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1.
Zhonghua Er Ke Za Zhi ; 57(7): 520-525, 2019 Jul 02.
Artigo em Chinês | MEDLINE | ID: mdl-31269551

RESUMO

Objective: To analyze the clinical and genotypic characteristics of infantile inflammatory bowel disease (IBD). Methods: The age of onset, family history, clinical manifestations, and treatment effect were retrospectively analyzed in 39 infants (male 23 cases, female 16 cases) with IBD who were admitted to the Department of Gastroenterology in Children's Hospital, Capital Institute of Pediatrics from January 2007 to December 2017. Next generation sequencing (NGS) based on target gene panel was used for gene analysis in 17 patients. Results: The median age of onset was 0.5 (0.5, 1.0) month. The most common clinical symptoms included diarrhea (39, 100%), malnutrition (38, 97%), hematochezia (34, 87%), fever (25, 64%), and perianal diseases (24, 61%). Four children had associated family history. Among the 17 patients whose gene was analyzed, 10 were found to have the pathogenic gene variation, within whom 7 had interleukin-10 receptor α subunit (IL-10RA) mutation, 2 had CYBB heterozygous mutation, 1 had interleukin-10 receptor ß subunit (IL-10RB) mutation. The therapeutic medicine included mesalazine, steroids, and thalidomide. Eighteen children (46%) reached clinical remission (10 cases) or partial remission (8 cases). Conclusions: The incidence of single gene mutation in infants with IBD is high, with IL-10RA mutation as the most common. Refractory diarrhea and malnutrition may indicate infantile IBD.


Assuntos
Doenças Inflamatórias Intestinais/diagnóstico , Subunidade alfa de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/genética , NADPH Oxidase 2/genética , Criança , Feminino , Marcadores Genéticos , Genótipo , Humanos , Lactente , Doenças Inflamatórias Intestinais/genética , Interleucina-10 , Masculino , Mutação , Estudos Retrospectivos , Análise de Sequência de DNA
2.
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
3.
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
4.
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
5.
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
6.
Allergol Immunopathol (Madr) ; 47(4): 372-377, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31176517

RESUMO

INTRODUCTION: Chronic granulomatous disease (CGD) is a disorder of phagocyte function, characterized by pyogenic infections and granuloma formation caused by defects in NADPH oxidase complex activity. Although the effect of CGD mainly reflects the phagocytic compartment, B cell responses are also impaired in patients with CGD. MATERIALS AND METHODS: Flow cytometric analysis was performed on peripheral blood samples from 35 CGD patients age-matched with healthy controls (HC). The target cells of our study were the naive (IgD+/CD27-), memory (IgD-/CD27+), and B1a (CD5+) cells. Immunoglobulins (Igs) were also measured. This study was performed in a Latin American cohort. RESULTS: We found significantly higher levels of naive B cells and B1a cells, but lower levels of memory B cells were found in CGD patients compared to HC. There was no significant difference of cell percentages per inheritance type. DISCUSSION: Our findings suggest that the deficiency of NADPH oxidase components can affect the differentiation of naive B cells to memory B cells. Consequently, memory cells will be low, which also influenced the expression of CD27 in memory B cells and as a result, the percentage of naive cells increases. An altered phenotype of B lymphocytes in CGD patients may contribute to the opportunistic infections and autoimmune disorders that are seen in this disease.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Doença Granulomatosa Crônica/imunologia , NADPH Oxidase 2/genética , Adolescente , Adulto , Separação Celular , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Citometria de Fluxo , Doença Granulomatosa Crônica/genética , Humanos , Memória Imunológica , Imunofenotipagem , Lactente , Masculino , México , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Adulto Jovem
7.
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
8.
Nat Commun ; 10(1): 1076, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842418

RESUMO

Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6ChiCX3CR1lo monocytes/macrophages to pro-resolving Ly6CloCX3CR1hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2-/- neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Regeneração Hepática/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Acetaminofen/toxicidade , Transferência Adotiva/métodos , Animais , Transplante de Medula Óssea , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Humanos , Fígado/imunologia , Fígado/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidase 2/imunologia , Neutrófilos/metabolismo , Neutrófilos/transplante , Cultura Primária de Células , Quimeras de Transplante
9.
Front Immunol ; 10: 73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30761141

