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1.
Acta Neurochir Suppl ; 127: 47-54, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31407062

RESUMO

BACKGROUND: Previously studies have shown that Nox2 and Nox4, as members of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, Nox), participate in brain damage caused by ischemia-reperfusion (I/R). The aim of this study is to investigate the effects of specific chemical inhibitors of Nox2 and Nox4 on cerebral I/R-induced brain injury in rats. METHODS: At 0.5 h before MCAO surgery, the rats were pretreated with vehicle, Nox2 inhibitor (gp91ds-tat), and Nox4 inhibitor (GKT137831), respectively. After reperfusion for 24 h, the infarct sizes of brain tissues in rats in various groups are determined. The penumbra (ischemic) tissues are collected to measure ROS levels, neuronal apoptosis, and degeneration, as well as the integrity of the blood-brain barrier (BBB) in brain tissues of rats. RESULTS: gp91ds-tat and GKT137831 pretreatment significantly reduced the infarct sizes in brain tissues of rats, effectively suppressed I/R-induced increase in ROS levels, neuronal apoptosis and degeneration, and obviously alleviated BBB damage. CONCLUSION: Under cerebral I/R conditions, Nox2 inhibitor (gp91ds-tat) and Nox4 inhibitor (GKT137831) can effectively play a protective role in the brain tissues of rats.


Assuntos
Lesões Encefálicas , Isquemia Encefálica , NADPH Oxidase 2 , NADPH Oxidase 4 , Traumatismo por Reperfusão , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , NADPH Oxidase 2/antagonistas & inibidores , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/metabolismo , NADPH Oxidases , Ratos , Espécies Reativas de Oxigênio , Traumatismo por Reperfusão/metabolismo
2.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 35(4): 300-303, 2019 Jul 28.
Artigo em Chinês | MEDLINE | ID: mdl-31701710

RESUMO

OBJECTIVE: To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25-VitD3) supplementation on cerebral injury after ischemia/reperfusion (I/R) in mice with middle cerebral artery occlusion (MCAO). METHODS: Male C57BL6 mice were randomly divided into Sham group, Vehicle group and 1,25-VitD3 group, with 10 mice in each group. Vehicle group and 1,25-VitD3 group were given MCAO for 1 hour, and then killed after reperfusion for 24 hours. Mice in 1,25-VitD3 group were treated with 1,25-VitD3 at the dose of 100 ng/(kg·d) by injected intraperitoneally for 5 days before MCAO operation. Cerebral ischemic penumbra areas of each group were collected for TTC staining, RT-PCR, TTC staining and immunohistochemistry assay. The function defect of mice was evaluated by using neurological function score. RESULTS: Compared with the sham group, the volume of cerebral infarction in Vehicle group was increased significantly, and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues were increased significantly (P<0.05); compared with Vehicle group, supplementation of 1,25-VitD3 reduced the volume of cerebral infarction by about 50% in I/R mice (P<0.05), and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues of 1,25-VitD3 group were decreased significantly (P<0.05). The expression of Foxp3, a T-regulatory cell marker, was significantly increased in the brain of mice (P<0.05), while the expression of Rorc, a transcription factor, was significantly decreased (P<0.05), suggesting that Th17/gamma Delta T-cell response was reduced and the number of neutrophils in the brain injury site of mice was significantly reduced (P<0.05). CONCLUSION: Vitamin D could alleviate the development of cerebral infarction after arterial occlusion (MCAO) reperfusion, and its mechanism may be through regulating the inflammatory response in mouse brain I/R.


Assuntos
Infarto da Artéria Cerebral Média/tratamento farmacológico , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Vitamina D/farmacologia , Animais , Encéfalo , Citocinas/metabolismo , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Linfócitos T , Células Th17
3.
Chem Biol Interact ; 313: 108818, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494106

