Efeitos da administração da proteína anexina a1 e do extrato da garcinia brasiliensis em modelo de doença pulmonar obstrutiva crônica induzida por exposição à fumaça de cigarro
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2021-03-05
Autores
Possebon, Lucas
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Universidade Estadual Paulista (Unesp)
Resumo
A proteína anti-inflamatória Anexina A1 (AnxA1) e o extrato da Garcinia brasiliensis têm mostrado resultados positivos em diferentes contextos de inflamações. Diante disso e da grande incidência da doença pulmonar obstrutiva crônica (DPOC), o objetivo desta investigação foi avaliar os efeitos da administração do peptídeo mimético da AnxA1, Ac2-26, e do extrato alcoólico da G. brasiliensis em modelo de DPOC. O extrato de G. brasiliensis foi submetido a análises fitoquímicas, microbiológicas e de citotoxicidade para escolha da concentração de uso adequada. As análises de padronização do extrato indicaram presença de tanino, glicosídeos flavanoídios e alcaloides e ausência de glicosídeos saponínicos. O extrato mostrou baixa toxicidade nas concentrações de 4%, 6% e 8% em testes de hemólise e in ovo. Ainda, indicou inibição de E. coli, Klebsiella, Proteus, Salmonella e Shigella. Para o desenvolvimento da DPOC, ratos (machos e fêmeas, Wistar) foram expostos à fumaça da queima de 10 cigarros comerciais, 2x/dia, por 8 semanas, em aparelho próprio para exposição ao fumo. Animais dos grupos tratados foram administrados via intraperitoneal com o Ac2-26 (1mg/Kg) e outros com solução de extrato da G. brasiliensis (4%), por gavagem, 3x/semana. Os grupos controles foram mantidos no mesmo regime, mas na ausência da fumaça do cigarro. Peso e pressão sanguínea arterial foram avaliados no início e no término do protocolo de exposição. Ao final do experimento, foram realizadas análises das imagens de raios X e estudos da ventilação pulmonar (pletismografia). Após eutanásia por dose excessiva de anestésico, sangue, lavado broncoalveolar (LBA), pulmões e traqueia foram coletados. As células inflamatórias do LBA foram quantificadas em câmara de Neubauer. As expressões das proteínas AnxA1, e ciclo-oxigenase-2 (COX-2), receptores de peptídeos formilados (FPR1 e FPR2) e metaloproteinases de matriz (MMP2 e MMP9) foram estudadas por imuno-histoquímica nos tecidos, enquanto os macrófagos foram quantificados por imunofluorescência. As expressões de AnxA1, também foram analisadas no sobrenadante do macerado pulmonar por Western blotting. A hemoglobina foi avaliada no sangue total e as citocinas interleucina (IL)-1β, IL-6, fator de necrose tumoral (TNF)-α e proteína quimioatractante para monócitos (MCP)-1 foram quantificadas no plasma sanguíneo e no sobrenadante pulmonar, pelo LUMINEX xMAP MAGPIX. Os ratos machos e fêmeas expostos ao fumo sem tratamento apresentaram reduções macroscópicas das dimensões pulmonares por imagens de raio-X, aumento de frequência, volume e ventilação pulmonares, bem como da concentração de hemoglobina comparados aos ratos tratados e controles. Nas análises do LBA, foi observada maior quantidade de linfócitos e macrófagos no grupo exposto à fumaça sem tratamento, comparado à redução significante dessas células nos grupos tratados. Nas avaliações morfológicas, maiores espaços intra-alveolares ocorreram no pulmão dos ratos expostos à fumaça do cigarro comparados aos grupos controle e tratados. Nos pulmões também foi observado aumento de macrófagos nos animais não tratados, comparados aos demais. Os estudos imuno-histoquímicos revelaram aumento significante da expressão das proteínas analisadas nos animais expostos sem tratamento, com redução de AnxA1 e MMP-2 após os tratamentos. Entretanto, nos grupos tratados com o peptídeo Ac2-26 ocorreu aumento de FPR1 e FPR2 comparados ao não tratados. Nas dosagens das citocinas, foram observados aumentos significantes de IL-1β, TNF-α e MCP-1 nos animais expostos à fumaça do cigarro, com redução após os tratamentos, bem como aumento de IL-10 nos animais que receberam tratamento com o peptídeo e extrato vegetal comparado aos animais não tratados. Nas fêmeas, de modo geral, o processo inflamatório foi mais exacerbado e a reversão dos danos menos pronunciada após os tratamentos. Associados, nossos dados mostram que a administração do peptídeo mimético Ac2- 26 da AnxA1 e do extrato vegetal da G. brasilisensis promoveu efeitos anti-inflamatórios protetores contra o desenvolvimento da DPOC no modelo proposto.
