Ação da piperlongumina em modelo de exposição pulmonar ao carcinógeno benzopireno
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Data
2020-03-05
Autores
Ashino, Tissiane Eid Barbosa
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Universidade Estadual Paulista (Unesp)
Resumo
A poluição do ar e o uso de cigarro são as principais fontes de inalação e exposição a agentes carcinógenos, como os hidrocarbonetos aromáticos policíclicos (HAP) resultantes da combustão incompleta de matéria orgânica e distribuídos amplamente no ambiente. O benzopireno é um dos principais HAP presentes no ambiente e com alta capacidade carcinogênica, sendo, portanto, usado em modelos experimentais in vivo. Investigações têm mostrado que os fármacos anti-inflamatórios podem reduzir a incidência de câncer de pulmão. Os compostos bioativos naturais (CBN) como os terpenos e flavonoides apresentam muitas propriedades, entre elas, redução da inflamação, estimulação do sistema imune e modulação de enzimas detoxificantes. Entre os CBNs destacamos a piperlongumina (PL), um alcaloide da Piper longum, que pode representar uma possibilidade de tratamento para o câncer de pulmão. Para o desenvolvimento do trabalho foram usados camundongos Balb/c divididos em 3 grupos (n=10/grupo): controle (sham), induzido por benzopireno (100 mg/kg, diluído em DMSO e administrado intraperitonealmente, i.p.) não tratado (BaP) e induzido por benzopireno e tratado com PL (2 mg/kg em DMSO a 10%, i.p.) a partir da oitava semana pós-indução (BaP/PL). Os animais foram pesados diariamente. A pletsmografia para verificação de volume, frequência e ventilação pulmonares foi realizada na última semana do protocolo (12ª semana). Após, os animais foram eutanasiados para coleta de sangue, lavado broncoalveolar e fragmentos dos pulmões. Os materiais foram processados para a quantificação de células inflamatórias no lavado broncoalveolar (LBA), genotoxicidade pelo teste do cometa em linfócitos e níveis de hemoglobina no sangue, análise histopatológica do pulmão, estudos imuno-histoquímicos da proteína anti-inflamatória anexina A1 (AnxA1), enzima pró-inflamatória ciclooxigenase 2 (COX-2), proteína anti-apoptótica Bcl-2 e fator de transcrição nuclear NF-kB, bem como dosagem das citocinas interleucina(IL)-1β, IL-17 e fator de necrose tumoral (TNF)-α. Os resultados mostraram que não houve diferença de peso entre os grupos. Os níveis de volume pulmonar, frequência respiratória e ventilação pulmonar foram elevados em animais BaP em comparação com Sham (p <0,001). Houve redução significativa desses parâmetros pulmonares foi observada em camundongos tratados com PL (p <0,001). Ocorreu aumento de linfócitos, monócitos e neutrófilos (p <0,001) no LBA do grupo BaP em comparação com o Sham, mas a PL reduziu o número dessas células (p <0,05). Da mesma forma, níveis mais altos de hemoglobina foram encontrados no grupo BaP (p <0,01) em comparação ao Sham, com redução nos animais BaP / PL (p <0,01). Ainda, maior dano ao DNA foi identificado no grupo BaP comparado ao sham. Houve redução significante desses danos no grupo tratado com a PL. O tratamento também preservou a arquitetura pulmonar em comparação com os animais não tratados. Além disso, expressões elevadas de AnxA1 (p <0,01), COX-2 (p <0,001), Bcl-2 (p <0,01) e NF-kB (p <0,001) foram observadas no grupo BaP em relação ao Sham. O efeito anti-inflamatório e protetor do PL foi evidenciado pela redução significativa de COX-2 (p <0,05), Bcl-2 (p <0,01) e NF-kB (p <0,001) e manutenção dos altos níveis de AnxA1. Ainda a administração da PL também reduziu os níveis de IL-1β, IL-17 e TNF-α próximos aos animais controle. Os resultados desse estudo mostram a potencialidade da PL para tratamento de condições anti-inflamatórias e antitumorais do pulmão, com implicações terapêuticas promissoras.
