Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.     

Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.     

Loperamide‐induced cardiotoxicity in rats: Evidence from cardiac and oxidative stress biomarkers

At therapeutic dose, loperamide is a safe over‐the‐counter antidiarrheal drug but could induce cardiotoxic effect at a supratherapeutic dose. In this study, we use cardiac and oxidative biomarkers to evaluate loperamide‐induced cardiotoxicity in rats. Rats were orally gavaged with 1.5, 3, or 6 mg/kg body weight (BW) of loperamide hydrochloride for 7 days. The results after 7 days administration of loperamide, revealed dose‐dependent increase (P < 0.05) in aspartate aminotransferase, lactate dehydrogenase, creatine kinase‐MB, and serum concentration of cardiac troponin I, total homocysteine, and nitric oxide. A 50% decrease in antioxidant enzymes activity was observed at 6 mg/kg BW. Furthermore, malondialdehyde and fragmented DNA also increased significantly in the heart of the treatment groups. Loperamide provoked cardiotoxicity through oxidative stress, lipid peroxidation, and DNA fragmentation in rats. This study has provided a possible biochemical explanation for the reported cardiotoxicity induced by loperamide overdose.     

Melatonin reduces protein and lipid oxidative damage induced by homocysteine in rat brain homogenates

Numerous data indicate that hyperhomocysteinemia is a risk factor for cardio- and cerebrovascular diseases. At least in part, homocysteine (HCY) impairs cerebrovascular function because it generates large numbers of free radicals. Since melatonin is a well-known antioxidant, which reduces oxidative stress and decreases HCY concentrations in plasma, the aim of this study was to investigate the effect of melatonin in preventing HCY-induced protein and lipid oxidation in rat brain homogenates. Brain homogenates were obtained from Sprague-Dawley rats and were incubated with or without HCY (0.01-5 mM) or melatonin (0.01-3 mM). Carbonyl content of proteins, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations in the brain homogenates were used as an index of protein and lipid oxidation, respectively. Under the experimental conditions used, the addition of HCY (0.01-5 mM) to the homogenates enhanced carbonyl protein and MDA+4-HDA formation. Melatonin reduced, in a concentration-dependent manner, protein and lipid oxidation due to HCY in the brain homogenates. These data suggest that preserving proteins from oxidative insults is an additional mechanism by which melatonin may act as an agent in potentially decreasing cardiovascular and cerebrovascular diseases related to hyperhomocysteinemia.     

Galangin,a dietary flavonoid,improves antioxidant status and reduces hyperglycemia-mediated oxidative stress in streptozotocin-induced diabetic rats

Objective: To examine the effect of galangin on hyperglycemia-mediated oxidative stress in streptozotocin (STZ)-induced diabetic rats.

Methods: Diabetes was induced by intraperitoneal administration of low-dose STZ (40?mg/kg body weight (BW)) into male albino Wistar rats. Galangin (8?mg/kg BW) or glibenclamide (600?µg/kg BW) was given orally, once daily for 45 days to normal and STZ-induced diabetic rats.

Results: Diabetic rats showed significantly increased levels of plasma glucose, thiobarbituric acid reactive substances, lipid hydroperoxides, and conjugated dienes. The levels of insulin and non-enzymatic antioxidants (vitamin C, vitamin E, reduced glutathione) and the activity of enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase (GST)) were decreased significantly in diabetic control rats. These altered plasma glucose, insulin, lipid peroxidation products, enzymatic and non-enzymatic antioxidants ions were reverted to near-normal level after the administration of galangin and glibenclamide.

Conclusion: The present study shows that galangin decreased oxidative stress and increased antioxidant status in diabetic rats, which may be due to its antidiabetic and antioxidant potential.     

