Late preconditioning by ethanol is initiated via an oxidant-dependent signaling pathway

Ingestion of alcoholic beverages at low to moderate levels 24 h prior to ischemia and reperfusion (I/R) prevents postischemic leukocyte/endothelial cell adhesive interactions, a phenomenon referred to as late ethanol preconditioning (EtOH-PC). The aim of this study was to determine whether oxidants act as initiators of late EtOH-PC. Ethanol was instilled into the stomachs of C57BL/6 mice as a bolus by gavage at a dose that produced a peak plasma concentration of 45 mg/dl 30 min after administration and returned to control levels 60 min after ingestion. Twenty four hours later, the superior mesenteric artery was occluded for 45 min followed by 70 min of reperfusion. The numbers of rolling and firmly adherent leukocytes were quantified in postcapillary venules of the small intestine in sham animals (no EtOH-PC, no I/R), in mice subjected to I/R alone or EtOH-PC + I/R, and in animals treated with Mn-TBAP (a cell-permeant superoxide dismutase mimetic), oxypurinol (a XO inhibitor), the NAD(P)H oxidase inhibitors PR-39 or apocynin, or oxypurinol plus PR39 during the period of EtOH-PC on Day 1 followed by I/R on Day 2. In separate groups of mice, oxypurinol or apocynin were also administered 1 h after ethanol ingestion on Day 1, with induction of I/R 24 h later. I/R induced marked increases in leukocyte rolling and adherence, effects that were completely prevented by EtOH-PC. Coincident treatment with Mn-TBAP, oxypurinol, PR-39, apocynin, or oxypurinol plus PR-39 with ethanol attenuated these anti-inflammatory actions of EtOH-PC. However, administration of oxypurinol or apocynin 1 h after ethanol ingestion failed to prevent these protective effects of EtOH-PC. Our results indicate that reactive oxygen species formed during the period of ethanol exposure on Day 1 trigger the development of an anti-inflammatory phenotype that renders the small bowel resistant to the proadhesive effects of I/R 24 h later.     

Preconditioning with ethanol prevents postischemic leukocyte-endothelial cell adhesive interactions

Long-term ethanol consumption at low to moderate levels exerts cardioprotective effects in the setting of ischemia and reperfusion (I/R). The aims of this study were to determine whether 1) a single orally administered dose of ethanol [ethanol preconditioning (EtOH-PC)] would induce a biphasic temporal pattern of protection (early and late phases) against the inflammatory responses to I/R and 2) adenosine and nitric oxide (NO) act as initiators of the late phase of protection. Ethanol was administered as a bolus to C57BL/6 mice at a dose that achieved a peak plasma concentration of ~45 mg/dl 30 min after gavage and returned to control levels within 60 min of alcohol ingestion. The superior mesenteric artery was occluded for 45 min followed by 60 min of reperfusion beginning 10 min or 1, 2, 3, 4, or 24 h after ethanol ingestion, and the numbers of fluorescently labeled rolling and firmly adherent (stationary) leukocytes in single postcapillary venules of the small intestine were quantified using intravital microscopic approaches. I/R induced marked increases in leukocyte rolling and adhesion, effects that were attenuated by EtOH-PC 2-3 h before I/R (early phase), absent when assessed after 10 min, 1 h, and 4 h of ethanol ingestion, with an even more powerful late phase of protection reemerging when I/R was induced 24 h later. The anti-inflammatory effects of late EtOH-PC were abolished by treatment with adenosine deaminase, an adenosine A(2) (but not A(1)) receptor antagonist, or a NO synthase (NOS) inhibitor during the period of EtOH-PC. Preconditioning with an adenosine A(2) (but not an A(1)) receptor agonist in lieu of ethanol 24 h before I/R mimicked the protective actions of late phase EtOH-PC. Like EtOH-PC, the effect of preconditioning with an adenosine A(2) receptor agonist was abrogated by coincident NOS inhibition. These findings suggest that EtOH-PC induces a biphasic temporal pattern of protection against the proinflammatory effects of I/R. In addition, our observations are consistent with the hypothesis that the late phase of EtOH-PC is triggered by NO formed secondary to adenosine A(2) receptor-dependent activation of NOS during the period of ethanol exposure.     

