RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Callicarpa japonica Thunb., as an herbal medicine has been used for the treatment of inflammatory diseases in China and Korea. MATERIALS AND METHODS: Ultra performance liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometer (UPLC-PDA-QTof MS) was used to detect the major phenylethanoid glycosides in the C. japonica extract. BALB/c mice were intraperitoneally sensitized by ovalbumin (OVA) (on days 0 and 7) and challenged by OVA aerosol (on days 11-13) to induce airway inflammatory response. The mice were also administered with C. japonica Thunb. (CJT) (20 and 40 mg/kg Per oral) on days 9-13. CJT pretreatment was conducted in lipopolysaccharide (LPS)-stimulated RAW264.7 or phorbol 12-myristate 13-acetate (PMA)-stimulated A549 cells. RESULTS: CJT administration significantly reduced the secretion of Th2 cytokines, TNF-α, IL-6, immunoglobulin E (IgE) and histamine, and the recruitment of eosinophils in an OVA-exposed mice. In histological analyses, the amelioration of inflammatory cell influx and mucus secretion were observed with CJT. The OVA-induced airway hyperresponsiveness (AHR), iNOS expression and NF-κB activation were effectively suppressed by CJT administration. In addition, CJT led to the upregulation of HO-1 expression. In an in vitro study, CJT pretreatment suppressed the LPS-induced TNF-α secretion in RAW264.7 cells and attenuated the PMA-induced IL-6, IL-8 and MCP-1 secretion in A549 cells. These effects were accompanied by downregulated NF-κB phosphorylation and by upregulated HO-1 expression. CONCLUSION: These results suggested that CJT has protective activity against OVA-induced airway inflammation via downregulation of NF-κB activation and upregulation of HO-1, suggesting that CJT has preventive potential for the development of allergic asthma.
Assuntos
Antiasmáticos/farmacologia , Anti-Inflamatórios/farmacologia , Asma/prevenção & controle , Hiper-Reatividade Brônquica/prevenção & controle , Callicarpa , Heme Oxigenase-1/metabolismo , Pulmão/efeitos dos fármacos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Células A549 , Animais , Antiasmáticos/isolamento & purificação , Anti-Inflamatórios/isolamento & purificação , Asma/induzido quimicamente , Asma/enzimologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição/efeitos dos fármacos , Callicarpa/química , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/enzimologia , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Extratos Vegetais/isolamento & purificação , Células RAW 264.7 , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Asthma is a chronic inflammatory disease characterized by airways hyper-responsiveness (AHR), reversible airway obstruction, and airway inflammation and remodeling. We previously showed that Syk modulates methacholine-induced airways contractility in naïve mice and in mice with allergic airways inflammation. We hypothesize that Syk plays a role in the pathogenesis of AHR; this was evaluated in a chronic 8-week mouse model of house dust mite (HDM)-induced allergic airways inflammation. METHODS: We used the Sykflox/flox//rosa26CreERT2 conditional Syk knock-out mice to assess the role of Syk prior to HDM exposure, and treated HDM-sensitized mice with the Syk inhibitor, GSK143, to evaluate its role in established allergic airways inflammation. Respiratory mechanics and methacholine (MCh)-responsiveness were assessed using the flexiVent® system. Lungs underwent bronchoalveolar lavage to isolate inflammatory cells or were frozen for determination of gene expression in tissues. RESULTS: MCh-induced AHR was observed following HDM sensitization in the Syk-intact (Sykflox/flox) and vehicle-treated BALB/c mice. MCh responsiveness was reduced to control levels in HDM-sensitized Sykdel/del mice and in BALB/c and Sykflox/flox mice treated with GSK143. Both Sykdel/del and GSK143-treated mice mounted appropriate immune responses to HDM, with HDM-specific IgE levels that were comparable to Sykflox/flox and vehicle-treated BALB/c mice. HDM-induced increases in bronchoalveolar lavage cell counts were attenuated in both Sykdel/del and GSK143-treated mice, due primarily to decreased neutrophil recruitment. Gene expression analysis of lung tissues revealed that HDM-induced expression of IL-17 and CXCL-1 was significantly attenuated in both Sykdel/del and GSK143-treated mice. CONCLUSION: Syk inhibitors may play a role in the management of neutrophilic asthma.
