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1.
J Immunol ; 207(3): 923-937, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34301842

RESUMO

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by genetic defects in leukocyte NADPH oxidase, which has both microbicidal and immunomodulatory roles. Hence, CGD is characterized by recurrent bacterial and fungal infections as well as aberrant inflammation. Fungal cell walls induce neutrophilic inflammation in CGD; yet, underlying mechanisms are incompletely understood. This study investigated the receptors and signaling pathways driving aberrant proinflammatory cytokine production in CGD neutrophils activated by fungal cell walls. Although cytokine responses to ß-glucan particles were similar in NADPH oxidase-competent and NADPH oxidase-deficient mouse and human neutrophils, stimulation with zymosan, a more complex fungal particle, induced elevated cytokine production in NADPH oxidase-deficient neutrophils. The dectin-1 C-type lectin receptor, which recognizes ß-glucans (1-3), and TLRs mediated cytokine responses by wild-type murine neutrophils. In the absence of NADPH oxidase, fungal pathogen-associated molecular patterns engaged additional collaborative signaling with Mac-1 and TLRs to markedly increase cytokine production. Mechanistically, this cytokine overproduction is mediated by enhanced proximal activation of tyrosine phosphatase SHP2-Syk and downstream Card9-dependent NF-κB and Card9-independent JNK-c-Jun. This activation and amplified cytokine production were significantly decreased by exogenous H2O2 treatment, enzymatic generation of exogenous H2O2, or Mac-1 blockade. Similar to zymosan, Aspergillus fumigatus conidia induced increased signaling in CGD mouse neutrophils for activation of proinflammatory cytokine production, which also used Mac-1 and was Card9 dependent. This study, to our knowledge, provides new insights into how NADPH oxidase deficiency deregulates neutrophil cytokine production in response to fungal cell walls.


Assuntos
Aspergillus fumigatus/fisiologia , Doença Granulomatosa Crônica/imunologia , Lectinas Tipo C/metabolismo , Antígeno de Macrófago 1/metabolismo , NADPH Oxidase 2/metabolismo , Neutrófilos/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Antígenos de Fungos/imunologia , Células Cultivadas , Citocinas/metabolismo , Doença Granulomatosa Crônica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/genética , NF-kappa B/metabolismo , Ativação de Neutrófilo , Padrões Moleculares Associados a Patógenos/imunologia , Receptor Cross-Talk , Transdução de Sinais , beta-Glucanas/imunologia
2.
Redox Biol ; 45: 102051, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34217063

RESUMO

Aberrant placental angiogenesis is associated with fetal intrauterine growth restriction (IUGR), but the mechanism underlying abnormal placental angiogenesis remains largely unknown. Here, lower vessel density and higher expression of NADPH oxidases 2 (Nox2) were observed in the placentae for low birth weight (LBW) fetuses versus normal birth weight (NBW) fetuses, with a negative correlation between Nox2 and placental vessel density. Moreover, it was revealed for the first time that Nox2 deficiency facilitates angiogenesis in vitro and in vivo, and vascular endothelial growth factor-A (VEGF-A) has an essential role in Nox2-controlled inhibition of angiogenesis in porcine vascular endothelial cells (PVECs). Mechanistically, Nox2 inhibited phospho-signal transducer and activator of transcription 3 (p-STAT3) in the nucleus by inducing the production of mitochondrial reactive oxygen species (ROS). Dual-luciferase assay confirmed that knockdown of Nox2 reduces the expression of VEGF-A in an STAT3 dependent manner. Our results indicate that Nox2 is a potential target for therapy by increasing VEGF-A expression to promote angiogenesis and serves as a prognostic indicator for fetus with IUGR.


Assuntos
NADPH Oxidase 2/metabolismo , Neovascularização Fisiológica , Placenta , Fator A de Crescimento do Endotélio Vascular , Animais , Células Endoteliais/metabolismo , Feminino , Placenta/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/genética , Suínos , Fator A de Crescimento do Endotélio Vascular/genética
3.
Front Immunol ; 12: 636954, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34168641