Angiotensin preconditioning of the heart

Angiotensin II (Ang II) has been found to exert preconditioning-like effect on mammalian hearts. Diverse mechanisms are known to exist to explain the cardioprotective abilities of Ang II preconditioning. The present study hypothesized, based on the recent report that Ang II generates reactive oxygen species (ROS) through NADPH oxidase, that Ang II preconditioning occurs through redox cycling. To test this hypothesis, a group of rat hearts was treated with Ang II in the absence or presence of an NADPH oxidase inhibitor, apocynin; or a cell-permeable ROS scavenger, N-acetyl cysteine (NAC). Ang II pretreatment improved postischemic ventricular recovery; reduced myocardial infarction; and decreased the number of cardiomyocyte apoptosis, indicating its ability to precondition the heart against ischemic injury. Both apocynin and NAC almost abolished the preconditioning ability of Ang II. Ang II resulted in increase in ROS activity in the heart, which was reduced by either NAC or apocynin. Ang II also increased both the NADPH oxidase subunits gp91 phox and p22phox mRNA expression, which was abolished with apocynin and NAC. Our results thus demonstrate that the Ang II preconditioning was associated with enhanced ROS activities and increased NADPH oxidase subunits p22phox and gp91phox expression. Both NAC and apocynin reduced ROS activities simultaneously abolishing preconditioning ability of Ang II, suggesting that Ang II preconditioning occurs through redox cycling. That both NAC and apocynin reduced ROS activities and abolished Ang II-mediated increase in p22phox and gp91phox activity further suggest that such redox cycling occurs via both NADPH oxidase-dependent and -independent pathways.     

Cdc42-dependent activation of NADPH oxidase is involved in ethanol-induced neuronal oxidative stress

It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47(phox) and p67(phox), the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47(phox) and p67(phox), which were prerequisites for NOX assembly and activation. Knocking down p47(phox) with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons.     

Role of calcitonin gene-related peptide in the postischemic anti-inflammatory effects of antecedent ethanol ingestion

The aim of this study was to determine the role of calcitonin gene-related peptide (CGRP) in the postischemic anti-inflammatory effects of antecedent ethanol ingestion. Ethanol was administered to wild-type C57BL/6 mice on day 1 as a bolus by gavage at a dose that produces a peak plasma ethanol of 45 mg/dl 30 min after administration. Twenty-four hours later (day 2), the superior mesenteric artery was occluded for 45 min followed by 70 min of reperfusion (I/R). Intravital fluorescence microscopy was used to quantify the numbers of rolling (LR) and adherent (LA) leukocytes labeled with carboxyfluorescein diacetate succinimidyl ester in postcapillary venules of the small intestine. I/R increased LR and LA, effects that were prevented by antecedent ethanol. The postischemic anti-inflammatory effects of ethanol consumption were abolished by administration of a specific CGRP receptor antagonist [CGRP-(8-37)] or after sensory nerve neurotransmitter depletion using capsaicin administered 4 days before ethanol ingestion, which initially induces rapid release of CGRP from sensory nerves, thereby depleting stored neuropeptide. Administration of exogenous CGRP or induction of endogenous CGRP release by treatment with capsaicin 24 h before I/R mimicked the postischemic anti-inflammatory effects of antecedent ethanol ingestion. Preconditioning with capsaicin 24 h before I/R was prevented by coincident treatment with CGRP-(8-37), while exogenous CGRP induced an anti-inflammatory phenotype in mice depleted of CGRP by capsaicin administration 4 days earlier. Our results indicate that the effect of antecedent ethanol ingestion to prevent postischemic LR and LA is initiated by a CGRP-dependent mechanism.     

