AT1-receptor blockade by telmisartan upregulates GTP-cyclohydrolase I and protects eNOS in diabetic rats

Several enzymatic sources of reactive oxygen species (ROS) were described as potential reasons of eNOS uncoupling in diabetes mellitus. In the present study, we investigated the effects of AT(1)-receptor blockade with chronic telmisartan (25 mg/kg/day, 6.5 weeks) therapy on expression of the BH(4)-synthesizing enzyme GTP-cyclohydrolase I (GCH-I), eNOS uncoupling, and endothelial dysfunction in streptozotocin (STZ, 60 mg/kg iv, 7 weeks)-induced diabetes mellitus (type I). Telmisartan therapy did not modify blood glucose and body weight. Aortas from diabetic animals had vascular dysfunction as revealed by isometric tension studies (acetylcholine and nitroglycerin potency). Vascular and cardiac ROS produced by NADPH oxidase, mitochondria, eNOS, and xanthine oxidase were increased in the diabetic group as was the expression of NADPH oxidase subunits at the protein level. The expression of GCH-I and the phosphorylation of eNOS at Ser1177 was decreased by STZ treatment. Therapy with telmisartan normalized these parameters. The present study demonstrates for the first time that AT(1)-receptor blockade by telmisartan prevents downregulation of the BH(4) synthase GCH-I and thereby eNOS uncoupling in experimental diabetes. In addition, telmisartan inhibits activation of superoxide sources like NADPH oxidase, mitochondria, and xanthine oxidase. These effects may explain the beneficial effects of telmisartan on endothelial dysfunction in diabetes.     

Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation

Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.     

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.     

Insights from the computational analysis of CD271 glycation in mescenchymal stem cells in diabetes mellitus as a predisposition to latent tuberculosis

Diabetes mellitus is considered as a predisposition factor for active tuberculosis and is known to activate the latent form of tuberculosis. However, the causative association of latent tuberculosis with diabetes is not conclusively established. Therefore, it is of interest to relate their predisposition. We describe the glycation pattern of mescenchymal stem cell surface markers as CD271+/CD45-mescenchymal stem cell is known to be associated with latent tuberculosis. We show that the lysine residues important for function of CD271 death domain are predicted to be and glycated. These observations help to discuss the role of CD271 and glycation to modulate the genesis of latent tuberculosis in chronic diabetic mellitus.     

NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytes

Reactive oxygen species (ROS) produced by activated astrocytes have been considered to be involved in the pathogenesis of neurodegenerative diseases, while NADPH oxidase is an essential enzyme involved in ROS-mediated signal transduction. The goal of the present study was to determine whether NADPH oxidase plays a role in ROS generation and cell survival in rat astrocytes. We found that the release of ROS in rat astrocytes was significantly increased by stimulation with calcium ionophore or opsonized zymosan, which are known to trigger a respiration burst in phagocytes by the NADPH oxidase pathway. Further study indicated that diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, significantly suppressed the increase of ROS release caused by the calcium ionophore or opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI dose- and time-dependently decreased the viability of normal astrocytes, whereas exogenous supplementation of H2O2 can reverse the survival of DPI-treated astrocytes. For the first time, our results suggest that NADPH oxidase is an important enzyme for the generation of ROS in astrocytes, and the ROS generated by NADPH oxidase play an essential role in astrocyte survival.     

质膜上的活性氧制造者--NOX家族

NADPH氧化酶特异存在于吞噬细胞质膜,能生成用于清除病原微生物的活性氧（reactive oxygen species,ROS）。NOX是NADPH氧化酶催化亚基gp91^phox的同源物,存在于多种非吞噬细胞。目前发现的NOX有NOX1、NOX3、NOX4及NOX5,虽然它们有一定的组织特异性,但与NADPH氧化酶一样均有催化生成ROS的能力。与吞噬细胞中NADPH氧化酶所制造的ROS不同,NOX所产生的ROS并不主要起细胞防御功能,而是作为第二信使,参与细胞增殖、分化、凋亡的调节。此外,NOX对血管生成及骨吸收也有一定的影响,同时还可作为氧感受器调节促红细胞生成素（EPO）的产生。     

Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE

Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.     

Annatto extract and β-carotene enhances antioxidant status and regulate gene expression in neutrophils of diabetic rats

