5种石斛及其组织培养物对活性氧的清除作用

采用化学发光法,以3种活性氧R0S(02、     

Imaging mitochondrial reactive oxygen species with fluorescent probes: Current applications and challenges

Abstract

Mitochondrial reactive oxygen species (ROS) is a key element in the regulation of several physiological functions and in the development or progression of multiple pathological events. A key task in the study of mitochondrial ROS is to establish reliable methods for measuring the ROS level in mitochondria with high selectivity, sensitivity, and spatiotemporal resolution. Over the last decade, imaging tools with fluorescent indicators from either small-molecule dyes or genetically encoded probes that can be targeted to mitochondria have been developed, which provide a powerful method to visualize and even quantify mitochondrial ROS level not only in live cells, but also in live animals. These innovative tools that have bestowed exciting new insights in mitochondrial ROS biology have been further promoted with the invention of new techniques in indicator design and fluorescent detection. However, these probes present some limitations in terms of specificity, sensitivity, and kinetics; failure to recognize these limitations often results in inappropriate interpretations of data. This review evaluates the recent advances in mitochondrial ROS imaging approaches with emphasis on their proper application and limitations, and highlights the future perspectives in the development of novel fluorescent probes for visualizing all species of ROS.     

Scavenging of reactive oxygen species by the plant phenols genistein and oleuropein.

The plant-derived phenolic compounds genistein and oleuropein are known to exhibit several biological properties, many of which may result from their antioxidant and free radical scavenger activity. In this paper we report the results of a complex study of antioxidant activity of genistein and oleuropein, using electron spin resonance (ESR), chemiluminescence, fluorescence and spectrophotometric techniques. Different reaction systems were applied to study the inhibitory effect of the phenolic compounds studied: (a) the potassium superoxide/18-crown-6 dissolved in DMSO system, which generates superoxide radical (O(2).(-)) and hydrogen peroxide (H(2)O(2)); (b) the Co(II)-EDTA-H(2)O(2) system (the Fenton-like reaction), which generates hydroxyl radical (HO.); (c) 2,2'-azobis(2-amidino-propane)dichloride (AAPH) as the peroxyl radical (ROO.) generator, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical test. Results showed that genistein and oleuropein decreased the chemiluminescence sum from the O(2).(-) generating system, an inhibitory effect that was dependent on their concentration. These compounds also reacted with ROO radicals and they showed activity about two-fold greater than the standard Trolox. The antioxidant effects were studied at different concentrations and reflected in protection against the fluorescence decay of beta-phycoerythrin (beta-PE), due to ROO. attack on this protein. Using the Fenton-like reaction and the spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the phenolic compounds examined were found to inhibit DMPO-.OH radical formation in the range 10-90% at concentrations of 0.1 mmol/L to 2 mmol/L. Furthermore, these compounds also inhibited HO.-dependent deoxyribose degradation; about 20% and 60% inhibitions were observed in the presence of 0.5 mmol/L genistein and oleuropein, respectively. It was also demonstrated that genistein had a weaker DPPH radical scavenging activity than oleuropein. Our results confirm good scavenging activity towards O(2).(-), HO. and ROO. and the antioxidant effect of genistein and oleuropein.     

Reactivity of pyruvic acid and its derivatives towards reactive oxygen species

Pyruvic acid and its derivatives occurring in most biological systems are known to exhibit several pharmacological properties, such as anti‐inflammatory, neuroprotective or anticancer, many of which are suggested to originate from their antioxidant and free radical scavenger activity. The therapeutic potential of these compounds is a matter of particular interest, due to their mechanisms of action, particularly their possible antioxidant behaviour. Here, we report the results of a study of the effect of pyruvic acid (PA), ethyl pyruvate (EP) and sodium pyruvate (SP) on reactions generating reactive oxygen species (ROS), such as superoxide anion radicals, hydroxyl radicals and singlet oxygen, and their total antioxidant capacity. Chemiluminescence (CL) and spectrophotometry techniques were employed. The pyruvate analogues studied were found to inhibit the CL signal arising from superoxide anion radicals in a dose‐dependent manner with IC₅₀ = 0.0197 ± 0.002 mM for EP and IC₅₀ = 69.2 ± 5.2 mM for PA. These compounds exhibited a dose‐dependent decrease in the CL signal of the luminol + H₂O₂ system over the range 0.5–10 mM with IC₅₀ values of 1.71 ± 0.12 mM for PA, 3.85 ± 0.21 mM for EP and 22.91 ± 1.21 mM for SP. Furthermore, these compounds also inhibited hydroxyl radical‐dependent deoxyribose degradation in a dose‐dependent manner over the range 0.5–200 mM, with IC₅₀ values of 33.2 ± 0.3 mM for SP, 116.1 ± 6.2 mM for EP and 168.2 ± 6.2 mM for PA. All the examined compounds also showed antioxidant capacity when estimated using the ferric–ferrozine assay. The results suggest that the antioxidant activities of pyruvate derivatives may reflect a direct effect on scavenging ROS and, in part, be responsible for their pharmacological actions. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Mitochondrial metabolism of reactive oxygen species

Oxidative stress is considered a major contributor to etiology of both normal senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 246–264.Original Russian Text Copyright © 2005 by Andreyev, Kushnareva, Starkov.This revised version was published online in April 2005 with corrections to the post codes.     

