Utilização da microscopia confocal para determinação de superóxido mediante estímulo com luteolina em células endoteliais
Data
2019-11-01
Autores
Cruz, Yan Milen Coelho 
Orientadores
Fernandes, Liliam
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: O estresse oxidativo endotelial afeta todo o sistema circulatório, sendo
marcado por diversos agentes reativos denominados espécies reativas de oxigênio.
A luteolina é um flavonóide com efeito antioxidante, mas seus efeitos sobre as
células endoteliais são pouco conhecidos. O presente trabalho utilizou a sonda
dihidroetidina (DHE) em microscopia confocal para investigar a geração de
superóxido (O2•-) em células endoteliais de ratos. Foram analisados os efeitos de
luteolina isolada ou em associação à Angiotensina II (Ang II), um peptídeo
hipertensor relacionado ao estresse oxidativo vascular.
Métodos: Células endoteliais cultivadas da veia cava de rato foram previamente
plaqueadas em lamínulas, pré-tratadas com a sonda fluorescente DHE [10 μM] por
30 minutos e expostas à luteolina [10, 20 e 50 μM] por 10 minutos, em presença ou
ausência de Ang II [1μM]. Controles positivo e negativo foram feitos pelo tratamento
das células com meio de cultura contendo alto nível de glicose (HG, 25 mM) ou
tempol (300 μM), respectivamente. As células foram fixadas e as lamínulas foram
analisadas em um microscópio confocal. Os comprimentos de onda utilizados foram
490 nm excitação e 590 nm emissão, e as configurações do sistema de aquisição
de imagem se mantiveram padronizadas para todas as capturas. A intensidade da
fluorescência foi quantificada célula a célula por densitometria, empregando-se
ferramentas do software ImageJ para delimitação automática do contorno celular
individualmente.
Resultados: As imagens obtidas no microscópio confocal apresentaram alto grau
de qualidade e foram analisadas com sucesso. A identificação do estresse oxidativo
através da sonda DHE foi comprovada nas células incubadas com meio HG. Houve
redução significativa da geração de O2•- após os tratamentos com luteolina 10 μM,
20 μM e 50 μM isolada e em associação com Ang II 1 μM em comparação às células
não tratadas. (* P < .05)
Conclusão: O ensaio de microscopia confocal mostrou que a luteolina reduz a
geração de O2•- pelas células endoteliais em relação ao basal e na presença de Ang
II. Esses resultados sugerem que o efeito antioxidante da luteolina no endotélio deve
ser mais profundamente avaliado para melhor compreensão dos mecanismos de
interação com Ang II. Esses dados também mostram consistência na metodologia
empregada para utilização da sonda DHE como ferramenta para quantificação
relativa de O2•- em culturas celulares.
Introduction: Endothelial oxidative stress affects the entire circulatory system, being marked by several reactive agents called reactive oxygen species. Luteolin is a flavonoid with antioxidant effect, but its effects on endothelial cells are poorly known. The present work used the dihydroethidine probe (DHE) in confocal microscopy to investigate the generation of superoxide (O2•-) in rat endothelial cells. The effects of luteolin alone or in association with Angiotensin II (Ang II), a hypertensive peptide related to vascular oxidative stress, were analyzed. Methods: Cultured rat vena cava endothelial cells were previously plated on coverslips, pretreated with the fluorescent probe DHE [10 μM] for 30 minutes and exposed to luteolin [10, 20 and 50 μM] for 10 minutes in the presence or absence of Ang II [1μM]. Positive and negative controls were made by treating cells with culture medium containing high glucose (HG, 25 mM) or tempol (300 μM), respectively. Cells were fixed and coverslips were analyzed under a confocal microscope. The wavelengths used were 490 nm excitation and 590 nm emission, and the image acquisition system settings remained standard for all captures. The fluorescence intensity was quantified cell by cell by densitometry, using ImageJ software tools to automatically delimit the cell contour individually. Results: Confocal microscope images were of high quality and were successfully analyzed. The identification of oxidative stress through the DHE probe was proven in cells incubated with HG medium. There was a significant reduction in O2•- generation after treatments with luteolin 10 μM, 20 μM and 50 μM alone and in association with Ang II 1 μM in comparison with untreated cells. (* P <.05) Conclusion: The confocal microscopy assay showed that luteolin reduces the generation of O2•- by endothelial cells in relation to basal values and in the presence of Ang II. These results suggest that the antioxidant effect of luteolin on the endothelium must be further evaluated to better understand the mechanisms of interaction with Ang II. These data also show consistency in the methodology employed to use the DHE probe as a tool for relative quantification of O2•- in cell cultures.
Introduction: Endothelial oxidative stress affects the entire circulatory system, being marked by several reactive agents called reactive oxygen species. Luteolin is a flavonoid with antioxidant effect, but its effects on endothelial cells are poorly known. The present work used the dihydroethidine probe (DHE) in confocal microscopy to investigate the generation of superoxide (O2•-) in rat endothelial cells. The effects of luteolin alone or in association with Angiotensin II (Ang II), a hypertensive peptide related to vascular oxidative stress, were analyzed. Methods: Cultured rat vena cava endothelial cells were previously plated on coverslips, pretreated with the fluorescent probe DHE [10 μM] for 30 minutes and exposed to luteolin [10, 20 and 50 μM] for 10 minutes in the presence or absence of Ang II [1μM]. Positive and negative controls were made by treating cells with culture medium containing high glucose (HG, 25 mM) or tempol (300 μM), respectively. Cells were fixed and coverslips were analyzed under a confocal microscope. The wavelengths used were 490 nm excitation and 590 nm emission, and the image acquisition system settings remained standard for all captures. The fluorescence intensity was quantified cell by cell by densitometry, using ImageJ software tools to automatically delimit the cell contour individually. Results: Confocal microscope images were of high quality and were successfully analyzed. The identification of oxidative stress through the DHE probe was proven in cells incubated with HG medium. There was a significant reduction in O2•- generation after treatments with luteolin 10 μM, 20 μM and 50 μM alone and in association with Ang II 1 μM in comparison with untreated cells. (* P <.05) Conclusion: The confocal microscopy assay showed that luteolin reduces the generation of O2•- by endothelial cells in relation to basal values and in the presence of Ang II. These results suggest that the antioxidant effect of luteolin on the endothelium must be further evaluated to better understand the mechanisms of interaction with Ang II. These data also show consistency in the methodology employed to use the DHE probe as a tool for relative quantification of O2•- in cell cultures.