Assuntos
Hiper-Reatividade Brônquica/enzimologia , Modelos Animais de Doenças , Quinase Syk/metabolismo , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pyroglyphidae/imunologia , Quinase Syk/genéticaRESUMO
The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γKOδD910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/deficiência , Eosinofilia/enzimologia , Eosinofilia/imunologia , Imunoglobulina E/biossíntese , Ovalbumina/imunologia , Pneumonia/enzimologia , Pneumonia/imunologia , Animais , Linfócitos B/imunologia , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Contagem de Células , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Eosinofilia/sangue , Eosinofilia/complicações , Eosinófilos/imunologia , Células Caliciformes/patologia , Hipersensibilidade/sangue , Hipersensibilidade/complicações , Hipersensibilidade/enzimologia , Hipersensibilidade/imunologia , Interleucina-5/sangue , Pulmão/imunologia , Pulmão/patologia , Metaplasia , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Pneumonia/sangue , Pneumonia/complicações , Linfócitos T/imunologiaRESUMO
Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41-2272 and BAY 60-2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41-2272 and BAY 60-2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.
Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Broncodilatadores/farmacologia , Guanilato Ciclase/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Antiasmáticos/uso terapêutico , Asma/enzimologia , Asma/fisiopatologia , Benzoatos/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/enzimologia , Broncodilatadores/uso terapêutico , Técnicas de Cocultura , GMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Hidrocarbonetos Fluorados/uso terapêutico , Pulmão/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Óxido Nítrico/farmacologia , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Solubilidade , Traqueia/efeitos dos fármacosRESUMO
BACKGROUND: In allergen-induced asthma, activated mast cells start the lung inflammatory process with degranulation, cytokine synthesis, and mediator release. Bruton's tyrosine kinase (Btk) activity is required for the mast cell activation during IgE-mediated secretion. METHODS: This study characterized a novel inhaled Btk inhibitor RN983 in vitro and in ovalbumin allergic mouse models of the early (EAR) and late (LAR) asthmatic response. RESULTS: RN983 potently, selectively, and reversibly inhibited the Btk enzyme. RN983 displayed functional activities in human cell-based assays in multiple cell types, inhibiting IgG production in B-cells with an IC50 of 2.5 ± 0.7 nM and PGD2 production from mast cells with an IC50 of 8.3 ± 1.1 nM. RN983 displayed similar functional activities in the allergic mouse model of asthma when delivered as a dry powder aerosol by nose-only inhalation. RN983 was less potent at inhibiting bronchoconstriction (IC50(RN983) = 59 µg/kg) than the ß-agonist salbutamol (IC50(salbutamol) = 15 µg/kg) in the mouse model of the EAR. RN983 was more potent at inhibiting the antigen induced increase in pulmonary inflammation (IC50(RN983) = <3 µg/kg) than the inhaled corticosteroid budesonide (IC50(budesonide) = 27 µg/kg) in the mouse model of the LAR. CONCLUSIONS: Inhalation of aerosolized RN983 may be effective as a stand-alone asthma therapy or used in combination with inhaled steroids and ß-agonists in severe asthmatics due to its potent inhibition of mast cell activation.