RESUMO

Neutrophil extracellular traps (NETs) consist of decondensed nuclear chromatin that is associated with proteins and are released by neutrophils during an inflammatory response. Released NETs are able to capture pathogens, prevent their dissemination and potentially kill them via antimicrobial peptides and proteins that are associated with the decondensed chromatin. In addition to their antimicrobial functions, NETs have also been shown to exert immunomodulatory effects by activation and differentiation of macrophages, dendritic cells and T cells. However, the effect of NETs on neutrophil functions is poorly understood. Here we report the first comprehensive study regarding the effects of NETs on human primary neutrophils in vitro. NETs were isolated from cultures of PMA-exposed neutrophils. Exposure of neutrophils to isolated NETs resulted in the activation of several neutrophil functions in a concentration-dependent manner. NETs induced exocytosis of granules, the production of reactive oxygen species (ROS) by the NADPH oxidase NOX2, NOX2-dependent NET formation, increased the phagocytosis and killing of microbial pathogens. Furthermore, NETs induced the secretion of the proinflammatory chemokine IL-8 and the B-cell-activating cytokine BAFF. We could show that the NET-induced activation of neutrophils occurs by pathways that involve the phosphorylation of Akt, ERK1/2 and p38. Taken together our results provide further insights into the proinflammatory role of NETs by activating neutrophil effector function and further supports the view that NETs can amplify inflammatory events. On the one hand the amplified functions enhance the antimicrobial defense. On the other hand, NET-amplified neutrophil functions can be involved in the pathophysiology of NET-associated diseases. In addition, NETs can connect the innate and adaptive immune system by inducing the secretion of the B-cell-activating cytokine BAFF.


Assuntos
Armadilhas Extracelulares/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Fator Ativador de Células B/metabolismo , Células Cultivadas , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Sistema de Sinalização das MAP Quinases , NADPH Oxidase 2/metabolismo , Ativação de Neutrófilo , Fagocitose , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo
4.
Sci Rep ; 11(1): 11970, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099836

RESUMO

The NADPH oxidase enzymes Nox2 and 4, are important generators of Reactive oxygen species (ROS). These enzymes are abundantly expressed in cardiomyocytes and have been implicated in ischemia-reperfusion injury. Previous attempts with full inhibition of their activity using genetically modified animals have shown variable results, suggesting that a selective and graded inhibition could be a more relevant approach. We have, using chemical library screening, identified a new compound (GLX481304) which inhibits Nox 2 and 4 (with IC50 values of 1.25 µM) without general antioxidant effects or inhibitory effects on Nox 1. The compound inhibits ROS production in isolated mouse cardiomyocytes and improves cardiomyocyte contractility and contraction of whole retrogradely (Langendorff) perfused hearts after a global ischemia period. We conclude that a pharmacological and partial inhibition of ROS production by inhibition of Nox 2 and 4 is beneficial for recovery after ischemia reperfusion and might be a promising venue for treatment of ischemic injury to the heart.


Assuntos
Antioxidantes/química , Inibidores Enzimáticos/química , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Oxirredução , Estresse Oxidativo , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
5.
J Oleo Sci ; 70(7): 947-954, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34121036

RESUMO

Lysophospholipids (LPLs) are small bioactive lipid molecules characterized by a single carbon chain and a polar head group. LPLs have recently shown to be involved in many physiological and pathological processes such as nervous system regulation. In our previous studies, a porcine liver decomposition product (PLDP) has been identified as a substance that improves cognitive function at old ages. This PLDP is a rich source of LPLs, including lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). This study was designed to evaluate the anti-inflammatory effect of these LPLs on lipopolysaccharide (LPS)-stimulated SIM-A9 microglial cells in terms of cytokine expression and oxidative stress and to investigate the potential mechanisms underlying these effects. SIM-A9 cells were pretreated with LPLs prior to LPS stimulation, and the anti-inflammatory potential of the LPLs in LPS-induced SIM-A9 cells was examined. Pretreatment with LPLs significantly inhibited the LPS-induced expression of IL-6 in SIM-A9 cells. Furthermore, oxidative-related protein, NADPH oxidase 2 (Nox2) levels were markedly increased in the LPS-treated cells, and pretreatment with LPC and LPE significantly reduced to basal levels. In addition, LPS-induced ROS production was eliminated in apocynin-treated cells, indicating that ROS production was dependent on Nox2. Our findings revealed that pretreatment with LPC and LPE decreased LPS-stimulated ROS production. These results indicated that LPC and LPE exerted significant protective effects against LPS-induced inflammation and oxidative stress in SIM-A9 cell.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Lisofosfatidilcolinas/farmacologia , Lisofosfolipídeos/farmacologia , Microglia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Camundongos , NADPH Oxidase 2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
6.
Toxicol Lett ; 349: 1-11, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34052309

