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1.
Anticancer Res ; 40(9): 5071-5079, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878795

RESUMO

BACKGROUND/AIM: Liver cancer has extremely poor prognosis. The cancerous tissues contain hypoxic regions, and the available drugs are poorly effective in hypoxic environments. NADPH oxidase 4 (NOX4), producing reactive oxygen species (ROS), may contribute to cancer malignancy under hypoxic conditions. However, its role in liver cancer has not been examined in detail. Our aim was to explore the effects of setanaxib, a recently developed selective NOX4 inhibitor, in liver cancer cells under hypoxic conditions. MATERIALS AND METHODS: Liver cancer cell lines (HepG2, HLE and Alexander) were treated with hypoxia-mimetic agent cobalt chloride. Cytotoxicity assays, immunoblot analysis and ROS detection assay were performed to detect the effect of setanaxib under hypoxic conditions. RESULTS: Setanaxib exhibited hypoxia-selective cytotoxicity and triggered apoptosis in cancer cells. Moreover, setanaxib caused mitochondrial ROS accumulation under hypoxic conditions. Treatment with antioxidants markedly attenuated setanaxib-induced cytotoxicity and apoptosis under hypoxic conditions. CONCLUSION: Setanaxib caused mitochondrial ROS accumulation in a hypoxia-selective manner and evoked cancer cell cytotoxicity by inducing apoptosis. Thus, setanaxib has a great potential as a novel anticancer compound under hypoxic conditions.


Assuntos
Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
2.
Life Sci ; 257: 118072, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659367

RESUMO

AIMS: Sunitinib (Su), a tyrosine kinase inhibitor, is one of the most commonly used anti-angiogenic drugs. Some studies have described retinal detachment and photoreceptor damage following systemic exposure to Su, despite beneficial effects achieved with local treatment of ocular pathologies. The aim of this study was to explore the role of NADPH oxidase system and oxidative stress in eyes from Su-treated animals. MAIN METHODS: Male Wistar rats were administered 25 mg Su/kg body weight/day incorporated in the chow for 3 weeks. Upon treatment completion, NADPH oxidase activity and ROS levels were measured in ocular tissue by chemiluminescence and dihydroethidium (DHE) staining, respectively. The expression of NADPH oxidase isoforms (NOX1, NOX2 and NOX4), antioxidant enzymes and endothelial/inducible nitric oxidase isoforms (eNOS/iNOS) in the eyecup and/or retina were measured via immunofluorescence, immunoblotting and RT-qPCR. KEY FINDINGS: NADPH oxidase activity/expression increased in eyecup and retinas from Su-treated rats. Immunohistofluorescence studies in retinal layer confirmed a higher signal of NADPH oxidase isoforms after Su treatment. Treated animals also presented with reductions in NO levels and eNOS expression, whereas iNOS was upregulated. Finally, a significant depletion of antioxidant enzyme glutathione peroxidase was measured in eyecups of rats following Su exposure, and the opposite pattern was seen for glutathione reductase and superoxide dismutase. SIGNIFICANCE: This study demonstrates that Su treatment is associated with NADPH oxidase-derived oxidative stress in the eye. Long-term treatment of Su should be properly monitored to avoid retinotoxic effects that might result in ocular pathologies and sight-threatening conditions.


Assuntos
Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/toxicidade , Retina/efeitos dos fármacos , Sunitinibe/toxicidade , Animais , Antioxidantes/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Masculino , NADPH Oxidases/metabolismo , Ratos , Ratos Wistar , Retina/patologia , Superóxido Dismutase/metabolismo
3.
Toxicol Lett ; 332: 140-145, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32659472