Assuntos
Antiasmáticos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/prevenção & controle , Broncoconstrição/efeitos dos fármacos , Broncodilatadores/administração & dosagem , Inaladores de Pó Seco , Pulmão/efeitos dos fármacos , Ovalbumina , Ftalazinas/administração & dosagem , Pneumonia/prevenção & controle , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridazinas/administração & dosagem , Administração por Inalação , Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Tirosina Quinase da Agamaglobulinemia , Albuterol/administração & dosagem , Animais , Antiasmáticos/farmacocinética , Anti-Inflamatórios/farmacocinética , Asma/enzimologia , Asma/imunologia , Asma/fisiopatologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/imunologia , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Broncodilatadores/farmacocinética , Budesonida/administração & dosagem , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Glucocorticoides/administração & dosagem , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/fisiopatologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Mastócitos/imunologia , Camundongos Endogâmicos BALB C , Ftalazinas/farmacocinética , Pneumonia/enzimologia , Pneumonia/imunologia , Pneumonia/fisiopatologia , Prostaglandina D2/imunologia , Prostaglandina D2/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Tirosina Quinases/metabolismo , Piridazinas/farmacocinéticaRESUMO
Obesity is a major risk factor for asthma, which is characterized by airway hyperreactivity (AHR). In obesity-associated asthma, AHR may be regulated by non-TH2 mechanisms. We hypothesized that airway reactivity is regulated by insulin in the CNS, and that the high levels of insulin associated with obesity contribute to AHR. We found that intracerebroventricular (ICV)-injected insulin increases airway reactivity in wild-type, but not in vesicle acetylcholine transporter knockdown (VAChT KD(HOM-/-)), mice. Either neutralization of central insulin or inhibition of extracellular signal-regulated kinases (ERK) normalized airway reactivity in hyperinsulinemic obese mice. These effects were mediated by insulin in cholinergic nerves located at the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA), which convey parasympathetic outflow to the lungs. We propose that increased insulin-induced activation of ERK in parasympathetic pre-ganglionic nerves contributes to AHR in obese mice, suggesting a drug-treatable link between obesity and asthma.
Assuntos
Tronco Encefálico/enzimologia , Hiper-Reatividade Brônquica/complicações , Neurônios Colinérgicos/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hiperinsulinismo/complicações , Sistema de Sinalização das MAP Quinases , Animais , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição , Neurônios Colinérgicos/patologia , Dieta Hiperlipídica , Ativação Enzimática , Hiperinsulinismo/enzimologia , Hiperinsulinismo/fisiopatologia , Inflamação/patologia , Injeções Intraventriculares , Insulina/metabolismo , Cloreto de Metacolina , Camundongos Endogâmicos C57BL , Camundongos Obesos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Receptor de Insulina/metabolismoRESUMO
BACKGROUND/AIMS: We sought to increase our understanding of the rhinitis-asthma relationship and improve strategies for the treatment of patients with these diseases. The aim of this study was to identify a connection between upper airway inflammation and lower airway responsiveness. METHODS: We counted eosinophils on nasal smears, and performed spirometry, allergic skin tests, and methacholine challenge tests in 308 schoolchildren plus a questionnaire on respiratory symptoms. The methacholine concentration causing a 20% fall in forced expiratory volume in 1 second (PC20 < 25 mg/mL) was used as the threshold of bronchial hyperresponsiveness (BHR). RESULTS: In total, 26% of subjects had positive nasal eosinophils on a smear, and 46.2% of subjects had BHR at < 25 mg/mL methacholine PC20. Nasal symptoms were higher in subjects with than without nasal eosinophils (p = 0.012). Asthma symptoms did not differ between subjects with and without nasal eosinophils. Nasal eosinophils were higher in subjects with atopy than those without (p = 0.006), and there was no difference in PC20 methacholine according to atopy (15.5 ± 1.07 vs. 17.5 ± 0.62; p > 0.05). No difference in BHR was detected when comparing subjects with and without nasal eosinophils. There were significant differences in the PC20 between subjects with greater than 50% nasal eosinophils and without nasal eosinophils (11.01 ± 2.92 mg/mL vs. 17.38 ± 0.61 mg/mL; p < 0.001). CONCLUSIONS: These findings demonstrated that nasal eosinophilic inflammation might contribute to lower airway responsiveness in schoolchildren, based on an epidemiological survey.
