Thymoquinone is a potent superoxide anion scavenger

The antioxidant and pro-oxidant effects of thymoquinone (TQ), a natural main constituent of the volatile oil of Nigella saliva seeds, and a synthetic structurally-related tert-butylhydroquinone (TBHQ), were examined in vitro. Both TQ and TBHQ efficiently inhibited iron-dependent microsomal lipid peroxidation in a concentration-dependent manner with median inhibitory concentration (IC50) values of 16.8 and 14.9 microM, respectively. TBHQ was stronger than TQ as a scavenger of 2,2'-diphenyl-p-picrylhydrazyl radical (DPPH) (IC50 = 5 microM, 200 times more active than TQ) and as a scavenger of hydroxyl radical (OH*) with an IC50 of 4.6 microM (approximately 10 times more active than TQ). TQ was more active than TBHQ as a superoxide anion scavenger with IC50 of 3.35 microM compared to 18.1 microM for TBHQ. Only TBHQ significantly promoted DNA damage in the bleomycin-Fe(III) system. The results suggest that both TQ and TBHQ have strong antioxidant potentials through scavenging ability of different free radicals. Moreover, the data indicate that TQ is acting mainly as a potent superoxide anion scavenger.     

Traditional Chinese medicine formula Qing Huo Yi Hao as superoxide anion scavenger in high glucose-treated endothelial cells

Aim:

To investigate the effects of a traditional Chinese medicine formula Qing Huo Yi Hao (QHYH) and its components on hydroxyl radical (HO^•) production in vitro and the activity of QHYH against free radicals in cultured endothelial cells induced by high glucose.

Methods:

Hydroxyl radicals (HO^•) were generated through Fenton reactions in vitro, and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a spin trap to form DMPO/HO^• adducts detected using electron paramagnetic resonance (EPR). Immortalized mouse cerebral microvascular endothelial (bEnd.3) cells were treated with high glucose (35 mmol/L). The free radical scavenging ability of QHYH in the cells was evaluated using EPR. Superoxide dismutase (SOD) was used to identify the free radicals scavenged by QHYH in the cells.

Results:

QHYH and its 8 components concentration-dependently reduced DMPO/HO^• signaling. The DMPO/HO^• adduct scavenging ability of QHYH was 82.2%, which was higher than each individual component. The free radical scavenging ability of 1% QHYH in high glucose-treated bEnd.3 cells was approximately 70%. In these cells, the free radicals were also specifically reduced by SOD (400 U/mL), implying that the free radicals were primarily superoxide anions.

Conclusion:

The results demonstrate that the QHYH formula is potent antioxidant acting as scavenge of superoxide anions in high glucose-treated endothelial cells.     

Effect of dimethylthiourea, a hydroxyl radical scavenger, on cigarette smoke-induced bronchoconstriction in guinea pigs

Cigarette smoke exposure causes bronchoconstriction in guinea pigs by stimulating cholinergic and excitatory nonadrenergic, noncholinergic (eNANC)-nerves in vagus system. The aim of this study is to elucidate the role of hydroxyl radical (OH(-)), contained in cigarette smoke, in bronchoconstriction. Anaesthetized animals were exposed to 80 puffs of smoke for 4 min. Pretreatment with dimethylthiourea, a OH(-) scavenger, significantly inhibited cigarette smoke-induced bronchoconstriction. To investigate its site of action, effects of dimethylthiourea were examined on vagally mediated bronchcoconstriction by electrical stimulation and on the bronchoconstriction by intravenous acetylcholine and neurokinin-A. Dimethylthiourea did not inhibit bronchoconstriction evoked by vagal stimulation, acetylcholine or neurokinin-A. These results suggest that dimethylthiourea inhibits cigarette smoke-induced bronchoconstriction by scavenging the smoke-derived OH(-), but not by inhibiting airway nerve function.     

Analysis of agonist-evoked nitric oxide release from human endothelial cells: role of superoxide anion

1. Dichlorofluorescein oxidation and electrochemical monitoring of in situ nitric oxide (NO) release from cultured human endothelial cells reveals that agonists such as thrombin and histamine simultaneously stimulate transient superoxide production. 2. The duration of *NO release was increased only in the simultaneous presence of extracellular L-arginine and exogenous superoxide dismutase. In contrast, the inhibition of membrane reduced nicotinamide adenine dinucleotide (phosphate) oxidases, the major source of *O2- in endothelial cells, did not prolong *NO release, although extracellular L-arginine was also present. Comparison of these two experimental conditions suggested that H2O2 was involved in the extension of the *NO signal. 3. The present study demonstrates that, in the absence of external L-arginine, *O2- production does not constitute the major pathway controlling the duration of agonist-induced *NO signal. These results suggest that L-arginine and H2O2 act jointly to maintain nitric oxide synthase in an activated form.     