Effect of NADPH oxidase inhibition on cardiopulmonary bypass-induced lung injury

Cardiopulmonary bypass (CPB) causes acute lung injury. Reactive oxygen species (ROS) from NADPH oxidase may contribute to this injury. To determine the role of NADPH oxidase, we pretreated pigs with structurally dissimilar NADPH oxidase inhibitors. Low-dose apocynin (4-hydroxy-3-methoxy-acetophenone; 200 mg/kg, n = 6), high-dose apocynin (400 mg/kg, n = 6), or diphenyleneiodonium (DPI; 8 mg/kg) was compared with diluent (n = 8). An additional group was treated with indomethacin (10 mg/kg, n = 3). CPB was performed for 2 h with deflated lungs, complete pulmonary artery occlusion, and bronchial artery ligation to maximize lung injury. Parameters of pulmonary function were evaluated for 25 min following CPB. Blood chemiluminescence indicated neutrophil ROS production. Electron paramagnetic resonance determined the effect of apocynin and DPI on in vitro pulmonary endothelial ROS production following hypoxia-reoxygenation. Both apocynin and DPI attenuated blood chemiluminescence and post-CPB hypoxemia. At 25 min post-CPB with Fi(O(2)) = 1, arterial Po(2) (Pa(o(2))) averaged 52 +/- 5, 162 +/- 54, 335 +/- 88, and 329 +/- 119 mmHg in control, low-dose apocynin, high-dose apocynin, and DPI-treated groups, respectively (P < 0.01). Indomethacin had no effect. Pa(O(2)) correlated with blood chemiluminescence measured after drug administration before CPB (R = -0.60, P < 0.005). Neither apocynin nor DPI prevented the increased tracheal pressure, plasma cytokine concentrations (tumor necrosis factor-alpha and IL-6), extravascular lung water, and pulmonary vascular protein permeability observed in control pigs. NADPH oxidase inhibition, but not xanthine oxidase inhibition, significantly blocked endothelial ROS generation following hypoxia-reoxygenation (P < 0.05). NADPH oxidase-derived ROS contribute to the severe hypoxemia but not to the increased cytokine generation and pulmonary vascular protein permeability, which occur following CPB.     

NADPH oxidase modulates myocardial Akt, ERK1/2 activation, and angiogenesis after hypoxia-reoxygenation

Recent studies have demonstrated that reactive oxygen species (ROS) mediate myocardial ischemia-reperfusion (I/R) and angiogenesis via the mitogen-activated protein kinases and the serine-threonine kinase Akt/protein kinase B pathways. NADPH oxidases are major sources of ROS in endothelial cells and cardiomyocytes. In the present study, we investigated the role of NADPH oxidase-derived ROS in hypoxia-reoxygenation (H/R)-induced Akt and ERK1/2 activation and angiogenesis using porcine coronary artery endothelial cells (PCAECs) and a mouse myocardial I/R model. Our data demonstrate that exposure of PCAECs to hypoxia for 2 h followed by 1 h of reoxygenation significantly increased ROS formation. Pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI, 10 microM) and apocynin (Apo, 200 and 600 microM), significantly attenuated H/R-induced ROS formation. Furthermore, exposure of PCAECs to H/R caused a significant increase in Akt and ERK1/2 activation. Exposure of PCAEC spheroids and mouse aortic rings to H/R significantly increased endothelial spheroid sprouting and vessel outgrowth, whereas pharmacological inhibition of NADPH oxidase or genetic deletion of the NADPH oxidase subunit, p47(phox) (p47(phox-/-)), significantly suppressed these changes. With the use of a mouse I/R model, our data further show that the increases in myocardial Akt and ERK1/2 activation and vascular endothelial growth factor (VEGF) expression were markedly blunted in the p47(phox-/-) mouse subjected to myocardial I/R compared with the wild-type mouse. Our findings underscore the important role of NADPH oxidase and its subunit p47(phox) in modulating Akt and ERK1/2 activation, angiogenic growth factor expression, and angiogenesis in myocardium undergoing I/R.     

Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress

Aging is associated with blunted endothelium-dependent relaxations and vascular oxidative stress. Our previous study has indicated that daily intake of red wine polyphenols (RWPs) by young rats retards aging-related endothelial dysfunction in middle-aged rats. The aim of the present study is to determine whether intake of RWPs also improves an established endothelial dysfunction in middle-aged rats and, if so, to determine the underlying mechanism. Middle-aged rats (51 weeks) received either solvent (3% ethanol), RWPs extract (100mg/kg/day) or the antioxidant and NADPH oxidase inhibitor apocynin (100mg/kg/day) in the drinking water for 4 weeks. Vascular reactivity of mesenteric artery rings from control young (12 weeks) and middle-aged rats was assessed in organ chambers. The expression level of endothelial NO synthase (eNOS), arginase I, angiotensin II receptors (AT1R and AT2R), NADPH oxidase subunits and nitrotyrosines was assessed by immunohistochemistry, and the vascular formation of reactive oxygen species (ROS) by dihydroethidine. Aging is associated with blunted endothelium-dependent relaxations, an excessive vascular formation of ROS and peroxynitrites, and an up-regulation of eNOS, arginase I, NADPH oxidase subunits (nox-1, p22phox), and AT1R and AT2R expression. RWPs and apocynin treatments improved endothelial dysfunction, normalized oxidative stress and the expression of the different proteins in the mesenteric artery of middle-aged rats. The present findings indicate that aging is associated with blunted endothelium-dependent relaxations involving an increased oxidative stress, and that these responses are improved by the intake of RWPs or apocynin for 4weeks most likely by normalizing the expression of eNOS, arginase I, NADPH oxidase and angiotensin receptors.     

