Inhibition Of NADPH Oxidase Activity Research Articles

Glucose-Dependent Insulinotropic Polypeptide Inhibits AGE-Induced NADPH Oxidase-Derived Oxidative Stress Generation and Foam Cell Formation in Macrophages Partly via AMPK Activation.

Glucose-dependent insulinotropic polypeptide (GIP) of the incretin group has been shown to exert pleiotropic actions. There is growing evidence that advanced glycation end products (AGEs), senescent macromolecules formed at an accelerated rate under chronic hyperglycemic conditions, play a role in the pathogenesis of atherosclerotic cardiovascular disease in diabetes. However, whether and how GIP could inhibit the AGE-induced foam cell formation of macrophages, an initial step of atherosclerosis remains to be elucidated. In this study, we address these issues. We found that AGEs increased oxidized low-density-lipoprotein uptake into reactive oxygen species (ROS) generation and Cdk5 and CD36 gene expressions in human U937 macrophages, all of which were significantly blocked by [D-Ala2]GIP(1-42) or an inhibitor of NADPH oxidase activity. An inhibitor of AMP-activated protein kinase (AMPK) attenuated all of the beneficial effects of [D-Ala2]GIP(1-42) on AGE-exposed U937 macrophages, whereas an activator of AMPK mimicked the effects of [D-Ala2]GIP(1-42) on foam cell formation, ROS generation, and Cdk5 and CD36 gene expressions in macrophages. The present study suggests that [D-Ala2]GIP(1-42) could inhibit the AGE-RAGE-induced, NADPH oxidase-derived oxidative stress generation in U937 macrophages via AMPK activation and subsequently suppress macrophage foam cell formation by reducing the Cdk5-CD36 pathway.

TaBAS1 encoding a typical 2-Cys peroxiredoxin enhances salt tolerance in wheat.

Efficient antioxidant enzymatic system contributes to salt tolerance of plants via avoiding ROS over-accumulation. Peroxiredoxins are crucial components of the reactive oxygen species (ROS) scavenging machinery in plant cells, but whether they offer salt tolerance with potential for germplasm improvement has not been well addressed in wheat. In this work, we confirmed the role of a wheat 2-Cys peroxiredoxin gene TaBAS1 that was identified through the proteomic analysis. TaBAS1 overexpression enhanced the salt tolerance of wheat at both germination and seedling stages. TaBAS1 overexpression enhanced the tolerance to oxidative stress, promoted the activities of ROS scavenging enzymes, and reduced ROS accumulation under salt stress. TaBAS1 overexpression promoted the activity of ROS production associated NADPH oxidase, and the inhibition of NADPH oxidase activity abolished the role of TaBAS1 in salt and oxidative tolerance. Moreover, the inhibition of NADPH-thioredoxin reductase C activity erased the performance of TaBAS1 in the tolerance to salt and oxidative stress. The ectopic expression of TaBAS1 in Arabidopsis exhibited the same performance, showing the conserved role of 2-Cys peroxiredoxins in salt tolerance in plants. TaBAS1 overexpression enhanced the grain yield of wheat under salt stress but not the control condition, not imposing the trade-offs between yield and tolerance. Thus, TaBAS1 could be used for molecular breeding of wheat with superior salt tolerance.

Distinct effects of form selective cytochrome P450 inhibitors on cytochrome P450-mediated monooxygenase and hydrogen peroxide generating NADPH oxidase

