Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UFPB |
Texto Completo: | https://repositorio.ufpb.br/jspui/handle/123456789/22027 |
Resumo: | Inflammation is a physiological response of the body, essentially beneficial. However, when exacerbated, inflammation is involved in the pathogenesis of numerous diseases, and drugs with anti-inflammatory properties are routinely used in the clinic. Currently, anti-inflammatory drugs available on the market have numerous adverse effects. Thus, it is necessary to search for new therapeutic strategies with safer anti-inflammatory drugs. 2- (3-Hydroxy-2-oxoindolin-3-yl) acrylonitrile (ISACN) is a Morita-Baylis-Hillman’s adduct derived from isatin that has anti-tumor and antibacterial action in addition to low acute toxicity, low risk of genotoxicity and good theoretical bioavailability by the oral route. Therefore, the aim of this work was to investigate the anti-inflammatory effect of ISACN and to determine its mechanism of action. For that, we used zimosan-induced peritonitis, acute lung injury (ALI) induced by bacterial lipopolysaccharide (LPS) and the culture of murine peritoneal macrophages stimulated with zimosan or LPS as experimental models. Initially, female Swiss mice were treated intraperitoneally (i.p.) with different doses of ISACN (1.5 mg/kg; 6mg/kg and 24 mg/kg) and subjected to experimental peritonitis through the challenge (ip) with zimosan (2mg/mL). The treatment with ISACN reduced the migration of neutrophils and the production of cytokines IL-1β, IL-6 and TNF-α. Male Balb/c mice were treated with 24 mg/kg of ISACN (i.p.) and subjected to experimental ALI through intranasal challenge (i.n) with LPS (2.5 mg/kg). The treatment with ISACN reduced the migration of neutrophils, the formation of pulmonary edema and the production of cytokines IL-1β, IL-6 and TNF-α, and reversed the histopathological changes characteristic of ALI. For in vitro experiments, murine peritoneal macrophages were cultured in the presence of different concentrations of ISACN (20μM, 10μM, 5μM) and stimulated with zimosan (0.2 mg/mL) or with LPS (1 μg / mL). The concentration of 20 μM was shown to be cytotoxic while the concentrations of 10 μM and 5 μM did not promote considerable changes in the viability of macrophages. During the culture of zimosan-stimulated murine peritoneal macrophages, ISACN considerably reduced the production of cytokines IL-1β, IL-6 and TNF-α. In the culture of macrophages stimulated with LPS, ISACN was effective in reducing the production of nitric oxide, the inflammatory cytokines IL-1β, IL-6 and TNF-α and the anti-inflammatory cytokine IL-10. To explore the mechanism of action of ISACN, experiments were performed with flow cytometry in which the expression of the CD69 molecules, toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS) and the active form of the protein kinases activated by mitogen ERK 1/2, JNK1/2 and p38, in addition to experiments with quantitative polymerase chain reaction where the gene expression of and iNOS in macrophages stimulated with LPS were determined. The treatment of macrophages with ISACN (10μM) reduced the expression of all analyzed molecules and reduced the gene expression of CD69 and iNOS. Considering the results obtained, we can suggest that ISACN had an anti-inflammatory effect and that this effect is associated with negative regulation of the TLR4/MAPK-ERK-JNK-p38 pathway. |
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Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitroISACNInflamaçãoLPAMacrófagosLPSInflammationALIMacrophagesCNPQ::CIENCIAS BIOLOGICAS::FARMACOLOGIAInflammation is a physiological response of the body, essentially beneficial. However, when exacerbated, inflammation is involved in the pathogenesis of numerous diseases, and drugs with anti-inflammatory properties are routinely used in the clinic. Currently, anti-inflammatory drugs available on the market have numerous adverse effects. Thus, it is necessary to search for new therapeutic strategies with safer anti-inflammatory drugs. 