RESUMO

Fumonisin B1 (FB1) is a congener of fumonisins produced by Fusarium species that may be found as corn contaminants threatening health of humans and animals. FB1 causes a variety of toxicity effects, including hepatotoxic, nephrotoxic and cytotoxic effects. However, detailed mechanisms associated with FB1 immunotoxicity in neutrophils are still unclear. To accomplish this, we utilized neutrophils to study the mechanisms of FB1 immunotoxicity. In the current study, we found that FB1 induced the formation of neutrophil extracellular traps (NETs), increased reactive oxygen species (ROS) levels, and decreased SOD and CAT activities. Concurrently, FB1 treatment led to the concentration-dependent phosphorylation of ERK-1/2 and p38 in neutrophils. Moreover, we demonstrated that FB1-induced NET formation was dependent of NADPH oxidase activity. Pretreatment of neutrophils with DPI, U0126 and SB202190 significantly reduced ROS generation, and prevented NET formation, further suggesting that ROS dependent activation of ERK 1/2 and p38 pathways, which possibly mediate FB1-induced NET release in neutrophils. Thus, NET formation and ROS production could be attributed to FB1 immunotoxicity, which might enrich the toxicological mechanisms of FB1.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Fumonisinas/toxicidade , Imunotoxinas/toxicidade , Micotoxinas/toxicidade , Neutrófilos/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Catalase/sangue , Bovinos , Armadilhas Extracelulares/imunologia , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/sangue , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Open Heart ; 7(1)2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-595177

RESUMO

The high rate of thrombotic complications associated with COVID-19 seems likely to reflect viral infection of vascular endothelial cells, which express the ACE2 protein that enables SARS-CoV-2 to invade cells. Various proinflammatory stimuli can promote thrombosis by inducing luminal endothelial expression of tissue factor (TF), which interacts with circulating coagulation factor VII to trigger extrinsic coagulation. The signalling mechanism whereby these stimuli evoke TF expression entails activation of NADPH oxidase, upstream from activation of the NF-kappaB transcription factor that drives the induced transcription of the TF gene. When single-stranded RNA viruses are taken up into cellular endosomes, they stimulate endosomal formation and activation of NADPH oxidase complexes via RNA-responsive toll-like receptor 7. It is therefore proposed that SARS-CoV-2 infection of endothelial cells evokes the expression of TF which is contingent on endosomal NADPH oxidase activation. If this hypothesis is correct, hydroxychloroquine, spirulina (more specifically, its chromophore phycocyanobilin) and high-dose glycine may have practical potential for mitigating the elevated thrombotic risk associated with COVID-19.


Assuntos
Betacoronavirus/patogenicidade , Coagulação Sanguínea , Infecções por Coronavirus/virologia , Endossomos/virologia , Células Endoteliais/virologia , NADPH Oxidases/metabolismo , Pneumonia Viral/virologia , Tromboplastina/metabolismo , Trombose/virologia , Animais , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Coagulação Sanguínea/efeitos dos fármacos , Infecções por Coronavirus/sangue , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática , Fibrinolíticos/uso terapêutico , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Transdução de Sinais , Trombose/sangue , Trombose/enzimologia , Trombose/prevenção & controle
6.
PLoS One ; 15(6): e0233785, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32521542

RESUMO

This study evaluated the impact of combined exercise training on the development of cardiovascular and neuroimmune complications induced by fructose consumption (10% in the drinking water) in hypertensive rats (SHR). After weaning, SHR were divided into 3 groups: SHR (H), SHR+fructose (HF) and SHR+fructose+combined exercise training (treadmill+ladder, 40-60% of maximum capacity) (HFTC). Metabolic, hemodynamic, autonomic, inflammatory and oxidative stress parameters were evaluated in the subgroups (n = 6 group/time) at 7, 15, 30 and 60 days of protocol. Fructose consumption (H vs. HF groups) decreased spontaneous baroreflex sensitivity and total variance of pulse interval at day 7 (7 to 60); increased IL-6 and TNFα in the heart (at day 15, 30 and 60) and NADPH oxidase activity and cardiac lipoperoxidation (LPO) (day 60); increased white adipose tissue weight, reduced insulin sensitivity and increased triglycerides (day 60); induced an additional increase in mean arterial pressure (MAP) (days 30 and 60). Combined exercise training prevented such dysfunctions and sustained increased cardiac IL-10 (day 7) and glutathione redox balance (GSH/GSSG) for the entire protocol. In conclusion, combined exercise training performed simultaneously with exacerbated fructose consumption prevented early cardiovascular autonomic dysfunction, probably trigging positive changes in inflammation and oxidative stress, resulting in a better cardiometabolic profile in rats genetically predisposed to hypertension.