RESUMO

Background: Disseminated Bacillus Calmette-Guérin disease (D-BCG) in children with chronic granulomatous disease (CGD) can be fatal, while its clinical characteristics remain unclear because both diseases are extremely rare. The patients with CGD receive BCG vaccination, because BCG vaccination is usually performed within 24 h after delivery in China. Methods: We prospectively followed-up Chinese patients with CGD who developed D-BCG to characterize their clinical and genetic characteristics. The diagnoses were based on the patients' clinical, genetic, and microbiological characteristics. Results: Between September 2009 and September 2016, we identified 23 patients with CGD who developed D-BCG. Their overall 10-year survival rate was 34%. We created a simple dissemination score to evaluate the number of infected organ systems and the survival probabilities after 8 years were 62 and 17% among patients with simple dissemination scores of ≤3 and >3, respectively (p = 0.0424). Survival was not significantly associated with the CGD stimulation index or interferon-γ treatment. Eight patients underwent umbilical cord blood transplantation and 5 of them were successfully treated. The genetic analyses found mutations in CYBB (19 patients), CYBA (1 patient), NCF1 (1 patient), and NCF2 (1 patient). We identified 6 novel highly likely pathogenic mutations, including 4 mutations in CYBB and 2 mutations in NCF1. Conclusions: D-BCG is a deadly complication of CGD. The extent of BCG spreading is strongly associated with clinical outcomes, and hematopoietic stem cell transplantation may be a therapeutic option for this condition.


Assuntos
Doença Granulomatosa Crônica/complicações , Doença Granulomatosa Crônica/genética , Mycobacterium bovis/imunologia , Tuberculose/etiologia , Tuberculose/genética , Vacina BCG/efeitos adversos , Criança , Pré-Escolar , China , Feminino , Seguimentos , Testes Genéticos , Genótipo , Doença Granulomatosa Crônica/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas , Humanos , Interferon gama/uso terapêutico , Estimativa de Kaplan-Meier , Masculino , Mutação , NADPH Oxidase 2/genética , NADPH Oxidases/genética , Estudos Prospectivos , Rodaminas/análise , Taxa de Sobrevida , Resultado do Tratamento , Tuberculose/tratamento farmacológico , Tuberculose/mortalidade , Vacinação/efeitos adversos
10.
Biol Trace Elem Res ; 191(2): 426-434, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30666592

RESUMO

Lead has heavy metal toxicity which endangers human and animal health. Salidroside (SDS) is a natural antioxidant that has extensive pharmacological usage. However, its protective effects on lead-induced oxidative stress and hepatotoxicity has not been reported. In this study, we established an animal model to evaluate the protective effects of SDS on chronic lead exposure induced oxidative stress and hepatotoxicity. Forty healthy Sprague-Dawley (SD) rats were assigned to control group (control, animals were provided with distilled water, n = 10); lead acetate-exposed group (PbAc, animals received lead acetate solution of 500 ppm for 60 days, n = 10); low dosage of SDS-treated group (PbAc-SDS-L, lead acetate exposed animals were given intragastric SDS 150 mg/kg body weight for 60 days, n = 10); and high dosage of SDS-treated group (PbAc-SDS-H, lead acetate exposed animals were given intragastric SDS 300 mg/kg body weight for 60 days, n = 10). The results showed that lead exposure caused a significant increase in serum ALP, AST, ALT, and TB (P < 0.01), and these were reversed after treatment with salidroside for 60 days. Compared to the control, the liver GSH, SOD, and GSH-Px were decreased significantly after lead acetate exposure (P < 0.01). However, after treatment with SDS for 60 days, those were dose-dependently reversed. Similarly, MDA was significantly increased in the PbAc group (P < 0.01), and it was significantly decreased in SDS treatment group. Moreover, SDS ameliorated lead-induced congestion and necrosis of hepatocytes. In addition, the RT-PCR and immunohistochemistry results revealed that the PbAc group showed a significant increase in the protein and mRNA of cytochrome P450 2E1 (CYP2E1) and NADPH oxidase 2 (NOX2) in rat liver. Treatment with SDS significantly reversed CYP2E1 and NOX2 expressions in the liver of lead-exposed rats. The results above indicated that SDS has obvious antioxidant activity; it can cure liver injury caused by lead acetate by inhibiting oxidative stress and increasing the antioxidant stress activity, thus improving the liver tissue structure.


