胡椒碱减轻兔原代左心房肌细胞氧化应激损伤的作用

目的:利用低浓度过氧化氢（H2O2）建立兔原代左心房肌细胞氧化损伤模型,探讨胡椒碱减轻氧化应激损伤的保护作用。方法: 将18只新西兰白兔随机分为3组,每组6只:即正常对照(NC)组、H2O2组和胡椒碱组,均分别进行左心房肌细胞的原代培养。NC组对培养的心房细胞直接进行检测,H2O2组直接在培养的原代心房肌细胞中加入终浓度为100 μmol/L的H2O2培养2 h,胡椒碱组以7×10-6 mol/L浓度的胡椒碱处理细胞1 h之后,加入终浓度为100 μmol/L的H2O2共同培养2 h。检测氧化和抗氧化指标的变化。MTT法检测三组原代细胞活力,黄嘌呤氧化酶法检测超氧化物歧化酶（SOD）活力、比色法检测丙二醛（MDA）含量及还原型谷胱甘肽（GSH）含量,Fura-2 AM检测细胞内钙离子浓度,RT-PCR对线粒体mRNA进行定量分析。结果: 与NC组相比较,H2O2组细胞的活力、SOD的活力及GSH的含量明显下降（P<0.05）; MDA的含量、钙离子浓度和线粒体mRNA的表达均明显增加（P<0.05）。胡椒碱组和H2O2组比较,上述指标均有显著改善（P<0.05）。结论: 胡椒碱能够在氧自由基的产生清除等环节,减轻兔原代左心房肌细胞的氧化应激损伤。     

Maturation-associated increase in sensitivity of cultured guinea pig gastric pit cells to hydrogen peroxide

Guinea pig gastric pit cells in primary culture undergo serum-dependent cell maturation, which mimics their maturation in vivo. In this study we compared the sensitivities of these cells, as a function of early- and late-stage cell maturation, to various gastric stressors. Gastric pit cells in the early stage of maturation (two days of culture) were more resistant to apoptotic cell death induced by exposure to hydrogen peroxide than those cells in late-stage maturation (three days of culture). This maturation-associated difference was not observed for the sensitivities of cells to necrotic cell death induced by hydrogen peroxide, nor for necrotic and apoptotic cell death induced by other gastric stressors (ethanol and hydrochloric acid). The activities of catalase and glutathione peroxidase were specifically decreased in cells at the late stage of maturation compared to those at the early stage. The relative gastric pit cell phenotype sensitivity to hydrogen peroxide at the late stage of maturation may be due to a decrease in the activities of catalase and glutathione peroxidase, which are antioxidants for hydrogen peroxide.     

The occurrence of oxidative stress during reperfusion in experimental animals and men

Reperfusion is the prerequisite for the ischemic myocardium to recover its metabolic and mechanical function. However, reperfusion after a prolonged period of ischemia in the experimental animal may exacerbate, or at least accelerate, the occurrence of ischemic injury, whilst in humans at the least it is not beneficial. This entity has been called reperfusion damage, since much of the damage is believed to be caused by events occurring at the moment of reperfusion rather than by changes occurring during ischemia. The existence of reperfusion damage, however, has been questioned, and evidence in favour of the concept is sparse. At the moment the molecular events occurring at the time of reperfusion are not completely understood, and the relative importance of several proposed deleterious mechanisms is not yet established. One of the most fashionable ideas for the cause of reperfusion damage is that the function of cell membrane is modified by oxygen radicals generated at the moment of reperfusion. Evidence in favour of and against this hypothesis is described in detail in the present article.     