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H2O2, either directly or indirectly via superoxide anion, a reaction referred to as “NADPH oxidase” activity. H2O2 production by CYPs can lead to the accumulation of cytotoxic reactive oxygen species which can compromise cellular functioning and contribute to tissue injury. Herein we determined if form selective CYP inhibitors could distinguish between the activities of the monooxygenase and NADPH oxidase activities of rat recombinant CYP1A2, CYP2E1, CYP3A1 and CYP3A2 and CYP1A1/2-enriched β-naphthoflavone-induced rat liver microsomes, CYP2E1-enriched isoniazide-induced rat liver microsomes and CYP3A subfamily-enriched dexamethasone-induced rat liver microsomes. In the presence of 7,8-benzoflavone (2.0 μM) for CYP1A2 and 4-methylpyrazole (32 μM) or DMSO (16 mM) for CYP2E1, monooxygenase activity was blocked without affecting NADPH oxidase activity for both the recombinant enzymes and microsomal preparations. Ketoconazole (1.0 μM), a form selective inhibitor for CYP3A subfamily enzymes, completely inhibited monooxygenase activity of rat recombinant CYP3A1/3A2 and CYP3A subfamily in rat liver microsomes; it also partially inhibited NADPH oxidase activity. 7,8-benzoflavone is a type I ligand, which competes with substrate binding, while 4-methylpyrazole and DMSO are type II heme binding ligands. Interactions of heme with these type II ligands was not sufficient to interfere with oxygen activation, which is required for NADPH oxidase activity. Ketoconazole, a type II ligand known to bind multiple sites on CYP3A subfamily enzymes in close proximity to heme, also interfered, at least in part, with oxygen activation. These data indicate that form specific inhibitors can be used to distinguish between monooxygenase reactions and H2O2 generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined.

TBHQ attenuates podocyte injury in diabetic nephropathy by inhibiting NADPH oxidase-derived ROS generation via the Nrf2/HO-1 signalling pathway

AimsOxidative stress plays a crucial role in podocyte injury in diabetic nephropathy (DN). tert-Butylhydroquinone (tBHQ) is an activator of Nrf2 that exerts protective effects in diabetic mice, but the underlying mechanism of tBHQ in the podocytes of DN is not fully understood. Materials and methodsA high glucose (HG)-induced HK2 cell model and streptozotocin-induced rat model of DN were established and treated with tBHQ or apocynin. The expression levels of Nrf2, HO-1, NOX2 and NOX4 were determined by Western blot or immunohistochemical staining. The level of oxidative stress in podocytes or kidney tissues was assessed using DCFH-DA or dihydroethidium (DHE) staining. Cell injury was assessed by F-actin staining and flow cytometry analysis. Key findingsWe showed that HG treatment increased the expressions of NOX2 and NOX4 and enhanced ROS production in podocytes. Inhibition of NADPH oxidase activity by apocynin dramatically attenuated HG-induced ROS production and further alleviated cell injury and apoptosis in podocytes. Moreover, we found that HG inhibited the Nrf2/HO-1 signalling pathway in podocytes; however, tBHQ treatment significantly activated the Nrf2 signalling pathway, inhibited NADPH oxidase activity, and attenuated ROS production and cell injury in HG-treated podocytes. Furthermore, we observed that tBHQ treatment partially attenuated renal injury, activated the Nrf2 signalling pathway, inhibited NADPH oxidase activity and reduced ROS generation in the kidneys of STZ-induced diabetic rats. SignificanceThese results suggest that tBHQ exerts a protective role in hyperglycaemia-induced podocyte injury, and that the potential protective mechanism of tBHQ involves inhibiting NADPH oxidase-derived ROS generation by activating the Nrf2/HO-1 signalling pathway.

Chicken Muscle Protein-Derived Peptide VVHPKESF Reduces TNFα-Induced Inflammation and Oxidative Stress by Suppressing TNFR1 Signaling in Human Vascular Endothelial Cells.

This study aims to investigate the protective effects of four chicken muscle-derived peptides Val-Arg-Pro (VRP), Leu-Lys-Tyr (LKY), Val-Arg-Tyr (VRY), and Val-Val-His-Pro-Lys-Glu-Ser-Phe [VVHPKESF (V-F)] on tumor necrosis factor alpha (TNFα)-induced endothelial inflammation and oxidative stress in human vascular endothelial EA.hy926 cells. Inflammation and oxidative stress are induced in EA.hy926 cells by TNFα (10ngmL-1 ) treatment for different periods of time. Inflammatory proteins and signaling molecules including inducible nitric oxide synthase, intracellular cell adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase 2 (COX2), nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), and TNFα receptor 1 (TNFR1) are measured by qRT-PCR or western blotting; soluble TNFR1 level and nicotinamide adenine dinucleotide phosphate NADPH)oxidase activity are determined by Elisa kits; superoxide is measured by dihydroethidium staining. Only V-F treatment inhibits the expression of VCAM-1 and COX2, via suppressing NF-κB and p38 MAPK signaling, respectively, while reduced oxidative stress via the inhibition of NADPH oxidase activity; V-F treatment attenuates both gene and protein expressions of TNFR1. V-F treatment ameliorates TNFα-induced endothelial inflammation and oxidative stress likely via the inhibition of TNFR1 signaling, suggesting its potential as a functional food ingredient or nutraceutical in the prevention and treatment of hypertension and cardiovascular diseases.