活性氧在气孔运动中的作用

水分代谢是植物基础代谢的重要组成部分,气孔开关精细地调节着植物水分散失和光合作用。气孔运动受到多种因子的调控,保卫细胞内大量的第二信使分子是响应外界刺激、调节保卫细胞代谢方式、改变保卫细胞水势进而引起气孔开关的重要功能组分。细胞内的活性氧就是其中重要的成员之一。保卫细胞中的活性氧包括过氧化氢、超氧阴离子自由基和羟自由基等,这些活性氧可以通过光合作用、呼吸作用产生或通过专门的酶催化合成,在触发下游生理反应、完成信号转导后由专门的酶将其清除。在植物激素(脱落酸、水杨酸)、一氧化氮、质外体钙调素、细胞外ATP等因子调节气孔运动的过程中,活性氧都发挥了介导作用。该文对于近年来活性氧在气孔运动过程中发挥的作用方面的研究进展进行了综述。     

两株乳酸杆菌SY13和LJJ对活性氧的耐受性

【目的】从传统发酵乳制品中筛选具有抗氧化能力的乳酸菌菌株并对其抗氧化特性进行评价。【方法】分别利用乳酸杆菌SY13和LJJ完整细胞和无细胞提取物对亚油酸过氧化的抑制效果,对DPPH自由基、羟自由基、超氧阴离子自由基清除能力,对过氧化氢的耐受性以及对亚铁离子的螯合能力和还原活性进行了研究。【结果】结果表明,SY13和LJJ对亚油酸过氧化的最大抑制率分别达到了62.95%和66.16%;两菌株的无细胞提取物清除超氧阴离子和羟自由基的的效果较好,LJJ完整细胞对超氧阴离子和羟自由基没有清除能力;SY13和LJJ对DPPH自由基的清除能力及对亚铁离子的螯合能力都是完整细胞优于无细胞提取物,还原活性分别相当于305 μmol/L和294 μmol/L的L-cysteine。【结论】以上指标测定的结果说明,这两株乳酸杆菌具有较好的抗氧化能力,具有潜在的应用价值。     

Role of Ca2+ in activation of reactive oxygen species production in polymorphonuclear leukocytes during tumour growth in rats.

The role of Ca(2+) ions in PMA-induced generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) was studied during Zajdela hepatoma growth in the peritoneal cavity of rats. In PMNL from control healthy animals, a manifold Ca(2+)-induced enhancement of ROS generation and its significant reduction in the presence of Ca(2+) binding agent (BAPTA-AM) were observed. In contrast, ROS generation by PMNL from tumour-carrying animals dramatically increased in Ca(2+)-free medium, being practically insensitive to the agents, which can increase or decrease intracellular Ca(2+) levels. Free cytosolic Ca(2+) ([Ca(2+)](i)) in control PMNL was found to be relatively low ( approximately 250 nmol/L), rising slowly after Ca(2+) addition and further to two-fold in the presence of Ca(2+) and ionomycin in the incubating medium. Tumour growth in animals was accompanied with a significant [Ca(2+)](i) elevation. In Ca(2+)-free medium, [Ca(2+)](i) elevation was up to 480 nmol/L in tPMNL with the additions of Ca(+) and ionomycin as well as EGTA and ionomycin being able to increase [Ca(2+)](i) to 700-900 nmol/L onward. It was concluded that a higher Ca(2+) permeability of the plasma membrane and higher Ca(2+) accumulation in intracellular pools of PMNL was developed at the advanced stages of malignant disease. These results indicate the primed state of circulating PMNL and the independence of PMA-induced ROS generation at intra- and extracellular Ca(2+) levels at the advanced stages of tumour growth in animals.     