RESUMO

Pesticides exposure can lead to damage of dopaminergic neurons, which are associated with increased risk of Parkinson's disease (PD). However, the etiology of PD remains poorly understood and no therapeutic strategy is available. Previous studies suggested the involvement of NLRP3 inflammasome in the onset of PD. This study was designed to investigate whether glibenclamide, an inhibitor of NLRP3 inflammasome, could offer a reliable protective strategy for PD in a mouse PD model induced by paraquat and maneb. We found that glibenclamide exerted potent neuroprotection against paraquat and maneb-induced upregulation of α-synuclein, dopaminergic neurodegeneration and motor impairment in brain of mice. Mechanistically, glibenclamide treatment blocked NLRP3 inflammasome activation evidenced by reduced expressions of NLRP3, activated caspase-1 and mature interleukin-1ß in glibenclamide co-treated mice compared with those in paraquat and maneb group mice. Furthermore, glibenclamide treatment mitigated paraquat and maneb-induced microglial M1 proinflammatory response and nuclear factor-κB activation in mice. Finally, the increased superoxide production, lipid peroxidation, protein levels of NADPH oxidase 2 (NOX2) and inducible nitric oxide synthase (iNOS) induced by paraquat and maneb were all attenuated by glibenclamide. Overall, our findings demonstrated that glibenclamide protected dopaminergic neurons in a mouse PD model induced by combined exposures of paraquat and maneb through suppression of NLRP3 inflammasome activation, microglial M1 polarization and oxidative stress.


Assuntos
Antiparkinsonianos/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Glibureto/farmacologia , Inflamassomos/antagonistas & inibidores , Atividade Motora/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Degeneração Neural , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos/prevenção & controle , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Inflamassomos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Maneb , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , NADPH Oxidase 2/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Paraquat , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia
7.
Neurochem Res ; 46(8): 2089-2096, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34008119

RESUMO

Repeated morphine administration results in analgesic tolerance. However, the underlying mechanism of morphine analgesic tolerance remains unclear. NADPH-oxidase 2 (NOX2) is the first discovered NADPH oxidase, which mainly functions to produce reactive oxygen species. Its specific role in morphine tolerance has not been fully investigated. In this work, we found that chronic morphine administration significantly increased the expression of NOX2 in spinal cord. Pretreatment of NOX2 inhibitor blocked the upregulation of NOX2 and autophagy markers, including LC3B and P62, and consequently the development of morphine tolerance. NOX2 and LC3B were both colocalized with NeuN in spinal dorsal horn in morphine-tolerant rats. Our results suggest that the increased autophagy activity in spinal neurons promoted by NOX2 activation contributes to the development of morphine tolerance. NOX2 may be considered as a new therapeutic target for morphine tolerance.


Assuntos
Analgésicos Opioides/farmacologia , Autofagia/efeitos dos fármacos , Tolerância a Medicamentos/fisiologia , Morfina/farmacologia , NADPH Oxidase 2/metabolismo , Neurônios/efeitos dos fármacos , Animais , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Oxidase 2/antagonistas & inibidores , Oniocompostos/farmacologia , Ratos Sprague-Dawley , Corno Dorsal da Medula Espinal/citologia
9.
Biochemistry (Mosc) ; 86(3): 262-274, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33838628