Air pollution and cigarette use are the main sources of inhalation and exposure to carcinogenic agents, such as polycyclic aromatic hydrocarbons (PAH) resulting from incomplete combustion of organic matter and widely distributed in the environment. Benzopyrene is one of the main PAHs present in the environment and has a high carcinogenic capacity, being therefore used in experimental models in vivo. Research has shown that anti-inflammatory drugs can reduce the incidence of lung cancer. Natural bioactive compound (NBC) such as terpenes and flavonoids have many properties, including reducing inflammation, stimulating the immune system and modulating detoxifying enzymes. Among the NBC we highlight piperlongumine (PL), an alkaloid of Piper longum, which may represent a possibility of treatment for lung cancer. For the development of the work, Balb / c mice divided into 3 groups (n = 10 / group) were used: control (sham), induced by benzopyrene (100 mg / kg, diluted in DMSO and administered intraperitoneally, ip) untreated (BaP ) and induced by benzopyrene and treated with PL (2 mg / kg in 10% DMSO, ip) from the eighth week post-induction (BaP / PL). The animals were weighed daily. Pletsmography to check lung volume, frequency and ventilation was performed in the last week of the protocol (12th week). Afterwards, the animals were euthanized for blood collection, bronchoalveolar lavage and fragments of the lungs. The materials were processed for the quantification of inflammatory cells in the bronchoalveolar lavage (BAL), genotoxicity by the comet test in lymphocytes and blood hemoglobin levels, histopathological analysis of the lung, immunohistochemical studies of the anti-inflammatory protein annexin A1 (AnxA1) , proinflammatory enzyme cyclooxygenase 2 (COX-2), anti-apoptotic protein Bcl-2 and nuclear transcription factor NF-kB, as well as measurement of cytokines interleukin (IL) -1β, IL-17 and tumor necrosis factor ( TNF) -α. Our results showed that there was no difference in weight between the groups. Levels of lung volume, respiratory rate and lung ventilation were high in BaP animals compared to Sham (p <0.001). There was a significant reduction in these pulmonary parameters in mice treated with PL (p <0.001). There were increases in lymphocytes, monocytes and neutrophils (p <0.001) in the BAL of the BaP group compared to the Sham. But PL reduced the number of these cells (p <0.05). Likewise, higher levels of hemoglobin were found in the BaP group (p <0.01) compared to Sham, with a reduction in the BaP / PL animals (p <0.01). In addition, greater damage to DNA was identified in the BaP group compared to sham. There was a significant reduction in these damages in the group treated with PL. The treatment also preserved the lung architecture compared to untreated animals. In addition, high expressions of AnxA1 (p <0.01), COX-2 (p <0.001), Bcl-2 (p <0.01) and NF-kB (p <0.001) were observed in the BaP group in relation to to Sham. The anti-inflammatory and protective effect of PL was evidenced by the significant reduction of COX-2 (p <0.05), Bcl-2 (p <0.01) and NF-kB (p <0.001) and maintenance of high levels of AnxA1. PL administration also reduced the levels of IL-1β, IL-17 and TNF-α close to the control animals. The results of this study show the potential of PL to treat anti-inflammatory and anti-tumor conditions of the lung, with promising therapeutic implications.
Air pollution and cigarette use are the main sources of inhalation and exposure to carcinogenic agents, such as polycyclic aromatic hydrocarbons (PAH) resulting from incomplete combustion of organic matter and widely distributed in the environment. Benzopyrene is one of the main PAHs present in the environment and has a high carcinogenic capacity, being therefore used in experimental models in vivo. Research has shown that anti-inflammatory drugs can reduce the incidence of lung cancer. Natural bioactive compound (NBC) such as terpenes and flavonoids have many properties, including reducing inflammation, stimulating the immune system and modulating detoxifying enzymes. Among the NBC we highlight piperlongumine (PL), an alkaloid of Piper longum, which may represent a possibility of treatment for lung cancer. For the development of the work, Balb / c mice divided into 3 groups (n = 10 / group) were used: control (sham), induced by benzopyrene (100 mg / kg, diluted in DMSO and administered intraperitoneally, ip) untreated (BaP ) and induced by benzopyrene and treated with PL (2 mg / kg in 10% DMSO, ip) from the eighth week post-induction (BaP / PL). The animals were weighed daily. Pletsmography to check lung volume, frequency and ventilation was performed in the last week of the protocol (12th week). Afterwards, the animals were euthanized for blood collection, bronchoalveolar lavage and fragments of the lungs. The materials were processed for the quantification of inflammatory cells in the bronchoalveolar lavage (BAL), genotoxicity by the comet test in lymphocytes and blood hemoglobin levels, histopathological analysis of the lung, immunohistochemical studies of the anti-inflammatory protein annexin A1 (AnxA1) , proinflammatory enzyme cyclooxygenase 2 (COX-2), anti-apoptotic protein Bcl-2 and nuclear transcription factor NF-kB, as well as measurement of cytokines interleukin (IL) -1β, IL-17 and tumor necrosis factor ( TNF) -α. Our results showed that there was no difference in weight between the groups. Levels of lung volume, respiratory rate and lung ventilation were high in BaP animals compared to Sham (p <0.001). There was a significant reduction in these pulmonary parameters in mice treated with PL (p <0.001). There were increases in lymphocytes, monocytes and neutrophils (p <0.001) in the BAL of the BaP group compared to the Sham. But PL reduced the number of these cells (p <0.05). Likewise, higher levels of hemoglobin were found in the BaP group (p <0.01) compared to Sham, with a reduction in the BaP / PL animals (p <0.01). In addition, greater damage to DNA was identified in the BaP group compared to sham. There was a significant reduction in these damages in the group treated with PL. The treatment also preserved the lung architecture compared to untreated animals. In addition, high expressions of AnxA1 (p <0.01), COX-2 (p <0.001), Bcl-2 (p <0.01) and NF-kB (p <0.001) were observed in the BaP group in relation to to Sham. The anti-inflammatory and protective effect of PL was evidenced by the significant reduction of COX-2 (p <0.05), Bcl-2 (p <0.01) and NF-kB (p <0.001) and maintenance of high levels of AnxA1. PL administration also reduced the levels of IL-1β, IL-17 and TNF-α close to the control animals. The results of this study show the potential of PL to treat anti-inflammatory and anti-tumor conditions of the lung, with promising therapeutic implications.
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Palavras-chave
Câncer de pulmão, Tabagismo, Compostos bioativos naturais, Lung cancer, Smoking, Natural bioactive compound