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。     

Anatomically specific reactive oxygen species production participates in Marfan syndrome aneurysm formation

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1^C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin‐enhanced chemiluminescence (LGCL), Verhoeff's elastin‐Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1^C1039G/+ AS‐ or DES‐derived smooth muscle cells (SMC) were treated with anti‐TGF‐β antibody, angiotensin II (AngII), anti‐TGF‐β antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1^C1039G/+ AS aorta, but absent in normal‐sized DES aorta. Fbn1^C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1^C1039G/+‐derived AS SMC had increased NADPH activity compared to DES‐derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF‐β dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF‐β dependent.     

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH₂) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH₂-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

Abstract:

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

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)).     

Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals

In this study reactive oxygen species (ROS) generated in the respiratory chain were measured and the quantitative relationship between inhibition of the respiratory chain complexes and ROS formation was investigated in isolated nerve terminals. We addressed to what extent complex I, III and IV,respectively, should be inhibited to cause ROS generation. For inhibition of complex I, III and IV, rotenone, antimycin and cyanide were used, respectively, and ROS formation was followed by measuring the activity of aconitase enzyme. ROS formation was not detected until complex III was inhibited by up to 71 +/- 4%, above that threshold inhibition, decrease in aconitase activity indicated an enhanced ROS generation. Similarly, threshold inhibition of complex IV caused an accelerated ROS production. By contrast, inactivation of complex I to a small extent (16 +/- 2%) resulted in a significant increase in ROS formation, and no clear threshold inhibition could be determined. However, the magnitude of ROS generated at complex I when it is completely inhibited is smaller than that observed when complex III or complex IV was fully inactivated. Our findings may add a novel aspect to the pathology of Parkinson's disease, showing that a moderate level of complex I inhibition characteristic in Parkinson's disease leads to significant ROS formation. The amount of ROS generated by complex I inhibition is sufficient to inhibit in situ the activity of endogenous aconitase.     

The effect of electromagnetic field on reactive oxygen species production in human neutrophils in vitro

The present study was undertaken in order to determine the effect of low frequency electromagnetic field (EMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. We investigated how differently generated EMF and several levels of magnetic induction affect ROS production. To evaluate the level of ROS production, two fluorescent dyes were used: 2′7′-dichlorofluorscein-diacetate and dihydrorhodamine. Phorbol 12-myristate 13-acetate (PMA), known as strong stimulator of the respiratory burst, was also used. Alternating magnetic field was generated by means of Viofor JPS apparatus. Three different levels of magnetic induction have been analyzed (10, 40 and 60 μT). Fluorescence of dichlorofluorescein and 123 rhodamine was measured by flow cytometry. The experiments demonstrated that only EMF tuned to the calcium ion cyclotron resonance frequency was able to affect ROS production in neutrophils. Statistical analysis showed that this effect depended on magnetic induction value of applied EMF. Incubation in EMF inhibited cell activity slightly in unstimulated neutrophils, whereas the activity of PMA-stimulated neutrophils has increased after incubation in EMF.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Reactive oxygen species stimulate homologous recombination in plants

Coping with the continuous production of free radicals is a daily routine of the cell. Despite their toxicity, the reactive oxygen species (ROS) are involved in dual physiological action – signal transduction and immune response. We analysed the influence of oxidative stress‐generating compounds, rose Bengal (RB), paraquat (PQ) and amino‐triazole (ATZ) on the genome stability of Arabidopsis using transgenic recombination‐monitoring plants. Homologous recombination frequencies in plants were increased upon the treatment with RB and PQ but not ATZ. Application of the N‐acetyl‐L ‐cysteine (NAC), radicals scavenging compound, decreased the DNA damage caused by RB. Interestingly, the incubation of plants with very low concentration of RB (less than 0.2 µM ) led to the subsequent increase in plant tolerance to methyl methane sulfonate (MMS): stronger plants with a lower increase of homologous recombination frequency. In contrast, the incubation of plants with 0.5 µM of RB resulted in the potentiation of the MMS effect: the weaker plants with higher frequency of recombination. The data of the present study suggest the existence of a dual concentration‐dependent role of ROS in plants.     

植物线粒体、活性氧与信号转导

活性氧(ROS)的产生是需氧代谢不可避免的结果。在植物细胞中,线粒体电子传递链(ETC)的复合物Ⅰ和Ⅱ是ROS产生的主要的部位。交替氧化酶和可能的内源鱼藤酮不敏感的NADH脱氢酶通过保持ETc的相对氧化状态限制线粒体产生ROS。线粒体基质中的抗氧化酶系统与小分子量的抗氧化剂一道起ROS的解毒作用。ROS除了引起细胞的伤害外,在植物中还能够作为一种普遍存在的信号分子起作用。在低浓度时,ROS能诱导防御基因的表达和引起适应反应;在高浓度时,引起细胞死亡。一氧化氮是植物合成和释放的一种气体,也可作为信号分子调节植物的生长和发育。