RESUMO

Altered expression of cellular redox genes and proteins contributes to invasion, metastasis, and drug resistance in cancer. NADPH oxidase (NOX) isoforms are the pro-oxidant enzymes that generate ROS as a primary product. Dysregulation of NOX activity and expression alters ROS generation, which either directly or indirectly modulates cell death and survival signaling during the progression of cancer. Nuclear factor erythroid 2-related factor 2 (Nrf-2) is an inducible transcription factor, which transcribes an array of antioxidant genes and protects cancer cells from the oxidative stress. Both NOXs and Nrf-2 participate in the regulation of cellular redox homeostasis; but their dysregulation promotes oxidative stress, which contributes to the progression of different types of cancer. Indeed, the role of NOX isoforms and Nrf-2 in developing the drug resistance in cancer is largely unknown. In the present study, we have explored the association of NOX isoforms and Nrf-2 signaling with the MDR1 gene expression in colon carcinoma cells (HCT-116/R). The MDR1 gene was overexpressed to develop resistant HCT-116/R cells and the NOX activation and ROS generation were monitored. We also assessed the role of NOX isoforms and Nrf-2 in the 5-fluorouracil (5-FU) mediated apoptotic cell death of HCT-116/R cells. The HCT-116/R cells demonstrated higher expression of HIF-1α, Nrf-2, and HO-1 and were highly resistant to 5-FU; they also displayed upregulated expression and activity of NOX-2, as well as elevated ROS levels. Interestingly, the treatment with HDC, a specific NOX-2 inhibitor, reduced the ROS levels in HCT-116/R cells. The treatment with HDC and ML-385 (specific inhibitor of Nrf-2) augmented the 5-FU-mediated apoptotic cell death of HCT-116/R cells, which suggests that NOX-2 and Nrf-2 are involved in the development of the chemoresistant phenotype of these cells. Taken together, NOX-2 and Nrf-2 are associated with developing drug resistance of colorectal cancer cells and might be potential targets to overcome drug resistance during cancer therapy.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , NADPH Oxidase 2/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/fisiopatologia , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Estresse Oxidativo , Isoformas de Proteínas , Transdução de Sinais
10.
Mol Biol Rep ; 48(3): 2335-2350, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33811574

RESUMO

Mounting evidences have shown that nicotinamide adenine dinucleotide phosphate oxidase-2 (Nox-2) pathway modifies glutamic-acid decarboxylase-67 (GAD67) (GABAergic enzyme) and cholinergic systems via oxidative-nitrergic mechanisms in schizophrenia pathology. Rutin, a neuroactive antioxidant compound, with proven neuroprotective property has been shown to reduce schizophrenic-like behavior in mice. This study sought to investigate the mechanisms of action of the psychopharmacological activity of rutin in the preventive and reversal effects of ketamine-induced schizophrenic-like behavior, oxidative-nitrergic stress, cholinergic and GABAergic derangements in mice. In the preventive treatment, male mice were given rutin (0.1, 0.2 and 0.4 mg/kg) or risperidone (0.5 mg/kg) orally for 14 days prior to ketamine (20 mg/kg, i.p.) treatment from the 8 to 14th day. However, in the reversal treatment, ketamine was given for 14 days prior to rutin and risperidone. Behavioral (open-field, social-interaction and Y-maze tests), biochemical (oxidative/nitrergic stress markers, acetylcholinesterase activity), immunohistochemical (GAD67, Nox-2) and neuronal cell deaths in the striatum, prefrontal cortex, and hippocampus were evaluated. Ketamine-induced behavioral impairments were prevented and reversed by rutin. Exposure of mice to ketamine increased malondialdehyde, nitrite contents, acetylcholinesterase activity, neuronal cell death and Nox-2 expressions in the striatum, prefrontal cortex and hippocampus. Conversely, these derangements were prevented and reversed by rutin. The decreased glutathione levels due to ketamine were marked increased by rutin. Rutin only prevented ketamine-induced decrease in GAD67 expression in the striatal-hippocampal region. Altogether, the study showed that the prevention and reversal treatments of mice with rutin attenuated ketamine-induced schizophrenic-like behaviors via reduction of Nox-2 expression, oxidative/nitrergic stresses, acetylcholinesterase activity, and increased GAD67 enzyme.


Assuntos
Colinérgicos/metabolismo , Glutamato Descarboxilase/metabolismo , NADPH Oxidase 2/metabolismo , Estresse Oxidativo , Rutina/uso terapêutico , Esquizofrenia/genética , Esquizofrenia/prevenção & controle , Acetilcolinesterase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Regulação para Baixo/efeitos dos fármacos , Glutationa/metabolismo , Ketamina , Locomoção/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Transtornos da Memória/complicações , Transtornos da Memória/tratamento farmacológico , Camundongos , Nitritos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Rutina/farmacologia , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico , Interação Social , Memória Espacial/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
11.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916948