Ventricular arrhythmias following exposure of failing hearts to oxidative stress in vitro

Introduction: There is experimental evidence that heart failure (HF) is an oxidative stress and that HF myocytes may be damaged by oxygen-derived free radicals. However, the arrhythmogenicity of these radicals has not been studied in HF.
Methods and Results: Isolated perfused hearts were obtained from sham-operated (SHAM, n = 6), and fast pacing (250 ms, 2 weeks)-induced heart failure porcines (HF, n = 8). Epicardial conduction was mapped in the longitudinal and transverse directions and ventricular arrhythmias were closely monitored after perfusion of 100, 300, and 1000 μmol/L H ₂ O ₂. Left ventricular epicardium was sampled for action potentials recordings in the same conditions. Myocardial levels of thiobarbituric acid reactive substances and antioxidant enzymatic capacity were also assessed. Epicardial conduction velocities were unaffected by H ₂ O ₂ in both groups. Isolated ventricular premature beats and runs of slow ventricular rhythm with H ₂ O ₂ more frequently occurred in HF compared to SHAM despite an increased antioxidant capacity including Cu/Zn and Mn superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase. Sustained arrhythmias were not observed. Higher thiobarbituric acid reactive substances levels were found in HF confirming endogenous oxidative stress. Action potential duration at plateau level was increased following H ₂ O ₂ in SHAM but not in HF epicardial fibers where a toxic effect developed at 1000 μmol/L.
Conclusion: Oxidative stress with concomitant increase in antioxidant capacity develops in this HF model. There is a greater proclivity to oxidative stress-mediated arrhythmias in HF. These arrhythmias are mainly extrasystoles or slow ventricular rhythms and not dependent on abnormal myocardial conduction.     

Mechanisms of streptozotocin- and alloxan-induced damage in rat B cells

Summary In studies to evaluate possible inhibitors of the B-cell toxin, streptozotocin, the superoxide scavenger, superoxide dismutase, did not prevent or reduce the toxic effects of streptozotocin as determined by loss of insulin secretion from rat pancreatic B cells in monolayer culture. However, 1,1-dimethyl urea, a scavenger of the hydroxyl radical, did afford significant protection. Both scavengers diminished the cytotoxic effects of alloxan. The inhibitors of poly (ADP-ribose) synthetase, 3-aminobenzamide and nicotinamide, also were effective in attenuating alloxan- and streptozotocin-induced B-cell toxicity. Tests of the hydroxyl-scavenging ability of the three streptozotocin antagonists revealed that 3-aminobenzamide, nicotinamide and 1,1-dimethyl urea were effective scavengers of this free radical. Conversely, 1,1-dimethyl urea, although not as potent as 3-aminobenzamide or nicotinamide, was found to inhibit poly (ADP-ribose) synthetase. These data indicate that these chemicals most likely attenuate alloxan-induced toxicity by scavenging the hydroxyl radical and diminish streptozotocin-induced toxicity by inactivation of the poly (ADP-ribose) system.     

Effects of deferoxamine on H2O2-induced oxidative stress in isolated rat heart

During myocardial reperfusion injury, iron has been implicated in the Fenton based generation of hydroxyl radical, ·OH, leading to further organ injury. Although previous studies have investigated the protective effect of iron chelators including deferoxamine (DFX) in myocardial reperfusion injury, there is little information regarding the role of iron chelation during oxidative stress produced by H₂O₂ on the heart. Isolated hearts from male Sprague-Dawley rats were retrograde-perfused with Krebs-Henseleit solution at 5 ml/min. After a 60-min equilibration, oxyradical challenge was instituted by the addition of H₂O₂ (200–600 M) to the perfusate for 60 min. A subgroup of animals received DFX (400 M) in the perfusate prior to challenge with 400 M H₂O₂. Contractility was continuously monitored; perfusate samples for glutathione (GSH) and lactate dehydrogenase (LDH) estimations were collected at 30-min intervals. Headspace ethane, an indicator of lipid peroxidation, was estimated at 30-min intervals by gas chromatography. Control hearts maintained contractility during the perfusion period. H₂O₂ perfusion caused a dose dependent decrease in myocardial contractility; DFX pretreatment was partialy protective. Headspace ethane slowly accumulated in control hearts; perfusion with H₂O₂ caused dose dependent increase in ethane accumulation indicative of enhanced lipid peroxidation. GSH and LDH in the perfusate remained low in control hearts. In contrast, H₂O₂ treated hearts had a dose dependent inclease in the efflux of GSH and LDH which was markedly increased by perfusion with 600 M H₂O₂. Pretreatment with DFX did not significantly reduce GSH or LDH efflux from hearts perfused with peroxide. While H₂O₂ perfused with peroxide. While H₂O₂ perfusion causes a dose dependent decrease in myocardial contractility with a corresponding increase in headspace ethane release with GSH & LDH efflux indicative of oxidative stress, concurrent treatment with DFX reduces myocardial dysfunction and ethane generation. However, sublethal damage of plasma membrane still continues as reflected by continuous enhancement of LDH efflux, possibly indicating involvement of other reactive species besides hydroxyl radical.     

