Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores

Detalhes bibliográficos
Autor(a) principal: Simão, Antonio Felipe Leite
Data de Publicação: 2010
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Institucional da Universidade Federal do Ceará (UFC)
dARK ID: ark:/83112/0013000024mkr
Texto Completo: http://www.repositorio.ufc.br/handle/riufc/2208
Resumo: Ischemic preconditioning (IPC) has been considered as a potent endogenous mechanism capable of inhibiting the inflammatory response. The migration of neutrophils (mn) is a central event in the development of inflammation. Our group has demonstrated that PCI inhibits mn in experimental models, however the mechanisms and mediators involved are not yet known. Aim: To study the involvement of mediators nitric oxide and carbon monoxide, adhesion proteins (ICAM-1 and β2-integrin) and expression of CXCR2 in the inhibitory effect of PCI on the distance min. Methods: The model of distance PCI was performed with a tourniquet in the right hind limb of mice for 10 minutes followed by 30 reperfusion. Involvement of NO and CO was investigated using inhibitors of iNOS (1400 W, 3 mg/kg or aminoguanidine (Amg), 50 mg/kg) and HO-1 (ZnPPIX, 10 mg/kg) as pretreatment 30 min. Later, peritonitis was induced by carrageenan (Cg) (500 mg/cav). Four hours later the peritoneal cavity (pc) was washed and leukocytes were counted. After that same procedure, the animals were subjected to intravital microscopy (IVM) to evaluate the effects of NO and CO in the mesenteric venules of 3rd order. Neutrophils from animals preconditioned and pre-treated or untreated, were used for testing chemotaxis in vitro, using the stimulus to chemokine KC (30ng/ml). The expression of GRK2 and CXCR2 in neutrophils was determined by flow cytometry and immunohistochemistry, respectively. The stakes and ICAM-1/CD54 β2-integrina/CD11b been investigated in knockout mice for genes of these molecules. The mn in these animals was evaluated according to protocol previously described by washed peritonela. In the investigation of the role of NO and CO in the modulation of cell adhesion protein (β2-integrin), we used iNOS inhibitor (1400W, 3 mg/kg or Amg, 50 mg/kg, sc), HO-1 (ZnPPIX, 10 mg/kg, sc) and guanylate cyclase (ODQ (5 µmol/kg, ip)) in pre-treatment 30 min before PCI. After peritonitis, blood was collected and the expression of CD11b on neutrophils was determined by flow cytometry. For statistical analysis, we used ANOVA/Bonferroni. P<0,05 was accepted. Results: The CO and NO inhibitors prevented the inhibitory effect of PCI on mn (P<0,05). Moreover, neutrophils from animals preconditioned showed reduced chemotaxis (p<0,05), a finding that correlated with decreased expression of CXCR2 in the membrane of neutrophils (p<0,05) and increased expression of GRK2. There was no change in chemotaxis or the expression of GRK2 when neutrophils were obtained from animals pre-conditioned and pretreated with inhibitors of CO, NO and sGC. Preconditioned animals showed a reduction in circulating neutrophils and the grip on IVM, which were prevented by pretreatment with inhibitors (P<0,05). The inhibitory effect of PCI was shown in knockout animals for β2-integrin, which was not observed in the knockout animals to ICAM-1. Moreover, neutrophils from animals preconditioned showed a significant reduction in the expression of CD11b, which was prevented in animals pretreated with inhibitors of iNOS, HO-1 and GCs. Conclusions: The results suggest that NO and CO act in the inhibitory effect of PCI via iNOS and HO-1 in place apart by modulating ICAM-1, β2-integrin and CXCR2 via GRK2.
