活性氧与勃起功能障碍

男性勃起功能障碍(ED)是男科常见疾病之一,当前全世界有15200万男性受到ED的困扰。近年来已经认识到活性氧(reactive oxygen species,ROS)在ED中起重要作用。本文总结近年来ROS在ED中作用的研究进展,并阐述ROS与ED中各种危险因子的关系以及抗氧化治疗进展,为开发治疗ED的药物提供新的方向。     

NADPH Oxidase NOX2 Defines a New Antagonistic Role for Reactive Oxygen Species and cAMP/PKA in the Regulation of Insulin Secretion

In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform–specific knockout mice. Both human and mouse β-cells express NOX, in particular NOX2. With use of Nox isoform–specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic β-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.NOX enzymes generate superoxide by transferring one electron from NADPH to oxygen (1). The best known NOX isoform is the phagocyte NADPH oxidase (NOX), a multicomponent complex comprising a membrane catalytic heterodimer, the flavocytochrome b_558, formed by gp91^phox (also referred to as NOX2) and p22^phox (where phox is phagocyte oxidase). The cytosolic regulatory subunits are composed of p40^phox, p47^phox, p67^phox, and GTPases Rac1 or Rac2 (1). Assembly of cytosolic elements to membrane catalytic core initiates the activation of NOX. To date, seven isoforms of NOX (NOX1–5 and dual oxidases DUOX1–2) have been identified with different activation mechanisms and heterogeneous tissue distribution (1). In addition to microbial attack by professional phagocytes, physiopathological roles of NOX have recently attracted attention in nonphagocytic cells, including pancreatic β-cells (2–7). Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, might participate in β-cell dysfunction (8). The redox imbalance favored by high metabolic rate and a relatively low detoxifying system has contributed to the general concept that β-cells are sensitive to ROS, although they can handle rather high concentrations of H₂O₂ (9).NOX family represents one of the potential sources of ROS in insulin-secreting cells (4). Both rat islets and insulinoma express membrane-associated catalytic components Nox1, Nox2, Nox4, and p22^phox, as well as cytosolic regulators p40^phox, p47^phox, and p67^phox and their homologs Noxo1 and Noxa1 (3,5,6). Regarding their putative function in the β-cell, NOXs have been implicated in glucose-induced ROS production in MIN-6 cells (10). Knockdown of p47^phox results in total inhibition of glucose-stimulated insulin secretion and lowers ROS (11). In animal models of type 2 diabetes, islets exhibit increased NOX components Nox2 and p22^phox, correlating with increased oxidative stress (12). Activation of Nox and accompanying ROS generation were demonstrated in Zucker diabetic fatty (ZDF) rat and diabetic human islets (13). However, inhibition of islet NOX using diphenyleneiodonium (DPI) impairs glucose-stimulated insulin secretion (6) along with blunted glucose-induced superoxide production (5,10). These conflicting findings regarding NOX activity and β-cell function might be attributed to poor specificity of old-generation NOX inhibitors, such as apocynin and DPI (14). The former has been shown to function as a general ROS scavenger, and the latter is a nonspecific inhibitor of electron transporters (1,15).In the current study, we first investigated relative expression levels of the different catalytic subunits of NOXs in both human and mouse pancreatic islets. Then, subcellular distribution of the identified predominant NOX isoform NOX2 was assessed in human islet cells. For avoidance of poor specificity of NOX inhibitors, islets isolated from Nox isoform–specific–deficient mice were used to investigate the contribution of NOXs in insulin secretory function.     