Protection by 6-aminonicotinamide against oxidative stress in cardiac cells.

Oxidative stress at the time of reperfusion is a major aspect of ischemia-reperfusion injury in heart as well as in other organs. There is a continuing interest in development of pharmacological approaches to alleviate this injury. 6-Aminonicotinamide (6AN) has been shown to diminish myocardial necrosis following global ischemia in an isolated rat heart, apparently by limiting the oxidative injury component. We therefore explored the antioxidative potential of 6AN in a model using H9C2(2-1) rat cardiac myoblasts exposed to H2O2 stress. Dependent on the specific protocol, 6AN pretreatment for 6-23 h resulted in a strongly increased cell survival: from 11% to 16% in untreated cells to 56-75% following 6AN treatment. This 6AN-mediated protection was associated with a modest increase (up to 55%) of the cytosolic free Ca2+, and was blocked by ryanodine, but not by verapamil or nifedipine. The protective effect of 6AN was associated with a decrease in total cell content of the reduced glutathione (GSH) by 15-44%, indicative of an oxidative shift in the GSH/GSSG system redox potential. We propose that this redox shift caused an increased Ca2+ leak through ryanodine receptors, reflecting their known sensitivity to redox modulation. In turn, this Ca2+ redistribution appeared to trigger a state of an enhanced antioxidative resistance, somewhat analogous to the phenomenon of Ca2+ preconditioning. Similar to some of the cases of Ca2+ preconditioning, this protected state involved the activity of Ca2+ -independent, but not of Ca2+ -dependent, isoform(s) of protein kinase C.     

Acteoside protects endothelial cells against free radical-induced oxidative stress

The protective effect of acteoside against membrane lipid oxidation and free radical-mediated impairment of endothelial function was investigated. Results showed that iron-mediated oxidative modification of the cell membrane in cultured bovine pulmonary endothelial cells (PAECs) was significantly attenuated by acteoside as measured by thiobarbituric acid-reactive substances (TBARS). Fenton's reagent (H2O2/Fe2+) was used to generate hydroxyl radicals (*OH) and induce oxidative stress. Acteoside not only effectively minimized the loss of cell viability induced by hydroxyl radicals in cultured endothelial cells but also countered the free radical-induced destruction of the endothelium-dependent relaxation to acetylcholine in rat aorta. Furthermore, acteoside showed a dose-dependent scavenging effect of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals and appeared to be the most efficient in comparison with the four reference compounds (alpha-tocopherol, vitamin C, probucol and resveratrol). These data suggested that acteoside protects the cell from oxidative stress and that scavenging of free radicals could be a key mechanism contributing to the cytoprotective effect of acteoside.     

超氧阴离子清除剂对家兔硝酸甘油耐受性影响的实验研究

目的探讨超氧阴离子自由基清除剂对家兔硝酸甘油耐受性的影响。方法取18只家兔按随机数字表分为硝酸甘油+维生素C组、硝酸甘油组、对照组各6只,剃净家兔胸部部分皮毛,对照组置0.9%氯化钠注射液(5mg)贴膜,硝酸甘油组置含5mg硝酸甘油贴膜,硝酸甘油+维生素C组置含5mg硝酸甘油贴膜,同时给予维生素C配10mL 0.9%氯化钠注射液灌胃。观察3组胸主动脉血管环对硝酸甘油的舒张反应、超氧阴离子水平和超氧化物歧化酶(SOD)活性。结果硝酸甘油组对不同浓度硝酸甘油血管舒张反应低于对照组、硝酸甘油+维生素C组(P<0.05);硝酸甘油组血管超氧阴离子含量高于对照组、硝酸甘油+维生素C组(P<0.05)。结论家兔持续经皮贴硝酸甘油72h可产生耐药性,超氧阴离子清除剂对硝酸甘油耐受性有明显改善作用。     