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spelling Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadoresInhibitory effect of ischemic preconditioning distance on migration of neutrophils : mechanisms and mediatorsMonóxido de CarbonoNeutrófilosPrecondicionamento Isquêmico MiocárdicoInibição de Migração CelularMediadores da InflamaçãoMoléculas de Adesão CelularIschemic preconditioning (IPC) has been considered as a potent endogenous mechanism capable of inhibiting the inflammatory response. The migration of neutrophils (mn) is a central event in the development of inflammation. Our group has demonstrated that PCI inhibits mn in experimental models, however the mechanisms and mediators involved are not yet known. Aim: To study the involvement of mediators nitric oxide and carbon monoxide, adhesion proteins (ICAM-1 and β2-integrin) and expression of CXCR2 in the inhibitory effect of PCI on the distance min. Methods: The model of distance PCI was performed with a tourniquet in the right hind limb of mice for 10 minutes followed by 30 reperfusion. Involvement of NO and CO was investigated using inhibitors of iNOS (1400 W, 3 mg/kg or aminoguanidine (Amg), 50 mg/kg) and HO-1 (ZnPPIX, 10 mg/kg) as pretreatment 30 min. Later, peritonitis was induced by carrageenan (Cg) (500 mg/cav). Four hours later the peritoneal cavity (pc) was washed and leukocytes were counted. After that same procedure, the animals were subjected to intravital microscopy (IVM) to evaluate the effects of NO and CO in the mesenteric venules of 3rd order. Neutrophils from animals preconditioned and pre-treated or untreated, were used for testing chemotaxis in vitro, using the stimulus to chemokine KC (30ng/ml). The expression of GRK2 and CXCR2 in neutrophils was determined by flow cytometry and immunohistochemistry, respectively. The stakes and ICAM-1/CD54 β2-integrina/CD11b been investigated in knockout mice for genes of these molecules. The mn in these animals was evaluated according to protocol previously described by washed peritonela. In the investigation of the role of NO and CO in the modulation of cell adhesion protein (β2-integrin), we used iNOS inhibitor (1400W, 3 mg/kg or Amg, 50 mg/kg, sc), HO-1 (ZnPPIX, 10 mg/kg, sc) and guanylate cyclase (ODQ (5 µmol/kg, ip)) in pre-treatment 30 min before PCI. After peritonitis, blood was collected and the expression of CD11b on neutrophils was determined by flow cytometry. For statistical analysis, we used ANOVA/Bonferroni. P<0,05 was accepted. Results: The CO and NO inhibitors prevented the inhibitory effect of PCI on mn (P<0,05). Moreover, neutrophils from animals preconditioned showed reduced chemotaxis (p<0,05), a finding that correlated with decreased expression of CXCR2 in the membrane of neutrophils (p<0,05) and increased expression of GRK2. There was no change in chemotaxis or the expression of GRK2 when neutrophils were obtained from animals pre-conditioned and pretreated with inhibitors of CO, NO and sGC. Preconditioned animals showed a reduction in circulating neutrophils and the grip on IVM, which were prevented by pretreatment with inhibitors (P<0,05). The inhibitory effect of PCI was shown in knockout animals for β2-integrin, which was not observed in the knockout animals to ICAM-1. Moreover, neutrophils from animals preconditioned showed a significant reduction in the expression of CD11b, which was prevented in animals pretreated with inhibitors of iNOS, HO-1 and GCs. Conclusions: The results suggest that NO and CO act in the inhibitory effect of PCI via iNOS and HO-1 in place apart by modulating ICAM-1, β2-integrin and CXCR2 via GRK2.O pré-condicionamento isquêmico (PCI) vem sendo considerado como um potente mecanismo endógeno capaz de inibir a resposta inflamatória. A migração de neutrófilos (mn) é um evento central no desenvolvimento da reação inflamatória. Nosso grupo tem demonstrado que o PCI inibe a mn em modelos experimentais, entretanto os mecanismos e mediadores envolvidos ainda não são conhecidos.Objetivo:Estudar a participação dos mediadores Óxido nítrico e Monóxido de carbono, proteínas de adesão (ICAM-1 e β2-integrina) e da expressão de CXCR2 no efeito inibitório do PCI a distância sobre a mn. Métodos:O modelo de PCI a distância foi realizado com um torniquete no membro posterior direito de camundongos durante 10 minutos seguidos de 30 de reperfusão. Participação do NO e CO foi investigada através de inibidores de iNOS (1400 W, 3 mg/kg ou Aminoguanidina (Amg), 50 