Acute Antioxidant and Cytoprotective Effects of Caffeine in the Model of Neurovascular Injury by Oxidative Stress

Caffeine is commonly used in the neonatal intensive care units for respiratory disorders, but the effects of this medication in the neonatal cerebral circulation remain largely unexplored. We used newborn pigs as a clinically relevant model of the neonatal human brain to investigate the effects of caffeine on cerebral arterioles and primary neurovascular cells. First, we examined dose‐dependent cerebrovascular and systemic effects of water‐soluble anhydrous caffeine in anesthetized newborn pigs equipped with closed cranial windows. Caffeine administered systemically at therapeutic concentrations (20‐40 mg/kg i.v.) had no significant effects on diameter of pial arterioles, mean arterial blood pressure (MABP), and heart rate within 10‐60 min after the injection. When used at a higher concentration (70 mg/kg i.v.), caffeine produced dilation of pial arterioles (15‐20%) accompanied by an increased heart rate, without any effect on MABP. Topical application of caffeine (10‐5‐10‐3M) under the cranial window produced a dose‐dependent dilation of pial cerebral arterioles (15‐30%) that was fully reversed by a brief flushing with artificial cerebrospinal fluid. These in vivo data indicate that cerebral vascular cells are potential targets for caffeine. Next, we tested the hypothesis that caffeine exhibits cytoprotective properties in cerebral circulation during oxidative stress conditions. We used an in vitro model of acute oxidative stress‐induced neurovascular injury caused by excitotoxic and inflammatory conditions concomitant with epileptic seizures, asphyxia, and cerebral ischemia. Endothelial cells and cortical astrocytes are the key components of the neurovascular unit that are most vulnerable to oxidative stress injury. Primary cultures of cerebral vascular endothelial cells (CVEC) and cortical astrocytes from newborn pigs responded to pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) and excitotoxic glutamate (1‐2 mM) by a surge in reactive oxygen species (ROS) production leading to apoptosis. NADPH oxidase is the major ROS‐generating system in the neurovascular unit during inflammation and excitotoxicity. We examined the acute effects of caffeine on oxidative stress‐induced neurovascular cell death caused by TNF‐alpha and excitotoxic glutamate. Caffeine (0.1‐10 µM) inhibited NADPH oxidase activation, reduced overall ROS generation, and attenuated major key events of apoptosis (caspase‐3 activation, DNA fragmentation, and loss of cell‐cell contacts) in CVEC and astrocytes exposed to TNF‐a and glutamate. These data demonstrate, for the first time, that caffeine exhibits acute antioxidant and cytoprotective effects in cerebral vascular endothelial cells and astrocytes. Overall, endothelial and astrocytic components of the neurovascular unit represent target cells for caffeine cytoprotective therapy in the neonatal cerebral circulation during oxidative stress.

He-Ne Laser Enhances Seed Germination and Salt Acclimation in Salvia officinalis Seedlings in a Manner Dependent on Phytochrome and H2O2.

In the current study the role of H2O2 in He-Ne laser-induced effects on seed germination and post-germinative performance of Salvia officinalis seedlings was assessed under both non-stress and saline conditions. Salinity had adverse impacts on seed germination and root length and decreased seed germination tolerance index. Seed priming with H2O2 and He-Ne laser impacted the seed germination and vigoration in a dose-dependent manner. The optimal effects were gathered by energy dose of 6J/cm2 laser and concentration of 5mM H2O2. These pre-treatments enhanced seed germination due to increasing contents of total soluble and reducing sugars and the amylase activity in seeds and improved seedling performance under saline and non-saline conditions. Furthermore, Phy B transcripts were upregulated, salt-accrued oxidative stress was mitigated, and the activities of POD and CAT increased in seedlings primed with H2O2 and laser. Interestingly, applying diphenyleneiodonium (DPI as an inhibitor of NADPH oxidase activity) and N, N-dimethyl thiourea (DMTU as a H2O2 scavenger) arrested the upregulation of phy B gene and abolished stimulatory impact of laser priming on the aforementioned attributes under both non-stress and saline conditions. These novel findings suggest that H2O2 as a downstream signal modulates the impacts of He-Ne laser on seed germination, seedling performance and salt acclimation in sage seedlings, and likely phy B also is involved in these responses.