2- (3-Hydroxy-2-oxoindolin-3-yl) acrylonitrile (ISACN) is a Morita-Baylis-Hillman’s adduct derived from isatin that has anti-tumor and antibacterial action in addition to low acute toxicity, low risk of genotoxicity and good theoretical bioavailability by the oral route. Therefore, the aim of this work was to investigate the anti-inflammatory effect of ISACN and to determine its mechanism of action. For that, we used zimosan-induced peritonitis, acute lung injury (ALI) induced by bacterial lipopolysaccharide (LPS) and the culture of murine peritoneal macrophages stimulated with zimosan or LPS as experimental models. Initially, female Swiss mice were treated intraperitoneally (i.p.) with different doses of ISACN (1.5 mg/kg; 6mg/kg and 24 mg/kg) and subjected to experimental peritonitis through the challenge (ip) with zimosan (2mg/mL). The treatment with ISACN reduced the migration of neutrophils and the production of cytokines IL-1β, IL-6 and TNF-α. Male Balb/c mice were treated with 24 mg/kg of ISACN (i.p.) and subjected to experimental ALI through intranasal challenge (i.n) with LPS (2.5 mg/kg). The treatment with ISACN reduced the migration of neutrophils, the formation of pulmonary edema and the production of cytokines IL-1β, IL-6 and TNF-α, and reversed the histopathological changes characteristic of ALI. For in vitro experiments, murine peritoneal macrophages were cultured in the presence of different concentrations of ISACN (20μM, 10μM, 5μM) and stimulated with zimosan (0.2 mg/mL) or with LPS (1 μg / mL). The concentration of 20 μM was shown to be cytotoxic while the concentrations of 10 μM and 5 μM did not promote considerable changes in the viability of macrophages. During the culture of zimosan-stimulated murine peritoneal macrophages, ISACN considerably reduced the production of cytokines IL-1β, IL-6 and TNF-α. In the culture of macrophages stimulated with LPS, ISACN was effective in reducing the production of nitric oxide, the inflammatory cytokines IL-1β, IL-6 and TNF-α and the anti-inflammatory cytokine IL-10. To explore the mechanism of action of ISACN, experiments were performed with flow cytometry in which the expression of the CD69 molecules, toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS) and the active form of the protein kinases activated by mitogen ERK 1/2, JNK1/2 and p38, in addition to experiments with quantitative polymerase chain reaction where the gene expression of and iNOS in macrophages stimulated with LPS were determined. The treatment of macrophages with ISACN (10μM) reduced the expression of all analyzed molecules and reduced the gene expression of CD69 and iNOS. Considering the results obtained, we can suggest that ISACN had an anti-inflammatory effect and that this effect is associated with negative regulation of the TLR4/MAPK-ERK-JNK-p38 pathway.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESA inflamação é uma resposta fisiológica do sistema imunológico, essencialmente benéfica. Entretanto, quando exacerbada, a inflamação está envolvida na patogênese de inúmeras doenças. Os anti-inflamatórios disponíveis no mercado apresentam inúmeros efeitos adversos quando utilizados a longo prazo. Dessa forma, faz-se necessária a busca por novas estratégias terapêuticas com fármacos anti-inflamatórios mais seguros. O 2-(3-Hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) é um aduto de Morita-Baylis-Hillman derivado da isatina que apresenta ação anti-tumoral e antibacteriana além de baixa toxicidade aguda, baixo risco de genotoxicidade e boa biodisponibilidade teórica por via oral. Diante disso, o objetivo desse trabalho foi investigar o efeito anti-inflamatório do ISACN e determinar o seu mecanismo de ação. Para isso utilizamos a peritonite induzida por zimosan, a lesão pulmonar aguda (LPA) induzida por lipopolissaacarídeo bacteriano (LPS) e a cultura de macrófagos peritoneais murinos estimulados com zimosan ou com LPS como modelos experimentais. Inicialmente, camundongos Swiss fêmeas foram tratados via intraperitoneal (i.p.) com diferentes doses de ISACN (1,5 mg/kg; 6mg/kg e 24 mg/kg) e submetidos a peritonite experimental através do desafio (i.p) com zimosan (2mg/mL). O tratamento com ISACN reduziu a migração de neutrófilos e a produção das citocinas IL-1β, IL-6 e TNF-α. Camundongos Balb/c machos foram tratados com 24 mg/kg de ISACN (i.p.) e submetidos a LPA experimental através do desafio intranasal (i.n) com LPS (2,5 mg/kg). O tratamento com ISACN reduziu a migração de neutrófilos, a formação de edema pulmonar e produção das citocinas IL-1β, IL-6 e TNF-α, e reverteu