Role of eNOS-derived NO in the postischemic anti-inflammatory effects of antecedent ethanol ingestion in murine small intestine

Ingestion of low levels of ethanol 24 h before [ethanol preconditioning (EPC)] ischemia and reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and adhesion (LA), effects that were abolished by adenosine A(2) receptor (ADO-A(2)R) antagonists or nitric oxide (NO) synthase (NOS) inhibitors. The aims of this study were to determine whether NO derived from endothelial NOS (eNOS) during the period of ethanol exposure triggered entrance into this preconditioned state and whether these events were initiated by an ADO-A(2)R-dependent mechanism. Ethanol or distilled water vehicle was administered to C57BL/6J [wild type (WT)] or eNOS-deficient (eNOS-/-) mice by gavage. Twenty-four hours later, the superior mesenteric artery was occluded for 45 min. LR and LA were quantified by intravital microscopy after 30 and 60 min of reperfusion. I/R increased LR and LA in WT mice, effects that were abolished by EPC or NO donor preconditioning (NO-PC). NO-PC was not attenuated by coincident administration of an ADO-A(2)R antagonist. I/R increased LR and LA in eNOS-/- mice to levels comparable with those noted in WT animals. However, EPC only slightly attenuated postischemic LR and LA, whereas NO-PC remained effective as a preconditioning stimulus in eNOS-/- mice. Preconditioning with an ADO-A(2)R agonist (which we previously demonstrated prevents I/R-induced LR and LA in WT animals) failed to attenuate these postischemic adhesive responses in eNOS-/- mice. Our results indicate that EPC is triggered by NO formed secondary to ADO-A(2)R-dependent eNOS activation during the period of ethanol exposure 24 h before I/R.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22(phox) and p47(phox) expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.     

Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH oxidase. Since it acts as a potent inhibitor in studies with neutrophils and macrophages, no inhibitory effect can often be found in non-phagocyte cells. In our experiments, apocynin even stimulated reactive oxygen species (ROS) production by vascular fibroblasts. Even when added to macrophages, apocynin initially caused an increase in ROS production. The inhibition of ROS formation followed, suggesting that in the presence of leukocyte myeloperoxidase and hydrogen peroxide, apocynin is converted to another compound. Apocynin pre-activated with H2O2 and horseradish peroxidase (HRP) inhibited ROS production immediately. In non-phagocytes, apocynin stimulated ROS production and no inhibition was observed even after 60 min. Apocynin treated with H2O2 and HRP, however, decreased ROS production in the same manner as in macrophages. The stimulatory effect on ROS production can be abolished by tiron and superoxide dismutase (SOD), suggesting that superoxide was the produced species. The effect of apocynin was inhibited by diphenylene iodinium (DPI), a non-scavenging NADPH oxidase inhibitor. It can be summarized that apocynin stimulates cell superoxide production. In the presence of peroxidase and hydrogen peroxide, however, it is converted into another compound that acts as an inhibitor of superoxide production. It strongly suggests that under conditions in vivo, apocynin can have opposite effects on phagocytes and non-phagocyte cells. It acts as an inhibitor of phagocyte NADPH oxidase but also as a ROS production stimulator in non-phagocyte cells.     

The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseases

Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca(2+)-dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A(2) (PLA(2)). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA(2), namely, the group IV cytosolic cPLA(2) and the group II secretory sPLA(2). A major focus is to elaborate the dual role of NADPH oxidase and PLA(2) in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA(2) will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases.     