Taurine preserves gap junctional intercellular communication in rat hepatocytes under oxidative stress

Gap junctional intercellular communication (GJIC) between hepatocytes is important for the maintenance of differentiated liver functions. Taurine is known to be cytoprotective, and is used clinically to improve liver functions. We evaluated the effect of taurine on GJIC in hepatocyte doublets under oxidative stress. Hepatocyte doublets were isolated from female Wistar rats, using a collagenase perfusion technique, and cultured in Leibovitz-15 medium containing fetal bovine serum (10%). H₂O₂ (2 mM) and/or taurine (0.1–1 mM) were added 2 h after inoculation, and the culture was incubated for 3 h. Fluorescent dye (Lucifer Yellow CH) coupling between adjacent cells was evaluated by microinjection. The distribution and quantity of connexin 32 (Cx32) in hepatocytes were detected using indirect immunofluorescence analysis and Western blotting. Steady state mRNA levels of Cx32 were detected by Northern blotting. The percentage of dye coupling 5 h after inoculation was 88 ± 6.3% in the control, however, this was decreased to almost half the control value by H₂O₂. Taurine prevented the decrease caused by H₂O₂ in a dose-dependent manner. Immunofluorescence analysis for Cx32 demonstrated numerous punctate fluorescent spots along the intercellular plasma membrane in controls, which were significantly decreased by H₂O₂. Taurine prevented the decrease of Cx32. Western blot analysis also showed the decrease of Cx32 protein levels by H₂O₂ treatment, which decrease was prevented by taurine. Interestingly, H₂O₂ and/or taurine treatments did not affect Cx32 mRNA levels. Our findings indicated that H₂O₂ treatment decreased GJIC between hepatocytes, most likely due to augmenting the degradation of Cx32 proteins, whereas taurine prevented this process. This effect of taurine is beneficial for the preservation of differentiated functions in the liver under oxidative stress. Received: August 12, 1999 / Accepted: November 26, 1999     

血管过氧化物酶1介导的氧化应激在心血管疾病中的研究进展

血管过氧化物酶1(VPO1)是心血管系统新近发现的血红素过氧化物酶超家族成员,可催化NADPH氧化酶(NOX)来源的过氧化氢(H₂O₂)生成次氯酸(HClO),进而加重氧化应激,在高血压、动脉粥样硬化、心肌梗死和肺动脉高压等多种心血管疾病的发生发展过程中具有重要作用。本文主要就VPO1介导的氧化应激在心血管疾病中的作用及潜在机制进行综述。     

Increased susceptibility of microcytic red blood cells to in vitro oxidative stress

Abstract: Oxidative damage to erythrocytes in thalassaemia has been related to generation of free radicals by an excess of denaturated α- or β-globin chains, intracellular iron overload and low concentration of normal haemoglobin (HGB). Two good indicators of such oxidative damage are the high red blood cell (RBC) malonyldialdehyde (MDA) production detected following exogenous oxidant stress and the decrease of pyrimidine 5′-nucleotidase (P5N), the most sensitive enzyme to SH-group damage in vivo. Conflicting data, however, have so far accumulated in the literature concerning differences in oxidative damage between the different forms of thalassaemia and iron deficiency anaemia (IDA). In the present study, oxidative susceptibility, as defined by the production of MDA in vitro and antioxidant capacity, as measured by the activity of RBC glutathione peroxidase (GPx), superoxide dismutase (SOD) and by reduced glutathione (GSH), have been studied in microcytic RBCs from patients with β-thalassaemia trait, Spanish (δβ)°-thalassaemia heterozygotes (δβ-thalassaemia trait) and iron deficiency anaemia (IDA). The results are consistent with the existence of significant differences in the severity and pattern of oxidative stress susceptibility between β-thalassaemia trait (increased MDA production and higher SOD and GPx activities) and the other two forms of microcytosis (δβ thalassaemia trait and IDA). Furthermore, the finding of normal P5′N activity in δβ thalassaemia trait, gives further support to the less intense peroxidative environment of RBCs in this form of thalassaemia when compared to β-thalassaemia trait, characterized by acquired RBC P5′N deficiency due to oxidative damage.     

