250-500 ng/ml). Thus, although the sequelae of TNF-alpha-receptor interaction on human PMN remain to be characterized in detail, they do not involve a pertussis toxin-sensitive GTP-binding protein.     

Study on the generation mechanism of reactive oxygen species on calcium peroxide by chemiluminescence and UV-visible spectra.

In the present work, the generation mechanism of reactive oxygen species (ROS) on calcium peroxide (CaO(2)) was studied. A very intense chemiluminescence (CL) signal was observed when adding an aqueous solution of luminol or 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2alpha]-pyrazin-3-one hydrochloride (MCLA) to a suspension of CaO(2). The ROS released on CaO(2) were thought to be oxidizing agents leading to CL, and were characterized by CL, UV-visible (UV-vis) spectra and the effective scavengers of the special ROS. From experimental results, the hydroxyl (.OH) and superoxide (.O(2) (-)) radicals were suggested to exist on the surface of CaO(2). A reaction scheme for the formation of the ROS on CaO(2) was also proposed and discussed. Of more interest was the finding that the CaO(2) which released the .OH and .O(2) (-) on the surface exhibited good transition properties compared with alkaline-earth metal peroxides of the same group (MgO(2), BaO(2)).     

The early effect of sublethal X-irradiation of phagocytic cells in mouse blood and the influence of cystamine as measured by chemiluminescence

The metabolic burst accompanying phagocytosis of granulocytes (PMN) leads to the generation of activated oxygen species such as O-2, H2O2, 1O2 and OH; which give rise to chemiluminescence (CL) in the presence of luminol. Reliable CL-measurements of stimulated PMN can be carried out in freshly drawn mouse blood, when photon counts are related to the number of PMN. Effects of low dose total body X-irradiation were studied using C57B1/6 mice. It was found that 24 and 48 hours after irradiation (0.24-0.95 Gy) CL of whole blood was slightly decreased. If however CL-counts were related to the number of PMN, an enhanced CL per single granulocyte was recorded. The administration of cystamine leads to an immune stimulating effect of unirradiated animals. In animals, who received 0.95 Gy a distinct radioprotective effect of cystamine can be observed.     

Comparative study of antioxidants as quenchers or scavengers of reactive oxygen species based on quenching of MCLA-dependent chemiluminescence.

The quenching or scavenging effect of non-enzymatic antioxidants against reactive oxygen species (ROS) was studied by comparing the degree of suppression of chemiluminescence (CL) caused by the oxidation of MCLA (methoxylated Cypridina luciferin analogue) by ROS. MCLA-dependent CL caused by O2- was effectively quenched by ascorbic acid, beta-carotene, lycopene and astaxanthin, while it was enhanced by alpha-tocopherol. The CL by 1O2 was quenched effectively by beta-carotene, lycopene and astaxanthin, moderately by ascorbic acid, and slightly by alpha-tocopherol. beta-Carotene and alpha-tocopherol remarkably suppressed the CL when ROS was HO*. The present study revealed that MCLA-dependent CL assay provides a simple and rapid method for the evaluation of antioxidants as a quencher or scavenger against any kind of ROS.     

Time-resolved chemiluminescence study of the TiO2 photocatalytic reaction and its induced active oxygen species.

The time-resolved chemiluminescence (CL) method has been applied to study the TiO(2) photocatalytic reaction on a micros-ms timescale. The experimental set-up for time-resolved CL was improved for confirmation of the unique luminol CL induced by the photocatalytic reaction. The third harmonic light (355 nm) from an Nd:YAG laser was used for the light source of the TiO(2) photocatalytic reaction. Luminol CL induced by this reaction was detected by a photomultiplier tube (PMT) and a preamplifier was used for amplifying the CL signal. Experimental conditions affecting the photocatalytically induced CL were discussed in detail. The involvement of active oxygen species such as .OH, O(2) (.-) and H(2)O(2) in the CL was examined by adding their scavengers. It is concluded that .OH was greatly involved in the CL on a micros-ms timescale, especially in time periods <100 micros after illumination of the pulse laser. On the other hand, CL generated by O(2) (.-) began to increase after 100 micros and became dominant after 2.5 ms. A small part of the CL might be generated by H(2)O(2) on the whole micros-ms timescale. A CL reaction mechanism related with .OH and dissolved oxygen was proposed to explain the photocatalytically induced luminol CL on a micros-ms timescale, especially in periods <100 micros.