RESUMO

In Hashimoto's thyroiditis (HT), oxidative stress (OS) is driven by Th1 cytokines' response interfering with the normal function of thyrocytes. OS results from an imbalance between an excessive production of reactive oxygen species (ROS) and a lowering of antioxidant production. Moreover, OS has been shown to inhibit Sirtuin 1 (SIRT1), which is able to prevent hypoxia-inducible factor (HIF)-1α stabilization. The aims of this study were to determine the involvement of NADPH-oxidases (NOX), SIRT1, and HIF-1α in HT pathophysiology as well as the status of antioxidant proteins such as peroxiredoxin 1 (PRDX1), catalase, and superoxide dismutase 1 (SOD1). The protein expressions of NOX2, NOX4, antioxidant enzymes, SIRT1, and HIF-1α, as well as glucose transporter-1 (GLUT-1) and vascular endothelial growth factor A (VEGF-A), were analyzed by Western blot in primary cultures of human thyrocytes that were or were not incubated with Th1 cytokines. The same proteins were also analyzed by immunohistochemistry in thyroid samples from control and HT patients. In human thyrocytes incubated with Th1 cytokines, NOX4 expression was increased whereas antioxidants, such as PRDX1, catalase, and SOD1, were reduced. Th1 cytokines also induced a significant decrease of SIRT1 protein expression associated with an upregulation of HIF-1α, GLUT-1, and VEGF-A proteins. With the exception of PRDX1 and SOD1, similar results were obtained in HT thyroids. OS due to an increase of ROS produced by NOX4 and a loss of antioxidant defenses (PRDX1, catalase, SOD1) correlates to a reduction of SIRT1 and an upregulation of HIF 1α, GLUT-1, and VEGF-A. Our study placed SIRT1 as a key regulator of OS and we, therefore, believe it could be considered as a potential therapeutic target in HT.


Assuntos
Doença de Hashimoto/etiologia , Doença de Hashimoto/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Estresse Oxidativo , Sirtuína 1/genética , Células Th1/imunologia , Células Th1/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Autoimunidade/genética , Biomarcadores , Citocinas/genética , Citocinas/metabolismo , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Doença de Hashimoto/diagnóstico , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/metabolismo , Superóxido Dismutase-1/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timócitos/imunologia , Timócitos/metabolismo , Testes de Função Tireóidea , Glândula Tireoide/imunologia , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
12.
J Leukoc Biol ; 110(2): 219-237, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33857329

RESUMO

p67phox fulfils a key role in the assembly/activation of the NADPH oxidase by direct interaction with Nox2. We proposed that Rac-GTP serves both as a carrier of p67phox to the membrane and an inducer of a conformational change enhancing its affinity for Nox2. This study provides evidence for the latter function: (i) oxidase activation was inhibited by p67phox peptides (106-120) and (181-195), corresponding to the ß hairpin and to a downstream region engaged in intramolecular bonds with the ß hairpin, respectively; (ii) deletion of residues 181-193 and point mutations Q115R or K181E resulted in selective binding of p67phox to Nox2 peptide (369-383); (iii) both deletion and point mutations led to a change in p67phox , expressed in increased apparent molecular weights; (iv) p67phox was bound to p67phox peptide (181-195) and to a cluster of peptides (residues 97-117), supporting the participation of selected residues within these sequences in intramolecular bonds; (v) p67phox failed to bind to Nox2 peptide (369-383), following interaction with Rac1-GTP, but a (p67phox -Rac1-GTP) chimera exhibited marked binding to the peptide, similar to that of p67phox deletion and point mutants; and (vi) size exclusion chromatography of the chimera revealed its partition in monomeric and polymeric forms, with binding to Nox2 peptide (369-383) restricted to polymers. The molecular basis of Rac-GTP action entails unmasking of a previously hidden Nox2-binding site in p67phox , following disengagement of the ß hairpin from more C-terminal residues. The domain in Nox2 binding the "modified" p67phox comprises residues within the 369-383 sequence in the cytosolic dehydrogenase region.


Assuntos
NADPH Oxidase 2/metabolismo , Fosfoproteínas/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Sítios de Ligação , Mutação , NADPH Oxidase 2/química , NADPH Oxidase 2/genética , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
13.
Arch Biochem Biophys ; 704: 108875, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33891961

