Avaliação da proteína anti-inflamatória anexina a1 em modelo de doença pulmonar obstrutiva crônica induzida por exposição à fumaça do cigarro
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Data
2017-02-20
Autores
Possebon, Lucas [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O processo inflamatório causado pelo tabagismo está relacionado a diferentes tipos de doenças como enfisema pulmonar, doença pulmonar obstrutiva crônica (DPOC) e aterosclerose. Neste cenário, a proteína anti-inflamatória AnxA1 pode representar uma alternativa terapêutica. Por essas razões, o objetivo da pesquisa foi avaliar os efeitos do peptídeo mimético Ac2-26 da proteína AnxA1, em modelo de tabagismo. Ratas Wistar foram divididas em 3 grupos (n=10/grupo): expostos ao fumo não tratados (F) e tratados com o peptídeo (F+Ac2-26) e controles (C). Os grupos de animais expostos ao fumo foram colocados em um equipamento específico da Unidade Didática e de Pesquisa Experimental (UDPE) das Faculdades Integradas Padre Albino (FIPA), e expostos à queima de 10 cigarros comerciais, um após o outro, 2x/dia, por 5 semanas. O grupo C foi mantido no mesmo regime, porém na ausência da fumaça do cigarro e tratamento. Para avaliar a eficácia do Ac2-26, animais F+Ac2-26 foram administrados intraperitonealmente com o peptídeo (1mg/kg), 1x/dia, antes da primeira exposição ao cigarro. As ratas foram pesadas e tiveram a pressão arterial aferida no início e final do experimento. No período final, a ventilação pulmonar foi verificada por meio da pletismografia e também foram realizadas imagens de raios-X. Os animais foram eutanasiados e coletados o lavado bronco alveolar (LBA) para quantificações de células inflamatórias e citocinas, o sangue para dosagens bioquímicas de citocinas e hemoglobina e os órgãos, pulmão e traqueia, para os estudos histopatológicos e imuno-histoquímicos. As análises fisiológicas mostraram perda de peso, aumento da pressão arterial, reduções da frequência e ventilação pulmonares, bem como alterações macroscópicas das dimensões pulmonares por imagens de raio-X no grupo F. Enquanto, nos F+Ac2-26, esses valores foram semelhantes aos controles. As análises histopatológicas mostraram maiores espaços intra-alveolares e aumento do tecido linfoide no pulmão e perda dos cílios no epitélio da traqueia no grupo F comparado as F+Ac2-26 e C. Nas análises do LBA, foi observado aumento na quantidade de linfócitos e macrófagos em F, com redução significante dessas células promovida após o tratamento. Nas quantificações de células inflamatórias nos tecidos, os macrófagos e mastócitos foram observados aumentados no grupo F comparado aos C e F+Ac2-26. As análises imuno-histoquímicas do pulmão e da traqueia mostraram menor expressão de AnxA1, COX-2 e MMP-9 nos animais C e F+Ac2-26. As dosagens de citocinas e quimiocina indicaram aumento no sobrenadante do macerado do pulmão, plasma sanguíneo e LBA no grupo F e redução nos níveis desses mediadores em C e F+Ac2-26. Ainda, as dosagens bioquímicas do sangue mostraram que o tratamento com o peptídeo ocasionou aumento da concentração de hemoglobina e glicose e redução do colesterol total e transaminase glutâmico oxalacética (TGO) comparados aos animais não tratados. Nossos resultados evidenciaram a ação protetora do peptídeo mimético Ac2-26 no modelo de DPOC, atenuando o processo inflamatório causado pela exposição à fumaça do cigarro e abre novas perspectivas para o tratamento das doenças relacionadas ao tabagismo.