Pharmacological inhibition of Rac1 attenuates myocardial abnormalities in tail-suspended mice.

Microgravity conditions cause myocardial abnormalities with limited therapeutic approaches. We reported that NADPH oxidase-derived reactive oxygen species contribute to microgravity-induced myocardial abnormalities. This study investigated whether pharmacological inhibition of Rac1 protected the heart during microgravity. Simulated microgravity was induced by tail-suspension in mice. Tail-suspension for 28days increased Rac1 activity in hearts, reduced heart weight and cross-sectional areas of cardiomyocytes, indicative of myocardial atrophy, and myocardial dysfunction. Administration of NSC23766, a selective inhibitor of Rac1, or atorvastatin reported to inhibit Rac1 activation, attenuated myocardial atrophy and preserved myocardial function in tail-suspended mice. These protective effects of Rac1 inhibition were associated with inhibition of NADPH oxidase activation and a reduction of oxidative stress. Our finding may inform a future clinical trial using atorvastatin to prevent myocardial abnormalities under microgravity conditions.

Glucose-6-phosphate dehydrogenase and abscisic acid mediate programmed cell death induced by aluminum toxicity in soybean root tips

Programmed cell death (PCD) induced by aluminum (Al) is considered an important reason of Al phytotoxicity. However, the underlying mechanism of how Al induces PCD remains largely unknown in plants. The roles of glucose-6-phosphate dehydrogenase (G6PDH) and abscisic acid (ABA) in regulating Al-induced PCD were investigated in soybean roots. Al treatment increased G6PDH activity, while inhibition of G6PDH activity alleviated PCD occurrence and reactive oxygen species (ROS) accumulation under Al stress. Overexpression of cytosolic G6PDH1 enhanced G6PDH activity, thus promoting ROS production and cell death under Al exposure. Inhibition of NADPH oxidase activity mitigated ROS generation and cell death under Al stress. Further investigation demonstrated that G6PDH positively regulated the activity of NADPH oxidase under Al treatment using pharmacological and transgenic approach. Furthermore, Al stress increased ABA production, while inhibition of ABA biosynthesis alleviated PCD occurrence and ROS accumulation under Al stress. Interestingly, ABA upregulated G6PDH1 expression and G6PDH activity under Al stress. These results suggest that G6PDH mediates Al-induced PCD occurrence through the activation of NADPH oxidase-dependent ROS production, and ABA acts upstream of G6PDH in this process. This study will provide novel clues for the improvement of Al phytotoxicity in acidic soils.

C-Phycocyanin-derived Phycocyanobilin as a Potential Nutraceutical Approach for Major Neurodegenerative Disorders and COVID-19- induced Damage to the Nervous System.

The edible cyanobacterium Spirulina platensis and its chief biliprotein C-Phycocyanin have shown protective activity in animal models of diverse human health diseases, often reflecting antioxidant and anti-inflammatory effects. The beneficial effects of C-Phycocyanin seem likely to be primarily attributable to its covalently attached chromophore Phycocyanobilin (PCB). Within cells, biliverdin is generated from free heme and it is subsequently reduced to bilirubin. Although bilirubin can function as an oxidant scavenger, its potent antioxidant activity reflects its ability to inactivate some isoforms of NADPH oxidase. Free bilirubin can also function as an agonist for the aryl hydrocarbon receptor (AhR); this may explain its ability to promote protective Treg activity in cellular and rodent models of inflammatory disease. AhR agonists also promote transcription of the gene coding for Nrf-2, and hence can up-regulate phase 2 induction of antioxidant enzymes, such as HO-1. Hence, it is proposed that C-Phycocyanin/PCB chiefly exert their protective effects via inhibition of NADPH oxidase activity, as well as by AhR agonism that both induces Treg activity and up-regulates phase 2 induction. This simple model may explain their potent antioxidant/anti-inflammatory effects. Additionally, PCB might mimic biliverdin in activating anti-inflammatory signaling mediated by biliverdin reductase. This essay reviews recent research in which C-Phycocyanin and/or PCB, administered orally, parenterally, or intranasally, have achieved marked protective effects in rodent and cell culture models of Ischemic Stroke and Multiple Sclerosis, and suggests that these agents may likewise be protective for Alzheimer’s disease, Parkinson’s disease, and in COVID-19 and its neurological complications.

