Avaliação dos mecanismos de ação dos compostos sakuranetina, eugenol e bis-eugenol na atenuação das alterações pulmonares em modelo de asma experimental
Data
2019-03-28
Autores
Santana, Fernanda Paula Roncon 
Orientadores
Prado, Carla Maximo
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Os avanços na fisiopatologia e a melhora do tratamento da asma ainda não foram suficientes para reduzir a alta prevalência, e principalmente a elevada morbidade e os altos custos gerados para o Sistema Único de Saúde. Muitos pacientes, em especial os que tem asma grave, não respondem ao tratamento convencional. Embora uma gama de novas substâncias venha sendo estudadas para o tratamento da asma, pouco se sabe sobre os exatos mecanismos de ação e possíveis efeitos adversos destes compostos na redução da inflamação pulmonar, o que é essencial para que possa ser um dia utilizado na clínica. Assim, os objetivos do presente estudo foram: 1. Avaliar os mecanismos de ação envolvidos no efeito anti-inflamatório da sakuranetina, isolada das partes aéreas de Baccharis retusa (Asteraceae) em modelo experimental de inflamação crônica alérgica de vias aéreas; 2. Avaliar os efeitos antiinflamatórios e os mecanismos de ação do eugenol e de seu dímero, bis-eugenol, em modelo experimental de inflamação crônica alérgica de vias aéreas. Para tanto, animais foram submetidos a exposições à ovoalbumina (OVA) ou salina intraperitoneal (dias 0 e 14) e inalatória (dias 22, 24, 26 e 28). O tratamento com sakuranetina, eugenol, bis-eugenol, dexametasona ou veículo foi administrado por 8 dias consecutivos a partir do 22o dia. No dia 28, os animais foram anestesiados, coletado o lavado broncoalveolar e o pulmão. Nos animais tratados com eugenol e deidrodieugenol foi avaliado a função pulmonar. Foram avaliadas, por ELISA e
Western Blotting, as citocinas e a vias da MAPK, da JAK2/STAT3/SOCS3 e o VAChT, um componente do sistema colinérgico. Possíveis alterações patológicas no fígado dos animais e os efeitos in vitro destes compostos em macrófagos RAW 264,7 também foram elucidados. A sakuranetina atenuou a inflamação pulmonar por inibição da fosforilação de STAT3, da MAPK e de IL-17, além de reduzir os níveis de VAChT no pulmão de animais sensibilizados. Em um segundo momento, observamos que o bis-eugenol apresentou um efeito melhor em relação ao eugenol e a dexametasona, reduzindo a resposta inflamatória, incluindo o recrutamento de neutrófilos a hiperresponsividade brônquica. Os efeitos destes dois compostos foram associados a inibição das vias da MAPK, de STAT3, SOCS3 e de VAChT. Não houve efeito tóxico no fígado in vivo, e em células RAW 264,7 in vitro. Ainda estes compostos inibiram a liberação de citocinas pró-inflamatórias e óxido nítrico produzidos pelas RAW 264,7 in vitro. Em conjunto, nossos dados sugerem que a sakuranetina e o bis-eugenol tem efeito anti-inflamatório, além do antioxidante já associado a eles. Esses compostos atuam na inibição de diferentes vias de sinalização celular, como MAPK e STAT3, envolvidas no controle da resposta inflamatória. Ainda, parecem ter um efeito importante na via Th17, que é um importante mecanismo envolvido na asma grave. Estes achados reforçam que estes compostos podem ser ferramentas terapêuticas importantes a serem utilizadas em pacientes com asma.