Annatto (Bixa orellana L.) contains a mixture of orange-yellowish pigments due to the presence of various carotenoids that have antioxidant effect. The immune system is especially vulnerable to oxidative damage because many immune cells, such as neutrophils, produce reactive oxygen and nitrogen species (ROS and RNS) as part of the body's defence mechanisms to destroy invading pathogens. It is well known that the function of neutrophils is altered in diabetes; one of the major functional changes in neutrophils in diabetes is the increased generation of extracellular superoxide via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system. The purpose of this study is to evaluate the production of ROS and nitric oxide (NO) as well as the expression of NADPH oxidase subunits, inducible nitric oxide (iNOS), superoxide dismutase (SOD) and catalase (CAT) in neutrophils from diabetic rats treated with annatto extract and β-carotene. Forty-eight female Fisher rats were distributed into six groups according to the treatment received. All animals were sacrificed 7 days after treatment, and the neutrophils were isolated using two gradients of different densities. The ROS and NO were quantified by a chemiluminescence and spectrophotometric assays, respectively. Analyses of gene expression were performed using quantitative real time polymerase chain reaction (qRT-PCR). The results show that treatment with annatto extract and β-carotene was able to decrease ROS production and the mRNA levels of p22(phox) and p47(phox) and increase the mRNA levels of SOD and CAT in neutrophils from diabetic rats. These data suggest that annatto extract and β-carotene exerts antioxidant effect via inhibition of expression of the NADPH oxidase subunits and increase expression/activity of antioxidant enzymes.     

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ.

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.     

Advanced glycation end products enhance reactive oxygen and nitrogen species generation in neutrophils in vitro

Increased oxidative stress (OS) in diabetes mellitus is one of the major factors leading to diabetic pathology. However, the mediators and mechanism that provoke OS in diabetes is not fully understood, and it is possible that accumulation of advanced glycation end products (AGEs) formed secondary to hyperglycemic conditions may incite circulating polymorphonuclear neutrophils (PMN) to generate reactive oxygen species (ROS). In this report, we aim to investigate the effect of AGE on reactive oxygen and nitrogen species generation and subsequent OS in PMN. AGE-HSA exert dose- and time-dependent enhancement of ROS and reactive nitrogen intermediates (RNI) generation by PMN. Increased ROS and RNI generation were found to be mediated through the upregulation of NADPH oxidase and inducible nitric oxide synthase (iNOS), respectively, as evident from the fact that AGE-treated neutrophils failed to generate ROS and RNI in presence of diphenyleneiodonium, a flavoprotein inhibitor for both enzymes. Further increased generation of ROS and RNI ceased when the cells were incubated with anti-RAGE antibody suggesting the involvement of AGE-RAGE interaction. Also increased malondialdehyde (MDA) and protein carbonyl formation in AGE-exposed PMN suggest induction of OS by AGE. This study provides evidence that AGEs may play a key role in the induction of oxidative stress through the augmentation of PMN-mediated ROS and RNI generation and this may be in part responsible for development of AGE-induced diabetic pathology.     

Nox4 and diabetic nephropathy: With a friend like this,who needs enemies?

Oxidative stress has been linked to the pathogenesis of diabetic nephropathy, a complication of diabetes in the kidney. NADPH oxidases of the Nox family are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. Here, we present an overview of the current understanding of the roles of Nox catalytic and regulatory subunits in the processes that control mesangial cell, podocyte, and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin–angiotensin system and transforming growth factor-β. The role of the Nox isoform Nox4 in the redox processes that alter renal biology in diabetes is highlighted.     

NADPH氧化酶在电磁辐射生物效应中的作用

电磁辐射是一种复合电磁波,而人体生命活动包含一系列的生物电活动,这些生物电对环境的电磁波敏感,因此,电磁辐射可对人体造成危害.关于电磁辐射诱导的生物效应研究虽多,然而其具体机制尚不清楚.近年来,发现烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH氧化酶),又称呼吸爆发氧化酶(respiratory burst oxidase homologue,Rboh)在电磁辐射产生的生物学效应中扮演重要角色,电磁辐射可直接或间接活化NADPH氧化酶复合体,然后NADPH氧化酶可以将细胞内NADPH的电子转移而形成活性氧,或者通过一系列炎症因子和相关基质金属蛋白酶等参与炎症、防御以及组织修复等生命过程.本文对NADPH氧化酶在电磁生物学效应中的作用进行了综述.     

Therapeutic targeting of oxidative stress with coenzyme Q10 counteracts exaggerated diabetic cardiomyopathy in a mouse model of diabetes with diminished PI3K(p110α) signaling

Diabetes-induced cardiac complications include left ventricular (LV) dysfunction and heart failure. We previously demonstrated that LV phosphoinositide 3-kinase p110α (PI3K) protects the heart against diabetic cardiomyopathy, associated with reduced NADPH oxidase expression and activity. Conversely, in dominant negative PI3K(p110α) transgenic mice (dnPI3K), reduced cardiac PI3K signaling exaggerated diabetes-induced cardiomyopathy, associated with upregulated NADPH oxidase. The goal was to examine whether chronic supplementation with the antioxidant coenzyme Q₁₀ (CoQ₁₀) could attenuate LV superoxide and diabetic cardiomyopathy in a setting of impaired PI3K signaling. Diabetes was induced in 6-week-old nontransgenic and dnPI3K male mice via streptozotocin. After 4 weeks of diabetes, CoQ₁₀ supplementation commenced (10 mg/kg ip, 3 times/week, 8 weeks). At study end (12 weeks of diabetes), markers of LV function, cardiomyocyte hypertrophy, collagen deposition, NADPH oxidase, oxidative stress (3-nitrotyrosine), and concentrations of CoQ₉ and CoQ₁₀ were determined. LV NADPH oxidase (Nox2 gene expression and activity, and lucigenin-enhanced chemiluminescence), as well as oxidative stress, were increased by diabetes, exaggerated in diabetic dnPI3K mice, and attenuated by CoQ₁₀. Diabetes-induced LV diastolic dysfunction (prolonged deceleration time, elevated end-diastolic pressure, impaired E/A ratio), cardiomyocyte hypertrophy and fibrosis, expression of atrial natriuretic peptide, connective tissue growth factor, and β-myosin heavy chain were all attenuated by CoQ₁₀. Chronic CoQ₁₀ supplementation attenuates aspects of diabetic cardiomyopathy, even in a setting of reduced cardiac PI3K protective signaling. Given that CoQ₁₀ supplementation has been suggested to have positive outcomes in heart failure patients, chronic CoQ₁₀ supplementation may be an attractive adjunct therapy for diabetic heart failure.     

Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes

The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.     

Combined effect of testosterone and apocynin on nitric oxide and superoxide production in PMA-differentiated THP-1 cells

Human inducible nitric oxide synthase (iNOS) is most readily observed in macrophages from patients with inflammatory diseases like atherosclerosis. The aim of the present study was to find out the combined effect of male sex hormone; testosterone and apocynin (NADPH oxidase inhibitor) on cytokine-induced iNOS production. THP-1 cells were differentiated into macrophages by phorbol myristate acetate (PMA). Expression of iNOS was induced by the addition of cytokine mixture? Testosterone was added at different concentrations (10(-6)-10(-12) M) with apocynin (1 mM). Testosterone (10(-8), 10(-10) M) inhibited NOx production in cytokine-added THP-1 cells which was further confirmed by quantikine assay of iNOS protein and RT-PCR analysis. Testosterone treatment decreased 40% of superoxide anion production. Testosterone showed inhibition of NADPH oxidase, especially expression of p67phox and p47phox (cytosol subunits). Addition of testosterone with apocynin further decreased the expression of p67phox and p47phox subunits of NADPH oxidase. The findings of the present study suggest that, testosterone; the male androgen plays an important role in the prevention of atherogenesis. Even though apocynin does not have any role on NO production, addition of apocynin together with testosterone is effective in suppressing iNOS activity.     

Neutrophil dysfunction induced by hyperglycemia: modulation of myeloperoxidase activity

Our data suggest that impaired activity of myeloperoxidase (MPO) may play an important role in the dysfunction of neutrophils from hyperglycemic rats. Neutrophil biochemical pathways include the NADPH oxidase system and the MPO enzyme. They both play important role in the killing function of neutrophils. The effect of hyperglycemia on the activity of these enzymes and the consequences with regard to Candida albicans phagocytosis and the microbicidal property of rat peritoneal neutrophils is evaluated here. The NADPH oxidase system activity was measured using chemiluminescence and cytochrome C reduction assays. MPO activity was measured by monitoring HOCl production, and MPO protein expression was analysed using Western blot and immunofluorescence. C. albicans phagocytosis and death were evaluated by optical microscopy using the May-Grunwald-Giemsa staining method. ROS generation kinetic was slightly delayed in the diabetic group. MPO expression levels were higher in diabetic neutrophils; however, MPO activity was decreased in these same neutrophils compared with the controls. C. albicans phagocytosis and killing were lower in the diabetic neutrophils. Based on our experimental model, the phagocytic and killing functions of neutrophil phagocytosis are impaired in diabetic rats because of the decreased production of HOCl, highlighting the importance of MPO in the microbicidal function of neutrophils. Copyright ? 2012 John Wiley & Sons, Ltd.     

NADPH oxidase is required for NMDA receptor-dependent activation of ERK in hippocampal area CA1

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor activation results in production of reactive oxygen species (ROS) and activation of extracellular signal-regulated kinase (ERK) in hippocampal area CA1. In addition, application of ROS to hippocampal slices has been shown to result in activation of ERK in area CA1. To determine whether these events were linked causally, we investigated whether ROS are required for NMDA receptor-dependent activation of ERK. In agreement with previous studies, we found that treatment of hippocampal slices with NMDA resulted in activation of ERK in area CA1. The NMDA receptor-dependent activation of ERK was either blocked or attenuated by a number of antioxidants, including the general antioxidant N-acetyl-L-cysteine (L-NAC), the superoxide-scavenging enzyme superoxide dismutase (SOD), the membrane-permeable SOD mimetic Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), the hydrogen peroxide-scavenging enzyme catalase, and the catalase mimetic ebselen. The NMDA receptor-dependent activation of ERK also was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI) and was absent in mice that lacked p47(phox), one of the required protein components of NADPH oxidase. Taken together, our results suggest that ROS production, especially superoxide production via NADPH oxidase, is required for NMDA receptor-dependent activation of ERK in hippocampal area CA1.