Atypical antipsychotic drugs deregulate the cholesterol metabolism of macrophage-foam cells by activating NOX-ROS-PPARγ-CD36 signaling pathway

BackgroundClinical reports indicate that schizophrenia patients taking atypical antipsychotic drugs suffer from metabolism diseases including atherosclerosis. However, the mechanisms underlying the detrimental effect of atypical antipsychotic drugs on atherosclerosis remain to be explored. MethodsIn this study, we used apolipoprotein E-deficient (apoe−/−) hyperlipidemic mice and apoe−/−cd36−/− mice to investigate the underlying mechanism of atypical antipsychotic drugs on atherosclerosis and macrophage-foam cells. ResultsIn vivo studies showed that genetic deletion of cd36 gene ablated the pro-atherogenic effect of olanzapine in apoe−/− mice. Moreover, in vitro studies revealed that genetic deletion or siRNA-mediated knockdown of cd36 or pharmacological inhibition of CD36 prevented atypical antipsychotic drugs-induced oxLDL accumulation in macrophages. Additionally, olanzapine and clozapine activated NADPH oxidase (NOX) to generate reactive oxygen species (ROS) which upregulated the activity of peroxisome proliferator-activated receptor γ (PPARγ) and subsequently elevated CD36 expression. Inhibition of NOX activity, ROS production or PPARγ activity suppressed CD36 expression and abolished the detrimental effects of olanzapine and clozapine on oxLDL accumulation in macrophages. ConclusionCollectively, our results suggest that atypical antipsychotic drugs exacerbate atherosclerosis and macrophage-foam cell formation by activating the NOX-ROS-PPARγ-CD36 pathway.

Exogenous Nitric Oxide Confers Tolerance to Cr(VI) in Maize (Zea mays L.) Seedlings by Modulating Endogenous Oxido-Nitrosative Events

The use of exogenous compounds such as ‘gasotransmitter’ molecules is a well-established agronomic strategy to improve the crop tolerance to environmental stresses. In this current work, when Cr (200 µM) was combined with the nitric oxide (NO) generator sodium nitroprusside SNP, 500 µM) there was a suppression of metal-induced alterations in embryo growth. Exogenous NO produced by SNP reduced the accumulation of toxic hydrogen peroxide and methylglyoxal and stress-linked proline in Cr-treated seedlings. Chromium increased thiol and S-nitrosothiol levels but this was restored to control levels by SNP, in spite of a competing NO reaction leading to increase of S-nitrosoglutathione content. However, added complexity was indicated by addition of arginine analogue N (ω)-nitro-L-arginine methyl ester (L-NAME, 500 µM) in the germinating medium to suppress endogenous NO production. This suppressed endogenous NO production but superoxide dismutase (SOD) was suppressed not enhanced in Cr + L-NAME treatments. In addition, Cr + L-NAME significantly decreased the content of spermidine and spermine in epicotyls as compared to Cr treatment alone. Similarly, exposure to Cr + SNP decreased spermidine and spermine levels in both radicles and epicotyls. This is important as polyamines have been suggested as route for NO production. Thus, our observations suggest that exogenous NO mitigates Cr-induced damage and confers seedling tolerance to Cr through suppression of NADPH oxidase activity and increased GSNO contents. This may act to prevent an excess of methylglyoxal and hydrogen peroxide. However, the reduction in polyamine mediated cellular NO generation could also promote increased viability under Cr stress.

Involvement of the Microglial Aryl Hydrocarbon Receptor in Neuroinflammation and Vasogenic Edema after Ischemic Stroke.