A high-fat and cholesterol diet causes fatty liver in guinea pigs. The role of iron and oxidative damage

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease. Iron, cholesterol, and oxidative damage are frequently suggested to be related to the progression of NAFLD, but the precise relationship between them remains unclear. Guinea pigs fed on a high cholesterol and fat diet (without oxidized lipids) generated a disease model of NAFLD with hallmark observations in liver histology and increased liver damage markers. Hepatic cholesterol and iron levels were found to be significantly elevated and directly correlated. Plasma hepcidin and transferrin levels were decreased. Plasma iron concentrations were found to be elevated, likely due to an increased intestinal iron absorption caused by the decrease in plasma hepcidin. However, hepatic transferrin receptor-2 levels were unchanged. No significant increase in hepatic lipid peroxidation was detected using F₂-isoprostanes as a reliable biomarker, nor was there a rise in protein carbonyls, a general index of oxidative protein damage. Some increases in cholesterol oxidation products were observed, but largely negated after normalizing for the elevated hepatic cholesterol content. Indeed, increased hemosiderin deposition and unchanged ferritin levels in liver suggested that the excess iron mainly existed as hemosiderin, which is redox-inactive.     

Attenuation of oxidative stress and alteration of hepatic tissue ultrastructure by D-pinitol in streptozotocin-induced diabetic rats

Abstract

The present study was aimed to investigate the effect of D-pinitol on hyperglycaemia mediated oxidative stress by analysing the hepatic antioxidant competence, pro-inflammatory cytokines and ultrastructural changes in liver tissues of streptozotocin-induced diabetic rats. Oral administration of D-pinitol (50 mg/kg b.w.) resulted in significant (p < 0.05) attenuation in blood glucose, glycosylated haemoglobin and pro-inflammatory markers such as TNF-α, IL-1β, IL-6, NF-κB p65 unit and NO and significant (p < 0.05) elevation in the plasma insulin level. In addition, D-pinitol instigated a significant escalation in the levels of hepatic tissue non-enzymatic antioxidants and the activities enzymatic antioxidants of diabetic rats with significant (p < 0.05) decrease in lipid peroxides and hydroperoxides formation, thus demonstrating the protective role of D-pinitol on the hepatic tissues from oxidative stress-induced liver damage. These biochemical observations were complemented by histological and ultrastructural examination of liver section. Thus, the present study demonstrates the hepatoprotective nature of D-pinitol by attenuating hyperglycaemia-mediated pro-inflammatory cytokines and oxidative stress.     

Differential oxidative modification of proteins in MRL+/+ and MRL/lpr mice: Increased formation of lipid peroxidation-derived aldehyde-protein adducts may contribute to accelerated onset of autoimmune response

Even though reactive oxygen species (ROS) have been implicated in SLE pathogenesis, the contributory role of ROS, especially the consequences of oxidative modification of proteins by lipid peroxidation-derived aldehydes (LPDAs) such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) in eliciting an autoimmune response and disease pathogenesis remains largely unexplored. MRL/lpr mice, a widely used model for SLE, spontaneously develop a condition similar to human SLE, whereas MRL+/+ mice with the same MRL background, show much slower onset of SLE. To assess if the differences in the onset of SLE in the two substrains could partly be due to differential expression of LPDAs and to provide evidence for the role of LPDA-modified proteins in SLE pathogenesis, we determined the serum levels of MDA-/HNE-protein adducts, anti-MDA-/HNE-protein adduct antibodies, MDA-/HNE-protein adduct specific immune complexes, and various autoantibodies in 6-, 12- and 18-week old mice of both substrains. The results show age-related increases in the formation of MDA-/HNE-protein adducts, their corresponding antibodies and MDA-/HNE-specific immune complexes, but MRL/lpr mice showed greater and more accelerated response. Interestingly, a highly positive correlation between increased anti-MDA-/HNE-protein adduct antibodies and autoantibodies was observed. More importantly, we further observed that HNE-MSA caused significant inhibition in antinuclear antibodies (ANA) binding to nuclear antigens. These findings suggest that LPDA-modified proteins could be important sources of autoantibodies and CICs in these mice, and thus contribute to autoimmune disease pathogenesis. The observed differential responses to LPDAs in MRL/lpr and MRL+/+ mice may, in part, be responsible for accelerated and delayed onset of the disease, respectively.     