The anti-inflammatory protein Anexin A1 (AnxA1) and the extract of Garcinia brasiliensis have shown positive results in different contexts of inflammation. Given this and the high incidence of chronic obstructive pulmonary disease (COPD), the objective of this investigation was to evaluate the effects of administering the AnxA1 mimetic peptide, Ac2-26, and the alcoholic extract of the leaves of G. brasiliensis in a COPD model. The extract of G. brasiliensis was submitted to phytochemical, microbiological and cytotoxicity analyses to choose the appropriate concentration for use. The standardization analyses indicated the presence of tannins, flavanoid and alkaloids glycosides and absence of saponin glycosides. The extract showed low toxicity at concentrations of 4%, 6% and 8% in hemolysis and in ovo tests. Besides, it indicated inhibition of E. coli, Klebsiella, Proteus, Salmonella and Shigella. For the development of COPD, rats (male and female, Wistar) were exposed to smoke of 10 commercial cigarettes, 2x / day, for 8 weeks, in a device for smoke exposure. Animals in the treated groups were administered intraperitoneally with Ac2-26 (1mg / Kg) and others with solution of G. brasiliensis extract, by gavage, 3x / week. Control groups were maintained in the same regime, but in the absence of cigarette smoke. Arterial blood pressure and weight were assessed at the beginning and at the end of the exposure protocol. At the end of the experiment, analyses of X-ray images and studies of pulmonary ventilation (plethysmography) were performed. After euthanasia due to an overdose of anesthetic, blood, bronchoalveolar lavage (BAL), lungs and trachea were collected. Inflammatory BAL cells were quantified in a Neubauer chamber. The expressions of AnxA1, and cyclooxygenase-2 (COX-2), formylated peptide receptors- 1 and 2 (FPR1 and FPR2) and matrix metalloproteinase (MMP2 and MMP9) were studied by immuno -histochemistry in tissues, while macrophages were quantified by immunofluorescence. AnxA1expression was also analyzed in the pulmonary macerate supernatant by Western blotting. Hemoglobin was evaluated in whole blood and the cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and Monocyte Chemoattractant Protein (MCP)-1were quantified in blood plasma and pulmonary supernatant, by LUMINEX xMAP MAGPIX. Untreated-exposed-to-smoke rats showed macroscopic reductions in lung dimensions by X-ray images, increased pulmonary frequency, volume and ventilation, as well as hemoglobin concentration compared to treated and control rats. In the BAL analyses, a greater amount of lymphocytes and macrophages was observed in the untreated-exposed-to-smoke group, compared to the significant reduction of these cells in the treated groups. In morphological evaluations, larger intra-alveolar spaces occurred in the lungs of rats exposed to cigarette smoke compared to the control and treated groups. In lung tissues, an increase in macrophages was also observed in untreated animals, compared to the others. Immunohistochemical studies revealed a significant increase in the expression of the proteins analyzed in animals exposed to smoke without treatment, with a reduction in AnxA1 and MMPs after treatments. However, in the groups treated with the Ac2-26 peptide, there was an increase in FPR1 and FPR2 compared to untreated ones. In cytokine measurements, significant increases in cytokine IL-1β, TNF-α and MCP-1 were observed in animals exposed to cigarette smoke, with reduction after treatments, as well as an increase in IL-10 in animals that received treatment with peptides and plant extract compared to untreated animals. In females, in general, the inflammatory process was more exacerbated and the reversal of the damage was less pronounced after treatments. Taken together, our data showed that the administration of the AnxA1 mimetic peptide Ac2-26 and of the G. brasilisensis leaves extract promoted protective anti-inflammatory effects against the development of COPD in the proposed model.