Assuntos
Asma/epidemiologia , Hiper-Reatividade Brônquica/enzimologia , Eosinofilia/epidemiologia , Pulmão/fisiopatologia , Mucosa Nasal/imunologia , Rinite/epidemiologia , Adolescente , Distribuição por Idade , Fatores Etários , Asma/diagnóstico , Asma/fisiopatologia , Hiper-Reatividade Brônquica/diagnóstico , Hiper-Reatividade Brônquica/fisiopatologia , Testes de Provocação Brônquica , Criança , Eosinofilia/diagnóstico , Eosinofilia/imunologia , Eosinófilos/imunologia , Feminino , Inquéritos Epidemiológicos , Humanos , Testes Intradérmicos , Contagem de Leucócitos , Masculino , República da Coreia/epidemiologia , Rinite/diagnóstico , Rinite/imunologia , Espirometria , Inquéritos e QuestionáriosRESUMO
BACKGROUND: Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection. METHODS: Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression. RESULTS: RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice. CONCLUSIONS: MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Hiper-Reatividade Brônquica/enzimologia , Proteínas do Citoesqueleto/metabolismo , Interferon gama/metabolismo , Pulmão/enzimologia , Metaloproteinase 12 da Matriz/metabolismo , Pneumonia/enzimologia , Infecções por Vírus Respiratório Sincicial/enzimologia , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/imunologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/virologia , Líquido da Lavagem Broncoalveolar/imunologia , Broncoconstrição , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Imunidade Celular , Interferon gama/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/fisiopatologia , Pulmão/virologia , Metaloproteinase 12 da Matriz/imunologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/fisiopatologia , Pneumonia/virologia , Interferência de RNA , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/patogenicidade , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/virologia , Fatores de TempoRESUMO
BACKGROUND: Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma. METHODS: We prepared a mouse model of allergic asthma using PLCε+/+ mice and PLCεΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells. RESULTS: After OVA challenge, the OVA-immunized PLCεΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCεΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCεΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency. CONCLUSIONS: PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.
Assuntos
Asma/enzimologia , Brônquios/enzimologia , Hiper-Reatividade Brônquica/enzimologia , Células Epiteliais/enzimologia , Fosfoinositídeo Fosfolipase C/imunologia , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Brônquios/imunologia , Brônquios/patologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Knockout , Ovalbumina , Fosfoinositídeo Fosfolipase C/deficiência , Fosfoinositídeo Fosfolipase C/genética , Cultura Primária de Células , Mucosa Respiratória/enzimologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Transdução de Sinais , Equilíbrio Th1-Th2/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Células Th2/patologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/imunologia , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/imunologiaRESUMO
CONTEXT: Nodakenin is a major coumarin glucoside in the root of Peucedanum decursivum Maxim, a commonly used traditional Chinese medicine for the treatment of asthma and chronic bronchitis for thousands of years. OBJECTIVE: In this work, the anti-asthma potential of nodakenin was studied by investigation of its effect to suppress airway inflammation, hyper-responsiveness and remodeling in a murine model of chronic asthma. MATERIALS AND METHODS: BALB/c mice sensitized to ovalbumin (OVA) were challenged with aerosolized OVA for 8 weeks, orally administered with nodakenin at doses of 5, 10 and 20 mg/kg before each OVA challenge. RESULTS: Compared with the model group, nodakenin treatment markedly inhibited airway inflammation, hyper-responsiveness and remodeling, showing improvement in subepithelial fibrosis, smooth muscle hypertrophy, and goblet cell hyperplasia, and decreased levels of interleukin (IL)-4, IL-5, IL-13 and matrix metalloproteinase-2/-9 in bronchoalveolar lavage fluid, and the level of OVA-specific IgE in serum. In addition, the NF-κB DNA-binding activity in lung tissues was also reduced by nodakenin treatment. CONCLUSIONS: These data indicated that nodakenin might mitigate the development of chronic experimental allergic asthma.
Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Bronquite/tratamento farmacológico , Cumarínicos/uso terapêutico , Glucosídeos/uso terapêutico , Hipersensibilidade/tratamento farmacológico , Animais , Asma/enzimologia , Asma/patologia , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/patologia , Bronquite/patologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Feminino , Hipersensibilidade/enzimologia , Hipersensibilidade/patologia , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/efeitos dos fármacosRESUMO
With a view to study the effect of genes GSTT1 and GSTM1 deletion on the non-specific bronchial hyperresponsiveness in children with neutrophilic bronchial asthma (BA) 46 school age children having neutrophilic BA (1st clinical group) and their 48 coevals with eosinophilic phenotype of the disease (2nd clinical group) were subjected to a complex examination at the pulmo-allergologic department of the regional child clinical hospital of Chernivtsi. The study proved that genotype T1+M1del was more frequently registered in patients with the neutrophilic phenotype of the disease, and genotype T1delM1del was equifrequent in patients with different types of the inflammation of the respiratory ways. In patients with neutrophilic BA and deletion polymorphism of genes GSTT1 and GSTM1, there was a tendency to decreasing of the bronchial lability index through the decrease of bronchodilation, and bronchial response to histamine occurred to be higher than in children with the absence of polymorphism of the referred genes of the xenobiotics biotransformation system.
