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Doctoral Thesis
DOI
https://doi.org/10.11606/T.10.2019.tde-28062019-163214
Document
Author
Full name
Mariá Munhoz Evangelinellis
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Castelucci, Patricia (President)
Kfoury Junior, José Roberto
Russo, Cristina Eusébio Mendes
Sá, Paulo Correia de
Silva, Marcos Vinicius da
Title in Portuguese
Estudo do antagonista do receptor P2X7, Brilliant Blue G, sobre os neurônios entéricos do colo distal de ratos submetidos à colite ulcerativa experimental
Keywords in Portuguese
Antagonista Brilliant Blue G
Colite ulcerativa
Plexo mioentérico
Receptor P2X7
Abstract in Portuguese
As doenças inflamatórias intestinais (DII) são doenças crônicas do trato gastrointestinal e incluem a colite ulcerativa e a Doença de Crohn. A colite ulcerativa caracteriza-se como uma inflamação limitada ao colo, normalmente iniciando no reto e espalhando de maneira contínua. A inflamação intestinal pode levar a alterações nos neurônios entéricos. Estudos em modelos animais têm demonstrado o efeito da inflamação intestinal sobre o sistema nervoso entérico, após a indução de colite com substâncias ácidas, como o 2,4,6 Trinitrobenzeno sulfônico (TNBS). Tem sido descrito a presença de receptores purinérgicos P2X1-P2X7 no sistema nervoso entérico. O receptor P2X7 participa na regulação de diversos processos, como a permeabilidade células, apoptose e liberação de citocinas. Este projeto visa avaliar o efeito do Brilliant Blue G (BBG), antagonista do receptor P2X7, no sistema nervoso entérico do colo distal de animais submetidos à colite ulcerativa. Foram analisados o colo distal de ratos Wistar submetidos a indução da colite ulcerativa pela administração TNBS no intestino grosso. O tratamento com BBG foi feito 1 hora após a indução da colite e por 5 dias consecutivos, os colos distais foram coletados 24 horas e 7 dias após a indução da colite. Os grupos estudados são grupo controle (não manipulado), grupo Sham (indução feita com veículo), grupos Colite 24h e 7 dias, e grupos BBG 24h e BBG 7 dias. Foi avaliado o código químico, a densidade neuronal e área dos neurônios entéricos imunorreativos a óxido nítrico sintase neuronal (NOS), colina acetil Transferase (ChAT) e o receptor P2X7. O material foi analisado através das técnicas de imunohistoquímica por dupla marcação quanto a imunorreatividade à P2X7 com a ChAT e a NOS. Foi feita análise histológica, utilizando a coloração de Hematoxilina-Eosina para avaliar a integridade da parede intestinal e a classificação da colite. Os resultados demonstraram diminuição dos neurônios positivos ao NOS, ao ChAT e ao receptor P2X7 na colite e recuperação na densidade neuronal nos grupos tratados com o BBG. A área do perfil neuronal demonstrou aumento da área do perfil nos grupos colites e recuperação nos grupos tratados com o BBG. Conclui-se que o BBG foi efetivo na colite ulcerativa experimental podendo ser o receptor P2X7 um alvo terapêutico.
Title in English
Study of the P2X7 receptor antagonist, Brilliant Blue G, on enteric neurons of the rats distal colon submitted to experimental ulcerative colitis
Keywords in English
Antagonist Blue Brilliant G
Myenteric plexus
P2X7 receptor
Ulcerative colitis.
Abstract in English
Inflammatory bowel diseases (IBD) are chronic diseases of the gastrointestinal tract that include ulcerative colitis and Crohn's disease. Ulcerative colitis is characterized as a limited inlammation of the colon, usually beginning in the rectum, spreading continuously. The intestinal inflammation, whether caused by infection or abnormal immune response may lead to structural changes in enteric neurons. Studies in animal models have demonstrated the effect of intestinal inflammation on enteric nervous system, following colitis induction with acidic substances such as 2,4,6-trinitrobenzene sulfonic (TNBS). It has been reported the presence of P2X1-P2X7 purinergic receptors in the enteric nervous system. The P2X7 receptor participates in the regulation of several processes such as cell permeability, apoptosis and cytokine release. A recent study has shown that ulcerative colitis reduces the number of P2X7 immunoreactive neurons in myenteric plexus. This project aims to evaluate the effect of Brilliant Blue G (BBG), a P2X7 receptor antagonist, in the enteric nervous system of the large intestine in animals with ulcerative colitis. The distal colon of Wistar rats was submitted to induction of ulcerative colitis by TNBS administration in the large intestine. Treatment with BBG was given 1 hour after induction of colitis and for 5 consecutive days, distal colons are collected 24 hours and 7 days after colitis induction. The animals was divided in control group (not manipulated), Sham 24 h and 7 days (induction was done with vehicle), Colitis 24h, BBG 24h, Colitis 7 days and BBG 7 days. The chemical code, neuronal density and area of enteric neurons reactive to neuronal nitric oxide synthase (NOS, choline acetyl transferase (ChAT) and the P2X7 receptor. The distal colon was analyzed through immunohistochemical techniques for double labelling as the immunoreactivity to P2X7, ChAT and NOS. Histological analysis using staining for Hematoxylin and Eosin was done to evaluate the integrity of the intestinal wall. The results showed a decrease in neurons positive to NOS, ChAT and P2X7 receptor in colitis groups and recovery in neuronal density in the groups treated with BBG. The neural profile area showed an increase in the profile area in the colitis groups and recovery in the groups treated with BBG. In conclusion, BBG was effective in experimental ulcerative colitis and the P2X7 receptor may be a therapeutic target.
 
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Publishing Date
2019-10-16
 
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