mg/kg) e HO-1 (ZnPPIX, 10 mg/kg) como pré-tratamento de 30 min. Posteriormente, induziu-se peritonite comCarragenina(Cg) (500 mg /cav). Quatro horas após, a cavidade peritoneal (cp) era lavada e leucócitos eram contados. Após aquele mesmo procedimento, os animais foram submetidos a microscopia intravital (miv) para avaliar os efeitos do NO e CO nas vênulas mesentéricas de 3ª ordem.Neutrófilos de animais pré-condicionados e pré-tratados ou não, foram utilizados para o ensaio de quimiotaxiain vitro, usandocomo estímulo a quimiocina KC(30ng/ml). Aexpressão de CXCR2 e GRK2 dos neutrófilos foi determinada por citometria de fluxo e imunofluorescência, respectivamente.Asparticipações de ICAM-1/CD54 e β2-integrina/CD11b foram investigadas em camundongos nocautes para os genes dessas moléculas. A mn nesses animais foi avaliada segundo protocolo já descrito pelo lavado peritonela.Na investigação do papel do NO e CO na modulação da proteína de adesão celular (β2-integrina), utilizou-se inibidores da iNOS (1400W, 3 mg/kg ou Amg, 50 mg/kg, sc), HO-1 (ZnPPIX, 10 mg/kg, sc) eGuanilato Ciclase (ODQ (5 µmol/kg, ip)) em pré-tratamento de 30 min antes do PCI. Após peritonite, o sangue foi colhido e a expressão de CD11bem neutrófilos foi determinada por citometria de fluxo. Para análise estatística, utilizou-se ANOVA/Bonferroni. P<0,05 foi aceito. Resultados:Os inibidores de CO e NO preveniram o efeito inibitório do PCIsobre a mn (p <0,05). Além disso, os neutrófilos de animais pré-condicionados apresentaram redução de quimiotaxia (p <0,05),achado que se correlacionou com a diminuição da expressãode CXCR2 na membrana dos neutrófilos (p <0,05) e comaumento da expressão de GRK2. Não houve alteração da quimiotaxia, nem da expressão de GRK2 quando os neutrófilos foram obtidos a partir de animais pré-condicionados e pré-tratados com inibidores de de CO, NO e GCs. Os animais pré-condicionados apresentaram redução nos neutrófilos circulantes e da aderênciana miv, as quais foram prevenidas pelo pré-tratamento com os inibidores (p <0,05). O efeito inibitório do PCI persistiu em animais nocautes para β2-integrina, o que não se observou nos animais nocautes para ICAM-1. Além disso, os neutrófilos de animais pré-condicionados apresentaram redução significativa na expressão de CD11b, que foi prevenida nos animais pré-tratados com os inibidores de iNOS, HO-1 e GCs.Conclusões: Os resultados sugerem que o NO e CO atuam no efeito inibitório do PCI via iNOS e HO-1 em sitio distante, modulando ICAM-1, β2-integrina e CXCR2 via GRK2.Ribeiro , Ronaldo de AlbuquerqueSimão, Antonio Felipe Leite2012-03-07T11:38:41Z2012-03-07T11:38:41Z2010info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfSIMÃO, A. F. L. Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos: mecanismos e mediadores. 2010. 140 f. Dissertação (Mestrado em Farmacologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2010.http://www.repositorio.ufc.br/handle/riufc/2208ark:/83112/0013000024mkrporreponame:Repositório Institucional da Universidade Federal do Ceará (UFC)instname:Universidade Federal do Ceará (UFC)instacron:UFCinfo:eu-repo/semantics/openAccess2021-07-15T12:22:11Zoai:repositorio.ufc.br:riufc/2208Repositório InstitucionalPUBhttp://www.repositorio.ufc.br/ri-oai/requestbu@ufc.br || repositorio@ufc.bropendoar:2024-09-11T19:00:07.088034Repositório Institucional da Universidade Federal do Ceará (UFC) - Universidade Federal do Ceará (UFC)false
dc.title.none.fl_str_mv Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
Inhibitory effect of ischemic preconditioning distance on migration of neutrophils : mechanisms and mediators
title Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
spellingShingle Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
Simão, Antonio Felipe Leite
Monóxido de Carbono
Neutrófilos
Precondicionamento Isquêmico Miocárdico
Inibição de Migração Celular
Mediadores da Inflamação
Moléculas de Adesão Celular
title_short Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
title_full Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
title_fullStr Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
title_full_unstemmed Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
title_sort Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos : mecanismos e mediadores
author Simão, Antonio Felipe Leite
author_facet Simão, Antonio Felipe Leite
author_role author
dc.contributor.none.fl_str_mv Ribeiro , Ronaldo de Albuquerque
dc.contributor.author.fl_str_mv Simão, Antonio Felipe Leite