RESUMO

Diabetic nephropathy (DN) is a common complication of diabetes that remains the major cause of end-stage renal disease (ESRD). Forkhead box P1 (FOXP1) is a member of FOX family involved in the progression of diabetes. However, the pathogenic role of FOXP1 in DN remains unclear. This study was aimed to explore the effects of FOXP1 on glomerular mesangial cells (MCs) in response to high glucose (HG) stimulation. We found that HG stimulation markedly inhibited the FOXP1 expression in MCs in dose-and time-dependent manner. CCK-8 assay proved that FOXP1 overexpression attenuated HG-induced cell proliferation in MCs. FOXP1 exhibited anti-oxidative activity in HG-induced MCs, as proved by the decreased production of ROS and expressions of ROS producing enzymes, NADPH oxidase (NOX) 2 and NOX4. Besides, FOXP1 suppressed the expression and secretion of extracellular matrix (ECM) proteins including collagen IV (Col IV) and fibronectin (FN). Furthermore, FOXP1 overexpression significantly prevented HG-induced activation of Akt/mTOR signaling in MCs, and Akt activator blocked FOXP1-mediated cell proliferation, ROS production and ECM accumulation in MCs. Collectively, FOXP1 prevented HG-induced proliferation, oxidative stress, and ECM accumulation in MCs via inhibiting the activation of Akt/mTOR signaling pathway. The findings suggested that FOXP1 might be a therapeutic target for the treatment of DN.


Assuntos
Matriz Extracelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Células Mesangiais/metabolismo , Proteínas Repressoras/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/metabolismo , Fatores de Transcrição Forkhead/genética , Glucose/administração & dosagem , Glucose/farmacologia , Humanos , Células Mesangiais/efeitos dos fármacos , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Serina-Treonina Quinases TOR/metabolismo
4.
Microbiol Immunol ; 63(10): 438-443, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31329291

RESUMO

The effects of chalcone and butein on the induction of the superoxide anion (O2 - )-generating system were studied in U937 cells by all-trans retinoic acid (RA). The chalcone skeleton, a common structural motif in them, significantly enhanced the transcription of gp91-phox in an epigenetic manner. In contrast, chalcone and butein showed opposite effects on the induction of the O2 - -generating activity by RA and the expression of gp91-phox protein. Chalcone inhibited, whereas butein promoted, the induction of O2 - -generating activity by RA and the expression of gp91-phox protein. These data raise the possibility that modification of the chalcone skeleton could produce more effective differentiation-promoting agents.


Assuntos
Chalcona/farmacologia , Chalconas/farmacologia , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Superóxidos/metabolismo , Humanos , Tretinoína/química , Células U937
5.
J Pineal Res ; 67(2): e12589, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31155748

RESUMO

It has been found that remote organ/limb temporary ischemia, known as remote ischemic conditioning, can provide protection against the formation of lethal ischemic outcome. Current evidence suggests that aging and age-releated comorbidities impair the cardioprotective effects of conditionings. In conjuction with aging, decrease in melatonin synthesis from pineal gland can have role in the pathogenesis of aging and age-related cardiovascular diseases. In this study, we investigated the effects of remote ischemic perconditioning (RIPerC) and physiological and pharmacological concentrations of melatonin on the infarct size, Fas gene, cytochrome b-245 beta chain (Cybb) gene, nuclear factor-kappa B (NfκB), and irisin using an in vivo model of myocardial ischemia/reperfusion (I/R) injury. Sprague-Dawley rats that were divided into two groups first as non-pinealectomized (Non-Px) and pinealectomized (Px), and then (a) Control; (b) I/R (30-minute ischemia, 120-minute reperfusion caused by left coronary artery ligation); (c) I/R + RIPerC (when myocardial ischemia initiated, three cycles of 5-minute occlusion followed by 5-minute reperfusion); (d) I/R + Mel; (e) Px; (f) Px + I/R; (g) Px + I/R + RIPerC; (h) Px + I/R + RIPerC + Mel groups. The infarct size was determined by TTC staining and analyzed by the ImageJ program. Molecular parameters were evaluated by qRT-PCR and Western blot. Results showed that increased infarct size in Non-Px groups decreased with RIPerC and melatonin. However, increased infarct size in Px groups was decreased minimally with RIPerC and significantly decreased with RIPerC + Melatonin. Fold change in Fas gene was associated with the infarct size. RIPerC and melatonin reduced expressions of Cybb, NfκB, and irisin genes. The physiological release and pharmacological concentration of melatonin may improve protective effect of RIPerC against I/R-induced infarct size by modulating Cybb, Fas, NfκB, Irisin signaling pathways.