Hormone sensitivity of gynecological tumor cells in tissue culture

Summary Proliferating tumor cells obtained from ovarian, mammary, and endometrial tumors in tissue culture were tested for the influence of proteohormones and steroid hormones on cellular DNA synthesis and cell growth. The gonadotropic hormones stimulated DNA synthesis of ovarian tumor cells by single administration, or in combination with cortisol, up to the 11-fold of the comparable controls. The hormone sensitivity of the cell lines was variable, resulting in individual reaction patterns. There was no correlation to the histological diagnosis of the primary tumors with respect to the grade of differentiation. The results suggest that ovarian tumor cells in tissue culture can maintain sensitivity to organotropic hormones. Compared to the ovarian carcinoma lines, mammary or endometrial tumor cells did not respond to a similar extent. Progesterone decreased DNA synthesis of endometrial carcinoma cells.     

Hemeoxygenase-1 expression in response to arecoline-induced oxidative stress in human umbilical vein endothelial cells

Background

Arecoline, the most abundant areca alkaloid, has been reported to stimulate reactive oxygen species (ROS) production in several cell types. Overproduction of ROS has been implicated in atherogenesis. Hemeoxygenase-1 (HO-1) has cytoprotective activities in vascular tissues. This study investigated the effect of arecoline on adhesion molecule expression and explored the role of HO-1 in this process.

Methods

Human umbilical vein endothelial cells (HUVECs) were treated with arecoline, then ROS levels and the expression of adhesion molecules and HO-1 were analyzed and potential signaling pathways investigated.

Results

After 2 h of arecoline treatment, ROS production was stimulated and reached a maximum at 12 h. Expression of the adhesion molecules ICAM and VCAM was also induced. Glutathione pretreatment completely blocked arecoline-stimulated ROS production and VCAM expression, but not ICAM expression. Arecoline also induced HO-1 expression and this effect was partly due by ROS stimulation. Inhibition of c-jun N-terminal kinase (JNK) by SP600125, p38 by SB 203580, or tyrosine kinase by genistein reduced arecoline-induced HO-1 expression. In contrast, inhibition of ERK (extracellular signal-related MAP kinase) by PD98059 had no effect. Transfection of HUVECs with the GFP/HO-1 gene, which resulted in a 5-fold increase in HO-1 activity, markedly, but not completely, inhibited the decrease in cell viability caused by arecoline.

Conclusions

This study demonstrates that, in HUVECs, arecoline stimulates ROS production and ICAM and VCAM expression. HO-1 expression is also upregulated through the ROS, tyrosine kinase, and MAPK (JNK and p38) signaling pathways.     

Influence of insulin on sensitivity to tetrodotoxin of isolated chick embryo heart cells in culture

A progressive evolution of sensitivity to TTX and to Mn²⁺ was observed in isolated chick embryo heart cells in culture. TTX-insensitivity progressively develops and affects the whole cell population after 48 h of cultivation. At the same time, spontaneously beating cells become sensitive to Mn ions which inhibit their activity. Insulin prevents the decrease of TTX-sensitivity of the cell population during the 4 to 48 h time of cultivation. It is assumed that the artificial conditions of the tissue culture method are partly responsible for the changes of the cell membrane properties. An improvement of these conditions by adding insulin to the culture medium can inhibit the loss of normal properties such as TTX-sensitivity. Whether insulin acts by influencing the value of the resting potential or by controlling protein synthesis in the cultured cells remain unknown.     