AGE与RAGE相互作用通过活性氧引起足细胞凋亡

目的：探讨晚期糖基化终末产物（AGE）对足细胞凋亡的影响,及氧化应激在其中的作用。方法：小鼠足细胞株由美国纽约西奈山医学院Peter Mundel教授馈赠。用钙磷脂结合蛋白Ⅴ-荧光异硫氰酸盐（FITC）和碘化物（PI）标记细胞,采用荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。Dharmacon On TargetPlus SMARTpool si RNA试剂和Amaxa RNAi nucleofection试剂盒成功转染si RNA到足细胞。绿荧光蛋白载体证明转染的有效性,分别采用Western Blot和实时定量PCR（RT-PCR）方法来检测si RNA转染足细胞后AGE受体蛋白（RAGE）靶基因蛋白质和mRNA的表达。用LS50B型荧光分光光度计测活性氧,根据波长485nm在530nm发射的荧光来判断活性氧（ROS）的产生。观察活性氧的清除剂N-乙酰基-半胱氨酸（NAC）能否减少AGE-BSA诱导的足细胞凋亡。结果：AGE引起足细胞的凋亡呈剂量依赖性,随AGE浓度的增大,凋亡的发生率逐渐升高;RAGE siRNA能减少60%~70%RAGE mRNA和蛋白质的表达;ROS的清除剂NAC可明显减少AGE-BSA引起的ROS的产生和足细胞凋亡。结论：AGE与RAGE作用后活性氧产生增加,活性氧的增加可能是AGE引起足细胞凋亡的途径之一,可通过抗氧化减少ROS的产生延缓糖尿病肾病的进展。     

Source of Reactive Oxygen Species in Anti-Thy1 Nephritis

In proliferative glomerulonephritis, both macrophages and mesangial cells generate reactive oxygen species (ROS), contributing to the development of glomerular injury. We have attempted to determine which cell produces ROS during anti-Thyl nephritis (ATN) in rats. The generation of ROS was studied using lutninol amplified chemiluminescence (GCL) on isolated glomeruli. Immunohistochemical studies used avidin-biotin complex (ABC) to label macrophages and mesangial cells. Immediately after ATN induction, mesangiolysis and infiltration with ED-1 positive cells (referred to as macrophage) was noted with a peak at day 1. After day 4, mesangial proliferation appeared with a decrease of the ED-1 positive cells and a prominent increase of PCNA positive cells (regarded as mesangial cells). In the early phase of ATN, GCL, reflecting ROS generation, increased along with the appearance of ED-1 positive cells. GCL subsequently decreased as mesangial cells increased. This suggested that macrophage were the principal participants in ROS generation in the early phase of ATN although mesangial cells cannot be completely disregarded in the generation of ROS and development of glomerular injury.     

Reactive Oxygen Species Play a Biphasic Role in Brain Ischemia

Objective: Reactive oxygen species (ROS) are the essential mechanism involving in the ischemic process. Due to their complex characteristics, the precise effects of ROS on post-ischemic neurons remain uncertain. This study aimed to investigate the potential role of ROS in brain ischemia. Methods: Dynamic ROS levels in the perifocal cortex were evaluated after right middle cerebral artery occlusion (MCAO) of SD rats. Furthermore the role of ROS was assessed following delayed treatment with the ROS scavenger dimethylthiourea (DMTU) after brain ischemia. Results: ROS levels markedly increased at 1 hr after reperfusion and then gradually decreased as the post-reperfusion time interval increased. ROS levels reached their lowest point at 3 days after reperfusion before increasing and showing a second peak at 7 days after reperfusion. ROS levels negatively correlated with neurological function scores. Delayed DMTU treatment after stroke worsened neurological outcomes, decreased microvessel density and inhibited stress-activated protein kinase activation. Conclusion: ROS may play a biphasic role in cerebral ischemia. Namely, ROS may induce damage during the injury phase of brain ischemia and participate in improving neurological function during the recovery phase.     

Mitochondrial Reactive Oxygen Species Are Obligatory Signals for Glucose-Induced Insulin Secretion