木犀草素减轻氧化应激引起的血管内皮损伤

目的：探讨木犀草素对叔丁基过氧化氢致血管内皮损伤的保护作用及相关机制。方法：首先通过制备大鼠胸主动脉环,观察木犀草素对叔丁基过氧化氢所致血管张力变化的影响;再采用叔丁基过氧化氢诱导血管内皮细胞氧化损伤模型,观察木犀草素对其细胞形态学变化及细胞活力的影响,并用RT-PCR检测eNOS和COX-1 mRNA的含量变化。结果：木犀草素能够浓度依赖性地对抗叔丁基过氧化氢导致的血管舒张功能损伤及细胞损伤作用,且浓度依赖性地减弱叔丁基过氧化氢对内皮细胞eNOS mRNA表达抑制的影响。结论：木犀草素是一种有效的舒血管物质,它可以起到抗氧化的作用,减轻氧化应激反应,并可能通过维持eNOS活性等血管内皮途径舒张血管。     

牛磺酸对高糖诱导的大鼠血管内皮细胞氧化应激与凋亡的保护作用

目的观察牛磺酸对高糖培养诱导大鼠血管内皮细胞氧化应激与凋亡的影响。方法组织块法原代培养大鼠胸主动脉内皮细胞,取对数生长期的第4代内皮细胞进行试验。培养体系中的葡萄糖浓度保持30 mmol/L,加入终浓度为0,2.5,5,10,20 mmol/L的牛磺酸共培养24 h,流式细胞仪检测细胞内活性氧(ROS)水平及细胞凋亡率,比色法测定细胞培养基中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果与0 mmol/L比较,2.5~20 mmol/L组细胞内ROS水平依次降低,组间比较差异均有统计学意义(P<0.05);5和10 mmol/L牛磺酸抑制细胞凋亡作用明显。5~20 mmol/L组MDA含量逐渐降低,而SOD活性逐渐增强。结论牛磺酸能削弱高糖诱导大鼠血管内皮细胞的氧化应激,抑制细胞凋亡。     

Protein-specific S-thiolation in human endothelial cells during oxidative stress.

Confluent human umbilical vein endothelial cells were treated with diamide, t-butyl hydroperoxide (t-BH) or the hydrogen peroxide generating system glucose/glucose oxidase and the effects on glutathione oxidation and protein S-thiolation were examined. In the presence of all three oxidants glutathione was rapidly oxidized to a similar extent and S-thiolation of a limited number of proteins occurred. Diamide caused considerable S-thiolation of proteins with molecular masses of 44, 34, 24 and 14 kDa, of which the protein with molecular mass of 44 kDa was most extensively modified. t-BH caused extensive modification of proteins with molecular masses of 24 and 14 kDa whilst hydrogen peroxide caused S-thiolation of proteins of 39, 24 and 14 kDa. This study shows that S-thiolation of proteins is an important metabolic response to oxidant insult in human endothelial cells and that the specificity of the response depends on the chemical nature of the oxidant.     

Inhibitory effects of azelastine on superoxide anion generation from activated inflammatory cells measured by a simple chemiluminescence method

Blood was drawn from healthy human volunteers and neutrophils and eosinophils were purified on a Conray-Ficoll and a Percoll gradient, respectively. Rat mast cells were also purified on a Percoll gradient. Superoxide anion (O2-) generation from the cells were measured by 2-methyl-6-[p-methoxy-phenyl]-3,7-dihydroimidazo[1,2-a]pyrazin+ ++-3-one (MCLA)-dependent luminescence. Addition of 0.5 mumol/l MCLA and a stimulatory agent, such as phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and compound 48/80, to a suspension of each cell caused a marked luminescence which was inhibited by 0.5 mumol/l superoxide dismutase (SOD). Azelastine (A-5610) significantly inhibited the O2- generation from each activated inflammatory cell in a dose-dependent manner. When eosinophils were activated by fMLP in the presence of cytochalasine (CB), azelastine abolished the luminescence stronger than that from the fMLP-stimulated cells in the absence of CB.     

TEMPOL, a membrane-permeable radical scavenger, attenuates peroxynitrite- and superoxide anion-enhanced carrageenan-induced paw edema and hyperalgesia: a key role for superoxide anion