Assuntos
Glucosídeos/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/uso terapêutico , Animais , Antioxidantes/metabolismo , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Feminino , Glucosídeos/farmacologia , Humanos , Imuno-Histoquímica , Chumbo/toxicidade , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Fenóis/farmacologia , Ratos , Ratos Sprague-Dawley , Temperatura Ambiente
11.
Food Funct ; 10(1): 26-32, 2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30604799

RESUMO

Inflammation involves the activation of redox-sensitive transcription factors, e.g., nuclear factor κB (NF-κB). Administration of (-)-epicatechin to high-fructose-fed rats prevented NF-κB activation and up-regulation of the NADPH oxidase 4 (NOX4) in the kidney cortex. These results add mechanistic insights into the action of (-)-epicatechin diminishing inflammatory responses.


Assuntos
Catequina/metabolismo , Frutose/metabolismo , Córtex Renal/enzimologia , NADPH Oxidase 1/metabolismo , NADPH Oxidase 4/metabolismo , NF-kappa B/metabolismo , Animais , Córtex Renal/metabolismo , Masculino , NADPH Oxidase 1/genética , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/genética , NF-kappa B/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
12.
Scand J Immunol ; 89(2): e12737, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30506560

RESUMO

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by susceptibility to bacterial and fungal infections resulting from the inadequacy of phagocytic leucocytes to produce reactive oxygen radicals. CGD is a genetically heterogeneous disease with an X-linked recessive (XR-CGD) form caused by mutations in the CYBB (OMIM #300481) gene encoding the gp91(phox) protein, and an autosomal recessive (AR-CGD) form caused by mutations in the CYBA (OMIM #608508), NCF1 (OMIM #608512), NCF2 (OMIM #608515) and NCF4 (OMIM #601488) genes encoding p22(phox), p47(phox), p67(phox) and p40(phox), respectively. The genetic mutation of one of the cytosolic p47phox/p67phox proteins and membrane-bound gp91phox/p22phox proteins, which constitutes the NADPH oxidase enzyme complex, causes the disease. In this study, we evaluated the clinical, laboratory and genetic findings and the prognostic effects of molecular inheritance of our 24 CGD cases (14 XR, 10 autosomal recessive-AR). Consanguinity (three XR and all AR cases) showed statistically significant relationship with the type of hereditary inheritance (P < 0.001). 83% patients had an infection since early infancy. The mean age of initiation of symptoms was earlier in XR cases, and 78% patients had respiratory tract infections. Bone marrow transplantation was performed in five XR cases (two ex) and four AR (one ex) cases. Three of nine XR and two of six AR cases deceased on medical follow-up. In countries especially with high consanguinity rates, the early diagnosis for appropriate prophylactic treatment of CGD is quietly important to avoid from recurrent severe infections, early death and fatal complications of late transplantation.


Assuntos
Consanguinidade , Doença Granulomatosa Crônica/imunologia , NADPH Oxidases/metabolismo , Adolescente , Idade de Início , Transplante de Medula Óssea , Criança , Pré-Escolar , Feminino , Seguimentos , Genes Recessivos/genética , Genes Ligados ao Cromossomo X/genética , Doença Granulomatosa Crônica/epidemiologia , Doença Granulomatosa Crônica/genética , Humanos , Masculino , Mutação/genética , NADPH Oxidase 2/genética , Espécies Reativas de Oxigênio/metabolismo , Estudos Retrospectivos , Resultado do Tratamento , Turquia/epidemiologia
13.
FASEB J ; 33(2): 2422-2434, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30260700

RESUMO

The importance of proteostasis in preventing cellular senescence has been well recognized. However, the exact mechanism by which the loss of proteostasis or endoplasmic reticulum (ER) stress induces cellular senescence remains unclear. We report that ER stress mediates cellular senescence through the activating transcription factor (ATF)6α branch of the unfolded protein response (UPR). Cellular senescence was induced by the abrogation of neighbor of breast cancer (BRCA)1 gene (NBR1). NBR1 abrogation-induced senescence was p53 dependent and observed in both transformed and nontransformed human cell lines: MCF-7, Caki-1, and MRC-5. NBR1 bound to p38 MAPK, preferentially to an active form, and upon NBR1 abrogation, the activity of p38 increased. NADPH oxidase was activated in turn by p38, and the resulting oxidative stress triggered ER stress. It was found that ER stress mediated cellular senescence through the UPR sensor ATF6α. Knockdown of ATF6α prevented senescence, whereas ATF6α overexpression triggered it. The transcriptional activity of ATF6α was important. The ER stress-ATF6α axis also mediated cellular senescence induced by H-RasV12 overexpression and UV irradiation, suggesting a common role of this axis in senescence induction. In summary, we presented an evidence for the novel role of the ER stress-ATF6α axis in cellular senescence.-Kim, H. S., Kim, Y., Lim, M. J., Park, Y.-G., Park, S. I., Sohn, J. The p38-activated ER stress-ATF6α axis mediates cellular senescence.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Senescência Celular , Estresse do Retículo Endoplasmático , Proteínas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 6 Ativador da Transcrição/genética , Humanos , Células MCF-7 , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Proteínas/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética
14.
Life Sci ; 216: 271-278, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500550

