Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Manancial - Repositório Digital da UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/18790 |
Resumo: | The skin is the largest organ in the human body and acts as a barrier protecting the body from the environment. The rupture of its integrity caused by an injury culminates in the activation of a complex healing process, which seeks the closure of the injured area and involves three overlapping stages over time: inflammatory, proliferative and remodeling. Often, the healing process is not efficient, necessitating the use of drugs or herbal medicines. A medicinal plant that has important healing properties is the Stryphnodendron adstringens (Mart.) Coville, popularly known as barbatimão and native of the Brazilian Cerrado biome. However, it is still unclear how this plant acts on the steps of the healing process. Within this context, the present study aimed to evaluate the in vitro effect of Stryphnodendron adstringens on the modulation of inflammatory and proliferative markers of the wound healing process. For this, an independent bi-cellular experimental model was developed using the commercial cell lines of macrophages (RAW 264.7) and dermal fibroblasts (HFF-1). Cells were maintained in cell culture separately, under sterile conditions at 37°C and 5% CO2. Macrophages were activated with the mitogenic agent phytohemagglutinin (PHA) and, after 24 hours, were treated with different concentrations of the barbatimão bark extract (0.24; 0.49; 0.99; 1.99; 3.98 mg/mL). After 72 hours, cell proliferation was evaluated by the test of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolic bromide (MTT). With the result of this test, the concentrations of 0.49 and 0.99 mg/mL of the barbatimão extract were defined for use in the subsequent analyzes. It was then evaluated, after 72 hours of treatment with both concentrations, the effect of the plant on protein levels (via immunoassay) and levels of gene expression (via Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)) of the proinflammatory cytokines interleukin one beta (IL-1β), interleukin six (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-ү) and the anti-inflammatory cytokine interleukin ten (IL-10) in activated and non-activated macrophages, to investigate the inflammatory phase. An additional protocol, which mimics in vitro healing processes, was also used. For this, cultures of fibroblasts with 80% of monolayer confluence were scratched with the aid of a 200 μL tip to mimic an injury and subsequently these cells were treated with both concentrations of the barbatimão extract. Then, after 24 and 72 hours of treatment, the levels of fibroblast growth factor one (FGF-1) and keratinocyte growth factor (KGF) were analyzed in scratched and integral fibroblasts to evaluate the proliferative state. The results showed anti-inflammatory effect of barbatimão on the activated macrophages, through a decrease of the protein levels and the gene expression levels of the proinflammatory cytokines and increase of the levels of the anti-inflammatory cytokine IL-10. In addition, barbatimão also increased the levels of both growth factors in scratched and integral fibroblasts after 24 and 72 hours of treatment. Despite the methodological limitations related to the in vitro studies, the results indicate that barbatimão has an effective action in the inflammatory modulation of macrophages, as well as it is able to induce the increase of the levels of growth factors related to proliferative events of fibroblasts during the process of wound healing. |
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Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitroEffect of Stryphnodendron adstringens (Mart.) Coville in inflammatory and proliferative markers of wound healing: a bi-cellular model in vitroPeleMacrófagoFibroblastoPlanta medicinalSkinMacrophageFibroblastMedicinal plantCNPQ::CIENCIAS DA SAUDE::FARMACIAThe skin is the largest organ in the human body and acts as a barrier protecting the body from the environment. The rupture of its integrity caused by an injury culminates in the activation of a complex healing process, which seeks the closure of the injured area and involves three overlapping stages over time: inflammatory, proliferative and remodeling. Often, the healing process is not efficient, necessitating the use of drugs or herbal medicines. A medicinal plant that has important healing