Assuntos
Asma/genética , Hiper-Reatividade Brônquica/genética , Glutationa Transferase/genética , Neutrófilos/imunologia , Polimorfismo Genético , Xenobióticos/farmacocinética , Asma/enzimologia , Asma/imunologia , Asma/fisiopatologia , Biotransformação , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/imunologia , Broncoconstrição/genética , Estudos de Casos e Controles , Criança , Eosinófilos/citologia , Eosinófilos/imunologia , Frequência do Gene , Genótipo , Humanos , Contagem de Leucócitos , Masculino , Muco/citologia , Neutrófilos/citologia , Escarro/citologiaRESUMO
Innate airway hyperresponsiveness (AHR) is well modeled by two strains of rat, the hyperresponsive Fischer 344 rat and the normoresponsive Lewis rat. Arginase has been implicated in AHR associated with allergic asthma models. We addressed the role of arginase in innate AHR using the Fischer-Lewis model. In vivo arginase inhibition with N(ω)-hydroxy-nor-arginine (nor-NOHA) was evaluated on methacholine-induced bronchoconstriction in the Fischer and the Lewis rats. Arginase activity and mRNA expression were quantified in structural and resident cells of the proximal airway tree. The effect of nor-NOHA was evaluated on cultured tracheal smooth muscle proliferation. Fischer rats exhibited significantly greater changes in respiratory resistance and elastance in response to methacholine compared with Lewis rats. nor-NOHA reduced the methacholine-induced bronchoconstriction in the central airways of Lewis rats, while it did not change the innate AHR of Fischer rats. Lewis rats exhibited greater arginase activity in tracheal smooth muscle but a lower proliferation rate compared with Fischer rats. Smooth muscle proliferation was not affected by nor-NOHA in either strain of rats. The strain-specific arginase expression in the smooth muscle may contribute to the differences in sensitivity of the methacholine challenged airways of Lewis and Fischer rats to inhibition of arginase.
Assuntos
Arginase/antagonistas & inibidores , Arginina/análogos & derivados , Hiper-Reatividade Brônquica/prevenção & controle , Testes de Provocação Brônquica , Broncoconstrição/efeitos dos fármacos , Broncoconstritores , Inibidores Enzimáticos/farmacologia , Pulmão/efeitos dos fármacos , Cloreto de Metacolina , Animais , Arginase/genética , Arginase/metabolismo , Arginina/farmacologia , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica , Pulmão/enzimologia , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Especificidade da EspécieRESUMO
BACKGROUND: Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined. OBJECTIVE: We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. METHODS: Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. RESULTS: SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs. CONCLUSION: S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.
Assuntos
Amino Álcoois/farmacologia , Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Inflamação/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Asma/induzido quimicamente , Asma/enzimologia , Asma/metabolismo , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Quimiocina CCL2/metabolismo , Feminino , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Humanos , Hiperplasia/tratamento farmacológico , Hiperplasia/metabolismo , Imunoglobulina E/metabolismo , Inflamação/metabolismo , Interferon gama/metabolismo , Interleucinas/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lisofosfolipídeos/metabolismo , Mastócitos/metabolismo , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Ovalbumina/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: IL-4 and signal transducer and activator of transcription 6 (STAT6) play an important role in the progression of allergic airway disease (AAD) or asthma. IL-4 and STAT6 mediate T(H)2 responses in T cells and immunoglobulin class-switching to IgE in B cells. Both T(H)2 responses and IgE promote the asthmatic condition. We have previously demonstrated that poly (ADP-ribose) polymerase (PARP) 14, a member of the PARP family of proteins, regulates the transcription function of STAT6. However, the role of PARP-14 in AAD is not known. OBJECTIVE: Here we investigate the role of PARP-14 and the enzyme activity associated with it in a model of AAD dependent on airway hyperresponsiveness and lung inflammation. We also elucidate the mechanism by which PARP-14 regulates AAD. METHODS: The role of PARP-14 and its enzyme activity in AAD and T(H)2 differentiation were examined by using a murine model of AAD and in vitro T(H) cell differentiation. RESULTS: PARP-14-deficient animals show reduced lung pathology and IgE levels when compared with control animals. Treating mice with a pharmacologic inhibitor for PARP activity reduced the severity of airway hyperresponsiveness and lung inflammation. Mechanistically, our data indicate that PARP-14 and its enzyme activity aid in the differentiation of T cells toward a T(H)2 phenotype by regulating the binding of STAT6 to the Gata3 promoter. CONCLUSION: PARP-14 and the catalytic activity associated with it promote T(H)2 differentiation and AAD in a murine model, and targeting PARP-14 might be a potential new therapy for allergic asthma.