Assuntos
Fibronectinas/metabolismo , Precondicionamento Isquêmico , Melatonina/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , NADPH Oxidase 2/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor fas/metabolismo , Animais , Masculino , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Sprague-Dawley
6.
Eur J Pharmacol ; 857: 172459, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31216444

RESUMO

Previous studies have demonstrated that NADPH oxidase (NOX)/vascular peroxidase (VPO1) pathway - mediated oxidative stress plays an important role in the pathogenesis of multiple cardiovascular diseases. This study aims to evaluate the correlation between NOX/VPO1 pathway and endothelial progenitor cells (EPCs) dysfunctions in hypoxia-induced pulmonary hypertension (PH). The rats were exposed to 10% hypoxia for 3 weeks to establish a PH model, which showed increases in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, acceleration in apoptosis and impairment in functions of the peripheral blood derived - EPCs (the reduced abilities in adhesion, migration and tube formation), accompanied by up-regulation of NOX (NOX2 and NOX4) and VPO1. Next, normal EPCs were cultured under hypoxia to induce apoptosis in vitro. Consistent with the in vivo findings, hypoxia enhanced the apoptosis and dysfunctions of EPCs concomitant with an increase in NOX and VPO1 expression, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production; these phenomena were attenuated by NOX2 or NOX4 siRNA. Knockdown of VPO1 showed similar results to that of NOX siRNA except no effect on NOX expression and H2O2 production. Based on these observations, we conclude that NOX/VPO1 pathway-derived reactive oxygen species promote the oxidative injury and dysfunctions of EPCs in PH, which may contribute to endothelial dysfunctions in PH.


Assuntos
Células Progenitoras Endoteliais/patologia , Hemeproteínas/metabolismo , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/patologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Peroxidases/metabolismo , Animais , Apoptose , Hipóxia Celular , Técnicas de Silenciamento de Genes , Hemeproteínas/deficiência , Hemeproteínas/genética , Hipertensão Pulmonar/genética , Masculino , NADPH Oxidase 2/deficiência , NADPH Oxidase 2/genética , NADPH Oxidase 4/deficiência , NADPH Oxidase 4/genética , Peroxidases/deficiência , Peroxidases/genética , Fenótipo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
7.
Int J Mol Sci ; 20(11)2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151207

RESUMO

We observed that on long-term breeding, gp91phox-knockout (gp91phox-/-) mice developed white hair. Here, we investigate the origin of this hitherto unexplained phenomenon. Moreover, we investigated the effect of tranexamic acid administration on the hair color in gp91phox-/- mice. We administered tranexamic acid (about 12 mg/kg/day) orally to 9-week-old C57BL/6j (control) and gp91phox-/- mice, thrice a week for 12 months. Compared to control mice, gp91phox-/- mice showed more white hair. However, the concentrations of reactive oxygen species and the levels of interleukin (IL)-1ß and transforming growth factor (TGF)-ß in the skin were lower than those in the control group. Furthermore, increase in white hair was observed in the control mice upon administration of the IL-1ß antagonist. On the other hand, administration of tranexamic acid led to brown colored hair on gp91phox-/- mice. Although tranexamic acid treatment did not alter the expression levels of melanocortin receptor 1 and agouti signaling protein on hair follicles, it increased the expression of mahogunin ring finger protein 1 (MGRN1) and collagen XVII. These results suggested that retention of black hair requires the gp91phox/ROS/IL-1ß/TGF-ß pathway and that elevated levels of MGRN1 and collagen XVII lead to brown hair in gp91phox-/- mice.


Assuntos
Antifibrinolíticos/administração & dosagem , Cor de Cabelo , NADPH Oxidase 2/genética , Ácido Tranexâmico/administração & dosagem , Animais , Biomarcadores , Colágeno/metabolismo , Imunofluorescência , Expressão Gênica , Técnicas de Inativação de Genes , Estudos de Associação Genética , Masculino , Camundongos , Camundongos Knockout , NADPH Oxidase 2/metabolismo , Fenótipo
8.
Methods Mol Biol ; 1982: 153-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172472

RESUMO

Structure-function analysis of specific regions of NOX2 can be carried out after stable expression of site-directed mutagenesis-modified NOX2 in the X0-CGD PLB-985 cell model. Indeed, the generation of this human cellular model by Prof. MC Dinauer's team gave researchers the opportunity to gain a deeper understanding of functional regions of NOX2. With this model cell line, the functional impact of X+-CGD or of new mutations in NOX2 can be highlighted, as the biological material is not limited. PLB-985 cells transfected with various NOX2 mutations can be easily cultured and differentiated into neutrophils or monocytes/macrophages. Several measurements in intact mutated NOX2 PLB-985 cells can be carried out such as NOX2 expression, cytochrome b 558 spectrum, enzymatic activity, and assembly of the NADPH oxidase complex. Purified membranes or purified cytochrome b 558 from mutated NOX2 PLB-985 cells can be used for the study of the impact of specific mutations on NADPH oxidase or diaphorase activity, FAD incorporation, and NADPH or NADH binding in a cell-free assay system. Here, we describe a method to generate mutated NOX2 PLB-985 cells in order to analyze NOX2 structure-function relationships.