properties is the Stryphnodendron adstringens (Mart.) Coville, popularly known as barbatimão and native of the Brazilian Cerrado biome. However, it is still unclear how this plant acts on the steps of the healing process. Within this context, the present study aimed to evaluate the in vitro effect of Stryphnodendron adstringens on the modulation of inflammatory and proliferative markers of the wound healing process. For this, an independent bi-cellular experimental model was developed using the commercial cell lines of macrophages (RAW 264.7) and dermal fibroblasts (HFF-1). Cells were maintained in cell culture separately, under sterile conditions at 37°C and 5% CO2. Macrophages were activated with the mitogenic agent phytohemagglutinin (PHA) and, after 24 hours, were treated with different concentrations of the barbatimão bark extract (0.24; 0.49; 0.99; 1.99; 3.98 mg/mL). After 72 hours, cell proliferation was evaluated by the test of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolic bromide (MTT). With the result of this test, the concentrations of 0.49 and 0.99 mg/mL of the barbatimão extract were defined for use in the subsequent analyzes. It was then evaluated, after 72 hours of treatment with both concentrations, the effect of the plant on protein levels (via immunoassay) and levels of gene expression (via Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)) of the proinflammatory cytokines interleukin one beta (IL-1β), interleukin six (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-ү) and the anti-inflammatory cytokine interleukin ten (IL-10) in activated and non-activated macrophages, to investigate the inflammatory phase. An additional protocol, which mimics in vitro healing processes, was also used. For this, cultures of fibroblasts with 80% of monolayer confluence were scratched with the aid of a 200 μL tip to mimic an injury and subsequently these cells were treated with both concentrations of the barbatimão extract. Then, after 24 and 72 hours of treatment, the levels of fibroblast growth factor one (FGF-1) and keratinocyte growth factor (KGF) were analyzed in scratched and integral fibroblasts to evaluate the proliferative state. The results showed anti-inflammatory effect of barbatimão on the activated macrophages, through a decrease of the protein levels and the gene expression levels of the proinflammatory cytokines and increase of the levels of the anti-inflammatory cytokine IL-10. In addition, barbatimão also increased the levels of both growth factors in scratched and integral fibroblasts after 24 and 72 hours of treatment. Despite the methodological limitations related to the in vitro studies, the results indicate that barbatimão has an effective action in the inflammatory modulation of macrophages, as well as it is able to induce the increase of the levels of growth factors related to proliferative events of fibroblasts during the process of wound healing.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESA pele é o maior órgão do corpo humano e atua como uma barreira de proteção do corpo ao meio ambiente. O rompimento da sua integridade provocado por uma lesão culmina na ativação de um processo complexo de cicatrização, que busca o fechamento da área lesionada e envolve três etapas sobrepostas ao longo do tempo: inflamatória, proliferativa e de remodelação. Muitas vezes, o processo de cicatrização não é eficiente, necessitando fazer-se uso de fármacos ou fitoterápicos auxiliares. Uma planta medicinal que apresenta importante propriedade cicatrizante é o Stryphnodendron adstringens (Mart.) Coville, popularmente conhecido como barbatimão e nativo do bioma Cerrado brasileiro. No entanto, ainda não está claro de que maneira esta planta atua sobre as etapas do processo cicatricial. Dentro deste contexto, o presente estudo objetivou avaliar o efeito in vitro do Stryphnodendron adstringens na modulação de marcadores inflamatórios e proliferativos do processo de cicatrização de feridas. Para isso, foi desenvolvido um modelo experimental bi-celular independente utilizando as linhagens celulares comerciais de macrófagos (RAW 264.7) e fibroblastos dérmicos (HFF-1). As células foram mantidas em cultivo celular separadamente, sob condições de esterilidade a 37ºC e a 5% de CO2. Os macrófagos foram ativados com o agente mitogênico fitohemaglutinina (PHA) e, após 24 horas, foram tratados com diferentes concentrações do extrato da casca de barbatimão (0,24; 0,49; 