OBJECTIVE—Insulin secretion involves complex events in which the mitochondria play a pivotal role in the generation of signals that couple glucose detection to insulin secretion. Studies on the mitochondrial generation of reactive oxygen species (ROS) generally focus on chronic nutrient exposure. Here, we investigate whether transient mitochondrial ROS production linked to glucose-induced increased respiration might act as a signal for monitoring insulin secretion.RESEARCH DESIGN AND METHODS—ROS production in response to glucose was investigated in freshly isolated rat islets. ROS effects were studied using a pharmacological approach and calcium imaging.RESULTS—Transient glucose increase from 5.5 to 16.7 mmol/l stimulated ROS generation, which was reversed by antioxidants. Insulin secretion was dose dependently blunted by antioxidants and highly correlated with ROS levels. The incapacity of β-cells to secrete insulin in response to glucose with antioxidants was associated with a decrease in ROS production and in contrast to the maintenance of high levels of ATP and NADH. Then, we investigated the mitochondrial origin of ROS (mROS) as the triggering signal. Insulin release was mimicked by the mitochondrial-complex blockers, antimycin and rotenone, that generate mROS. The adding of antioxidants to mitochondrial blockers or to glucose was used to lower mROS reversed insulin secretion. Finally, calcium imaging on perifused islets using glucose stimulation or mitochondrial blockers revealed that calcium mobilization was completely reversed using the antioxidant trolox and that it was of extracellular origin. No toxic effects were present using these pharmacological approaches.CONCLUSIONS—Altogether, these complementary results demonstrate that mROS production is a necessary stimulus for glucose-induced insulin secretion.Elucidating the mechanisms by which pancreatic β-cells couple glucose sensing to insulin secretion, a vital process in energy homeostasis, is of prime importance. Although ATP production is considered the main mitochondrial signal, detailed studies show that insulin secretion cannot be restricted to ATP synthesis, and numerous experimental clues show that additional mitochondrial factors involved in glucose-secretion coupling are necessary, although not yet identified (1).Transient increases in glucose metabolism generate NADH and FADH₂, leading rapidly to increased superoxide anion (O₂^·) production; obligatorily associated with the respiratory chain function, superoxide anion will be converted into H₂O₂ (2). This production of mitochondrial reactive oxygen species (mROS)—transient because H₂O₂-inactivating enzymes rapidly quench it before a damage to the physiological conditions of the cell occurs—is now recognized as an intracellular messenger (3,4). These features make mROS a good candidate for rapidly regulating pathways that depend directly on metabolic fluxes. Based on such a view, we recently demonstrated that mROS production is required for hypothalamic glucose and lipid sensing (5,6). These results lead us to speculate that O₂^· might operate more generally in nutrient-sensitive cells and also to look for the role of mROS as a signal involved in glucose-stimulated insulin secretion (GSIS). Recently, a study revealed that H₂O₂ is effectively a signal of GSIS (7). Here, we provide clues that glucose-induced mitochondrial O₂^· production is an obligatory stimulus for insulin secretion.     

白细胞精子症病人精浆活性氧与细胞因子检测及评价

目的 :研究不育症病人精浆中活性氧 (ROS)、超氧化物歧化酶 (SOD)及白介素 8(IL 8)的水平及其之间的关系。　方法 :收集 15例生育男性 (生育组 )、16例非感染性不育症者 (非感染组 )及 11例白细胞精子症者 (感染组 )的精液 ,进行常规精液分析 ,检测其精浆中IL 8、丙二醛 (MDA)、SOD及白细胞 (WBC)含量 ,并进行相关分析。　结果 :感染组与另两组相比 ,MDA、WBC及IL 8明显升高 (P <0 .0 0 1)。相关分析发现 ,IL 8与MDA显著相关 (r =0 .85 2 ,P<0 .0 0 1) ;WBC与IL 8间显著相关 (r=0 .818,P <0 .0 1)。　结论 :感染性不育症病人精液中 ,ROS与IL 8的增加有关 ,抗氧化系统缺陷是ROS增加的原因之一。     

Cyclic Tensile Stretch Stimulates the Release of Reactive Oxygen Species from Osteoblast-like Cells

It is known that the excessive generation of reactive oxygen species (ROS) is a significant factor in tissue injury observed in many disease states. To determine whether extreme levels of mechanical stress applied to osteoblasts enhances ROS synthesis, we loaded cyclic tensile stretch on osteoblast-like HT-3 cells. Cyclic tensile stretch loaded on these cells clearly enhanced ROS synthesis in a time- and magnitude-dependent fashion. Cyclic tensile stretch also enhanced superoxide dismutase (SOD) activity. The disruption of microfilaments with cytochalasin D abolished the stress-induced ROS synthesis. Rotenone, an inhibitor of the mitochondrial electron transport chain, enhanced stress-induced ROS synthesis. These data suggest that actin filament and mitochondria are involved in this action.     