RESUMO

AIMS: The roles of miR-141 in various types of cancers and inflammatory bowel diseases are researched, whereas, little information about its function in lung inflammation is available. This study was designed to explore the effect of miR-141 on inflammation injury in WI-38 cells, possibly providing basis for targeted therapeutic strategy for treatment of infantile pneumonia. MAIN METHODS: WI-38 cells were treated with LPS to construct cell model with inflammation injury. Expressions of miR-141 and NOX2 were altered by transfection assay and expressions of them were detected by qRT-PCR or Western blot. Cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively. The tested pro-inflammatory factors were analyzed by qRT-PCR and Western blot; and their productions were quantified by ELISA. Main proteins participating in regulation of apoptosis, p38 MAPK pathway and NF-κB pathway were analyzed by Western blot. KEY FINDINGS: miR-141 was down-regulated in LPS-treated cells and elevating miR-141 level reduced inflammation extent of WI-38 cells by promoting viability, inhibiting apoptosis, and inhibiting production of tested pro-inflammatory cytokines. NOX2 was up-regulated by miR-141 overexpression. NOX2 silence impaired the cell-protective effect of miR-141. miR-141 inhibited LPS-induced activations of p38 MAPK and NF-κB pathways, which was also mediated by NOX2. SIGNIFICANCE: miR-141 alleviated LPS-induced inflammation injury in WI-38 fibroblasts by up-regulating NOX2 and further inhibiting p38 MAPK and NF-κB pathways.


Assuntos
Fibroblastos/patologia , Inflamação/patologia , MicroRNAs/genética , NADPH Oxidase 2/genética , Pneumonia/patologia , Apoptose/genética , Western Blotting , Linhagem Celular , Sobrevivência Celular/genética , Citocinas/metabolismo , Regulação para Baixo , Humanos , Inflamação/genética , Lipopolissacarídeos/toxicidade , NF-kappa B/metabolismo , Pneumonia/genética , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Oncogene ; 38(9): 1534-1543, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30323311

RESUMO

Mutations leading to constitutive RAS activation contribute in myeloid leukemogenesis. RAS mutations in myeloid cells are accompanied by excessive formation of reactive oxygen species (ROS), but the source of ROS and their role for the initiation and progression of leukemia have not been clearly defined. To determine the role of NOX2-derived ROS in RAS-driven leukemia, double transgenic LSL-KrasG12D × Mx1-Cre mice expressing oncogenic KRAS in hematopoietic cells (M-KrasG12D) were treated with Nα-methyl-histamine (NMH) that targeted the production of NOX2-derived ROS in leukemic cells by agonist activity at histamine H2 receptors. M-KrasG12D mice developed myeloid leukemia comprising mature CD11b+Gr1+ myeloid cells that produced NOX2-derived ROS. Treatment of M-KrasG12D mice with NMH delayed the development of myeloproliferative disease and prolonged survival. In addition, NMH-treated M-KrasG12D mice showed reduction of intracellular ROS along with reduced DNA oxidation and reduced occurence of double-stranded DNA breaks in myeloid cells. The in vivo expansion of leukemia was markedly reduced in triple transgenic mice where KRAS was expressed in hematopoietic cells of animals with genetic NOX2 deficiency (Nox2-/- × LSL-KrasG12D × Mx1-Cre). Treatment with NMH did not alter in vivo expansion of leukemia in these NOX2-deficient transgenic mice. We propose that NOX2-derived ROS may contribute to the progression of KRAS-induced leukemia and that strategies to target NOX2 merit further evaluation in RAS-mutated hematopoietic cancer.