Advances in the pathophysiology and improvement of asthma treatment have not yet been sufficient to reduce the high prevalence, and especially the high morbidity and high costs generated for the Sistema Único de Saúde. Many patients, especially those with severe asthma, do not respond to conventional treatment. Although a range of new substances have been studied for the treatment of asthma, little is known about the exact mechanisms of action and possible adverse effects of these compounds in the reduction of pulmonary inflammation, which is essential for them to be used in the clinic. Thus, the aims of the present study were: 1. To evaluate the mechanisms of action involved in the anti-inflammatory effect of sakuranetin, isolated from the aerial parts of Baccharis retusa (Asteraceae), in an experimental model of chronic allergic airway inflammation; 2. To evaluate the anti-inflammatory effects and mechanisms of action of eugenol and its dimer, dehydrodieugenol, in an experimental model of chronic allergic airway inflammation. For this, animals were exposed to ovalbumin (OVA) or saline. Treatment with sakuranetin, eugenol, dehydrodieugenol, dexamethasone or vehicle was given for 8 consecutive days from the 22nd day. On day 28, the animals were anesthetized, the bronchoalveolar lavage and lung were collected. In the animals treated with eugenol and dehydrodieugenol, pulmonary function was evaluated. By ELISA and Western Blot, cytokines, and VAChT, a component of the cholinergic system, the MAPK pathway and the JAK2/STAT3/SOCS3 pathway were evaluated. Possible pathological changes in the liver of the animals and in vitro effects of these compounds on RAW 264,7 macrophages were also elucidated. Sakuranetin attenuated lung inflammation by inhibiting the phosphorylation of STAT3, MAPK and reducing IL-17 and VAChT levels in the lungs of sensitized animals. Then, we observed that dehydrodieugenol had a better effect than eugenol and dexamethasone, reducing the inflammatory response, including the recruitment of neutrophils and also bronchial hyperresponsiveness. The effects of these two compounds were associated with MAPK, STAT3 and SOCS3 and VAChT pathways inhibition. There was no toxic effect on the liver in vivo, and RAW 264,7 cells in vitro. These compounds also inhibited the release of proinflammatory cytokines and nitric oxide produced by these cells. In conclusion, taken together our data suggest that sakuranetin and dihydrodieugenol have an anti-inflammatory effect, in addition to the anti-oxidant effect already associated with them. They act in the inhibition of different cellular signaling pathways, such as MAPK and STAT3, involved in the control of the inflammatory response. Still, they appear to have a significant effect on the Th17 pathway, which is an important mechanism involved in severe asthma. These findings reinforce that these compounds may be important therapeutic tools to be used in patients with asthma.
Advances in the pathophysiology and improvement of asthma treatment have not yet been sufficient to reduce the high prevalence, and especially the high morbidity and high costs generated for the Sistema Único de Saúde. Many patients, especially those with severe asthma, do not respond to conventional treatment. Although a range of new substances have been studied for the treatment of asthma, little is known about the exact mechanisms of action and possible adverse effects of these compounds in the reduction of pulmonary inflammation, which is essential for them to be used in the clinic. Thus, the aims of the present study were: 1. To evaluate the mechanisms of action involved in the anti-inflammatory effect of sakuranetin, isolated from the aerial parts of Baccharis retusa (Asteraceae), in an experimental model of chronic allergic airway inflammation; 2. To evaluate the anti-inflammatory effects and mechanisms of action of eugenol and its dimer, dehydrodieugenol, in an experimental model of chronic allergic airway inflammation. For this, animals were exposed to ovalbumin (OVA) or saline. Treatment with sakuranetin, eugenol, dehydrodieugenol, dexamethasone or vehicle was given for 8 consecutive days from the 22nd day. On day 28, the animals were anesthetized, the bronchoalveolar lavage and lung were collected. In the animals treated with eugenol and dehydrodieugenol, pulmonary function was evaluated. By ELISA and Western Blot, cytokines, and VAChT, a component of the cholinergic system, the MAPK pathway and the JAK2/STAT3/SOCS3 pathway were evaluated. Possible pathological changes in the liver of the animals and in vitro effects of these compounds on RAW 264,7 macrophages were also elucidated. Sakuranetin attenuated lung inflammation by inhibiting the phosphorylation of STAT3, MAPK and reducing IL-17 and VAChT levels in the lungs of sensitized animals. Then, we observed that dehydrodieugenol had a better effect than eugenol and dexamethasone, reducing the inflammatory response, including the recruitment of neutrophils and also bronchial hyperresponsiveness. The effects of these two compounds were associated with MAPK, STAT3 and SOCS3 and VAChT pathways inhibition. There was no toxic effect on the liver in vivo, and RAW 264,7 cells in vitro. These compounds also inhibited the release of proinflammatory cytokines and nitric oxide produced by these cells. In conclusion, taken together our data suggest that sakuranetin and dihydrodieugenol have an anti-inflammatory effect, in addition to the anti-oxidant effect already associated with them. They act in the inhibition of different cellular signaling pathways, such as MAPK and STAT3, involved in the control of the inflammatory response. Still, they appear to have a significant effect on the Th17 pathway, which is an important mechanism involved in severe asthma. These findings reinforce that these compounds may be important therapeutic tools to be used in patients with asthma.