dc.subject.por.fl_str_mv Monóxido de Carbono
Neutrófilos
Precondicionamento Isquêmico Miocárdico
Inibição de Migração Celular
Mediadores da Inflamação
Moléculas de Adesão Celular
topic Monóxido de Carbono
Neutrófilos
Precondicionamento Isquêmico Miocárdico
Inibição de Migração Celular
Mediadores da Inflamação
Moléculas de Adesão Celular
description Ischemic preconditioning (IPC) has been considered as a potent endogenous mechanism capable of inhibiting the inflammatory response. The migration of neutrophils (mn) is a central event in the development of inflammation. Our group has demonstrated that PCI inhibits mn in experimental models, however the mechanisms and mediators involved are not yet known. Aim: To study the involvement of mediators nitric oxide and carbon monoxide, adhesion proteins (ICAM-1 and β2-integrin) and expression of CXCR2 in the inhibitory effect of PCI on the distance min. Methods: The model of distance PCI was performed with a tourniquet in the right hind limb of mice for 10 minutes followed by 30 reperfusion. Involvement of NO and CO was investigated using inhibitors of iNOS (1400 W, 3 mg/kg or aminoguanidine (Amg), 50 mg/kg) and HO-1 (ZnPPIX, 10 mg/kg) as pretreatment 30 min. Later, peritonitis was induced by carrageenan (Cg) (500 mg/cav). Four hours later the peritoneal cavity (pc) was washed and leukocytes were counted. After that same procedure, the animals were subjected to intravital microscopy (IVM) to evaluate the effects of NO and CO in the mesenteric venules of 3rd order. Neutrophils from animals preconditioned and pre-treated or untreated, were used for testing chemotaxis in vitro, using the stimulus to chemokine KC (30ng/ml). The expression of GRK2 and CXCR2 in neutrophils was determined by flow cytometry and immunohistochemistry, respectively. The stakes and ICAM-1/CD54 β2-integrina/CD11b been investigated in knockout mice for genes of these molecules. The mn in these animals was evaluated according to protocol previously described by washed peritonela. In the investigation of the role of NO and CO in the modulation of cell adhesion protein (β2-integrin), we used iNOS inhibitor (1400W, 3 mg/kg or Amg, 50 mg/kg, sc), HO-1 (ZnPPIX, 10 mg/kg, sc) and guanylate cyclase (ODQ (5 µmol/kg, ip)) in pre-treatment 30 min before PCI. After peritonitis, blood was collected and the expression of CD11b on neutrophils was determined by flow cytometry. For statistical analysis, we used ANOVA/Bonferroni. P<0,05 was accepted. Results: The CO and NO inhibitors prevented the inhibitory effect of PCI on mn (P<0,05). Moreover, neutrophils from animals preconditioned showed reduced chemotaxis (p<0,05), a finding that correlated with decreased expression of CXCR2 in the membrane of neutrophils (p<0,05) and increased expression of GRK2. There was no change in chemotaxis or the expression of GRK2 when neutrophils were obtained from animals pre-conditioned and pretreated with inhibitors of CO, NO and sGC. Preconditioned animals showed a reduction in circulating neutrophils and the grip on IVM, which were prevented by pretreatment with inhibitors (P<0,05). The inhibitory effect of PCI was shown in knockout animals for β2-integrin, which was not observed in the knockout animals to ICAM-1. Moreover, neutrophils from animals preconditioned showed a significant reduction in the expression of CD11b, which was prevented in animals pretreated with inhibitors of iNOS, HO-1 and GCs. Conclusions: The results suggest that NO and CO act in the inhibitory effect of PCI via iNOS and HO-1 in place apart by modulating ICAM-1, β2-integrin and CXCR2 via GRK2.
publishDate 2010
dc.date.none.fl_str_mv 2010
2012-03-07T11:38:41Z
2012-03-07T11:38:41Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
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dc.identifier.uri.fl_str_mv SIMÃO, A. F. L. Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos: mecanismos e mediadores. 2010. 140 f. Dissertação (Mestrado em Farmacologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2010.
http://www.repositorio.ufc.br/handle/riufc/2208
dc.identifier.dark.fl_str_mv ark:/83112/0013000024mkr
identifier_str_mv SIMÃO, A. F. L. Efeito inibitório do pré-condicionamento isquêmico a distância sobre a migração de neutrófilos: mecanismos e mediadores. 2010. 140 f. Dissertação (Mestrado em Farmacologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2010.
ark:/83112/0013000024mkr
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