Assuntos
Hematopoese/genética , Transtornos Mieloproliferativos/genética , NADPH Oxidase 2/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Transtornos Mieloproliferativos/patologia , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida
16.
Methods Mol Biol ; 1874: 139-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30353512

RESUMO

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are an immunodeficient strain that enables human cell xenografts. However, NSG mice possess a complex genetic background that would complicate cross-breeding with other inbred transgenic or knockout mouse strains to establish a congenic strain with a desired genetic modification in the NSG background. Newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology enables modification of the mouse genome at the zygote stage without the need for extensive cross-breeding or the use of embryonic stem cells. In this chapter, we use the knockout of the X-linked Cybb gene as an example to describe our procedures for genetically modifying NSG mice using the CRISPR/Cas9 method. Briefly, two sgRNAs were designed and made to target exon 1 and exon 3 of the Cybb gene, and either sgRNA was then microinjected together with Cas9 mRNA into fertilized eggs collected from NSG mice. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. Offspring born to the foster mothers were genotyped by PCR and DNA sequencing. In this chapter, we describe our experiment procedures in detail and report our genotyping results for demonstrating that NSG mice can be genetically modified using the CRISPR/Cas9 technology in a highly efficient manner.


Assuntos
Sistemas CRISPR-Cas , Transferência Embrionária/métodos , NADPH Oxidase 2/genética , Animais , Éxons , Hospedeiro Imunocomprometido , Camundongos , Camundongos Knockout , Microinjeções , RNA Guia/genética
17.
Inflammation ; 42(1): 185-198, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30288635

RESUMO

Acute lung injury (ALI), developing as a component of the systemic inflammatory response syndrome (SIRS), leads to significant morbidity and mortality. Reactive oxygen species (ROS), produced in part by the neutrophil NADPH oxidase 2 (Nox2), have been implicated in the pathogenesis of ALI. Previous studies in our laboratory demonstrated the development of pulmonary inflammation in Nox2-deficient (gp91phox-/y) mice that was absent in WT mice in a murine model of SIRS. Given this finding, we hypothesized that Nox2 in a resident cell in the lung, specifically the alveolar macrophage, has an essential anti-inflammatory role. Using a murine model of SIRS, we examined whole-lung digests and bronchoalveolar lavage fluid (BALf) from WT and gp91phox-/y mice. Both genotypes demonstrated neutrophil sequestration in the lung during SIRS, but neutrophil migration into the alveolar space was only present in the gp91phox-/y mice. Macrophage inflammatory protein (MIP)-1α gene expression and protein secretion were higher in whole-lung digest from uninjected gp91phox-/y mice compared to the WT mice. Gene expression of MIP-1α, MCP-1, and MIP-2 was upregulated in alveolar macrophages obtained from gp91phox-/y mice at baseline compared with WT mice. Further, ex vivo analysis of alveolar macrophages, but not bone marrow-derived macrophages or peritoneal macrophages, demonstrated higher gene expression of MIP-1α and MIP-2. Moreover, isolated lung polymorphonuclear neutrophils migrate to BALf obtained from gp91phox-/y mice, further providing evidence of a cell-specific anti-inflammatory role for Nox2 in alveolar macrophages. We speculate that Nox2 represses the development of inflammatory lung injury by modulating chemokine expression by the alveolar macrophage.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Macrófagos Alveolares/metabolismo , NADPH Oxidase 2/fisiologia , Neutrófilos/patologia , Lesão Pulmonar Aguda/patologia , Animais , Movimento Celular , Quimiocinas/metabolismo , Inflamação/prevenção & controle , Pulmão/enzimologia , Macrófagos Alveolares/enzimologia , Camundongos , NADPH Oxidase 2/deficiência , NADPH Oxidase 2/genética , Espécies Reativas de Oxigênio , Síndrome de Resposta Inflamatória Sistêmica/patologia
18.
Neurotox Res ; 35(1): 139-149, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30112693

RESUMO

Dexmedetomidine (Dex) is a widely used sedative in anesthesia and critical care units, and it exhibits neuroprotective activity. However, the precise mechanism of Dex-exerted neuroprotection is not clear. Increased neuronal NADPH oxidase 2 (NOX2) contributes to oxidative stress and neuronal damage in various hypoxia-related neurodegenerative disorders. The present study investigated whether Dex regulated neuronal NOX2 to exert its protective effects under hypoxic conditions. Well-differentiated PC12 cells were exposed to cobalt chloride (CoCl2) to mimic a neuronal model of chemical hypoxia-mediated neurotoxicity. The data showed that Dex pretreatment of PC12 cells significantly suppressed CoCl2-induced neurotoxicity, as evidenced by the enhanced cell viability, restoration of cellular morphology, and reduction in apoptotic cells. Dex improved mitochondrial function and inhibited CoCl2-induced mitochondrial apoptotic pathways. We further demonstrated that Dex attenuated oxidative stress, downregulated NOX2 protein expression and activity, and inhibited intracellular calcium ([Ca2+]i) overload in CoCl2-treated PC12 cells. Moreover, knockdown of the NOX2 gene markedly improved mitochondrial function and attenuated apoptosis under hypoxic conditions. These results demonstrated that the protective effects of Dex against hypoxia-induced neurotoxicity in neural cells were mediated, at least partially, via inhibition of NOX2-mediated oxidative stress.