Assuntos
NADPH Oxidase 2/química , NADPH Oxidase 2/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Ativação Enzimática , Citometria de Fluxo , Expressão Gênica , Granulócitos/metabolismo , Humanos , Medições Luminescentes , Mutagênese Sítio-Dirigida , NADPH Oxidase 2/genética , Plasmídeos/genética , Proteínas Recombinantes , Relação Estrutura-Atividade
9.
Methods Mol Biol ; 1982: 341-352, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172483

RESUMO

The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22phox and gp91phox (renamed NOX2), which form the cytochrome b558, and four cytosolic proteins, p47phox, p67phox, p40phox, and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91phox/NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.


Assuntos
NADPH Oxidase 2/metabolismo , Neutrófilos/metabolismo , Western Blotting , Cromatografia em Camada Delgada , Humanos , Marcação por Isótopo , Neutrófilos/imunologia , Oxirredução , Fagócitos/imunologia , Fagócitos/metabolismo , Fagocitose/imunologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/imunologia
10.
Artif Cells Nanomed Biotechnol ; 47(1): 2139-2145, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31146598

RESUMO

Osteoarthritis (OA) is a common joint disease for which a safe and reliable treatment has yet to be developed. Here, we demonstrated the potential benefit of treatment with paeonol, a derivative of Paeonia suffruticosa, in the treatment and prevention of OA. Chondrogenic cell line ATDC5 cells were cultured with IL-1ß and the effects of paeonol were assessed through qRT-PCR, western blot analysis, MTT, ELISA, and NF-κB luciferase reporter gene assay. Our findings demonstrate a novel ability of paeonol to inhibit numerous factors of OA, including expressions of IL-6, TNF-α, NOX2, PTGS2, NUCB2/nesfatin-1, ICAM-1, VCAM-1, MMP-3/13, degradation of type II collagen, and NF-κB activation through the rescue of IκBα. Additionally, we assessed the effects of paeonol on cell viability to confirm its safety. These findings implicate a valuable potential role of paeonol in the treatment and prevention of OA.


Assuntos
Acetofenonas/farmacologia , Condrócitos/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteólise/efeitos dos fármacos , Acetofenonas/uso terapêutico , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , NADPH Oxidase 2/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Osteoartrite/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo
11.
Oxid Med Cell Longev ; 2019: 2717986, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182989

RESUMO

Hypoxia/reoxygenation (H/R) accelerates the process of cardiomyocyte apoptosis during ischemia-reperfusion. Excessive reactive oxygen species (ROS) are a critical driver of oxidative stress injury. Cyclophilin A (CyPA) is a major ROS-induced factor in atherosclerosis. There is a positive feedback mechanism between CyPA and ROS, which enables the oxidative stress response to continue and expand. However, it is unclear whether this positive feedback mechanism exists in cardiomyocytes. Through western blotting and flow cytometric assays and TUNEL assay, we found that CyPA inhibited the apoptosis of H9c2 cardiomyocytes under H/R conditions. By dihydroethidium (DHE) staining and electron spin resonance (ESR) assays, we demonstrated that CyPA reduced ROS production and suppressed O2 - production dependent on reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. By western blotting, we showed that CyPA inhibited the expression of NADPH oxidase 2 (Nox2) protein by the AKT pathway. Through confocal microscopy assay, we found that CyPA reduced the expression of Nox2 membrane-bound subunits. The current study shows that a positive feedback mechanism does not exist in H9c2 cardiomyoblasts. CyPA protects H9c2 cardiomyoblasts against H/R-induced apoptosis via the AKT/Nox2 pathway. This could be a potential target for ischemia-reperfusion injury therapy.