Microglia are activated after ischemic stroke and induce neuroinflammation. The expression of the aryl hydrocarbon receptor (AhR) has recently been reported to elicit cytokine expression. We previously reported that microglial activation mediates ischemic edema progression. Thus, the purpose of this study was to examine the role of AhR in inflammation and edema after ischemia using a mouse middle cerebral artery occlusion (MCAO) model. MCAO upregulated AhR expression in microglia during ischemia. MCAO increased the expression of tumor necrosis factor α (TNFα) and then induced edema progression, and worsened the modified neurological severity scores, with these being suppressed by administration of an AhR antagonist, CH223191. In THP-1 macrophages, the NADPH oxidase (NOX) subunit p47phox was significantly increased by AhR ligands, especially under inflammatory conditions. Suppression of NOX activity by apocynin or elimination of superoxide by superoxide dismutase decreased TNFα expression, which was induced by the AhR ligand. AhR ligands also elicited p47phox expression in mouse primary microglia. Thus, p47phox may be important in oxidative stress and subsequent inflammation. In MCAO model mice, P47phox expression was upregulated in microglia by ischemia. Lipid peroxidation induced by MCAO was suppressed by CH223191. Taken together, these findings suggest that AhR in the microglia is involved in neuroinflammation and subsequent edema, after MCAO via p47phox expression upregulation and oxidative stress.

Apocynin prevents reduced myocardial nerve growth factor, contributing to amelioration of myocardial apoptosis and failure.

Reduced nerve growth factor (NGF) is associated with cardiac sympathetic nerve denervation in heart failure (HF) which is characterized by increased oxidative stress. Apocynin is considered an antioxidant agent which inhibits NADPH oxidase activity and improves reactive oxygen species scavenging. However, it is unclear whether apocynin prevents reduced myocardial NGF, leading to improvement of cardiac function in HF. In this study, we tested the hypothesis that apocynin prevents reduced myocardial NGF, contributing to amelioration of myocardial apoptosis and failure. Rabbits with myocardial infarction (MI) or sham operation were randomly assigned to receive apocynin or placebo for 4weeks. MI rabbits exhibited left ventricular (LV) dysfunction, and elevation in oxidative stress, as evidenced by a decreased reduced-to-oxidized glutathione ratio and an increased 4-hydroxynonenal expression, and reduction in NGF and NGF receptor tyrosine kinase A (TrKA) expression in the remote non-infarcted myocardium. Apocynin treatment ameliorated LV dysfunction, reduced oxidative stress, prevented decreases in NGF and TrKA expression and reduced cardiomyocyte apoptosis after MI. In cultured H9C2 cardiomyocytes, hypoxia or hydrogen peroxide decreased NGF expression, and apocynin normalized hypoxia-induced reduction of NGF. Recombinant NGF attenuated hypoxia-induced apoptosis. Apocynin prevented hypoxia-induced apoptosis, and the suppressive effect of apocynin on apoptosis was abolished by NGF receptor TrKA inhibitor K252a. We concluded that apocynin prevented reduced myocardial NGF, leading to attenuation of cardiomyocyte apoptosis and LV remodelling and dysfunction in HF after MI. These findings suggest that strategies to prevent NGF reduction by inhibition of oxidative stress may be of value in amelioration of LV dysfunction in HF.

Supernatants of Bifidobacterium longum and Lactobacillus plantarum Strains Exhibited Antioxidative Effects on A7R5 Cells.

Vascular reactive oxygen species (ROS) play an essential role in cardiovascular diseases and the antioxidative effects of probiotics have been widely reported. To screen the probiotic strains that may prevent cardiovascular diseases, we tested the antioxidative effects of supernatants of different Bifidobacterium and Lactobacillus strains on A7R5 cells. Preincubation with supernatants of B. longum CCFM752, L. plantarum CCFM1149, or L. plantarum CCFM10 significantly suppressed the angiotensin II-induced increases in ROS levels and increased catalase (CAT) activity in A7R5, whereas CCFM752 inhibited NADPH oxidase activation and CCFM1149 enhanced the intracellular superoxide dismutase (SOD) activity simultaneously. Treatment with CCFM752, CCFM1149, or CCFM10 supernatants had no significant impact on transcriptional levels of Cat, Sod1, Sod2, Nox1, p22phox, or p47phox, but altered the overall transcriptomic profile and the expression of genes relevant to protein biosynthesis, and up-regulated the 60S ribosomal protein L7a (Rpl7a). A positive correlation between Rpl7a expression and intracellular CAT activity implied that Rpl7a may participate in CAT synthesis in A7R5. Supernatant of CCFM752 could also down-regulate the expression of NADPH oxidase activator 1 (Noxa1) and angiotensinogen in A7R5. Collectively, the probiotic strains CCFM752, CCFM1149, and CCFM10 exhibited antioxidative attributes on A7R5 cells and might help to reduce the risk of cardiovascular diseases.