Carrageenan produces both inflammation and pain when injected in rat paws via enhancement of the formation of reactive oxygen species. We have tested the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), a membrane-permeable superoxide dismutase (SOD) mimetic in carrageenan-induced rat paw edema. Treatment of rats with TEMPOL (15, 30, and 60 mg/kg, 15 min prior to carrageenan) inhibited the paw edema. Furthermore, treatment of rats with the SOD inhibitor diethylthiocarbamate (DETCA, 100 mg/kg, 1 h before carrageenan) enhanced the carrageenan-induced paw edema. Co-administration of peroxynitrite with carrageenan produced a similar fortification of the carrageenan-induced edema. Prior treatment of rats with TEMPOL (30 mg/kg) inhibited the enhancement produced by DETCA treatment (endogenous superoxide anion stress) as well as that produced by the peroxynitrite stress. The effect of TEMPOL as well as the influence of superoxide anion and peroxynitrite stresses was also tested in carrageenan-induced hyperalgesia model. Carrageenan (500 mug/paw) produced significant hyperalgesia presented as shortening of withdrawal latency times using hot plate (52 degrees C) starting 30 min after carrageenan and lasting for 3 h. TEMPOL (60 mg/kg, injected 15 min before carrageenan) ameliorated this hyperalgesia during the first 2 h. Concurrent administration of peroxynitrite promptly intensified the carrageenan hyperalgesia. TEMPOL (60 mg/kg, 15 min before peroxynitrite-carrageenan) inhibited the peroxynitrite enhancement of carrageenan hyperalgesia when tested at 60 min after injection of the cocktail. The present investigation gives the proof for the effectiveness of TEMPOL as anti-inflammation and analgesic agents in carrageenan-induced model of inflammation and hyperalgesia. It further indicated the importance of superoxide anion and peroxynitrite in acute inflammation and inflammatory pain. This raises the chances for considering pharmacologic interventions that interrupt superoxide anion and peroxynitrite stress for putative alternative agents as anti-inflammatory analgesic new medical strategies.     

Effects of phenolcarboxylic acids on superoxide anion and lipid peroxidation induced by superoxide anion.

The effects of phenolcarboxylic acids, caffeic acid, p-coumaric acid, and ferulic acid on the generation of superoxide anion and the production of lipid peroxide induced by superoxide anion were studied. Only ferulic acid anion among the phenolcarboxylic acids scavenged superoxide. Caffeic acid and ferulic acid inhibited lipid peroxidation induced by superoxide anion. These effects were comparable to those of superoxide dismutase or DL-alpha-tocopherol.     

Ascorbic acid: a scavenger of superoxide radical

The second order rate constant for the reaction between ascorbic acid and superoxide at pH 7.4 using the xanthine-xanthine oxidase system was estimated to be 5.4 x 10(6) M-1 sec-1. The results indicate that the efficacies of superoxide dismutase and ascorbic acid for catalyzing the decay of superoxide radical in animal tissues are similar. The significance of ascorbic acid as a scavenger of superoxide is discussed from the point of view of evolution of ascorbic acid synthesizing capacity in the terrestrial vertebrates.     

Single-wall carbon nanotubes induce oxidative stress in rat aortic endothelial cells

Oxidative stress is a major factor contributing to endothelial cell damage. Single-wall carbon nanotubes (SWCNTs) have oxidative properties; however, the oxidative effects of SWCNTs on endothelial cells are not fully understood. In the present study, we investigated the effects of oxidative stress induced by SWCNTs on rat aortic endothelial cells (RAECs). Various markers of cellular damage were assessed, such as biochemical and ES immunity indexes, and DNA and protein damage. Our findings suggest that RAEC endured oxidative damage following SWCNT exposure. Specifically, after SWCNTs exposure, non-enzymatic antioxidant glutathione was activated prior to superoxide dismutase activation in order to defend against oxidative stress. Additionally, it was found that as SWCNT concentration increased, so did the stress protein, heme oxygenase-1 (HO-1), expression levels. These changes may induce RAEC damage, and result in many serious diseases.     

Single-wall carbon nanotubes induce oxidative stress in rat aortic endothelial cells

Oxidative stress is a major factor contributing to endothelial cell damage. Single-wall carbon nanotubes (SWCNTs) have oxidative properties; however, the oxidative effects of SWCNTs on endothelial cells are not fully understood. In the present study, we investigated the effects of oxidative stress induced by SWCNTs on rat aortic endothelial cells (RAECs). Various markers of cellular damage were assessed, such as biochemical and ES immunity indexes, and DNA and protein damage. Our findings suggest that RAEC endured oxidative damage following SWCNT exposure. Specifically, after SWCNTs exposure, non-enzymatic antioxidant glutathione was activated prior to superoxide dismutase activation in order to defend against oxidative stress. Additionally, it was found that as SWCNT concentration increased, so did the stress protein, heme oxygenase-1 (HO-1), expression levels. These changes may induce RAEC damage, and result in many serious diseases.