Assuntos
Hipertensão/terapia , Condicionamento Físico Animal/métodos , Animais , Barorreflexo , Pressão Sanguínea , Frutose/efeitos adversos , Frequência Cardíaca , Hipertensão/etiologia , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Masculino , Miocárdio/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo , Ratos , Ratos Endogâmicos SHR , Fator de Necrose Tumoral alfa/metabolismo
7.
PLoS Genet ; 16(6): e1008892, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569316

RESUMO

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living organisms. The homeostasis of NAD is required for plant growth, development, and adaption to environmental cues. In this study, we isolated a chilling hypersensitive Arabidopsis thaliana mutant named qs-2 and identified the causal mutation in the gene encoding quinolinate synthase (QS) critical for NAD biosynthesis. The qs-2 mutant is also hypersensitive to salt stress and abscisic acid (ABA) but resistant to drought stress. The qs-2 mutant accumulates a reduced level of NAD and over-accumulates reactive oxygen species (ROS). The ABA-hypersensitivity of qs-2 can be rescued by supplementation of NAD precursors and by mutations in the ABA signaling components SnRK2s or RBOHF. Furthermore, ABA-induced over-accumulation of ROS in the qs-2 mutant is dependent on the SnRK2s and RBOHF. The expression of QS gene is repressed directly by ABI4, a transcription factor in the ABA response pathway. Together, our findings reveal an unexpected interplay between NAD biosynthesis and ABA and stress signaling, which is critical for our understanding of the regulation of plant growth and stress responses.


Assuntos
Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Complexos Multienzimáticos/genética , Reguladores de Crescimento de Planta/metabolismo , Estresse Fisiológico/genética , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Retroalimentação Fisiológica , Perfilação da Expressão Gênica , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Mutação , NAD/biossíntese , NADPH Oxidases/metabolismo , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo
8.
Life Sci ; 256: 117914, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512010

RESUMO

Hyperglycemia and oxidative stress are the primary stressors that elicit mitochondria specific cell stress in diabetes. Here we hypothesized that elevated level of ROS in high glucose (HG) environment, trigger mitochondrial stress by damaging mitochondrial DNA (mtDNA), altering inflammatory mediators, and neurodegenerative markers via stress signalling pathway in retinal ganglion cells (RGC-5). Mechanistically, our findings illustrated that the HG environment increases the ROS production in retinal cells leading to the disruption of antioxidant defence mechanism, and altering mitochondrial machinery such as an increase in loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, and increase in mtDNA fragmentation. Furthermore, fragmented mtDNA escape from mitochondria into the cytosol, where it engaged with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN gene (STING) phosphorylation and activate interferon regulatory factor 3 (IRF3) via ERK1/2-Akt-tuberin-mTOR dependent pathways. Our results further indicate that siRNA-mediated gene silencing of tuberin suppresses the strong downregulation of tuberin-mTOR-IRF3 activation. HG environment resulted in activation of IRF3, coinciding with the increased expression of inflammatory mediators and neurodegenerative markers. Pre-treatment of N-acetyl-l-cysteine (NAC) or ERK1/2 or phosphoinositide3-kinase (PI3-K)/Akt inhibitors in RGC-5 cells significantly reduced the HG-induced IRF3 expression and declined the expression of neurodegenerative markers. Collectively, our results demonstrates that HG-induced over production of ROS, disrupts the antioxidant defence mechanism and mitochondrial dysfunction, leading to alterations of inflammatory mediators and neurodegenerative markers through the ERK1/2-Akt-tuberin-mTOR dependent signalling pathway in RGC-5 cells.


Assuntos
Inibidores Enzimáticos/metabolismo , Glucose/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Ganglionares da Retina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Acetilcisteína/metabolismo , Animais , DNA Mitocondrial , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperglicemia/metabolismo , Inflamação/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
9.
Open Heart ; 7(1)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32532805

RESUMO

The high rate of thrombotic complications associated with COVID-19 seems likely to reflect viral infection of vascular endothelial cells, which express the ACE2 protein that enables SARS-CoV-2 to invade cells. Various proinflammatory stimuli can promote thrombosis by inducing luminal endothelial expression of tissue factor (TF), which interacts with circulating coagulation factor VII to trigger extrinsic coagulation. The signalling mechanism whereby these stimuli evoke TF expression entails activation of NADPH oxidase, upstream from activation of the NF-kappaB transcription factor that drives the induced transcription of the TF gene. When single-stranded RNA viruses are taken up into cellular endosomes, they stimulate endosomal formation and activation of NADPH oxidase complexes via RNA-responsive toll-like receptor 7. It is therefore proposed that SARS-CoV-2 infection of endothelial cells evokes the expression of TF which is contingent on endosomal NADPH oxidase activation. If this hypothesis is correct, hydroxychloroquine, spirulina (more specifically, its chromophore phycocyanobilin) and high-dose glycine may have practical potential for mitigating the elevated thrombotic risk associated with COVID-19.