Assuntos
Ciclofilina A/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NADPH Oxidase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Microscopia Confocal , Miócitos Cardíacos/citologia , Ratos , Espécies Reativas de Oxigênio/metabolismo
12.
Eur J Pharmacol ; 859: 172490, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31229536

RESUMO

Doxorubicin (DOX) is a classic anti-tumor chemotherapeutic used to treat a wide range of tumors. One major downfall of DOX treatment is it can induce fatal cardiotoxicity. Astragaloside IV (AS-IV) is one of the primary active ingredients that can be isolated from the traditional Chinese herbal medicine, Astragalus membranaceus. This study uses both in vitro and in vivo tools to investigate whether AS-IV alleviates DOX induced cardiomyopathy. We found that AS-IV supplementation alleviates body weight loss, myocardial injury, apoptosis of cardiomyocytes, cardiac fibrosis and cardiac dysfunction in DOX-treated mice. Also, DOX-induced cardiomyocyte injury and apoptosis were effectively improved by AS-IV treatment in vitro. NADPH oxidase (NOX) plays an important role in the progress of the oxidative signal transduction and DOX-induced cardiomyopathy. In this study, we found that AS-IV treatment relieves DOX-induced NOX2 and NOX4 expression and oxidative stress in cardiomyocytes. In conclusion, AS-IV, an antioxidant, attenuates DOX-induced cardiomyopathy through the suppression of NOX2 and NOX4.


Assuntos
Cardiomiopatias/induzido quimicamente , Cardiomiopatias/tratamento farmacológico , Doxorrubicina/efeitos adversos , NADPH Oxidase 2/antagonistas & inibidores , NADPH Oxidase 4/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiotoxicidade/tratamento farmacológico , Cardiotoxicidade/etiologia , Cardiotoxicidade/metabolismo , Cardiotoxicidade/patologia , Tamanho Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ratos , Saponinas/uso terapêutico , Triterpenos/uso terapêutico
13.
PLoS One ; 14(5): e0215482, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31048856

RESUMO

Although T cells play important roles in the pathophysiology of ischemic stroke, the dynamics of T cells remains unclear. In cancer, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) contribute to the maintenance of the tumor microenvironment by suppressing T cells. However, the presence of these cells has never been examined in ischemic brain. Therefore, we examined the temporal and spatial profiles of PMN-MDSCs, which are defined as the CD11b+Ly6ClowLy6G+ cells with higher expression levels of Nox2 and C/EBP Homologous Protein (CHOP) mRNA than normal neutrophil. Fluorescence-activated cell sorter (FACS) analysis showed that the count of CD11b+Ly6ClowLy6G+ cells was increased in the ischemic hemisphere and bone marrow at 72 hours, as well as in the spleen 24 hours after transient middle cerebral artery occlusion in mice. In contrast, the contralateral hemisphere, normal bone marrow, and normal spleen contained few CD11b+Ly6ClowLy6G+ cells. Real-time reverse transcription polymerase chain reaction revealed that CD11b+Ly6ClowLy6G+ cells sorted from brain and spleen 72 hours after ischemia had greater expression of Nox2 and CHOP mRNA than neutrophils in bone marrow, suggesting that these cells constitute PMN-MDSCs. Immunohistochemistry showed that CD11b+Ly6G+ cells were located in the ischemic core and border zone, indicating that PMN-MDSCs might be endemic to these regions. Although neutrophils are believed to invade infarct regions 48-72 hours after ischemia, the present study suggested that some of these cells are in fact PMN-MDSCs. Further studies on the function of PMN-MDSCs might unveil the unknown mechanisms of T cell activation and recruitment in ischemic stroke.


Assuntos
Isquemia Encefálica/patologia , Células Supressoras Mieloides/metabolismo , Neutrófilos/metabolismo , Animais , Antígenos Ly/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Isquemia Encefálica/imunologia , Isquemia Encefálica/veterinária , Antígeno CD11b/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/imunologia , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Neutrófilos/citologia , Neutrófilos/imunologia , Baço/citologia , Baço/imunologia , Baço/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
14.
Adv Exp Med Biol ; 1127: 85-95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31140173