Assuntos
Hiper-Reatividade Brônquica/patologia , Hipersensibilidade/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Doenças Respiratórias/patologia , Células Th2/enzimologia , Células Th2/patologia , Animais , Asma/genética , Asma/metabolismo , Asma/patologia , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Hipersensibilidade/genética , Hipersensibilidade/metabolismo , Imunoglobulina E/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/imunologia , Regiões Promotoras Genéticas/genética , Doenças Respiratórias/enzimologia , Doenças Respiratórias/genética , Doenças Respiratórias/metabolismo , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Células Th2/metabolismoRESUMO
OBJECTIVE: To determine whether a short-term protocol using subcutaneous sensitization with ovalbumin, without the use of adjuvants, would induce an eosinophilic response in the lungs of mice similar to that observed in previous, well-established protocols. METHODS: Adult female BALB/c mice were randomized and divided into groups according to the number of sensitizations with ovalbumin and the number/dosage of intranasal ovalbumin challenges. The short-term protocol (10 days) consisted of one sensitization with ovalbumin and three ovalbumin challenges (100 µg). Total and differential cell counts in BAL fluid, levels of eosinophil peroxidase in lung tissue, and histopathological examination of the lungs were performed 24 h after the last ovalbumin challenge. RESULTS: No significant differences were found among the groups regarding the variables studied. The short-term protocol, as well as the other protocols studied, induced an eosinophilic response similar to that obtained in the positive control. CONCLUSIONS: Subcutaneous sensitization with ovalbumin and without the use of adjuvants resulted in a significant allergic response in the lungs of mice, even in the short-term protocol group. Our findings suggest that this short-term protocol can be used as a first-line pre-clinical test for the study of new medications, reducing the costs and observation periods.
Assuntos
Asma/patologia , Hiper-Reatividade Brônquica/patologia , Peroxidase de Eosinófilo/metabolismo , Pulmão/patologia , Ovalbumina , Eosinofilia Pulmonar/imunologia , Doença Aguda , Animais , Asma/enzimologia , Hiper-Reatividade Brônquica/enzimologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Feminino , Pulmão/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Eosinofilia Pulmonar/patologia , Distribuição AleatóriaRESUMO
OBJETIVO: Determinar se um protocolo curto de sensibilização com ovalbumina subcutânea, sem adjuvante, induziria uma resposta pulmonar eosinofílica em pulmões de camundongos similar àquela encontrada em protocolos previamente estabelecidos. MÉTODOS: Fêmeas adultas de camundongos BALB/c foram randomizadas e divididas em grupos de acordo com o número de sensibilizações com ovalbumina e o número/dosagem de provocação intranasal. O protocolo curto (10 dias) consistiu de uma sensibilização e três provocações com ovalbumina (100 µg). A contagem total e diferencial de células no lavado broncoalveolar, o nível de peroxidase eosinofílica no tecido pulmonar e o exame histopatológico dos pulmões foram realizados 24 h após a última provocação. RESULTADOS: Não houve diferenças significativas entre os grupos em relação às variáveis estudadas. O protocolo curto, assim como os outros protocolos estudados, induziu uma resposta eosinofílica pulmonar semelhante àquela do grupo controle positivo. CONCLUSÕES: A sensibilização por ovalbumina subcutânea sem o uso de adjuvante resultou em uma significativa resposta pulmonar alérgica em ratos, mesmo no grupo de protocolo curto. Nossos achados sugerem que esse protocolo curto pode ser utilizado como teste pré-clínico de primeira linha para a pesquisa de novos fármacos, reduzindo custos e o tempo de observação.