Assuntos
Betacoronavirus/patogenicidade , Coagulação Sanguínea , Infecções por Coronavirus/virologia , Endossomos/virologia , Células Endoteliais/virologia , NADPH Oxidases/metabolismo , Pneumonia Viral/virologia , Tromboplastina/metabolismo , Trombose/virologia , Animais , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Coagulação Sanguínea/efeitos dos fármacos , Infecções por Coronavirus/sangue , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática , Fibrinolíticos/uso terapêutico , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Transdução de Sinais , Trombose/sangue , Trombose/enzimologia , Trombose/prevenção & controle
10.
Arch Biochem Biophys ; 690: 108416, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32502471

RESUMO

Alkaptonuria (AKU) is a rare metabolic disease correlated with the deficiency of homogentisate 1,2-dioxygenase and leading to an accumulation of the metabolite homogentisic acid (HGA) which can be subjected to oxidation and polymerization reactions. These events are considered a trigger for the induction of oxidative stress in AKU but, despite the large description of an altered redox status, the underlying pathogenetic processes are still unstudied. In the present study, we investigated the molecular mechanisms responsible for the oxidative damage present in an osteoblast-based cellular model of AKU. Bone, in fact, is largely affected in AKU patients: severe osteoclastic resorption, osteoporosis, even for pediatric cases, and an altered rate of remodeling biomarkers have been reported. In our AKU osteoblast cell model, we found a clear altered redox homeostasis, determined by elevated hydrogen peroxide (H2O2) levels and 4HNE protein adducts formation. These findings were correlated with increased NADPH oxidase (NOX) activity and altered mitochondrial respiration. In addition, we observed a decreased activity of superoxide dismutase (SOD) and reduced levels of thioredoxin (TRX) that parallel the decreased Nrf2-DNA binding. Overall, our results reveal that HGA is able to alter the cellular redox homeostasis by modulating the endogenous ROS production via NOX activation and mitochondrial dysfunctions and impair the cellular response mechanism. These findings can be useful for understanding the pathophysiology of AKU, not yet well studied in bones, but which is an important source of comorbidities that affect the life quality of the patients.


Assuntos
Alcaptonúria/metabolismo , Homeostase/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Ácido Homogentísico/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , NADPH Oxidases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Osteoblastos/citologia , Oxirredução , Estresse Oxidativo/fisiologia , Transdução de Sinais , Superóxido Dismutase/metabolismo , Tiorredoxinas/metabolismo
11.
J Pineal Res ; 69(3): e12676, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32597503

RESUMO

Melatonin is a chronobiotic hormone, which can regulate human diseases like cancer, atherosclerosis, respiratory disorders, and microbial infections by regulating redox system. Melatonin exhibits innate immunomodulation by communicating with immune system and influencing neutrophils to fight infections and inflammation. However, sustaining redox homeostasis and reactive oxygen species (ROS) generation in neutrophils are critical during chemotaxis, oxidative burst, phagocytosis, and neutrophil extracellular trap (NET) formation. Therefore, endogenous antioxidant glutathione (GSH) redox cycle is highly vital in regulating neutrophil functions. Reduced intracellular GSH levels and glutathione reductase (GR) activity in the neutrophils during clinical conditions like autoimmune disorders, neurological disorders, diabetes, and microbial infections lead to dysfunctional neutrophils. Therefore, we hypothesized that redox modulators like melatonin can protect neutrophil health and functions under GSH and GR activity-deficient conditions. We demonstrate the dual role of melatonin, wherein it protects neutrophils from oxidative stress-induced apoptosis by reducing ROS generation; in contrast, it restores neutrophil functions like phagocytosis, degranulation, and NETosis in GSH and GR activity-deficient neutrophils by regulating ROS levels both in vitro and in vivo. Melatonin mitigates LPS-induced neutrophil dysfunctions by rejuvenating GSH redox system, specifically GR activity by acting as a parallel redox system. Our results indicate that melatonin could be a potential auxiliary therapy to treat immune dysfunction and microbial infections, including virus, under chronic disease conditions by restoring neutrophil functions. Further, melatonin could be a promising immune system booster to fight unprecedented pandemics like the current COVID-19. However, further studies are indispensable to address the clinical usage of melatonin.


