Estudo dos mecanismos de ação do peptídeo Ac2-26 e da Piplartina nas células endoteliais de veias umbilicais humanas (HUVEC) ativadas pelo lipopolissacarídeo
Data
2018-06-21
Autores
Carvalho, Caroline de Freitas Zanon de
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os avanços recentes nos mecanismos de inflamação e a descoberta de vários mediadores endógenos anti-inflamatórios levaram a novas investigações sobre as possibilidades terapêuticas. A demonstração, em estudos científicos, da ação anti-inflamatória da proteína anexina A1 (ANXA1) e do seu peptídeo mimético Ac2-26, têm sido fonte de sucesso para o desenvolvimento de novos fármacos. A descoberta da interação biofísica da piplartina (PL) com a região N-terminal da ANXA1 abriu um novo e instigante campo de investigação para o nosso grupo de pesquisa. Diante destas considerações, o objetivo do presente trabalho foi investigar a atuação da PL nas células endoteliais da veia umbilical humana (HUVEC), ativadas pelo lipopolissacarídeo (LPS). Ainda, se a interação Ac2-26/PL influência nas ações anti-inflamatórias da PL, favorecendo ou atenuando os seus efeitos nos processos inflamatórios. Inicialmente foi utilizada a Espectroscopia de Absorbância UV-Vis e de Fluorescência para investigar as interações entre o ligante PL e Ac2-26. In vitro, as células HUVEC foram ativadas pelo LPS (10 μg/mL), nos tempos de 8, 24, 48 e 72 horas, seguido pelos tratamentos com Ac2-26 (1µM) e/ou PL (10µM), demonstrando resultados expressivos em 24 e 72 horas. Os efeitos foram avaliados nos seguintes aspectos: proliferação celular, viabilidade celular, dosagens da quimiocina MCP-1 e das citocinas IL-8 e IL-1β e expressão da proteína α-tubulina. A presença de um anel trimetoxiaromático na estrutura da PL, o que pode favorecer uma interação com a tubulina, motivou as análises de Western blotting, as quais demonstraram que a PL inibiu a síntese da proteína endógena α-tubulina, em todas as condições experimentais. Nossos resultados mostraram efeitos pró-proliferativos do peptídeo Ac2-26 e, por outro lado, antiproliferativos e pró-apoptóticos da PL. Os níveis das citocinas pró-inflamatórias confirmaram o papel anti-inflamatório do Ac2-26 nas células ativadas pelo LPS, com redução dos níveis de MCP-1 e IL-8, em 24 horas. O tratamento PL reduziu os níveis de MCP-1 nas células ativadas pelo LPS, e aumentou IL-8, inclusive no tratamento associado Ac2-26/PL. Em conjunto, os resultados com as células HUVEC indicam que a PL inibe a proliferação por meio da ativação da apoptose celular, regulada pela inibição da polimerização da α-tubulina e dos níveis elevados da citocina IL-8. A PL e o Ac2-26 mostraram ações anti-inflamatórias, e a interação Ac2-26/PL parece ser neutra em relação às propriedades anti-inflamatórias da PL.