as alterações histopatológicas características da LPA. Para os experimentos in vitro, macrófagos peritoneais murinos foram cultivados na presença de diferentes concentrações de ISACN (20μM, 10μM, 5μM) e estimulados com zimosan (0,2 mg/mL) ou com LPS (1μg/mL). A concentração de 20μM se mostrou citotóxica enquanto as concentrações de 10 μM e 5 μM não promoveram alterações consideráveis na viabilidade dos macrófagos. Durante a cultura de macrófagos peritoneais murinos estimulados com zimosan, o ISACN reduziu consideravelmente a produção das citocinas IL-1β, IL-6 e TNF-α. Na cultura de macrófagos estimulados com LPS, o ISACN reduziu a produção de óxido nítrico, das citocinas inflamatórias IL-1β, IL-6, TNF-α e da citocina anti-inflamatória IL-10. Para explorar o mecanismo de ação do ISACN, foram realizados experimentos para determinar a expressão das moléculas CD69, receptor toll-like 4 (TLR4), óxido nítrico sintase induzível (iNOS) e da forma ativa das proteínas quinases ativadas por mitógeno ERK 1/2, JNK1/2 e p38 por citometria de fluxo, além de experimentos com reação em cadeia polimerase quantitativa onde foram determinados a expressão gênica do CD69 e da iNOS em macrófagos estimulados com LPS. O tratamento de macrófagos com ISACN (10μM) reduziu a expressão de todas as moléculas analisadas por citometria e reduziu a expressão gênica do CD69 e da iNOS. Considerando os resultados obtidos podemos sugerir que ISACN apresentou efeito anti-inflamatório e que esse efeito está associado a regulação negativa da via TLR4/MAPK-ERK-JNK-p38.Universidade Federal da ParaíbaBrasilFarmacologiaPrograma de Pós-Graduação em Produtos Naturais e Sintéticos BioativosUFPBMascarenhas, Sandra Rodrigueshttp://lattes.cnpq.br/4300081489772959Silva, Juliane Santos de França da2022-02-03T18:17:14Z2021-09-092022-02-03T18:17:14Z2021-08-17info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesishttps://repositorio.ufpb.br/jspui/handle/123456789/22027porhttp://creativecommons.org/licenses/by-nd/3.0/br/info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UFPBinstname:Universidade Federal da Paraíba (UFPB)instacron:UFPB2022-04-28T17:21:33Zoai:repositorio.ufpb.br:123456789/22027Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufpb.br/PUBhttp://tede.biblioteca.ufpb.br:8080/oai/requestdiretoria@ufpb.br|| diretoria@ufpb.bropendoar:2022-04-28T17:21:33Biblioteca Digital de Teses e Dissertações da UFPB - Universidade Federal da Paraíba (UFPB)false |
dc.title.none.fl_str_mv |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro |
title |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro |
spellingShingle |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro Silva, Juliane Santos de França da ISACN Inflamação LPA Macrófagos LPS Inflammation ALI Macrophages CNPQ::CIENCIAS BIOLOGICAS::FARMACOLOGIA |
title_short |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro |
title_full |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro |
title_fullStr |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro |
title_full_unstemmed |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro |
title_sort |
Efeito do aduto de Morita-Baylis-Hillman 2-(3-hidroxi-2-oxoindolin-3-il)acrilonitrila (ISACN) na inflamação aguda experimental: abordagens in vivo e in vitro |
author |
Silva, Juliane Santos de França da |
author_facet |
Silva, Juliane Santos de França da |
author_role |
author |
dc.contributor.none.fl_str_mv |
Mascarenhas, Sandra Rodrigues http://lattes.cnpq.br/4300081489772959 |
dc.contributor.author.fl_str_mv |
Silva, Juliane Santos de França da |
dc.subject.por.fl_str_mv |
ISACN Inflamação LPA Macrófagos LPS Inflammation ALI Macrophages CNPQ::CIENCIAS BIOLOGICAS::FARMACOLOGIA |
topic |
ISACN Inflamação LPA Macrófagos LPS Inflammation ALI Macrophages CNPQ::CIENCIAS BIOLOGICAS::FARMACOLOGIA |
description |