Assuntos
Antioxidantes/uso terapêutico , Glutationa/metabolismo , Melatonina/uso terapêutico , Neutrófilos/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Feminino , Glutationa Redutase/metabolismo , Humanos , Masculino , Melatonina/farmacologia , Camundongos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
12.
Arch Biochem Biophys ; 689: 108453, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32524996

RESUMO

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.


Assuntos
Aterosclerose/terapia , Endotélio Vascular/patologia , NADPH Oxidases/metabolismo , Nitratos/uso terapêutico , Animais , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Nitratos/metabolismo , Nitritos/metabolismo , Estresse Oxidativo
13.
Life Sci ; 256: 117886, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32497631

RESUMO

AIMS: Reactive oxygen species (ROS) caused by high glucose (HG) is involved in a lot of diseases including diabetes. However, the underlying mechanism of ROS induction by HG remains unclear. Emerging evidence has shown the 8-oxoguanine glycosylase (OGG1) is the main DNA glycosylase responsible for atherosclerosis, obesity, hepatic steatosis, and insulin resistance, and so on. Our aim was to explore the role of OGG1 on HG-mediated endothelial ROS. MAIN METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to HG (30 mM) for different time periods. HG predominantly inhibited OGG1 expression in a time-dependent manner measured by western blotting, qPCR and immunofluorescence. Additionally, HUVECs were cultured with a fluorescent probe, DCFH and DHE, after being subjected to HG. Cell chemiluminescence and flow cytometry results revealed that HG caused endothelial ROS activation. KEY FINDINGS: High glucose remarkably decreased endothelial OGG1 expression. The overexpression of OGG1 significantly reversed HG-mediated PKC and NADPH oxidase activities and ROS levels. Moreover, manipulated expression of PKC significantly contacted the role of OGG1 on NADPH oxidase activation. SIGNIFICANCE: These results suggest that OGG1 downregulation promoted HG-induced endothelial ROS production and might be a potential clinical treatment target of diabetics.


Assuntos
DNA Glicosilases/metabolismo , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/metabolismo , NADPH Oxidases/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
15.
Adv Exp Med Biol ; 1246: 153-177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399830

RESUMO

The key purpose of phagocytosis is the destruction of pathogenic microorganisms. The phagocytes exert a wide array of killing mechanisms that allow mastering the vast majority of pathogens. One of these mechanisms consists in the production of reactive oxygen species inside the phagosome by a specific enzyme, the phagocyte NADPH oxidase. This enzyme is composed of 6 proteins that need to assemble to form a complex on the phagosomal membrane. Multiple signaling pathways tightly regulate the assembly. We briefly summarize key features of the enzyme and its regulation. We then focus on several related topics that address the activity of the NADPH oxidase during phagocytosis. Novel fluorescence microscopy techniques combined with fluorescent protein labeling of NADPH oxidase subunits opened the view on the structure and dynamics of these proteins in living cells. This combination revealed details of the role of anionic phospholipids in the control of phagosomal ROS production. It also added critical information to propose a 3D model of the complex between the cytosolic subunits prior to activation, in complement to other structural data on the oxidase.