Silicon increases boron tolerance and reduces oxidative damage of wheat grown in soil with excess boron

The effect of silicon on the growth, boron concentrations, malondialdehyde (MDA) content, lipoxygenase (LOX) activity, proline (PRO) and H₂O₂ accumulation, and the activities of major antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)] and non-enzymatic antioxidants (AA) of wheat grown in soil originally with toxic B concentrations were investigated. Applied of 5.0 and 10.0 mM Si to the B toxic soil significantly increased Si concentration of the wheat and counteracted the deleterious effects of B on shoot growth. The contents of PRO, H₂O₂, MDA, and LOX activity of wheat grown in B toxic soil were significantly reduced by Si treatments. Compared with control plants, the activities of SOD, CAT, APX and content of AA were decreased by applied Si. Based on the present work, it can be concluded that Si alleviates B toxicity of wheat by preventing oxidative membrane damage and also translocation of B from root to shoot and/or soil to plant.     

The role of oxidative stress in lung injury induced by cigarette smoke

Oxidative stress is a damaging process resulting from an imbalance between excessive generation of oxidant compounds and insufficient antioxidant defence mechanisms. Oxidative stress plays a crucial role in the initiation and progression of cigarette smoke-induced lung injury, deterioration in lung functions, and development of chronic obstructive pulmonary disease (COPD). In smokers and in patients with COPD, the increased oxidant burden derives from cigarette smoke per se, and from activated inflammatory cells releasing enhanced amounts of reactive oxygen and nitrogen species (ROS, RNS, respectively). Although mild oxidative stress resulting from cigarette smoking leads to the upregulation of the antioxidative enzymes synthesis in the lungs, high levels of ROS and RNS observed in patients with COPD overwhelm the antioxidant enzymes capacities, resulting in oxidant-mediated lung injury and cell death. In addition, depletion of antioxidative systems in the systemic circulation was consistently observed in such patients. The imbalance between the generation of ROS/RNS and antioxidant capacities — the state of “oxidative stress” — is one of the major pathophysiologic hallmarks in the development of COPD. Detrimental effects of oxidative stress include impairment of membrane functions, inactivation of membrane-bound receptors and enzymes, and increased tissue permeability. In addition, oxidative stress aggravates the inflammatory processes in the lungs, and contributes to the worsening of the protease-antiprotease imbalance. Several markers of oxidative stress, such as increases in lipid peroxidation products and reductions in glutathione peroxidase activity, have been shown to be related to the reductions in pulmonary functions. In the present article we review the current knowledge about the vicious cycle of cigarette smoking, oxidative stress, and inflammation in the pathogenesis of COPD.     

DSSylation,a novel protein modification targets proteins induced by oxidative stress,and facilitates their degradation in cells

Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.     

Induction of heat tolerance in wheat coleoptiles by calcium ions and its relation to oxidative stress

Effects of Ca²⁺ ions on the intensity of lipid peroxidation, activities of guaiacol peroxidase, superoxide dismutase (SOD), and catalase, as well as on heat resistance of winter wheat (Triticum aestivium L.) coleoptiles were examined. A preliminary incubation of coleoptile segments in a 5 mM CaCl₂ solution was shown to improve their survival rates after an injuring heat treatment (43.5°C). The effect of Ca²⁺ was suppressed by the inhibitor of Ca²⁺ channels (1 mM LaCl₃). An incubation of coleoptiles in the presence of 5 mM CaCl₂ prior to the stress treatment elevated the content of lipid peroxidation product, malondialdehyde (MDA) and stimulated the activities of guaiacol peroxidase, SOD, and catalase. After the heat exposure of untreated and Ca²⁺-treated seedlings, differential changes in MDA content and in activities of guaiacol peroxidase, SOD, and catalase were observed. It is concluded that a short-term oxidative stress arising in Ca²⁺-enriched plant tissues after the heat treatment is unrelated to their irreversible damage.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 227–232.Original Russian Text Copyright © 2005 by Kolupaev, Akinina, Mokrousov.This revised version was published online in April 2005 with a corrected cover date.     