Assuntos
Hipóxia Celular/efeitos dos fármacos , Dexmedetomidina/farmacologia , NADPH Oxidase 2/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cálcio/metabolismo , Hipóxia Celular/fisiologia , Cobalto/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADPH Oxidase 2/genética , Neuroproteção/efeitos dos fármacos , Neuroproteção/fisiologia , Estresse Oxidativo/fisiologia , Células PC12 , Ratos
19.
PLoS One ; 13(12): e0208602, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30532146

RESUMO

Macrophages are key inflammatory immune cells that display dynamic phenotypes and functions in response to their local microenvironment. Major advances have occurred in understanding the transcriptional, epigenetic, and functional differences in various macrophage subsets by in vitro modeling and gene expression and epigenetic profiling for biomarker discovery. However, there is still no standardized protocol for macrophage polarization largely due to the lack of thorough validation of macrophage phenotypes following polarization. In addition, transcriptional regulation is recognized as a major mechanism governing differential macrophage polarization programs and as such, many genes have been identified to be associated with each macrophage subset. However, the functional role of many of these genes in macrophage polarization is still unknown. Moreover, the role of other regulatory mechanisms, such as DNA methylation, in macrophage polarization remains poorly understood. Here, we employed an optimized model of human M1 and M2 macrophage polarization which we used for large-scale transcriptional and DNA methylation profiling. We were unable to demonstrate a role for DNA methylation in macrophage polarization, as no significant changes were identified. However, we observed significant changes in the transcriptomes of M1 and M2 macrophages. Additionally, we identified numerous novel differentially regulated genes involved in macrophage polarization, including CYBB and DHCR7 which we show as important regulators of M1 and M2 macrophage polarization, respectively. Taken together, our improved in vitro human M1 and M2 macrophage model provides new understandings of the regulation of macrophage polarization and candidate macrophage biomarkers.


Assuntos
Perfilação da Expressão Gênica , Macrófagos/citologia , Transcrição Genética , Quimiocina CCL17/deficiência , Metilação de DNA , Humanos , Interleucina-10/deficiência , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Macrófagos/imunologia , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fenótipo
20.
Molecules ; 23(11)2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30384445

RESUMO

The inflammatory response mediated by microglia plays a critical role in the progression of ischemic stroke. Phosphoinositide 3-kinase gamma (PI3Kγ) has been implicated in multiple inflammatory and autoimmune diseases, making it a promising target for therapeutic intervention. The aim of this study was to evaluate the efficacy of 8e, a hydrogen sulfide (H2S) releasing derivative of 3-n-butylphthalide (NBP), on brain damage and PI3Kγ signaling following cerebral ischemia injury. 8e significantly reduced sensorimotor deficits, focal infarction, brain edema and neural apoptosis at 72 h after transient middle cerebral artery occlusion (tMCAO). The NOX2 isoform of the NADPH oxidase family is considered a major enzymatic source of superoxide. We found that the release of superoxide, together with the expression of NOX2 subunits p47phox, p-p47phox, and the upstream PI3Kγ/AKT signaling were all down-regulated by 8e, both in the penumbral region of the rat brain and in the primary cultured microglia subjected to oxygen-glucose deprivation (OGD). With the use of siRNA and pharmacological inhibitors, we further demonstrated that 8e regulates the formation of superoxide in activated microglia through the PI3Kγ/AKT/NOX2 signaling pathway and subsequently prevents neuronal death in neighboring neurons. Our experimental data indicate that 8e is a potential candidate for the treatment of ischemic stroke and PI3Kγ-mediated neuroinflammation.


Assuntos
Benzofuranos/administração & dosagem , Isquemia Encefálica/tratamento farmacológico , Sulfeto de Hidrogênio/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Benzofuranos/química , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Humanos , Sulfeto de Hidrogênio/química , Sulfeto de Hidrogênio/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/fisiopatologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , NADPH Oxidase 2/genética , Neurônios/efeitos dos fármacos , Neurônios/patologia , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Superóxidos/química , Superóxidos/metabolismo
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