Assuntos
NADPH Oxidases/metabolismo , Fagossomos/enzimologia , Humanos , Fagócitos/citologia , Fagócitos/enzimologia , Fagócitos/metabolismo , Fagocitose , Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
16.
Am J Physiol Heart Circ Physiol ; 319(1): H51-H65, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32412791

RESUMO

Although there is a strong association between cigarette smoking exposure (CSE) and vascular endothelial dysfunction (VED), the underlying mechanisms by which CSE triggers VED remain unclear. Therefore, studies were performed to define these mechanisms using a chronic mouse model of cigarette smoking (CS)-induced cardiovascular disease mirroring that in humans. C57BL/6 male mice were subjected to CSE for up to 48 wk. CSE impaired acetylcholine (ACh)-induced relaxation of aortic and mesenteric segments and triggered hypertension, with mean arterial blood pressure at 32 and 48 wk of exposure of 122 ± 6 and 135 ± 5 mmHg compared with 99 ± 4 and 102 ± 6 mmHg, respectively, in air-exposed mice. CSE led to monocyte activation with superoxide generation in blood exiting the pulmonary circulation. Macrophage infiltration with concomitant increase in NADPH oxidase subunits p22phox and gp91phox was seen in aortas of CS-exposed mice at 16 wk, with further increase out to 48 wk. Associated with this, increased superoxide production was detected that decreased with Nox inhibition. Tetrahydrobiopterin was progressively depleted in CS-exposed mice but not in air-exposed controls, resulting in endothelial nitric oxide synthase (eNOS) uncoupling and secondary superoxide generation. CSE led to a time-dependent decrease in eNOS and Akt expression and phosphorylation. Overall, CSE induces vascular monocyte infiltration with increased NADPH oxidase-mediated reactive oxygen species generation and depletes the eNOS cofactor tetrahydrobiopterin, uncoupling eNOS and triggering a vicious cycle of oxidative stress with VED and hypertension. Our study provides important insights toward understanding the process by which smoking contributes to the genesis of cardiovascular disease and identifies biomarkers predictive of disease.NEW & NOTEWORTHY In a chronic model of smoking-induced cardiovascular disease, we define underlying mechanisms of smoking-induced vascular endothelial dysfunction (VED). Smoking exposure triggered VED and hypertension and led to vascular macrophage infiltration with concomitant increase in superoxide and NADPH oxidase levels as early as 16 wk of exposure. This oxidative stress was accompanied by tetrahydrobiopterin depletion, resulting in endothelial nitric oxide synthase uncoupling with further superoxide generation triggering a vicious cycle of oxidative stress and VED.


Assuntos
Endotélio Vascular/metabolismo , Leucócitos/metabolismo , Estresse Oxidativo , Lesão por Inalação de Fumaça/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Vasodilatação , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Pressão Sanguínea , Endotélio Vascular/fisiopatologia , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Lesão por Inalação de Fumaça/etiologia , Lesão por Inalação de Fumaça/fisiopatologia , Superóxidos/metabolismo
17.
Nat Commun ; 11(1): 1838, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296066

RESUMO

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection. The plant NADPH oxidase RBOHD is a primary player in ROS production during innate immunity. However, how RBOHD is negatively regulated remains elusive. Here we show that RBOHD is regulated by C-terminal phosphorylation and ubiquitination. Genetic and biochemical analyses reveal that the PBL13 receptor-like cytoplasmic kinase phosphorylates RBOHD's C-terminus and two phosphorylated residues (S862 and T912) affect RBOHD activity and stability, respectively. Using protein array technology, we identified an E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. Mimicking phosphorylation of RBOHD (T912D) results in enhanced ubiquitination and decreased protein abundance. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Thus, our study reveals an intricate post-translational network that negatively regulates the abundance of a conserved NADPH oxidase.


Assuntos
Proteínas de Arabidopsis/metabolismo , NADPH Oxidases/metabolismo , Imunidade Vegetal/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , NADPH Oxidases/genética , Fosforilação/genética , Fosforilação/fisiologia , Doenças das Plantas/genética , Imunidade Vegetal/genética , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitinação/genética , Ubiquitinação/fisiologia
18.
Nat Commun ; 11(1): 2031, 2020 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-32341348

RESUMO

Neutrophils employ several mechanisms to restrict fungi, including the action of enzymes such as myeloperoxidase (MPO) or NADPH oxidase, and the release of neutrophil extracellular traps (NETs). Moreover, they cooperate, forming "swarms" to attack fungi that are larger than individual neutrophils. Here, we designed an assay for studying how these mechanisms work together and contribute to neutrophil's ability to contain clusters of live Candida. We find that neutrophil swarming over Candida clusters delays germination through the action of MPO and NADPH oxidase, and restricts fungal growth through NET release within the swarm. In comparison with neutrophils from healthy subjects, those from patients with chronic granulomatous disease produce larger swarms against Candida, but their release of NETs is delayed, resulting in impaired control of fungal growth. We also show that granulocyte colony-stimulating factors (GCSF and GM-CSF) enhance swarming and neutrophil ability to restrict fungal growth, even during treatment with chemical inhibitors that disrupt neutrophil function.


