Ações do extrato quimicamente padronizado de C. glaziovii Snethl. na inflamação intestinal induzida em camundongos: efeitos na neurotransmissão colinérgica
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Data
2016-12-31
Autores
Bechara, Fabiana Moreira Nogueira [UNIFESP]
Orientadores
Souccar, Caden [UNIFESP]
Tipo
Tese de doutorado
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A doença inflamatória intestinal (DII) é um quadro de inflamação crônica e recorrente do trato gastrointestinal (TGI) que envolve alterações da resposta imunológica, associada à uma suscetibilidade genética e fatores ambientais. A inflamação intestinal pode afetar todo o TGI (doença de Crohn), ou ser restrita à mucosa do cólon (retocolite ulcerativa), mas os dois quadros apresentam sinais clínicos comuns caracterizados por diarreia, dor abdominal, edema e ulcerações. Estudos anteriores do grupo mostraram que o extrato aquoso (EA) e a fração butanólica (Fbu) de Cecropia glaziovii Snethl. apresentam atividades hipotensora/anti-hipertensiva, antiácida/anti-úlcera, broncodilatadora e efeitos do tipo ansiolítico e antidepressivo. O objetivo do presente trabalho foi avaliar os efeitos do EA e da FBu obtidos de C. glaziovii Snethl. e padronizados quimicamente, em um modelo de inflamação intestinal aguda induzida por sulfato de dextrana sódico (SDS) em camundongos e suas consequências nas respostas farmacológicas produzidas por ativação muscarínica e nicotínica, em preparações de cólon isolado. A colite aguda foi induzida em camundongos C57Bl/6 machos (8 a 12 semanas) pelo fornecimento de SDS (2,5%) no bebedouro, por 5 dias consecutivos, e água por mais dois dias (grupo SDS). O grupo controle (C) recebeu água durante o mesmo período. A inflamação intestinal foi avaliada pelos sinais clínicos, por análises histológicas do cólon e determinações da atividade da mieloperoxidase (MPO). Os animais dos grupos C e SDS foram tratados com o EA (1 e 2 g/kg) ou com a FBu (0,1 e 0,3 g/kg), v.o., por 7 dias. O estabelecimento da colite no grupo SDS foi confirmado pela presença de diarreia, sangue nas fezes e redução dos pesos corporais dos animais de 16% do valor inicial. Os pesos e os comprimentos do cólon foram reduzidos de 20% e 26% dos respectivos valores controles, enquanto que a atividade da mieloperoxidase (MPO) tecidual aumentou 3,2 vezes em relação ao valor controle. No cólon do grupo SDS, as análises histológicas revelaram a presença de lesões da mucosa, infiltrado de células inflamatórias e edema na mucosa e na submucosa. O tratamento do grupo SDS com o EA ou a FBu atenuou os sinais clínicos e as alterações histopatológicas e reduziu a atividade da MPO a valores controles. A FBu apresentou maior eficácia nesses ensaios e foi utilizada nos estudos funcionais. Registros de contração isométrica e curvas concentração-efeito de acetilcolina (ACh, 10-9 a 10-2 M), ou de carbacol (CCh, 10-9 a 10-2 M) na presença de hexametônio (C6, 300 µM), foram obtidos em cólon isolado dos diferentes grupos animais. No cólon do grupo C, a contração máxima produzida pela ACh ou CCh foi maior 3 e 5,5 vezes maior, respectivamente, na região distal do que na proximal. Nas preparações de cólon distal do grupo SDS, o Emax da ACh e do CCh foram reduzidos de 19% e 47%, respectivamente, dos valores controles. Esses efeitos foram revertidos pelo tratamento com a FBu (0,3 g/kg). Os valores de CE50 da ACh e do CCh não foram afetados no grupo SDS ou após o tratamento do grupo SDS com a FBu (grupo SDS+FBu). No entanto, o tratamento de animais intactos com a FBu (grupo FBu) reduziu a CE50 da ACh em relação ao valor controle, indicando aumento da afinidade dos receptores pelo agonista. Este efeito não foi observado nas curvas concentração-efeito do CCh obtidas na presença do bloqueio ganglionar com C6. No cólon proximal, os valores de Emax ou CE50 dos dois agonistas não diferiram entre os quatro grupos experimentais. As curvas concentração-efeito de nicotina (0,3 -100 µM) obtidas em cólon isolado do grupo C mostraram relaxamento relacionado às concentrações do agonista. O relaxamento máximo produzido