Aerobic training reduces oxidative stress in skeletal muscle of rats exposed to air pollution and supplemented with chromium picolinate

Objective: The purpose of this study was to investigate the effects of chromium picolinate (CrPic) supplementation associated with aerobic exercise using measures of oxidative stress in rats exposed to air pollution.

Methods: Sixty-one male Wistar rats were divided into eight groups: residual oil fly ash (ROFA) exposure and sedentary (ROFA-SED); ROFA exposure, sedentary and supplemented (ROFA-SED-CrPic); ROFA exposure and trained (ROFA-AT); ROFA exposure, supplemented and trained (ROFA-AT-CrPic); sedentary (Sal-SED); sedentary and supplemented (Sal-SED-CrPic); trained (Sal-AT); and supplemented and trained (Sal-AT-CrPic). Rats exposed to ROFA (air pollution) received 50?µg of ROFA daily via intranasal instillation. Supplemented rats received CrPic (1?mg/kg/day) daily by oral gavage. Exercise training was performed on a rat treadmill (5×/week). Oxidative parameters were evaluated at the end of protocols.

Results: Trained groups demonstrated lower gain of body mass (P?P?P?P?=?.0014), although CAT activity did not differ among groups (P?=?.4487).

Conclusion: Air pollution exposure did not lead to alterations in oxidative markers in lungs and heart, and exercise training was responsible for decreasing oxidative stress of the gastrocnemius.     

山楂总黄酮联合茶多酚对高脂膳食大鼠血脂及氧化应激的影响

为了探讨山楂总黄酮联合茶多酚对高脂膳食大鼠血脂及氧化应激的影响。将山楂总黄酮联合茶多酚灌胃高脂膳食大鼠5周,取血测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C);并测血清及肝组织丙二醛(MDA)、超氧化物歧化酶(S0D)、谷胱甘肽过氧化物酶(GSH-Px);计算肝指数及进行肝病理组织学观察。结果发现,与模型组相比,山楂总黄酮联合茶多酚组大鼠血清TC、TG、LDL-C水平显著下降,血清HDL-C水平显著升高,血清及肝组织MDA显著降低,S0D、GSH-Px活力明显升高,肝指数明显降低,肝细胞脂肪化程度明显减轻。表明山楂总黄酮联合茶多酚增强了高脂饮食大鼠的抗氧化水平,调节了脂质紊乱。     

多沙唑嗪光学异构体对高血脂家兔血脂水平的影响

目的:观察左旋多沙唑嗪(-)DOX、右旋多沙唑嗪(+)DOX和消旋多沙唑嗪(±)DOX对高血脂家兔血脂水平及动物死亡率的影响。方法:取普通级雄性新西兰大耳白兔,给予高脂饮食4周后,血清TC小于10mmol/L的8只家兔为普通饮食组,饲以标准饲料。血清TC大于10mmol/L的40只家兔随机分为4组(n=10):高脂模型组、高脂模型+(-)DOX组、高脂模型+(+)DOX组以及高脂模型+(±)DOX组。普通饮食组和高脂模型组家兔腹腔注射无菌双蒸水;其他3组家兔分别腹腔注射(-)DOX、(+)DOX和(±)DOX,连续9周。分析药物对兔血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)的影响。结果:饲以高脂饮食13周时模型组家兔死亡率达40%,远远高于普通饮食组家兔(10%),亦明显高于(±)DOX和(-)DOX处理组。模型组家兔随高脂饲养的时间延长,血清LDL-C水平进一步显著升高(P0.05和P0.01);而各药物处理组动物的血清LDL-C水平未显著升高(P0.05)。结论:(-)DOX和(±)DOX可提高高脂饮食家兔的生存率,并对高血脂家兔的血清LDL-C紊乱具有轻度的改善作用;该作用可能不是其提高高脂饮食家兔生存率的主要作用机制。     

A high-caloric diet rich in soy oil alleviates oxidative damage of skeletal muscles induced by dexamethasone in chickens

Objective: Glucocorticoids (GCs) can induce oxidative damage in skeletal muscles. The purpose of this study was to demonstrate a high caloric (HC) diet rich in soy oil would change the oxidative stress induced by a GC.