OBJECTIVE: To determine whether a short-term protocol using subcutaneous sensitization with ovalbumin, without the use of adjuvants, would induce an eosinophilic response in the lungs of mice similar to that observed in previous, well-established protocols. METHODS: Adult female BALB/c mice were randomized and divided into groups according to the number of sensitizations with ovalbumin and the number/dosage of intranasal ovalbumin challenges. The short-term protocol (10 days) consisted of one sensitization with ovalbumin and three ovalbumin challenges (100 µg). Total and differential cell counts in BAL fluid, levels of eosinophil peroxidase in lung tissue, and histopathological examination of the lungs were performed 24 h after the last ovalbumin challenge. RESULTS: No significant differences were found among the groups regarding the variables studied. The short-term protocol, as well as the other protocols studied, induced an eosinophilic response similar to that obtained in the positive control. CONCLUSIONS: Subcutaneous sensitization with ovalbumin and without the use of adjuvants resulted in a significant allergic response in the lungs of mice, even in the short-term protocol group. Our findings suggest that this short-term protocol can be used as a first-line pre-clinical test for the study of new medications, reducing the costs and observation periods.
Assuntos
Animais , Feminino , Camundongos , Asma/patologia , Hiper-Reatividade Brônquica/patologia , Peroxidase de Eosinófilo/metabolismo , Pulmão/patologia , Ovalbumina , Eosinofilia Pulmonar/imunologia , Doença Aguda , Asma/enzimologia , Testes de Provocação Brônquica , Hiper-Reatividade Brônquica/enzimologia , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Pulmão/enzimologia , Camundongos Endogâmicos BALB C , Eosinofilia Pulmonar/patologia , Distribuição AleatóriaRESUMO
BACKGROUND: Cigarette smoke exposure is strongly associated with airway hyperreactivity (AHR) which is the main characteristic seen in asthma. The intracellular MAPK signaling pathways are suggested to be associated with the airway damage to the AHR. In the present study, we hypothesize that secondhand cigarette smoke (SHS) exposure upregulates the bronchial contractile receptors via activation of the Raf/ERK/MAPK pathway. METHODOLOGY/PRINCIPAL FINDINGS: Rats were exposed to SHS for 3 h daily for up to 8 weeks. The receptor agonists-induced bronchial contractile reactivity was analyzed with a sensitive myograph system. The mRNA transcription and protein translation of the target receptors and the kinases in Raf/ERK/MAPK pathway were investigated by real-time PCR, Western blotting and immunofluorescence, respectively. Compared with exposure to fresh air, SHS induced enhanced bronchial contractile responses mediated by the 5-hydroxytryptamine 2A (5-HT(2A)) receptors as well as the endothelin type B (ET(B)) and type A (ET(A)) receptors. The response curves were shifted toward the left with an increased maximal contraction (E(max)) demonstrating that SHS induced AHR. Additionally, the mRNA and protein levels of the 5-HT(2A), ET(B) and ET(A) receptors were increased. Furthermore, SHS exposure increased the phosphorylation of Raf-1 and ERK1/2, but it did not alter p38 or JNK. A Raf-1 inhibitor (GW5074) suppressed the SHS-induced increase in the expression of 5-HT(2A) and ET(A) receptors and the receptor-mediated AHR. CONCLUSIONS/SIGNIFICANCE: Our findings show that SHS exposure induces transcriptional upregulation of the 5-HT(2A), ET(B) and ET(A) receptors in rat bronchial smooth muscle cells, which mediates AHR. The Raf/ERK/MAPK pathway is involved in SHS-associated receptor upregulation and AHR.
Assuntos
Hiper-Reatividade Brônquica/genética , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Receptor 5-HT2A de Serotonina/genética , Poluição por Fumaça de Tabaco , Transcrição Gênica , Regulação para Cima/genética , Animais , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/fisiopatologia , Exposição Ambiental , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas In Vitro , Indóis/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Fenóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Quinases raf/metabolismoRESUMO
The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/-)) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/-) and wild type mice. Pak1(-/-) mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/-) mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.