Inflammation is a physiological response of the body, essentially beneficial. However, when exacerbated, inflammation is involved in the pathogenesis of numerous diseases, and drugs with anti-inflammatory properties are routinely used in the clinic. Currently, anti-inflammatory drugs available on the market have numerous adverse effects. Thus, it is necessary to search for new therapeutic strategies with safer anti-inflammatory drugs. 2- (3-Hydroxy-2-oxoindolin-3-yl) acrylonitrile (ISACN) is a Morita-Baylis-Hillman’s adduct derived from isatin that has anti-tumor and antibacterial action in addition to low acute toxicity, low risk of genotoxicity and good theoretical bioavailability by the oral route. Therefore, the aim of this work was to investigate the anti-inflammatory effect of ISACN and to determine its mechanism of action. For that, we used zimosan-induced peritonitis, acute lung injury (ALI) induced by bacterial lipopolysaccharide (LPS) and the culture of murine peritoneal macrophages stimulated with zimosan or LPS as experimental models. Initially, female Swiss mice were treated intraperitoneally (i.p.) with different doses of ISACN (1.5 mg/kg; 6mg/kg and 24 mg/kg) and subjected to experimental peritonitis through the challenge (ip) with zimosan (2mg/mL). The treatment with ISACN reduced the migration of neutrophils and the production of cytokines IL-1β, IL-6 and TNF-α. Male Balb/c mice were treated with 24 mg/kg of ISACN (i.p.) and subjected to experimental ALI through intranasal challenge (i.n) with LPS (2.5 mg/kg). The treatment with ISACN reduced the migration of neutrophils, the formation of pulmonary edema and the production of cytokines IL-1β, IL-6 and TNF-α, and reversed the histopathological changes characteristic of ALI. For in vitro experiments, murine peritoneal macrophages were cultured in the presence of different concentrations of ISACN (20μM, 10μM, 5μM) and stimulated with zimosan (0.2 mg/mL) or with LPS (1 μg / mL). The concentration of 20 μM was shown to be cytotoxic while the concentrations of 10 μM and 5 μM did not promote considerable changes in the viability of macrophages. During the culture of zimosan-stimulated murine peritoneal macrophages, ISACN considerably reduced the production of cytokines IL-1β, IL-6 and TNF-α. In the culture of macrophages stimulated with LPS, ISACN was effective in reducing the production of nitric oxide, the inflammatory cytokines IL-1β, IL-6 and TNF-α and the anti-inflammatory cytokine IL-10. To explore the mechanism of action of ISACN, experiments were performed with flow cytometry in which the expression of the CD69 molecules, toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS) and the active form of the protein kinases activated by mitogen ERK 1/2, JNK1/2 and p38, in addition to experiments with quantitative polymerase chain reaction where the gene expression of and iNOS in macrophages stimulated with LPS were determined. The treatment of macrophages with ISACN (10μM) reduced the expression of all analyzed molecules and reduced the gene expression of CD69 and iNOS. Considering the results obtained, we can suggest that ISACN had an anti-inflammatory effect and that this effect is associated with negative regulation of the TLR4/MAPK-ERK-JNK-p38 pathway. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-09-09 2021-08-17 2022-02-03T18:17:14Z 2022-02-03T18:17:14Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufpb.br/jspui/handle/123456789/22027 |
url |
https://repositorio.ufpb.br/jspui/handle/123456789/22027 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
http://creativecommons.org/licenses/by-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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http://creativecommons.org/licenses/by-nd/3.0/br/ |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Universidade Federal da Paraíba Brasil Farmacologia Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos UFPB |
publisher.none.fl_str_mv |
Universidade Federal da Paraíba Brasil Farmacologia Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos UFPB |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da UFPB instname:Universidade Federal da Paraíba (UFPB) instacron:UFPB |
instname_str |
Universidade Federal da Paraíba (UFPB) |
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UFPB |
institution |
UFPB |
reponame_str |
Biblioteca Digital de Teses e Dissertações da UFPB |
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Biblioteca Digital de Teses e Dissertações da UFPB |
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Biblioteca Digital de Teses e Dissertações da UFPB - Universidade Federal da Paraíba (UFPB) |
repository.mail.fl_str_mv |
diretoria@ufpb.br|| diretoria@ufpb.br |
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1801842988215697408 |