Selective killing of cancer cells harboring mutant RAS by concomitant inhibition of NADPH oxidase and glutathione biosynthesis

Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.

Endothelium-Dependent Relaxation Effects of Actinidia arguta Extracts in Coronary Artery: Involvement of eNOS/Akt Pathway.

Cardiovascular diseases (CVD) are the major cause of death globally. Bioavailability of nitric oxide, antioxidative activity, and regulation of ionic homeostasis are the key targets for prevention of CVD. Actinidia arguta (AA) has shown promising effect for anticancer, anti-hypercholesterolemia, and antioxidant agents. However, the vascular effect of AA remains unclear. Therefore, we investigated the vascular relaxation of AA extract as well as the underlying mechanisms. Vascular reactivity was assessed in organ baths using porcine coronary arteries and antioxidant properties were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Methanol extract of AA stem (AASE) induced significantly vasorelaxation of porcine coronary artery and its effects is endothelium-dependent without cytotoxicity effects. In addition, ASSE scavenged reactive oxygen species (ROS) in vitro and strongly inhibited NADPH-oxidase activity, which is major source of ROS in vasculature. AASE strongly and dose-dependently activate endothelial nitric oxide synthase (eNOS), the major vascular protective enzyme, and Akt, the upstream signaling protein of eNOS, in porcine coronary artery endothelial cell. Altogether, these results have demonstrated that AASE is a potent endotheliumdependent vasodilator and this effect was involved in, at least in part, Akt/eNOS/NO pathway with strong anti-oxidant properties. The present findings indicate that AA stem could be a valuable candidate of herbal medicine for cardiovascular diseases associated with endothelial dysfunction and atherosclerosis.

Acute antioxidant and cytoprotective effects of sulforaphane in brain endothelial cells and astrocytes during inflammation and excitotoxicity.

Sulforaphane (SFN), a bioactive phytochemical isothiocyanate, has a wide spectrum of cytoprotective effects that involve induction of antioxidant genes. Nongenomic antioxidant effects of SFN have not been investigated. Brain oxidative stress during inflammation and excitotoxicity leads to neurovascular injury. We tested the hypothesis that SNF exhibits acute antioxidant effects and prevents neurovascular injury during oxidative stress. In primary cultures of cerebral microvascular endothelial cells (CMVEC) and cortical astrocytes from the newborn pig brain, a pro‐inflammatory cytokine TNF‐α and an excitotoxic glutamate elevate reactive oxygen species (ROS) and cause cell death by apoptosis. Nox4 NADPH oxidase is the main Nox isoform in CMVEC and cortical astrocytes that is acutely activated by TNF‐α and glutamate leading to ROS‐mediated cell death by apoptosis. The Nox4 inhibitor GKT137831 blocked NADPH oxidase activity and overall ROS elevation, and prevented apoptosis of CMVEC and astrocytes exposed to TNF‐α and glutamate, supporting the leading role of Nox4 in the neurovascular injury. Synthetic SFN (10−11‐10−6 mol/L) inhibited NADPH oxidase activity and reduced overall ROS production in CMVEC and astrocytes within 1‐hour exposure to TNF‐α and glutamate. Furthermore, in the presence of SFN, the ability of TNF‐α and glutamate to produce apoptosis in CMVEC and cortical astrocytes was completely prevented. Overall, SFN at low concentrations exhibits antioxidant and antiapoptotic effects in cerebral endothelial cells and cortical astrocytes via a via a nongenomic mechanism that involves inhibition of Nox4 NADPH oxidase activity. SFN may prevent cerebrovascular injury during brain oxidative stress caused by inflammation and glutamate excitotoxicity.