pela nicotina foi 5 vezes maior no cólon distal do que no cólon proximal. No cólon proximal, os efeitos da ativação nicotínica não diferiram entre os grupos C, SDS, FBu e SDS+FBu. No cólon distal, o efeito máximo produzido pela nicotina foi reduzido nos grupos SDS e FBu, observando-se somação dos efeitos no grupo SDS+FBu. Essas observações sugerem que o relaxamento produzido por nicotina no cólon do camundongo envolve a mediação de outros mecanismos além da liberação de NO. Os resultados mostraram que a FBu foi mais eficaz que o EA da planta em amenizar os efeitos macroscópicos e as alterações histológicas do cólon produzidos pela colite aguda por SDS. Além disso, o tratamento do grupo SDS com a FBu reverteu os efeitos da inflamação intestinal na contração máxima do cólon produzida por ativação muscarínica, mas não reverteu as alterações dos efeitos estimulados por ativação nicotínica. Em conjunto, os dados indicam que os efeitos da FBu na colite aguda parecem relacionados podem estar relacionados, em parte, às atividades anti-inflamatória e antioxidante atribuídas aos constituintes majoritários da FBu, os flavonoides e as procianidinas.
Inflammatory bowel diseases (IBD) are characterized by chronic and recurrent intestinal inflammation that result in altered immunological responses associated with genetic susceptibility and environmental conditions. The diseases may affect the entire gastrointestinal tract (Crohn’s disease - CD), or they may be restricted to the mucosal layer of the colon (ulcerative colitis – UC). However, both CD and UC present common clinical signs characterized by diarrhea, abdominal pain, edema and ulcerations. Previous studies from this laboratory have shown that a standardized aqueous extract (AE) and butanolic fraction (BuF) of Cecropia glaziovii Snethl are endowed with hypotensive/antihypertensive, bronchodilator, antacid/antiulcer, anxiolytic and antidepressant-like properties. Because of the antacid, antiulcer, anti-inflammatory and antioxidant activities attributed to the plant AE, this study was aimed to assess the effects of AE and BuF of C. glaziovii Snethl. on acute dextran sodium sulfate (DSS)-induced colitis in mice and its consequence on the cholinergic transmission of isolated colon preparations. The acute colitis was induced in male C57Bl/6 mice (8 to 12 weeks-old) by addition of 2.5% DSS in the drinking water for 5 days, followed by water for 2 more days (DSS group). The control group (C) was given filtered water during the same period. The intestinal inflammation was evaluated by clinical symptoms, histological analyzes and determination of the colon myeloperoxidase (MPO) activity. C and DSS animal groups were treated orally with AE (1 and 2 g/kg) or FBu (0,1 and 0,3 g/kg), for 7 days. Establishment of colitis in DSS group was confirmed by the presence of bloody diarrhea and decrease of their body weights by 16%. The colon weight and colon length were decreased by 20% and 26% of the respective control values, while the tissue MPO activity was increased by 3.2-fold. Histological analyzes of colon sections from DSS mice showed mucosal lesions, inflammatory cells infiltration and mucosal and submucosal edema. Treatment of DSS mice with AE or BuF attenuated the clinical symptoms and histopathological alterations, and reduced the MPO activity to control levels with the BuF being more active than AE. Concentration-response curves to acetylcholine (ACh, 10-9 to 10-2 M), or carbamylcholine (CCh, 10-9 to 10-2 M) in the presence of hexamethonium (C6, 300 µM) were obtained and isometric contractions of proximal and distal colon preparations from all four groups were recorded. In C group, the maximal contractions (Emax) produced by ACh and CCh were 3 and 5.5-fold greater in the distal than in the proximal colon. Emax or EC50 values of for ACh and CCh determined in the proximal colon did not differ among C, DSS, FBu and DSS+FBu groups. In distal colon of DSS-mice, Emax values of ACh and CCh were reduced 19 and 47%, respectively of control values. After treatment of DSS-mice with BuF (0,3 g/kg), the