RESUMO

Previous reports indicate that the central nervous system (CNS) is a target of air pollution, causing tissue damage and functional alterations. Oxidative stress and neuroinflammation have been pointed out as possible mechanisms mediating these effects. The aim of this work was to study the chronic effects of urban air pollution on mice brain cortex, focusing on oxidative stress markers, and mitochondrial function. Male 8-week-old BALB/c mice were exposed to filtered air (FA, control) or urban air (UA) inside whole-body exposure chambers, located in a highly polluted area of Buenos Aires city, for up to 4 weeks. Glutathione levels, assessed as GSH/GSSG ratio, were decreased after 1 and 2 weeks of exposure to UA (45% and 25% respectively vs. FA; p < 0.05). A 38% increase in lipid peroxidation was found after 1 week of UA exposure (p < 0.05). Regarding protein oxidation, carbonyl content was significantly increased at week 2 in UA-exposed mice, compared to FA-group, and an even higher increment was found after 4 weeks of exposure (week 2: 40% p < 0.05, week 4: 54% p < 0.001). NADPH oxidase (NOX) and glutathione peroxidase (GPx) activities were augmented at all the studied time points, while superoxide dismutase (Cu,Zn-SOD cytosolic isoform) and glutathione reductase (GR) activities were increased only after 4 weeks of UA exposure (p < 0.05). The increased NOX activity was accompanied with higher expression levels of NOX2 regulatory subunit p47phox, and NOX4 (p < 0.05). Also, UA mice showed impaired mitochondrial function due to a 50% reduction in O2 consumption in active state respiration (p < 0.05), a 29% decrease in mitochondrial inner membrane potential (p < 0.05), a 65% decrease in ATP production rate (p < 0.01) and a 30% increase in H2O2 production (p < 0.01). Moreover, respiratory complexes I-III and II-III activities were decreased in UA group (30% and 36% respectively vs. FA; p < 0.05). UA exposed mice showed alterations in mitochondrial function, increased oxidant production evidenced by NOX activation, macromolecules damage and the onset of the enzymatic antioxidant system. These data indicate that oxidative stress and impaired mitochondrial function may play a key role in CNS damage mechanisms triggered by air pollution.


Assuntos
Poluentes Atmosféricos/toxicidade , Poluição do Ar/efeitos adversos , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Encéfalo/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/patologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
14.
Front Immunol ; 12: 638381, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868265

RESUMO

While oxidative stress has been linked to multiple sclerosis (MS), the role of superoxide-producing phagocyte NADPH oxidase (Nox2) in central nervous system (CNS) pathogenesis remains unclear. This study investigates the impact of Nox2 gene ablation on pro- and anti-inflammatory cytokine and chemokine production in a mouse experimental autoimmune encephalomyelitis (EAE) model. Nox2 deficiency attenuates EAE-induced neural damage and reduces disease severity, pathogenic immune cells infiltration, demyelination, and oxidative stress in the CNS. The number of autoreactive T cells, myeloid cells, and activated microglia, as well as the production of cytokines and chemokines, including GM-CSF, IFNγ, TNFα, IL-6, IL-10, IL-17A, CCL2, CCL5, and CXCL10, were much lower in the Nox2-/- CNS tissues but remained unaltered in the peripheral lymphoid organs. RNA-seq profiling of microglial transcriptome identified a panel of Nox2 dependent proinflammatory genes: Pf4, Tnfrsf9, Tnfsf12, Tnfsf13, Ccl7, Cxcl3, and Cxcl9. Furthermore, gene ontology and pathway enrichment analyses revealed that microglial Nox2 plays a regulatory role in multiple pathways known to be important for MS/EAE pathogenesis, including STAT3, glutathione, leukotriene biosynthesis, IL-8, HMGB1, NRF2, systemic lupus erythematosus in B cells, and T cell exhaustion signaling. Taken together, our results provide new insights into the critical functions performed by microglial Nox2 during the EAE pathogenesis, suggesting that Nox2 inhibition may represent an important therapeutic target for MS.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Microglia/metabolismo , NADPH Oxidase 2/metabolismo , Estresse Oxidativo/fisiologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2/imunologia
15.
Theranostics ; 11(8): 3796-3812, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33664862