氧自由基诱导鸭乙型肝炎病毒DNA整合及其细胞传代研究

目的探讨氧自由基对鸭乙型肝炎病毒（DHBV）DNA在受感染细胞DNA上整合发生的影响以及整合后DHBVDNA的细胞传代性。方法选择鸡肝癌细胞株—LMHD21-6,在其由单个细胞培养至3×107～5×107个细胞（共23代）的过程中,用低浓度H2O2（10.0μmol/L）作用于细胞;TUNEL法检测细胞DNA链断裂情况;Southernblot技术观察细胞DNA上新的整合型DHBVDNA的发生情况。结果(1)低浓度H2O2（≤10.0μmol/L）导致的细胞死亡率仅为32.0%,但可诱导细胞DNA链断裂致细胞调亡,而H2O2浓度＞10.0μmol/L时其细胞死亡率＞50.0%;(2)低浓度H2O2作用的细胞DNA上新的DHBVDNA整合发生率为50.0%（6/12）,而无H2O2作用的发生率仅8.33%（1/12）,两者比较差异有显著意义（P＜0.05＝;(3)含新的整合型DHBADNA的细胞在无H2O2作用下,再由单个细胞培养至第23代,其子代细胞DNA上可见与其母代细胞DNA上新的整合型DHBVDNA碱基大小完全一致的DHBVDNA带。结论氧自由基为DHBVDNA整合发生的重要诱因之一,新的整合型DHBVDNA可有较稳定的细胞传代。     

The oxidative status of blood cells in a murine model of graft-versus-host disease

We studied the oxidative status of red and white blood cells during the development of graft vs host disease (GVHD) as well as the effects of treatment with antioxidants, both in vitro and in vivo. (BALB/c X C57BL/6) F1 mice were conditioned by total body radiation and, 1 day later, transplanted with semi-allogeneic C57BL/6 spleen cells. GVHD was followed by its clinical manifestations. Oxidative stress in red blood cells (RBC), neutrophils, and lymphocytes was assessed by measuring generation of reactive oxygen species and the content of reduced glutathione by flow cytometry after gating of the specific populations. Oxidative stress was noticed 3 weeks after transplantation. It was higher in mice receiving allogeneic spleen cells as compared with mice transplanted with syngeneic cells, suggesting that it was associated with GVHD. The results also demonstrated that treatment with the antioxidants N-acetylcysteine and a derivative of vitamin E (tocopherol succinate, propofol), both in vitro and in vivo, reduced the oxidative stress. The results indicate that various blood cells, including RBC, neutrophils, and lymphocytes, are under oxidative stress and that treatment with antioxidants reduced the stress and, thus, may be useful in ameliorating the severe consequences of GVHD. Johnny Amer and Lola Weiss contributed equally to this work.     

高浓度尿酸对人近端肾小管上皮细胞氧化应激的影响

目的观察高浓度尿酸对人近端肾小管上皮细胞氧化应激的影响,探讨其可能的作用途径。方法体外培养人近端肾小管上皮HK-2细胞,以720μmol/L的尿酸干预0、12、24、48 h后收集细胞;DCHF-DA染色检测细胞内活性氧（ROS）含量,分光光度计检测细胞上清液中的丙二醛（MDA）、总超氧化物岐化酶（T-SOD）,MitoSOXTM染色观察活细胞线粒体中ROS的生成情况。结果尿酸干预12 h时细胞内总ROS含量较未干预时无明显变化,细胞上清中MDA含量、T-SOD活性亦较未干预时无明显差异（P均〉0.05）;干预24 h时总ROS含量有所升高,MDA含量上升,T-SOD活性降低,48 h时上述变化更为明显（P均〈0.05）;线粒体内ROS也在尿酸干预24 h时合成上调,48 h时升高更为显著（P〈0.05）。结论高浓度尿酸可诱导HK-2细胞氧化应激反应,线粒体ROS合成增多可能是其作用基础。     

Helicobacter pylori-induced oxidative stress and DNA damage in a primary culture of human gastric mucosal cells