Methods: The effect of dexamethasone (DEX) and HC diet on oxidative stress in plasma, skeletal muscles (M. pectoralis major, PM; M. biceps femoris, BF), and mitochondria were determined. The biomarkers of oxidative damage and antioxidative enzyme activity were determined. The fatty acid profile of muscles and the activities of complex I and II in mitochondria were measured.

Results: The results showed that DEX increased the concentrations of oxidative damage markers in plasma, muscles, and mitochondria. The activity of complex I was significantly suppressed by DEX. DEX-chickens had higher proportions of polyunsaturated fatty acids and lower proportions of monounsaturated fatty acids in the PM. A HC diet decreased the levels of oxidative damage biomarkers in plasma, muscles, and mitochondria. The interaction between DEX and diet suppressed the activities of complex I and II in HC-chickens.

Discussion: Oxidative damage in skeletal muscles and mitochondria was the result of GC-induced suppression of the activity of mitochondrial complex I. A HC diet improved the antioxidative capacity and reduced the oxidative damage induced by the GC.     

Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures

Acid glycosaminoglycans (GAGs) antioxidant activity was assessed in a fibroblast culture system by evaluating reduction of oxidative system-induced damage. Three different methods to induce oxidative stress in human skin fibroblast cultures were used. In the first protocol cells were treated with CuSO₄ plus ascorbate. In the second experiment fibroblasts were exposed to FeSO₄ plus ascorbate. In the third system H₂O₂ was utilised. The exposition of fibroblasts to each one of the three oxidant systems caused inhibition of cell growth and cell death, increase of lipid peroxidation evaluated by the analysis of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and superoxide dismutase (SOD) levels, and rise of lactate dehydrogenase activity (LDH). The treatment with commercial GAGs at different doses showed beneficial effects in all oxidative models. Hyaluronic acid (HA) and chondroitin-4-sulphate (C4S) exhibited the highest protection. However, the cells exposed to CuSO₄ plus ascorbate and FeSO₄ plus ascorbate were better protected by GAGs compared to those exposed to H₂O₂. These outcomes confirm the antioxidant properties of GAGs and further support the hypothesis that these molecules may function as metal chelators. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of antioxidative and chelating effects of daidzein and daidzin on protein oxidative modification by copper in vitro

Daidzein and its glycoside daidzin are isoflavones. Their antioxidative effects were compared in vitro. Although both compounds inhibited protein oxidative modification by copper, the inhibitory effect of daidzein was stronger than that of daidzin. Because daidzein showed a greater affinity for Cu²⁺, the antioxidant effect of these isoflavones may be dependent on their respective copper-chelating abilities.     

Inhibitory effects of licoisoflavones A and B and sophoraisoflavone A of Sophra mooracroftiana Beth ex Baker on copper-ion-induced protein oxidative modification of mice brain homogenate, in vitro

We present the results of an in vitro investigation of the inhibitory effects of licoisoflavones A and B and sophoraisoflavone A isolated from Sophra mooracroftiana BETH ex BAKER on copper-induced protein oxidative modification of mice brain homogenate in vitro. Although inhibitory effect of sophoraisoflavone A was stronger than those of licoisoflavones A and B, genistein as a related isoflavone, and mannitol as a hydroxy radical scavenger, inhibitory effects of licoisoflavones A and B were weaker than those of genistein and mannitol. These results demonstrated that the difference of inhibitory effects are dependent on the relation between chemical structures of these isoflavones, such as hydroxy group or benzopyran, and oxidative stress.