The anti-inflammatory protein Anexin A1 (AnxA1) and the extract of Garcinia brasiliensis have shown positive results in different contexts of inflammation. Given this and the high incidence of chronic obstructive pulmonary disease (COPD), the objective of this investigation was to evaluate the effects of administering the AnxA1 mimetic peptide, Ac2-26, and the alcoholic extract of the leaves of G. brasiliensis in a COPD model. The extract of G. brasiliensis was submitted to phytochemical, microbiological and cytotoxicity analyses to choose the appropriate concentration for use. The standardization analyses indicated the presence of tannins, flavanoid and alkaloids glycosides and absence of saponin glycosides. The extract showed low toxicity at concentrations of 4%, 6% and 8% in hemolysis and in ovo tests. Besides, it indicated inhibition of E. coli, Klebsiella, Proteus, Salmonella and Shigella. For the development of COPD, rats (male and female, Wistar) were exposed to smoke of 10 commercial cigarettes, 2x / day, for 8 weeks, in a device for smoke exposure. Animals in the treated groups were administered intraperitoneally with Ac2-26 (1mg / Kg) and others with solution of G. brasiliensis extract, by gavage, 3x / week. Control groups were maintained in the same regime, but in the absence of cigarette smoke. Arterial blood pressure and weight were assessed at the beginning and at the end of the exposure protocol. At the end of the experiment, analyses of X-ray images and studies of pulmonary ventilation (plethysmography) were performed. After euthanasia due to an overdose of anesthetic, blood, bronchoalveolar lavage (BAL), lungs and trachea were collected. Inflammatory BAL cells were quantified in a Neubauer chamber. The expressions of AnxA1, and cyclooxygenase-2 (COX-2), formylated peptide receptors- 1 and 2 (FPR1 and FPR2) and matrix metalloproteinase (MMP2 and MMP9) were studied by immuno -histochemistry in tissues, while macrophages were quantified by immunofluorescence. AnxA1expression was also analyzed in the pulmonary macerate supernatant by Western blotting. Hemoglobin was evaluated in whole blood and the cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and Monocyte Chemoattractant Protein (MCP)-1were quantified in blood plasma and pulmonary supernatant, by LUMINEX xMAP MAGPIX. Untreated-exposed-to-smoke rats showed macroscopic reductions in lung dimensions by X-ray images, increased pulmonary frequency, volume and ventilation, as well as hemoglobin concentration compared to treated and control rats. In the BAL analyses, a greater amount of lymphocytes and macrophages was observed in the untreated-exposed-to-smoke group, compared to the significant reduction of these cells in the treated groups. In morphological evaluations, larger intra-alveolar spaces occurred in the lungs of rats exposed to cigarette smoke compared to the control and treated groups. In lung tissues, an increase in macrophages was also observed in untreated animals, compared to the others. Immunohistochemical studies revealed a significant increase in the expression of the proteins analyzed in animals exposed to smoke without treatment, with a reduction in AnxA1 and MMPs after treatments. However, in the groups treated with the Ac2-26 peptide, there was an increase in FPR1 and FPR2 compared to untreated ones. In cytokine measurements, significant increases in cytokine IL-1β, TNF-α and MCP-1 were observed in animals exposed to cigarette smoke, with reduction after treatments, as well as an increase in IL-10 in animals that received treatment with peptides and plant extract compared to untreated animals. In females, in general, the inflammatory process was more exacerbated and the reversal of the damage was less pronounced after treatments. Taken together, our data showed that the administration of the AnxA1 mimetic peptide Ac2-26 and of the G. brasilisensis leaves extract promoted protective anti-inflammatory effects against the development of COPD in the proposed model.
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Palavras-chave
Tabagismo, Anexina, Fitoterápico, Inflamação, DPOC, Smoking, Annexin, Phytotherapic, Inflammation, COPD