Assuntos
Candida albicans/crescimento & desenvolvimento , Neutrófilos/citologia , Neutrófilos/microbiologia , Sistemas CRISPR-Cas , Candidíase/microbiologia , Linhagem Celular , Armadilhas Extracelulares/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Doença Granulomatosa Crônica/microbiologia , Humanos , Processamento de Imagem Assistida por Computador , Análise em Microsséries , NADPH Oxidases/metabolismo , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Arch Biochem Biophys ; 685: 108355, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32268137

RESUMO

Psoriasis is a skin disease characterized by abnormal keratinocyte proliferation and inflammation. Currently, there are no cures for this disease, so the goal of treatment is to decrease inflammation and slow down the associated rapid cell growth and shedding. Recent advances have led to the usage of phosphodiesterase 4 (PDE4) inhibitors for treatment of this condition. For example, apremilast is an oral, selective PDE4 inhibitor that is able to reduce skin inflammation and is Food and Drug Administration (FDA)-approved to treat adults with moderate to severe psoriasis and/or psoriatic arthritis. However, common target-related adverse events, including diarrhea, nausea, headache, and insomnia limit the usage of this drug. To circumvent these effects, the usage of PDE4 inhibitors specifically designed for topical treatment, such as CHF6001, may combine local anti-inflammatory activity with limited systemic exposure, improving tolerability. In this study, we showed that CHF6001, currently undergoing clinical development for COPD, suppresses human keratinocyte proliferation as assessed via BrdU incorporation. We also observed decreased re-epithelialization in a scratch-wound model after CHF6001 treatment. At the molecular level, CHF6001 inhibited translocation of phosphorylated NF-κB subunit p65, promoting loss of nuclear cyclin D1 accumulation and an increase of cell cycle inhibitor p21. Furthermore, CHF6001 decreased oxidative stress, measured by assessing lipid peroxidation (4-HNE adduct formation), through the inactivation of the NADPH oxidase. These results suggest that CHF6001 has the potential to treat skin disorders associated with hyperproliferative keratinocytes, such as psoriasis by targeting oxidative stress, abnormal re-epithelization, and inflammation.


Assuntos
Proliferação de Células/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Sulfonamidas/farmacologia , para-Aminobenzoatos/farmacologia , Aldeídos/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Humanos , NADPH Oxidases/metabolismo , Inibidores da Fosfodiesterase 4/toxicidade , Psoríase/tratamento farmacológico , Sulfonamidas/toxicidade , Fator de Transcrição RelA/metabolismo , para-Aminobenzoatos/toxicidade
20.
Nat Commun ; 11(1): 1783, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286292

RESUMO

The microbial adaptations to the respiratory burst remain poorly understood, and establishing how the NADPH oxidase (NOX2) kills microbes has proven elusive. Here we demonstrate that NOX2 collapses the ΔpH of intracellular Salmonella Typhimurium. The depolarization experienced by Salmonella undergoing oxidative stress impairs folding of periplasmic proteins. Depolarization in respiring Salmonella mediates intense bactericidal activity of reactive oxygen species (ROS). Salmonella adapts to the challenges oxidative stress imposes on membrane bioenergetics by shifting redox balance to glycolysis and fermentation, thereby diminishing electron flow through the membrane, meeting energetic requirements and anaplerotically generating tricarboxylic acid intermediates. By diverting electrons away from the respiratory chain, glycolysis also enables thiol/disulfide exchange-mediated folding of bacterial cell envelope proteins during periods of oxidative stress. Thus, primordial metabolic pathways, already present in bacteria before aerobic respiration evolved, offer a solution to the stress ROS exert on molecular targets at the bacterial cell envelope.


Assuntos
Glicólise/fisiologia , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Salmonella typhimurium/enzimologia , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação/genética , Fermentação/fisiologia , Glicólise/genética , NADPH Oxidases/genética , Oxirredução , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Salmonella typhimurium/genética
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