The inflammatory process caused by smoking is related to different kinds of diseases such as pulmonary emphysema, chronic obstructive pulmonary disease (COPD) and atherosclerosis. In this scenario, an anti-inflammatory protein AnxA1 may represent a therapeutic alternative. For these reasons, the objective of this research was to analyze the effects of the mimetic peptide Ac2-26 of the AnxA1 protein, in a smoking model. Wistar rats were divided into 3 groups (n = 10/group). The groups of animals exposed to smoke were placed in a specific equipment of the Didactic and Experimental Research Unit (UDPE) of the Integrated Colleges Padre Albino (FIPA), and exposed to the burning of 10 cigarettes, one after another, 2x / day for 5 weeks. To evaluate the efficacy of Ac2-26, CS+Ac2-26 animals were administered intraperitoneally with peptide (1mg / kg), 1x / day, prior to first exposure to the cigarette. Group C was maintained in the same regimen, but in the absence of cigarette smoke or treatment. The rats were weighed and had blood pressure measured at the beginning and ending of the experiment. In the final period, pulmonary ventilation was verified through plethysmography and also performed ray-X images. The animals were euthanized and collected the alveolar bronchus (BAL) for quantification of inflammatory cells and cytokines, the blood for biochemical measurements of cytokines and hemoglobin and organs, lung and trachea, for histopathological and immunohistochemical studies. The physiological analyzes showed weight loss, increased blood pressure, reductions and in the pulmonary frequency and ventilation, as well as macroscopic alternative in the lung dimensions by X-ray images in group CS. While, in CS+Ac2-26, these values were similar to controls. The histopathological analyzes showed enlargement of the intra-alveolar spaces and increased lymphoid tissue (BALT), and loss of the cilia in the epithelium of the trachea in the CS group, compared to CS+Ac2-26 and C. Numerous lymphocytes and macrophages were observed in the BAL in CS, with significant reduction of the cells after treatment. In the quantifications of inflammatory cells in the tissues, macrophages and mast cells were increased in the CS+Ac2-26 group. The immunohistochemical analyzes of the lung and trachea showed lower expression of AnxA1, COX-2 and MMP-9 in C and CS+Ac2-26 animals. The dosages of cytokines and chemokine indicated incr ease in the supernatant of lung macerate, blood plasma and BAL in the F group and reduction of these mediators levels in C and CS+Ac2-26 groups. Also, the biochemical blood measurements showed that treatment with the peptide caused an increase in hemoglobin and glucose concentrations and reduction of total cholesterol and Glutamic oxaloacetic transaminase (GOT) compared to untreated animals. Our results evidenced a protective action of the Ac2-26 mimetic peptide in the COPD model, by attenuating the inflammatory process caused by exposure to cigarette smoke, which opens new perspectives for the treatment of smoking-related diseases.
The inflammatory process caused by smoking is related to different kinds of diseases such as pulmonary emphysema, chronic obstructive pulmonary disease (COPD) and atherosclerosis. In this scenario, an anti-inflammatory protein AnxA1 may represent a therapeutic alternative. For these reasons, the objective of this research was to analyze the effects of the mimetic peptide Ac2-26 of the AnxA1 protein, in a smoking model. Wistar rats were divided into 3 groups (n = 10/group). The groups of animals exposed to smoke were placed in a specific equipment of the Didactic and Experimental Research Unit (UDPE) of the Integrated Colleges Padre Albino (FIPA), and exposed to the burning of 10 cigarettes, one after another, 2x / day for 5 weeks. To evaluate the efficacy of Ac2-26, CS+Ac2-26 animals were administered intraperitoneally with peptide (1mg / kg), 1x / day, prior to first exposure to the cigarette. Group C was maintained in the same regimen, but in the absence of cigarette smoke or treatment. The rats were weighed and had blood pressure measured at the beginning and ending of the experiment. In the final period, pulmonary ventilation was verified through plethysmography and also performed ray-X images. The animals were euthanized and collected the alveolar bronchus (BAL) for quantification of inflammatory cells and cytokines, the blood for biochemical measurements of cytokines and hemoglobin and organs, lung and trachea, for histopathological and immunohistochemical studies. The physiological analyzes showed weight loss, increased blood pressure, reductions and in the pulmonary frequency and ventilation, as well as macroscopic alternative in the lung dimensions by X-ray images in group CS. While, in CS+Ac2-26, these values were similar to controls. The histopathological analyzes showed enlargement of the intra-alveolar spaces and increased lymphoid tissue (BALT), and loss of the cilia in the epithelium of the trachea in the CS group, compared to CS+Ac2-26 and C. Numerous lymphocytes and macrophages were observed in the BAL in CS, with significant reduction of the cells after treatment. In the quantifications of inflammatory cells in the tissues, macrophages and mast cells were increased in the CS+Ac2-26 group. The immunohistochemical analyzes of the lung and trachea showed lower expression of AnxA1, COX-2 and MMP-9 in C and CS+Ac2-26 animals. The dosages of cytokines and chemokine indicated incr ease in the supernatant of lung macerate, blood plasma and BAL in the F group and reduction of these mediators levels in C and CS+Ac2-26 groups. Also, the biochemical blood measurements showed that treatment with the peptide caused an increase in hemoglobin and glucose concentrations and reduction of total cholesterol and Glutamic oxaloacetic transaminase (GOT) compared to untreated animals. Our results evidenced a protective action of the Ac2-26 mimetic peptide in the COPD model, by attenuating the inflammatory process caused by exposure to cigarette smoke, which opens new perspectives for the treatment of smoking-related diseases.
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Palavras-chave
Tabagismo, AnxA1, LBA, Smoking, BAL