RESUMO

Arachidonic acid (AA) is a polyunsaturated fatty acid that participates in the inflammatory response mainly through bioactive-lipids formation in macrophages and also in the phagocytic NADPH oxidase 2 (NOX2) activation. NOX2 is the enzyme responsible for a huge superoxide formation in macrophages, essential to eliminate pathogens inside the phagosome. The oxidase is an enzymatic complex comprised of a membrane-bound flavocytochrome b 558 (gp91phox/p22phox), three cytosolic subunits (p47phox, p40phox and p67phox) and a Rac-GTPase. The enzyme becomes active when macrophages are exposed to appropriate stimuli that trigger the phosphorylation of cytosolic subunits and its migration to plasmatic membrane to form the active complex. It is proposed that AA stimulates NOX2 activity through AA interaction with different components of the NADPH oxidase complex. In inflammatory conditions, there is an increase in reactive oxygen and nitrogen species that results in the production of nitrated derivatives of AA, such as nitroarachidonic acid (NO2-AA). NO2-AA is capable to inhibit NOX2 activity by interfering with p47phox migration to the membrane without affecting phosphorylation of cytosolic proteins. Also, NO2-AA is capable to interact with protein disulfide isomerase (PDI), which is involved on NOX2 active complex formation. It has been demonstrated that NO2-AA forms a covalent adduct with PDI that could prevent the interaction with NOX2 and it would explain the inhibitory effects of the fatty acid upon NOX2. Together, current data indicate that AA is an important activator of NOX2 formed in the early events of the inflammatory response, leading to a massive production of oxidants that may, in turn, promote NO2-AA formation and shutting down the oxidative burst. Hence, AA and its derivatives could have antagonistic roles on NOX2 activity regulation.


Assuntos
Ácido Araquidônico/metabolismo , Inflamação/metabolismo , NADPH Oxidase 2/metabolismo , Humanos , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Superóxidos/metabolismo
15.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096551

RESUMO

We have previously derived three related peptides, based on a nine-amino acid sequence in human or rat/mouse surfactant protein A, that inhibit the phospholipase A2 activity of peroxiredoxin 6 (Prdx6) and prevent the activation of lung NADPH oxidase (type 2). The present study evaluated the effect of these Prdx6-inhibitory peptides (PIP) in a mouse (C57Bl/6) model of acute lung injury following lipopolysaccharide (LPS) administration. All three peptides (PIP-1, 2 and 3) similarly inhibited the production of reactive O2 species (ROS) in isolated mouse lungs as detected by the oxidation of Amplex red. PIP-2 inhibited both the increased phospholipase A2 activity of Prdx6 and lung reactive oxygen species (ROS) production following treatment of mice with intratracheal LPS (5 µg/g body wt.). Pre-treatment of mice with PIP-2 prevented LPS-mediated lung injury while treatment with PIP-2 at 12 or 16 h after LPS administration led to reversal of lung injury when evaluated 12 or 8 h later, respectively. With a higher dose of LPS (15 µg/g body wt.), mortality was 100% at 48 h in untreated mice but only 28% in mice that were treated at 12-24 h intervals, with PIP-2 beginning at 12 h after LPS administration. Treatment with PIP-2 also markedly decreased mortality after intraperitoneal LPS (15 µg/g body wt.), used as a model of sepsis. This study shows the dramatic effectiveness of a peptide inhibitor of Prdx6 against lung injury and mouse mortality in LPS models. We propose that the PIP nonapeptides may be a useful modality to prevent or to treat human ALI.


Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/prevenção & controle , Lipopolissacarídeos/efeitos adversos , NADPH Oxidase 2/metabolismo , Peptídeos/metabolismo , Peroxirredoxina VI/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/mortalidade , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Injeções Intraperitoneais , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Peroxirredoxina VI/metabolismo , Espécies Reativas de Oxigênio/metabolismo
16.
J Neuroinflammation ; 16(1): 91, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30995916