RESUMO

Rationale: Mechanisms underlying the compromised bone formation in type 1 diabetes mellitus (T1DM), which causes bone fragility and frequent fractures, remain poorly understood. Recent advances in organ-specific vascular endothelial cells (ECs) identify type H blood vessel injury in the bone, which actively direct osteogenesis, as a possible player. Methods: T1DM was induced in mice by streptozotocin (STZ) injection in two severity degrees. Bony endothelium, the coupling of angiogenesis and osteogenesis, and bone mass quality were evaluated. Insulin, antioxidants, and NADPH oxidase (NOX) inhibitors were administered to diabetic animals to investigate possible mechanisms and design therapeutic strategies. Results: T1DM in mice led to the holistic abnormality of the vascular system in the bone, especially type H vessels, resulting in the uncoupling of angiogenesis and osteogenesis and inhibition of bone formation. The severity of osteopathy was positively related to glycemic levels. These pathological changes were attenuated by early-started, but not late-started, insulin therapy. ECs in diabetic bones showed significantly higher levels of reactive oxygen species (ROS) and NOX 1 and 2. Impairments of bone vessels and bone mass were effectively ameliorated by treatment with anti-oxidants or NOX2 inhibitors, but not by a NOX1/4 inhibitor. GSK2795039 (GSK), a NOX2 inhibitor, significantly supplemented the insulin effect on the diabetic bone. Conclusions: Diabetic osteopathy could be a chronic microvascular complication of T1DM. The impairment of type H vessels by NOX2-mediated endothelial oxidative stress might be an important contributor that can serve as a therapeutic target for T1DM-induced osteopathy.


Assuntos
Osso e Ossos/irrigação sanguínea , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , NADPH Oxidase 2/metabolismo , Animais , Antioxidantes/farmacologia , Fenômenos Biomecânicos , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Células Endoteliais/fisiologia , Insulina/administração & dosagem , Insulina/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , NADPH Oxidase 2/antagonistas & inibidores , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Osteoporose/etiologia , Osteoporose/patologia , Osteoporose/fisiopatologia , Estresse Oxidativo , Medicina de Precisão
16.
Free Radic Biol Med ; 168: 1-5, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33775771

RESUMO

Type 2 diabetes mellitus (T2DM) is associated with oxidative stress but the underlying mechanisms promoting oxidative stress as well as its relationship with cardiovascular events is still unclear. In 375 T2DM patients who were followed-up for approximately 5 years we measured the serum levels of soluble NOX2-derived peptide (sNOX2-dp), a marker of Nox2 activation, and albumin, a powerful antioxidant protein. In the entire cohort soluble Nox2 and serum albumin were significantly correlated (r = -0.348, P < 0.0001). During the follow-up 49 cardiovascular events (CVE) were registered, of which 45 were non-fatal myocardial infarction (MI); patients with non-fatal MI had significantly higher soluble NOX2/albumin ratio compared to cardiovascular events-free patients. Cox regression analysis showed a significant association between sNox2-dp/serum albumin ratio and the incidental risk of non-fatal MI (HR 1.106, CI95% 1.020-1.198, P = 0.014). The study suggests that redox status imbalance negatively influences vascular outcomes in T2DM.


Assuntos
Diabetes Mellitus Tipo 2 , Hipoalbuminemia , Diabetes Mellitus Tipo 2/complicações , Humanos , Glicoproteínas de Membrana , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidases/genética , Regulação para Cima
18.
Nat Commun ; 12(1): 1508, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686057

RESUMO

LC3-associated phagocytosis (LAP) contributes to a wide range of cellular processes and notably to immunity. The stabilization of phagosomes by the macroautophagy machinery in human macrophages can maintain antigen presentation on MHC class II molecules. However, the molecular mechanisms involved in the formation and maturation of the resulting LAPosomes are not completely understood. Here, we show that reactive oxygen species (ROS) produced by NADPH oxidase 2 (NOX2) stabilize LAPosomes by inhibiting LC3 deconjugation from the LAPosome cytosolic surface. NOX2 residing in the LAPosome membrane generates ROS to cause oxidative inactivation of the protease ATG4B, which otherwise releases LC3B from LAPosomes. An oxidation-insensitive ATG4B mutant compromises LAP and thereby impedes sustained MHC class II presentation of exogenous Candida albicans antigens. Redox regulation of ATG4B is thereby an important mechanism for maintaining LC3 decoration of LAPosomes to support antigen processing for MHC class II presentation.