0,99; 1,99; 3,98 mg/mL). Após 72 horas, foi avaliada a proliferação celular, através do teste do brometo de 3-[4,5-dimetiltiazol-2-il]-2,5-difeniltetrazólio (MTT). Com o resultado deste teste, foram definidas as concentrações de 0,49 e 0,99 mg/mL do extrato de barbatimão para serem utilizadas nas análises subsequentes. Avaliou-se, então, após 72 horas de tratamento com ambas as concentrações, o efeito da planta sobre os níveis proteicos (via imunoensaio) e os níveis de expressão gênica (via Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)) das citocinas pró-inflamatórias interleucina um beta (IL-1β), interleucina seis (IL-6), fator de necrose tumoral alfa (TNF-α), interferon gama (IFN-ү) e da citocina anti-inflamatória interleucina dez (IL-10) em macrófagos ativados e não ativados, para a investigação da fase inflamatória. Um protocolo adicional, que imita processos cicatriciais in vitro, também foi utilizado. Para isso, culturas de fibroblastos com 80% de confluência de monocamada foram rasgadas com o auxílio de uma ponteira de 200 μL, para mimetizar uma lesão e, posteriormente, estas células foram tratadas com ambas as concentrações do extrato de barbatimão. Então, após 24 e 72 horas de tratamento, foram analisados os níveis do fator de crescimento de fibroblastos um (FGF-1) e do fator de crescimento de queratinócitos (KGF) em fibroblastos rasgados e íntegros, para avaliar o estado proliferativo. Os resultados mostraram efeito anti-inflamatório do barbatimão sobre os macrófagos ativados, via diminuição dos níveis proteicos e dos níveis de expressão gênica das citocinas pró-inflamatórias e aumento dos níveis da citocina anti-inflamatória IL-10. Além disso, o barbatimão também aumentou os níveis de ambos os fatores de crescimento em fibroblastos rasgados e íntegros, após 24 e 72 horas de tratamento. Apesar das limitações metodológicas relacionadas aos estudos in vitro, os resultados indicam que o barbatimão possui uma ação efetiva na modulação inflamatória de macrófagos, assim como é capaz de induzir o aumento dos níveis dos fatores de crescimento relacionados a eventos proliferativos de fibroblastos durante o processo de cicatrização de feridas.Universidade Federal de Santa MariaBrasilFarmacologiaUFSMPrograma de Pós-Graduação em FarmacologiaCentro de Ciências da SaúdeCruz, Ivana Beatrice Mânica dahttp://lattes.cnpq.br/3426369324110716Pavanato, Maria Amáliahttp://lattes.cnpq.br/8701892865724171Wagner, Glauberhttp://lattes.cnpq.br/8417542717418294Teixeira, Cibele Ferreira2019-11-04T20:43:14Z2019-11-04T20:43:14Z2018-07-19info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://repositorio.ufsm.br/handle/1/18790porAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessreponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSM2019-11-05T06:01:38Zoai:repositorio.ufsm.br:1/18790Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufsm.br/ONGhttps://repositorio.ufsm.br/oai/requestatendimento.sib@ufsm.br||tedebc@gmail.comopendoar:2019-11-05T06:01:38Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.none.fl_str_mv |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro Effect of Stryphnodendron adstringens (Mart.) Coville in inflammatory and proliferative markers of wound healing: a bi-cellular model in vitro |
title |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro |
spellingShingle |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro Teixeira, Cibele Ferreira Pele Macrófago Fibroblasto Planta medicinal Skin Macrophage Fibroblast Medicinal plant CNPQ::CIENCIAS DA SAUDE::FARMACIA |
title_short |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro |
title_full |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro |
title_fullStr |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro |
title_full_unstemmed |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro |
title_sort |
Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro |
author |
Teixeira, Cibele Ferreira |
author_facet |
Teixeira, Cibele Ferreira |
author_role |
author |
dc.contributor.none.fl_str_mv |
Cruz, Ivana Beatrice Mânica da http://lattes.cnpq.br/3426369324110716 Pavanato, Maria Amália http://lattes.cnpq.br/8701892865724171 Wagner, Glauber http://lattes.cnpq.br/8417542717418294 |
dc.contributor.author.fl_str_mv |
Teixeira, Cibele Ferreira |
dc.subject.por.fl_str_mv |
Pele Macrófago Fibroblasto Planta medicinal Skin Macrophage Fibroblast Medicinal plant CNPQ::CIENCIAS DA SAUDE::FARMACIA |