RESUMO

BACKGROUND: During excitotoxic damage, neuronal death results from the increase in intracellular calcium, the induction of oxidative stress, and a subsequent inflammatory response. NADPH oxidases (NOX) are relevant sources of reactive oxygen species (ROS) during excitotoxic damage. NADPH oxidase-2 (NOX-2) has been particularly related to neuronal damage and death, as well as to the resolution of the subsequent inflammatory response. As ROS are crucial components of the regulation of inflammatory response, in this work, we evaluated the role of NOX-2 in the progression of inflammation resulting from glutamate-induced excitotoxic damage of the striatum in an in vivo model. METHODS: The striata of wild-type C57BL/6 J and NOX-2 KO mice (gp91Cybbtm1Din/J) were stereotactically injected with monosodium glutamate either alone or in combination with IL-4 or IL-10. The damage was evaluated in histological sections stained with cresyl violet and Fluoro-Jade B. The enzymatic activity of caspase-3 and NOX were also measured. Additionally, the cytokine profile was identified by ELISA and motor activity was verified by the tests of the cylinder, the adhesive tape removal, and the inverted grid. RESULTS: Our results show a neuroprotective effect in mice with a genetic inhibition of NOX-2, which is partially due to a differential response to excitotoxic damage, characterized by the production of anti-inflammatory cytokines. In NOX-2 KO animals, the excitotoxic condition increased the production of interleukin-4, which could contribute to the production of interleukin-10 that decreased neuronal apoptotic death and the magnitude of striatal injury. Treatment with interleukin-4 and interleukin-10 protected from excitotoxic damage in wild-type animals. CONCLUSIONS: The release of proinflammatory cytokines during the excitotoxic event promotes an additional apoptotic death of neurons that survived the initial damage. During the subsequent inflammatory response to excitotoxic damage, ROS generated by NOX-2 play a decisive role in the extension of the lesion and consequently in the severity of the functional compromise, probably by regulating the anti-inflammatory cytokines production.


Assuntos
Corpo Estriado/enzimologia , Corpo Estriado/patologia , Inflamação/enzimologia , Inflamação/patologia , NADPH Oxidase 2/metabolismo , Animais , Corpo Estriado/imunologia , Progressão da Doença , Ácido Glutâmico/toxicidade , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
17.
PLoS One ; 14(4): e0215568, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31017961

RESUMO

The aim of this study was to evaluate the effects of exercise training (ET) on the aortic vascular reactivity of ovariectomized and infarcted rats. The animals were divided into 5 groups: Control, Ovariectomized + SHAM sedentary (OVX+SHAMSED), OVX+SHAM and ET (OVX+SHAMET), OVX + Myocardial Infarction sedentary (OVX+MISED), and OVX + MI and ET (OVX+MIET). ET protocol (60 minutes/day, 5x/week) in a motorized treadmill began 15 days after MI and lasted 8 weeks. The endothelium-dependent and endothelium-independent vascular reactivity were evaluated as well as the role of the reactive oxygen species (ROS). Superoxide and nitric oxide (NO) production were analyzed in situ using DHE and DAF-2 fluorescence, respectively. The expression of gp91phox and of the antioxidant enzymes were evaluated by western blotting in the thoracic aorta samples. MI promoted a significant increase in the contractile response and impaired endothelium-mediated relaxation. However, ET prevented the impairment in the vascular reactivity in MI animals. In addition, the protein expression of gp91phox and superoxide production increased and the NO production decreased in the OVX+MISED group but not in the OVX+MIET group. Therefore, ET improves vascular reactivity in MI ovariectomized rats by preventing the increase in the expression of gp91phox and the decrease in the antioxidant enzymes, resulting in a normal ROS and NO production. Thus, ET can be an effective therapeutic strategy for improving the MI-induced vascular alterations in estrogen deficiency condition.


Assuntos
Infarto do Miocárdio/terapia , Ovariectomia/efeitos adversos , Condicionamento Físico Animal , Animais , Antioxidantes/metabolismo , Aorta Torácica/fisiopatologia , Endotélio Vascular/fisiopatologia , Estrogênios/deficiência , Feminino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/fisiopatologia , NADPH Oxidase 2/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Vasodilatação/fisiologia
18.
Oxid Med Cell Longev ; 2019: 9296439, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019654

RESUMO

Cancer stem cells (CSCs) are known to mediate metastasis and recurrence and are therefore a promising therapeutic target. In this study, we found that dihydrotanshinone (DHTS) inhibits CSC formation. DHTS inhibited mammosphere formation in a dose-dependent manner and showed significant tumor growth inhibition in a xenograft model. This compound reduced the CD44high/CD24low- and aldehyde dehydrogenase- (ALDH-) expressing cell population and the self-renewal-related genes Nanog, SOX2, OCT4, C-Myc, and CD44. DHTS induced NOX5 activation by increasing calcium, and NOX5 activation induced reactive oxygen species (ROS) production. ROS production reduced the nuclear phosphorylation levels of Stat3 and secreted IL-6 levels in the mammospheres. DHTS deregulated the dynamic equilibrium from non-stem cancer cells to CSCs by dephosphorylating Stat3 and decreasing IL-6 secretion and inhibiting CSC formation. These novel findings showed that DHTS-induced ROS deregulated the Stat3/IL-6 pathway and induced CSC death. NOX5 activation by DHTS inhibits CSC formation through ROS/Stat3/IL-6 signaling, and DHTS may be a promising potential therapeutic agent against breast CSCs.