Helicobacter pylori has been identified in the pathogenesis of chronic active gastritis and peptic ulcer disease and is epidemiologically linked to gastric cancer and lymphoma. Our previous studies have demonstrated enhanced production of reactive oxygen species (ROS) in cultured gastric adenocarcinoma cells (ATCC CRL/1739) in association with H. pylori. Recently, we have isolated and cultured normal human gastric mucosal cells (GMC) from H. pylori-negative endoscopic biopsies. The integrity of these mucosal cells was determined by periodic acid–Schiff staining. We assessed the effects of various H. pylori strains including 60190 (a 87-kDa cytotoxin producing strain), ATCC 43504, and 60190-v1 (in which the cytotoxin gene has been disrupted) on the primary culture of human gastric mucosal cells. The induction of ROS and DNA fragmentation in the mucosal cells in association with these H. pylori strains were assessed by cytochrome c reduction (an index of superoxide anion production), hydroxyl radical production, and DNA fragmentation. Following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori strain 60190, approximately 6.2- and 9.9-fold increases were observed in cytochrome c reduction, respectively, as compared to mucosal cells in the absence of H. pylori, demonstrating the production of superoxide anion. The detection of hydroxyl radicals based on the formation of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was determined by using a high-performance liquid chromatograph equipped with an electrochemical detector. Approximately 3.5- and 7.7-fold increases in hydroxyl radical production were observed following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori, respectively. Approximately 3.6- and 4.5-fold increases in DNA fragmentation were observed in gastric mucosal cells following incubation with 1:0.5 and 1:1 ratios of H. pylori, respectively. The effects of culture supernatant preparations from H. pylori strains 60190 and 60190-v1 on the enhanced production of ROS and increased DNA fragmentation in mucosal cells were also investigated. Culture supernatant preparations, the prime source of the 87-kDa cytotoxin, from both H. pylori strains 60190 and 60190-v1 were extracted under identical conditions to determine the role of 87-kDa cytotoxin on the enhanced production of ROS and DNA fragmentation. The cytotoxin rich-H. pylori strain 60190 induced greater production of ROS and DNA fragmentation in mucosal cells as compared to the supernatant preparation from H. pylori strain 60190-v1, in which the cytotoxin gene has been disrupted. This study demonstrates that H. pylori induces enhanced production of ROS and DNA damage in association with human gastric mucosal cells andthat the 87-kDa cytotoxin protein plays a prime role in the induction of oxidative stress and DNA damage.     

The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking

Fanconi's anaemia (FA) is characterized by increased spontaneous and induced chromosome fragility. This has been widely regarded to be due to a defect in DNA crosslink repair, because of the sensitivity of cells to known DNA crosslinking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Although Fanconi cells are also sensitive to molecular oxygen, and may be protected by antioxidants, this has generally been considered to be a secondary phenomenon. However, it has recently been demonstrated that the FAC protein, coded for by the Fanconi anaemia gene for complementation group C, is strictly cytoplasmic and does not enter the nucleus even after DNA damage, which seems inconsistent with a role in DNA repair.
We have studied the effects of MMC and oxygen on apoptotic cell death in FA group C (FA-C) and normal lymphoblastoid cell lines. Hyperoxia alone failed to induce apoptosis in either FA-C or normal cells. At ambient oxygen, MMC is known to generate oxygen free radicals, whereas decreased oxygen tension facilitates the metabolic activation of MMC for DNA crosslinking. We therefore studied the effects of MMC at 20% and 5% oxygen to favour oxygen radical generation or DNA crosslinking respectively. FA-C cells showed increased sensitivity compared to normal cells for the induction of apoptosis by MMC at 20% oxygen. When cells were treated with MMC at 5% oxygen we found no increased sensitivity of Fanconi cells to MMC when compared to normal cells. These results imply a role for oxygen free radicals, but not for DNA crosslinking, in the sensitivity of FA cells to MMC.     

低浓度过氧化氢对内皮前体细胞的损伤效应及复方丹参注射液的保护作用

目的观察低浓度过氧化氢（H2O2）对内皮前体细胞（EPC）的损伤效应及复方丹参注射液（ISM）的保护作用。方法建立猪EPC体外培养模型,在培养液中加入低浓度H2O2（100、200、300、400μmol/L）及5 mg/L ISM,测定细胞增殖活力（MTT法）、乳酸脱氢酶（LDH）活性、丙二醛（MDA）含量及细胞凋亡率。结果H2O2使细胞增殖活力下降,使LDH、MDA含量增加,细胞凋亡率上升,并呈浓度依赖性,ISM可减轻上述变化。结论ISM对低浓度H2O2造成的EPC损伤具保护作用。