Recent advances in inflammation mechanisms and the discovery of several endogenous anti-inflammatory mediators have led to further investigations into the therapeutic possibilities. The demonstration, in scientific studies, of the annexin A1 protein (ANXA1) anti-inflammatory action and its peptide mimetic Ac2-26, have been a source of success for the development of new drugs. The discovery of the biophysical interaction of piplartin (PL) with the N-terminal region of ANXA1 opened a new and exciting field of investigation for our research group. In view of these considerations, the objective of the present study was to investigate the role of PL in lipopolysaccharide (LPS) activated human umbilical vein endothelial cells (HUVEC). Also, if the interaction Ac2-26/PL influences the anti-inflammatory actions of PL, favoring or attenuating its effects on inflammatory processes. Initially, UV-Vis Absorbance Spectroscopy and Fluorescence were used to investigate the interactions between the PL ligand and Ac2-26. In vitro, HUVEC cells were activated by LPS (10 μg/mL) at 8, 24, 48 and 72 hours, followed by treatments with Ac2-26 (1μM) and/or PL (10μM), demonstrating expressive results in 24 and 72 hours. The effects were evaluated in the following aspects: cell proliferation, cell viability, MCP-1 chemokine and IL-8 and IL-1β cytokines and α-tubulin protein expression. The presence of a trimethoxyaromatic ring in the PL structure, which may favor an interaction with tubulin, motivated Western blotting analyzes, which demonstrated that PL inhibited endogenous α-tubulin protein synthesis in all experimental conditions. Our results showed pro-proliferative effects of the peptide Ac2-26 and, on the other hand, antiproliferative and pro-apoptotic of PL. Proinflammatory cytokine levels confirmed the anti-inflammatory role of Ac2-26 in LPS-activated cells, reducing MCP-1 and IL-8 levels within 24 hours. PL treatment reduced MCP-1 levels in LPSactivated cells, and increased IL-8, including in the treatment associated with Ac226/PL.Taken together, the results with HUVEC cells indicate that PL inhibits proliferation through the activation of cellular apoptosis, regulated by the inhibition of α-tubulin polymerization and elevated IL-8 cytokine levels. PL and Ac2-26 showed anti-inflammatory actions, and the Ac2-26/PL interaction appears to be neutral in relation to the anti-inflammatory properties of PL.
Recent advances in inflammation mechanisms and the discovery of several endogenous anti-inflammatory mediators have led to further investigations into the therapeutic possibilities. The demonstration, in scientific studies, of the annexin A1 protein (ANXA1) anti-inflammatory action and its peptide mimetic Ac2-26, have been a source of success for the development of new drugs. The discovery of the biophysical interaction of piplartin (PL) with the N-terminal region of ANXA1 opened a new and exciting field of investigation for our research group. In view of these considerations, the objective of the present study was to investigate the role of PL in lipopolysaccharide (LPS) activated human umbilical vein endothelial cells (HUVEC). Also, if the interaction Ac2-26/PL influences the anti-inflammatory actions of PL, favoring or attenuating its effects on inflammatory processes. Initially, UV-Vis Absorbance Spectroscopy and Fluorescence were used to investigate the interactions between the PL ligand and Ac2-26. In vitro, HUVEC cells were activated by LPS (10 μg/mL) at 8, 24, 48 and 72 hours, followed by treatments with Ac2-26 (1μM) and/or PL (10μM), demonstrating expressive results in 24 and 72 hours. The effects were evaluated in the following aspects: cell proliferation, cell viability, MCP-1 chemokine and IL-8 and IL-1β cytokines and α-tubulin protein expression. The presence of a trimethoxyaromatic ring in the PL structure, which may favor an interaction with tubulin, motivated Western blotting analyzes, which demonstrated that PL inhibited endogenous α-tubulin protein synthesis in all experimental conditions. Our results showed pro-proliferative effects of the peptide Ac2-26 and, on the other hand, antiproliferative and pro-apoptotic of PL. Proinflammatory cytokine levels confirmed the anti-inflammatory role of Ac2-26 in LPS-activated cells, reducing MCP-1 and IL-8 levels within 24 hours. PL treatment reduced MCP-1 levels in LPSactivated cells, and increased IL-8, including in the treatment associated with Ac226/PL.Taken together, the results with HUVEC cells indicate that PL inhibits proliferation through the activation of cellular apoptosis, regulated by the inhibition of α-tubulin polymerization and elevated IL-8 cytokine levels. PL and Ac2-26 showed anti-inflammatory actions, and the Ac2-26/PL interaction appears to be neutral in relation to the anti-inflammatory properties of PL.
Descrição
Palavras-chave
HUVEC, Anexina A1, Lipopolissacarídeo, Inflamação, Proliferação, Apoptose, Piplartina, Piperlongumina, Annexin A1, Lipopolysaccharide, Inflammation, Proliferation, Apoptosis, Piplartine, Piperlongumine