Assuntos
Neoplasias da Mama/enzimologia , Medicamentos de Ervas Chinesas/farmacologia , NADPH Oxidase 5/metabolismo , Células-Tronco Neoplásicas/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Camundongos Nus , NADPH Oxidase 2/metabolismo , NADPH Oxidase 5/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Transcrição Genética/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Redox Biol ; 22: 101143, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30897521

RESUMO

First described as essential to the phagocytic activity of leukocytes, Nox2-derived ROS have emerged as mediators of a range of cellular and tissue responses across species from salubrious to deleterious consequences. Knowledge of their role in inflammation is limited, however. We postulated that TNFα-induced endothelial reactive oxygen species (ROS) generation and pro-inflammatory signaling would be ameliorated by targeting Nox2. Herein, we in silico-modelled two first-in-class Nox2 inhibitors developed in our laboratory, explored their cellular mechanism of action and tested their efficacy in in vitro and mouse in vivo models of inflammation. Our data show that these inhibitors (CPP11G and CPP11H) disrupted canonical Nox2 organizing factor, p47phox, translocation to Nox2 in the plasma membrane; and abolished ROS production, markedly attenuated stress-responsive MAPK signaling and downstream AP-1 and NFκB nuclear translocation in human cells. Consequently, cell adhesion molecule expression and monocyte adherence were significantly inhibited by both inhibitors. In vivo, TNFα-induced ROS and inflammation were ameliorated by targeted Nox2 inhibition, which, in turn, improved hind-limb blood flow. These studies identify a proximal role for Nox2 in propagated inflammatory signaling and support therapeutic value of Nox2 inhibitors in inflammatory disease.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , NADPH Oxidase 2/antagonistas & inibidores , Fluxo Sanguíneo Regional/efeitos dos fármacos , Vasculite/metabolismo , Animais , Biomarcadores , Adesão Celular , Linhagem Celular , Inibidores Enzimáticos/química , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NADPH Oxidase 2/química , NADPH Oxidase 2/metabolismo , NF-kappa B/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Fator de Transcrição AP-1/metabolismo , Vasculite/tratamento farmacológico , Vasculite/etiologia , Vasculite/patologia
20.
Planta Med ; 85(9-10): 708-718, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30822814

RESUMO

Right ventricle (RV) remodeling is a major pathological feature in pulmonary arterial hypertension (PAH). Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza and it possesses multiple pharmacological activities such as anti-inflammation and antioxidation. This study aims to investigate whether MLB is able to prevent RV remodeling in PAH and the underlying mechanisms. In vivo, SD rats were exposed to 10% O2 for 21 d to induce RV remodeling, which showed hypertrophic features (increases in the ratio of RV weight to tibia length, cellular size, and hypertrophic marker expression), accompanied by upregulation in expression of NADPH oxidases (NOX2 and NOX4) and vascular peroxidase 1 (VPO1), increases in hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production and elevation in phosphorylation levels of ERK; these changes were attenuated by treating rats with MLB. In vitro, the cultured H9c2 cells were exposed to 3% O2 for 24 h to induce hypertrophy, which showed hypertrophic features (increases in cellular size and hypertrophic marker expression). Administration of MLB or VAS2870 (a positive control for NOX inhibitor) could prevent cardiomyocyte hypertrophy concomitant with decreases in NOX (NOX2 and NOX4) and VPO1 expression, H2O2 and HOCl production, and ERK phosphorylation. Based on these observations, we conclude that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving a suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess the potential clinical value for PAH therapy.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Hemeproteínas/metabolismo , Hipertensão Pulmonar/fisiopatologia , NADPH Oxidases/metabolismo , Peroxidases/metabolismo , Salvia miltiorrhiza/química , Remodelação Ventricular/efeitos dos fármacos , Animais , Fator Natriurético Atrial/genética , Benzoxazóis/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/isolamento & purificação , Hemeproteínas/antagonistas & inibidores , Hipertensão Pulmonar/metabolismo , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , NADPH Oxidases/antagonistas & inibidores , Peptídeo Natriurético Encefálico/genética , Peroxidases/antagonistas & inibidores , Ratos Sprague-Dawley , Triazóis/farmacologia
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