Mechanisms of Podocyte Injury in Diabetes: Role of Cytochrome P450 and NADPH Oxidases

OBJECTIVE

We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes.

RESEARCH DESIGN AND METHODS

Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo.

RESULTS

Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N′-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria.

CONCLUSIONS

Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.Diabetic nephropathy in humans is characterized by increased urinary albumin excretion (microalbuminuria), which often progresses to proteinuria, one of the most important prognostic risk factors for kidney disease progression (1). Glomerular visceral epithelial cells, or podocytes, play a critical role in maintaining the structure and function of the glomerular filtration barrier. Careful morphometric analyses of renal biopsy in subjects with type 1 and type 2 diabetes (2–4) demonstrate that the density of podocytes is reduced not only in individuals with diabetic nephropathy, but also in patients with short duration of diabetes before the onset of microalbuminuria (4,5). Studies in experimental models of type 1 and type 2 diabetes have also documented that podocyte depletion represents one of the earliest cellular lesions affecting the diabetic kidney (6,7). Among various morphologic characteristics, the decreased number of podocytes in glomeruli is the strongest predictor of progression of diabetic nephropathy, where fewer cells predict more rapid progression (3,4). Although these observations identify podocyte depletion as one of the earliest cellular features of diabetic kidney disease, the mechanisms that underlie the loss of podocytes in diabetic nephropathy remain poorly understood.High glucose induces apoptosis (8), and there is evidence that podocyte apoptosis contributes to reduced podocyte number (9). High glucose, transforming growth factor-β (TGF-β), and angiotensin II (ANGII) induce apoptosis of cultured podocytes (9–12). ANGII appears to induce apoptosis in cultured rat glomerular epithelial cells at least partially via TGF-β because its apoptotic effect is attenuated by an anti–TGF-β antibody (12). There is also evidence that reactive oxygen species (ROS) contribute to podocyte apoptosis and depletion in cells exposed to high glucose and in experimental diabetic nephropathy (7). However, the sources of ROS and the kinetics of their generation have not been well characterized. We and others (13–15) have recently identified NADPH oxidases as major sources of ROS in kidney cortex and glomeruli of rats with type 1 diabetes. Six homologs of the cytochrome subunit of the phagocyte NADPH oxidase (Nox2/gp91^phox) have been cloned (16). At least three different Nox isoforms are expressed in the kidney cortex: Nox1, Nox2, and Nox4 (16). Cytochromes P450 (CYP450s) are significant sources of ROS in many tissues (17,18). CYP450 metabolizes arachidonic acid into hydroxyeicosatetraenoic acids (20-HETEs) and EETs (epoxyeicosatrienoic acids). 20-HETE, the ω-hydroxylation product of arachidonic acid, is one of the major CYP eicosanoids produced in the kidney cortex (19–21). The predominant CYP450 in the kidney cortex that synthesizes 20-HETE is cytochrome P450 of the 4A family (CYP4A) (19–21). 20-HETE has multiple and opposing functions depending on the site of production and target cells/tissues (19,22–24).In this study, we demonstrate that high glucose induces ROS production and apoptosis in cultured mouse podocytes through the upregulation of CYP4A with increased production of 20-HETE and upregulation of NADPH oxidases. Inhibition of 20-HETE production prevented podocyte apoptosis in vitro and decreased oxidative stress, podocyte apoptosis, and proteinuria in an in vivo model of type 1 diabetes.     

Inhibition of Reactive Oxygen Species by Lovastatin Downregulates Vascular Endothelial Growth Factor Expression and Ameliorates Blood-Retinal Barrier Breakdown in db/db Mice: Role of NADPH Oxidase 4

OBJECTIVE

Oxidative stress is a key pathogenic factor in diabetic retinopathy. We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in diabetic retinopathy.