topic |
Pele Macrófago Fibroblasto Planta medicinal Skin Macrophage Fibroblast Medicinal plant CNPQ::CIENCIAS DA SAUDE::FARMACIA |
description |
The skin is the largest organ in the human body and acts as a barrier protecting the body from the environment. The rupture of its integrity caused by an injury culminates in the activation of a complex healing process, which seeks the closure of the injured area and involves three overlapping stages over time: inflammatory, proliferative and remodeling. Often, the healing process is not efficient, necessitating the use of drugs or herbal medicines. A medicinal plant that has important healing properties is the Stryphnodendron adstringens (Mart.) Coville, popularly known as barbatimão and native of the Brazilian Cerrado biome. However, it is still unclear how this plant acts on the steps of the healing process. Within this context, the present study aimed to evaluate the in vitro effect of Stryphnodendron adstringens on the modulation of inflammatory and proliferative markers of the wound healing process. For this, an independent bi-cellular experimental model was developed using the commercial cell lines of macrophages (RAW 264.7) and dermal fibroblasts (HFF-1). Cells were maintained in cell culture separately, under sterile conditions at 37°C and 5% CO2. Macrophages were activated with the mitogenic agent phytohemagglutinin (PHA) and, after 24 hours, were treated with different concentrations of the barbatimão bark extract (0.24; 0.49; 0.99; 1.99; 3.98 mg/mL). After 72 hours, cell proliferation was evaluated by the test of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolic bromide (MTT). With the result of this test, the concentrations of 0.49 and 0.99 mg/mL of the barbatimão extract were defined for use in the subsequent analyzes. It was then evaluated, after 72 hours of treatment with both concentrations, the effect of the plant on protein levels (via immunoassay) and levels of gene expression (via Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)) of the proinflammatory cytokines interleukin one beta (IL-1β), interleukin six (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-ү) and the anti-inflammatory cytokine interleukin ten (IL-10) in activated and non-activated macrophages, to investigate the inflammatory phase. An additional protocol, which mimics in vitro healing processes, was also used. For this, cultures of fibroblasts with 80% of monolayer confluence were scratched with the aid of a 200 μL tip to mimic an injury and subsequently these cells were treated with both concentrations of the barbatimão extract. Then, after 24 and 72 hours of treatment, the levels of fibroblast growth factor one (FGF-1) and keratinocyte growth factor (KGF) were analyzed in scratched and integral fibroblasts to evaluate the proliferative state. The results showed anti-inflammatory effect of barbatimão on the activated macrophages, through a decrease of the protein levels and the gene expression levels of the proinflammatory cytokines and increase of the levels of the anti-inflammatory cytokine IL-10. In addition, barbatimão also increased the levels of both growth factors in scratched and integral fibroblasts after 24 and 72 hours of treatment. Despite the methodological limitations related to the in vitro studies, the results indicate that barbatimão has an effective action in the inflammatory modulation of macrophages, as well as it is able to induce the increase of the levels of growth factors related to proliferative events of fibroblasts during the process of wound healing. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-07-19 2019-11-04T20:43:14Z 2019-11-04T20:43:14Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/18790 |
url |
http://repositorio.ufsm.br/handle/1/18790 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Farmacologia UFSM Programa de Pós-Graduação em Farmacologia Centro de Ciências da Saúde |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Farmacologia UFSM Programa de Pós-Graduação em Farmacologia Centro de Ciências da Saúde |
dc.source.none.fl_str_mv |
reponame:Manancial - Repositório Digital da UFSM instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Manancial - Repositório Digital da UFSM |
collection |
Manancial - Repositório Digital da UFSM |
repository.name.fl_str_mv |
Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
atendimento.sib@ufsm.br||tedebc@gmail.com |
_version_ |
1805922016724779008 |