Avaliação do efeito de fitocistatinas sobre a expresão génica de catepsinas e citocinas pró-inflamatórias em células raw 264.7 de camundongos e sobre a produção de tnf-a em ratos
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Data
2017-02-20
Autores
Da Ponte Leguizamón, Natalia [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
As cisteíno-peptidases e as citocinas desempenham um papel importante no inicio e progressão dos processos imuno-inflamatórios, dentre estes, a doença periodontal. As fitocistatinas são cistatinas derivadas das plantas, inibidoras naturais das cisteíno- peptidases. Entre elas a Citrus CPI-2, derivada da laranja e a Cane CPI-4, derivada da cana-de-açúcar tem sido recentemente investigadas. O estudo das mesmas pode levar a novas terapias para o controle da doença periodontal, bem como outras doenças imuno- inflamatórias. O objetivo do presente estudo foi avaliar o efeito inibitório das fitocistatinas (Cane CPI-4 e Citrus CPI-2) sobre mediadores da inflamação. Em estudo in vitro, foi avaliado o efeito da atividade inibitória das fitocistatinas sobre a expressão gênica (RT-PCR em tempo real) das catepsinas B e K e citocinas pró-inflamatórias (IL- 1β, IL-6 e TNF-α) em células de linhagem macrofágica de camundongos (RAW 264.7), após 12 e 24h de estímulo com Porphyromonas gingivalis inativada por calor. Em estudo in vivo, foi avaliada a ação de diferentes concentrações das fitocistatinas sobre a produção de TNF- a por células sanguíneas de ratos submetidos à injeção de LPS de E.coli . Todos os animais foram submetidos à implementação de cânulas na veia femoral para à injeção de LPS de E.coli e na artéria femoral para a extração de sangue em 6 diferentes tempos (de 30 a 180 minutos) após o estimulo com LPS de E.coli (IV, 100 Ug/Kg). A quantificação de TNF-α foi realizada por teste ELISA. Os resultados in vitro demonstraram que a Citrus CPI-2 mostrou uma redução significativa na expressão gênica de catepsinas e citocinas, a Cane CPI-4 não apresentou uma redução dos genes testados. In vivo, a Citrus CPI-2 administrada IP (0,8 mg/kg) mostrou uma inibição da produção de TNF-α por células sanguíneas de ratos após 60 minutos do estímulo inflamatório, enquanto a Cane CPI-4 não apresentou uma redução significativa na produção de TNF- α. Os resultados sugerem que a nova fitocistatina derivada da laranja tem potencial para aplicação terapêutica coadjuvante no tratamento da doença periodontal e deve ser investigada em estudos pré-clínicos.
The cysteine proteinases and the cytokines play an important role in the initiation and progression of immune-inflammatory processes such as periodontal disease. The phytocystatins are cystatins derived from plants and they are natural inhibitors of cysteine proteinases. Among them, the Citrus CPI-2 derived from orange and Cane CPI- 4 derived from sugar cane have been investigated. The study of them may lead to new therapies for the control of periodontal disease and other immune inflammatory diseases. The overall objective of this study was to evaluate the effect of phytocystatins CaneCPI- 4 and CitrusCPI-2 on mediators of inflammation. The aim of the in vitro study was to assess the inhibitory activity of phytocystatins on gene expression (RT-PCR) of cathepsins B and K and pro-inflammatory cytokines (IL- 1β and IL-6 and TNF-α) in murine macrophage cells (RAW 264.7) after 12 and 24 hours of stimulation with heatinactivated bacteria Porphyromonas gingivalis. The aim of the in vivo study was to assess the inhibitory activity of both phytocystatins in the production of TNF-α by blood cells of rats submitted by an injection of lipopolysaccharide (LPS) of E.coli. All animals were subjected to the implementation of cannulas to femoral artery and vein for blood collection at 6 different time points (from 30 to 180 minutes) after LPS stimulus (IV, 100 ug/kg). Quantification of TNF-α was performed by ELISA. According to the results of the in vitro study, Citrus CPI-2 showed a significant reduction on the gene expression of cathepsins and cytokines, the Cane CPI-4 did not show a significant reduction on the tested genes. In the in vivo study, CitrusCPI-2 administrated IP (0,8 mg/kg) inhibited the TNF-α production by blood cells of rat 60 minutes after the inflammatory stimulus, the Cane CPI-4 didnot show a significant reduction in TNF-α production. The results suggest that the novel phytocystatin orange-derived has potential for application as adjunctive therapy treatment of periondontal disease and should be investigated in future preclinical studies.
The cysteine proteinases and the cytokines play an important role in the initiation and progression of immune-inflammatory processes such as periodontal disease. The phytocystatins are cystatins derived from plants and they are natural inhibitors of cysteine proteinases. Among them, the Citrus CPI-2 derived from orange and Cane CPI- 4 derived from sugar cane have been investigated. The study of them may lead to new therapies for the control of periodontal disease and other immune inflammatory diseases. The overall objective of this study was to evaluate the effect of phytocystatins CaneCPI- 4 and CitrusCPI-2 on mediators of inflammation. The aim of the in vitro study was to assess the inhibitory activity of phytocystatins on gene expression (RT-PCR) of cathepsins B and K and pro-inflammatory cytokines (IL- 1β and IL-6 and TNF-α) in murine macrophage cells (RAW 264.7) after 12 and 24 hours of stimulation with heatinactivated bacteria Porphyromonas gingivalis. The aim of the in vivo study was to assess the inhibitory activity of both phytocystatins in the production of TNF-α by blood cells of rats submitted by an injection of lipopolysaccharide (LPS) of E.coli. All animals were subjected to the implementation of cannulas to femoral artery and vein for blood collection at 6 different time points (from 30 to 180 minutes) after LPS stimulus (IV, 100 ug/kg). Quantification of TNF-α was performed by ELISA. According to the results of the in vitro study, Citrus CPI-2 showed a significant reduction on the gene expression of cathepsins and cytokines, the Cane CPI-4 did not show a significant reduction on the tested genes. In the in vivo study, CitrusCPI-2 administrated IP (0,8 mg/kg) inhibited the TNF-α production by blood cells of rat 60 minutes after the inflammatory stimulus, the Cane CPI-4 didnot show a significant reduction in TNF-α production. The results suggest that the novel phytocystatin orange-derived has potential for application as adjunctive therapy treatment of periondontal disease and should be investigated in future preclinical studies.
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Palavras-chave
Citocinas, Catepsinas, Cistatinas, Cytokines, Cathepsins, Cystatins