RESEARCH DESIGN AND METHODS

Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α; production of reactive oxygen species (ROS); and retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCECs) and in db/db mice treated with lovastatin.

RESULTS

Expressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCECs, hypoxia and high glucose upregulated mRNA and protein expression of Nox4, ROS generation, and VEGF level. These changes were abrogated by pretreatment with lovastatin or NADPH oxidase inhibitor diphenyleneiodonium chloride. Overexpression of Nox4 increased basal level of ROS generation, HIF-1α, and VEGF expression in RCECs. In contrast, blockade of Nox4 activity using adenovirus-expressing dominant-negative Nox4 abolished hypoxia- and high-glucose–induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1α and high-glucose–elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 small interfering RNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice.

CONCLUSIONS

Activation of Nox4 plays an important role in high-glucose– and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4, at least in part, contributes to the protective effects of lovastatin in diabetic retinopathy.Diabetic retinopathy is a common complication of diabetes and one of the most frequent causes of blindness in the U.S. (1–3). Hallmark sequential pathological changes in diabetic retinopathy include increased vascular permeability, pericyte and endothelial cell death, capillary occlusion and aberrant retinal new vessel growth, or neovascularization (4). Increased vascular permeability caused by the breakdown of the blood-retinal barrier (BRB) results in diabetic macular edema, a major cause of vision loss in diabetic patients (2,5,6). Vascular endothelial growth factor (VEGF) is a potent angiogenic factor playing a crucial role in angiogenesis (7,8). VEGF is also referred as vascular permeability factor (VPF) based on its ability to induce vascular hyperpermeability (9). Significantly elevated VEGF levels in the eye have been reported in diabetic patients with diabetic macular edema and correlated with the severity of vascular leakage (10). Overexpression of VEGF is also responsible for retinal hyperpermeability in streptozotocin (STZ)-induced diabetic rats (11). These findings suggest that VEGF is a key mediator of retinal vascular leakage in diabetic retinopathy.Oxidative stress plays an important role in vascular endothelial dysfunction in diabetes (12–15). Increased level of hydrogen peroxide, a reactive oxygen species (ROS), was colocalized with VEGF expression at the inner BRB and associated with vascular leakage in the retina in diabetic BBZ/Wor rats, suggesting a role of ROS in regulation of VEGF in diabetic retinopathy (16). In addition, suppression of ROS generation by NADPH oxidase inhibitor or antioxidants significantly attenuated retinal vascular leakage in diabetic animals, suggesting that activation of NADPH oxidase contributes to retinal ROS generation and vascular damage in diabetic retinopathy (17). NADPH oxidase (Nox) 4, which is originally identified in the kidney and termed renox (renal oxidase), is a novel isoform of NADPH oxidase expressed in nonphagocytes, such as vascular endothelial cells and smooth muscle cells (18,19). In aorta isolated from the STZ-induced diabetic apolipoprotein E–deficient mice or the db/db mice, Nox4 expression is significantly upregulated, associated with increased ROS production and inflammation, indicating a potential role of Nox4 in diabetic macrovascular disease (20). Moreover, inhibition of Nox4 expression using antisense oligonucleotides attenuates ROS generation and ameliorates glomerular hypertrophy in STZ-induced diabetic mice, suggesting that Nox4 is the major source of ROS in the diabetic kidney, contributing to renal hypertrophy in diabetic nephropathy (21). However, the role of Nox4 in diabetic retinopathy has not been investigated.3-Hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis commonly used in dyslipidemia and type 2 diabetes (22). Moreover, statins have demonstrated impressive beneficial effects, such as improvement of endothelial function, neuroprotection, and anti-inflammation, which are independent of their lipid-lowering effects (23). In a previous study, we have shown that lovastatin protects retinal tight junction and ameliorates BRB breakdown in db/db mice, a type 2 diabetes model (24). However, the mechanisms remain elusive. In the present study, we have tested the hypothesis that Nox4 is a key mediator of oxidative stress and BRB breakdown in diabetic retinopathy and that inhibition of Nox4 is, at least in part, responsible for the salutary effect of lovastatin on retinal endothelial function.