Assuntos
Hiper-Reatividade Brônquica/enzimologia , Quinases Ativadas por p21/fisiologia , Acetilcolina/administração & dosagem , Acetilcolina/efeitos adversos , Administração por Inalação , Administração Intravenosa , Animais , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/genética , Modelos Animais de Doenças , Dissulfetos/farmacologia , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Isoenzimas , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/fisiopatologia , Camundongos , Camundongos Knockout , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Naftóis/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genéticaRESUMO
Organic dust samples from swine confinement facilities elicit pro-inflammatory cytokine/chemokine release from bronchial epithelial cells and monocytes, dependent, in part, upon dust-induced activation of the protein kinase C (PKC) isoform, PKCε. PKCε is also rapidly activated in murine tracheal epithelial cells following in vivo organic dust challenges, yet the functional role of PKCε in modulating dust-induced airway inflammatory outcomes is not defined. Utilizing an established intranasal inhalation animal model, experiments investigated the biologic and physiologic responses following organic dust extract (ODE) treatments in wild-type (WT) and PKCε knock-out (KO) mice. We found that neutrophil influx increased more than twofold in PKCε KO mice following both a one-time challenge and 3 weeks of daily challenges with ODE as compared with WT mice. Lung pathology revealed increased bronchiolar and alveolar inflammation, lymphoid aggregates, and T cell influx in ODE-treated PKCε KO mice. Airway hyperresponsiveness to methacholine increased in PKCε KO + ODE to a greater magnitude than WT + ODE animals. There were no significant differences in cytokine/chemokine release elicited by ODE treatment between groups. However, ODE-induced nitric oxide (NO) production differed in that ODE exposure increased nitrate levels in WT mice but not in PKCε KO mice. Moreover, ODE failed to upregulate NO from ex vivo stimulated PKCε KO lung macrophages. Collectively, these studies demonstrate that PKCε-deficient mice were hypersensitive to organic dust exposure and suggest that PKCε is important in the normative lung inflammatory response to ODE. Dampening of ODE-induced NO may contribute to these enhanced inflammatory findings.
Assuntos
Hiper-Reatividade Brônquica/enzimologia , Poeira/imunologia , Mediadores da Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Proteína Quinase C-épsilon/biossíntese , Animais , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Testes de Provocação Brônquica , Quimiocinas/metabolismo , Modelos Animais de Doenças , Exposição Ambiental/efeitos adversos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Feminino , Pulmão/metabolismo , Pulmão/patologia , Masculino , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Proteína Quinase C-épsilon/deficiência , Proteína Quinase C-épsilon/genética , Linfócitos T/patologiaRESUMO
Protein kinase C variants (PKCs) have been involved in the control of airway smooth muscle (ASM) tone, and abnormalities in PKC-dependent signaling have been associated with respiratory diseases such as asthma. In this study, the role of atypical PKCζ in airway hyperresponsiveness was investigated, using an in-vitro model of TNFα-treated human bronchi and an in vivo guinea pig model of chronic asthma. Our results demonstrated that PKCζ-specific inhibition produced a significant increase in isoproterenol sensitivity in TNFα-treated bronchi and ovalbumin (OVA)-sensitized guinea pig bronchi. The role of epoxy-eicosanoids, known to exert anti-inflammatory effects in lung, on PKCζ expression and activity in these models was evaluated. An enhanced PKCζ protein expression was delineated in TNFα-treated bronchi when compared with control (untreated) and epoxy-eicosanoid-treated bronchi. Measurements of Ca(2+) sensitivity, performed in TNFα-treated bronchi, demonstrated that treatment with myristoylated (Myr) PKCζ peptide inhibitor resulted in significant reductions of pCa-induced tension. Epoxy-eicosanoid treatments had similar effects on Ca(2+) sensitivity in TNFα-treated bronchi. In control and epoxy-eicosanoid-treated bronchi, the phosphorylated forms of p38MAPK and CPI-17 were significantly decreased compared with the TNFα-treated bronchi. An enhanced expression of PKCζ was ascertained in our in-vivo model of allergic asthma. Hence an increased Ca(2+) sensitivity could be explained by the phosphorylation of p38-MAPK, which in turn leads to phosphorylation and activation of the CPI-17 regulatory protein. This process was reversed upon treatment with the Myr-PKCζ-peptide inhibitor. The present data provide relevant evidence regarding the role of PKCζ in human and rodent models of airways inflammation.