A Novel Domain of Amino-Nogo-A Protects HT22 Cells Exposed to Oxygen Glucose Deprivation by Inhibiting NADPH Oxidase Activity

This study aimed to investigate the protective effect of the M9 region (residues 290–562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia–reperfusion induced by oxygen–glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.     

Polyphenols restore endothelial function in DOCA-salt hypertension: role of endothelin-1 and NADPH oxidase

Red wine polyphenols (RWPs) have been reported to exert beneficial effects in preventing cardiovascular diseases, such as hypertension. We studied the effects of chronic treatment with RWPs and apocynin, an inhibitor of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, on blood pressure, endothelial function, and oxidative status in deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Rats were administered RWPs (40 mg/kg) or apocynin (33 microg/kg) daily by gavage for 5 weeks. Plasma catechin levels were detected only after RWP treatment. RWPs and apocynin prevented both the increase in systolic blood pressure and the proteinuria induced by DOCA-salt. Plasma malonyldialdehyde levels, urinary iso-prostaglandin F(2alpha) excretion, aortic superoxide production, and aortic NADPH oxidase activity were found to be increased in animals of the DOCA group. RWP and apocynin treatments reduced these parameters in DOCA-salt rats, having no effect on control rats. However, only RWPs reduced the increase in plasma endothelin-1 (ET-1) levels and aortic p22(phox) gene overexpression found in DOCA-salt animals. RWPs and apocynin also improved the blunted endothelium-dependent relaxation response to acetylcholine in noradrenaline-precontracted aortic rings. All these results suggest that chronic treatment with RWPs prevents hypertension and vascular dysfunction. RWPs prevent vascular oxidative stress by inhibiting NADPH oxidase activity and/or by reducing ET-1 release.     

Ischemia-activated microglia induces neuronal injury via activation of gp91phox NADPH oxidase

Although glial cells play a major role in the pathogenesis of many neurological diseases by exacerbating neuronal and non-neuronal cell death, the mechanisms involved are unclear. We examined the effects of microglia-(MCM) or astrocyte-(ACM) conditioned media obtained by chemical ischemia on the neuronal injury in SH-SY5Y cells. Chemical ischemia was induced by the treatment with NaN₃ and 2-deoxy-d-glucose for 2 h. MCM-treated SH-SY5Y cells showed reduced the viability, increased caspase-3 activity, decreased Bcl-2/Bax ratio, and increased cytochrome c release, increased inflammatory cytokines, and increased reactive oxygen species (ROS) generation. MCM also increased gp91phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which was inhibited by NADPH oxidase inhibitor, apocynin, and gp91phox siRNA. However, ACM did not show any significant changes. The results suggest that microglia activated by ischemic insult may increase reactive oxygen species generation via activation of gp91phox NADPH oxidase, resulting in neuronal injury.     

Apocynin attenuates oxidative stress and hypertension in young spontaneously hypertensive rats independent of ADMA/NO pathway

Both NADPH oxidase-derived reactive oxygen species (ROS) and asymmetric dimethylarginine (ADMA) are increased in hypertension. Apocynin, an NADPH oxidase inhibitor, could inhibit ROS, thus we tested whether apocynin can block NADPH oxidase and prevent increases of ADMA and blood pressure (BP) in spontaneously hypertensive rats (SHRs). SHRs and Wistar Kyoto (WKY) rats, aged 4 weeks, were assigned to four groups: untreated SHRs and WKY rats, SHRs and WKY rats that received 2.5 mM apocynin for 8 weeks. BP was significantly higher in SHRs compared to WKY rats, which was attenuated by apocynin. Apocynin prevented p47phox translocation in SHR kidneys, but not the increase of superoxide and H(2)O(2). Additionally, apocynin did not protect SHRs against increased ADMA. Apocynin blocks NADPH oxidase to attenuate hypertension, but has little effect on the ADMA/nitric oxide (NO) pathway in young SHRs. The reduction of ROS and the preservation of NO simultaneously might be a better approach to restoring ROS-NO balance to prevent hypertension.     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.     

Green Tea Polyphenols Precondition against Cell Death Induced by Oxygen-Glucose Deprivation via Stimulation of Laminin Receptor,Generation of Reactive Oxygen Species,and Activation of Protein Kinase C?

As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKCϵ from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKCϵ and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKCϵ, resulting in preconditioning against cell death induced by OGD/R.