Assuntos
Apresentação do Antígeno/fisiologia , Autofagia/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Fagossomos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos de Fungos , Proteínas Relacionadas à Autofagia , Candida albicans , Classe III de Fosfatidilinositol 3-Quinases , Cisteína Endopeptidases/metabolismo , Células HEK293 , Humanos , Macroautofagia , Macrófagos/metabolismo , NADPH Oxidase 2/metabolismo , Oxirredução , Fagocitose/fisiologia , Espécies Reativas de Oxigênio/metabolismo
19.
Neuromolecular Med ; 23(1): 25-46, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33547562

RESUMO

Neuro-inflammation accompanies numerous neurological disorders and conditions where it can be associated with a progressive neurodegenerative pathology. In a similar manner, alterations in sphingolipid metabolism often accompany or are causative features in degenerative neurological conditions. These include dementias, motor disorders, autoimmune conditions, inherited metabolic disorders, viral infection, traumatic brain and spinal cord injury, psychiatric conditions, and more. Sphingolipids are major regulators of cellular fate and function in addition to being important structural components of membranes. Their metabolism and signaling pathways can also be regulated by inflammatory mediators. Therefore, as certain sphingolipids exert distinct and opposing cellular roles, alterations in their metabolism can have major consequences. Recently, regulation of bioactive sphingolipids by neuro-inflammatory mediators has been shown to activate a neuronal NADPH oxidase 2 (NOX2) that can provoke damaging oxidation. Therefore, the sphingolipid-regulated neuronal NOX2 serves as a mechanistic link between neuro-inflammation and neurodegeneration. Moreover, therapeutics directed at sphingolipid metabolism or the sphingolipid-regulated NOX2 have the potential to alleviate neurodegeneration arising out of neuro-inflammation.


Assuntos
NADPH Oxidase 2/metabolismo , Doenças Neurodegenerativas/metabolismo , Esfingolipídeos/fisiologia , Complexo AIDS Demência/metabolismo , Animais , Produtos Biológicos/uso terapêutico , Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/metabolismo , Encefalopatias Metabólicas Congênitas/terapia , Descoberta de Drogas , Encefalite Viral/metabolismo , Ativação Enzimática , Terapia de Reposição de Enzimas , Humanos , Inflamação , Naftalenos/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/terapia , Neurônios/metabolismo , Oxirredução , Pirimidinonas/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Infecção por Zika virus/metabolismo
20.
Int J Nanomedicine ; 16: 1231-1244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33633448

RESUMO

Background and Aim: Acute myeloid leukemia (AML), initiated and maintained by leukemia stem cells (LSCs), is often relapsed or refractory to therapy. The present study aimed at assessing the effects of nanozyme-like Fe3O4 nanoparticles (IONPs) combined with cytosine arabinoside (Ara-C) on LSCs in vitro and in vivo. Methods: The CD34+CD38-LSCs, isolated from human AML cell line KG1a by a magnetic activated cell sorting method, were treated with Ara-C, IONPs, and Ara-C+ IONPs respectively in vitro. The cellular proliferation, apoptosis, reactive oxygen species (ROS), and the related molecular expression levels in LSCs were analyzed using flow cytometry, RT-qPCR, and Western blot. The nonobese diabetic/severe combined immune deficiency mice were transplanted with LSCs or non-LSCs via tail vein, and then the mice were treated with Ara-C, IONPs and IONPs plus Ara-C, respectively. The therapeutic effects on the AML bearing mice were further evaluated. Results: LSCs indicated stronger cellular proliferation, more clone formation, and more robust resistance to Ara-C than non-LSCs. Compared with LSCs treated with Ara-C alone, LSCs treated with IONPs plus Ara-C showed a significant increase in apoptosis and ROS levels that might be regulated by nanozyme-like IONPs via improving the expression of pro-oxidation molecule gp91-phox but decreasing the expression of antioxidation molecule superoxide dismutase 1. The in vivo results suggested that, compared with the AML bearing mice treated with Ara-C alone, the mice treated with IONPs plus Ara-C markedly reduced the abnormal leukocyte numbers in peripheral blood and bone marrow and significantly extended the survival of AML bearing mice. Conclusion: IONPs combined with Ara-C showed the effectiveness on reducing AML burden in the mice engrafted with LSCs and extending mouse survival by increasing LSC's ROS level to induce LSC apoptosis. Our findings suggest that targeting LSCs could control the AML relapse by using IONPs plus Ara-C.


Assuntos
Citarabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Nanopartículas Magnéticas de Óxido de Ferro/química , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Separação Celular , Citarabina/farmacologia , Hemoglobinas/metabolismo , Humanos , Leucemia Mieloide Aguda/sangue , Contagem de Leucócitos , Camundongos Endogâmicos NOD , Camundongos SCID , NADPH Oxidase 2/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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