Dietary nitrate attenuated endothelial dysfunction and atherosclerosis in apolipoprotein E knockout mice fed a high-fat diet: A critical role for NADPH oxidase

Nitric oxide (NO) deficiency and NADPH oxidase plays key roles in endothelial dysfunction and atherosclerotic plaque formation. Recent evidence demonstrates that nitrate-nitrite-NO pathway in vivo exerts beneficial effects upon the cardiovascular system. We aimed to investigate the effects of dietary nitrate on endothelial function and atherosclerosis in apolipoprotein E knockout (ApoE−/-) mice fed a high-fat diet. It was shown that dietary nitrate significantly attenuated aortic endothelial dysfunction and atherosclerosis in ApoE−/- mice. Mechanistic studies revealed that dietary nitrate significantly improved plasma nitrate/nitrite, inhibited vascular NADPH oxidase activity and oxidative stress in ApoE−/- mice, while xanthine oxidoreductase (XOR) expression and activity was enhanced in ApoE−/- mice in comparison with wide type animals. These beneficial effects of nitrate in ApoE−/- mice were abolished by PTIO (NO scavenger) and significantly prevented by febuxostat (XOR inhibitor). In the presence of nitrate, no further effect of apocynin (NADPH oxidase inhibitor) was observed, suggesting NADPH oxidase as a possible target. In vitro, NO donor significantly inhibited NADPH oxidase activity in vascular endothelial cells via the induction of heme oxygenase-1. Altogether, boosting this nitrate-nitrite-NO signaling pathway resulted in the decreases of vascular NADPH oxidase-derived oxidative stress and endothelial dysfunction, and consequently protected ApoE−/- mice against atherosclerosis. These findings may have novel nutritional implications for the preventive and therapeutic strategies against vascular endothelial dysfunction in atherosclerotic disease.

P0988THE PROTECTIVE EFFECT OF RED ORANGE AND LEMON EXTRACT (RLE) IN DIABETIC OBESE ZUCKER RATS IS RELATED TO ITS ACTION ON THE NADPH OXIDASE SUBUNIT

Abstract Background and Aims Type 2 diabetes mellitus is a pathological and chronical condition that induce a rearrangement in carbohydrate metabolism (Kaneto H et al. Antioxid Redox Signal. 2007). Even if the pathological mechanism of DN is not yet clear, NADPH oxidase (NOX) has been demonstrated to be involved in kidney ROS (Reactive Oxygen Species) generation during diabetic state (Palicz A. et al. J Biol Chem. 2001). Therefore, NOX-4, whose p22-phox subunit have shown a pivotal role in diabetes development, has been considered a potential pharmacological target in DN (Gill PS and Wilcox CS. Antioxid Redox Signal. 2006). In previous experiments performed on diabetic rat animal model with DN, we have found a beneficial effect of an extract rich in anthocyanins (ANT), members of flavonoids group (Shih PH. J Agric Food Chem. 2007). The aim of the present work was to investigate the molecular mechanisms involved in the DN and the protective effects of the new extract particularly abundant in ANT, the Red orange and Lemon Extract (RLE). Method Zucker diabetic fatty rats were divided into three groups and treated by gavage daily until 30 weeks of age, as follows: control group (ZF rats) received 1 mL of normal saline; ZDF Group (ZDF rats) received 1mL of normal saline; ZDF + RLE group (ZDF rats) treated with 90 mg/ kg of RLE dissolved in 1mL of normal saline. We examined the gene and protein expression of NOX4 catalytic subunit and its regulatory subunit p22-phox through Real Time PCR and western blot analysis. Results The expression gene levels of NOX4 significantly increased in diabetic rats ZDF, compared to control ZF (***p<0.001). Exposure of ZDF rats with RLE induces a decreased expression of NOX4 (Figure 1) compared to ZDF. On the contrary, there were not significant expression levels variations for regulatory subunit p22-phox. Western blot results are in agreement with Real Time PCR data, in fact an increase in NOX4 protein levels (*p<0.05) is evident, but no significant increase in p22-phox subunit was observed. Conclusion In conclusion, we hypothesize that the protective effect of RLE on the development of DN is related to its ability to inhibit NADPH oxidase activity and opens new perspectives for the treatment of DN.