Emax and EC50 values of ACh and CCh did not differ from those determined C group. Concentration-response curves constructed to nicotine (0,3 -100 µM) in colon preparations from C group showed relaxation effect related to the agonist concentration. The maximum relaxation produced by nicotine xvi was greater by 5-fold in distal colon than the proximal colon. Nicotine-induced relaxation effects in the proximal colon did not differ among C, DSS, BuF or DSS+BuF groups. In distal colon, nicotine-induced maximal relaxation was reduced in both DSS and BuF groups, and both effects were summed up in DSS+BuF group. These observations indicate that nicotine-induced colon relaxation in mice is mediated by other mechanisms in addition to nitric oxide (NO) release. The results showed that BuF was more effective than AE in reducing the macroscopic effects and histopathological alterations of the colon induced by acute DSS-colitis. Treatment of DSS-mice with BuF resulted in the increase of the maximal contractile effects of muscarinic agonists on colon preparations to control values, while it did not influence the altered nicotine-induced muscle relaxation. Together, the data indicate that the effects of Buf on DSS-colitis appear to be related mainly to a local action exerted on the colon, possibly related to the anti-inflammatory and antioxidant activities of its major constituents, the flavonoids and procyanidins.
Inflammatory bowel diseases (IBD) are characterized by chronic and recurrent intestinal inflammation that result in altered immunological responses associated with genetic susceptibility and environmental conditions. The diseases may affect the entire gastrointestinal tract (Crohn’s disease - CD), or they may be restricted to the mucosal layer of the colon (ulcerative colitis – UC). However, both CD and UC present common clinical signs characterized by diarrhea, abdominal pain, edema and ulcerations. Previous studies from this laboratory have shown that a standardized aqueous extract (AE) and butanolic fraction (BuF) of Cecropia glaziovii Snethl are endowed with hypotensive/antihypertensive, bronchodilator, antacid/antiulcer, anxiolytic and antidepressant-like properties. Because of the antacid, antiulcer, anti-inflammatory and antioxidant activities attributed to the plant AE, this study was aimed to assess the effects of AE and BuF of C. glaziovii Snethl. on acute dextran sodium sulfate (DSS)-induced colitis in mice and its consequence on the cholinergic transmission of isolated colon preparations. The acute colitis was induced in male C57Bl/6 mice (8 to 12 weeks-old) by addition of 2.5% DSS in the drinking water for 5 days, followed by water for 2 more days (DSS group). The control group (C) was given filtered water during the same period. The intestinal inflammation was evaluated by clinical symptoms, histological analyzes and determination of the colon myeloperoxidase (MPO) activity. C and DSS animal groups were treated orally with AE (1 and 2 g/kg) or FBu (0,1 and 0,3 g/kg), for 7 days. Establishment of colitis in DSS group was confirmed by the presence of bloody diarrhea and decrease of their body weights by 16%. The colon weight and colon length were decreased by 20% and 26% of the respective control values, while the tissue MPO activity was increased by 3.2-fold. Histological analyzes of colon sections from DSS mice showed mucosal lesions, inflammatory cells infiltration and mucosal and submucosal edema. Treatment of DSS mice with AE or BuF attenuated the clinical symptoms and histopathological alterations, and reduced the MPO activity to control levels with the BuF being more active than AE. Concentration-response curves to acetylcholine (ACh, 10-9 to 10-2 M), or carbamylcholine (CCh, 10-9 to 10-2 M) in the presence of hexamethonium (C6, 300 µM) were obtained and isometric contractions of proximal and distal colon preparations from all four groups were recorded. In C group, the maximal contractions (Emax) produced by ACh and CCh were 3 and 5.5-fold greater in the distal than in the proximal colon. Emax or EC50 values of for ACh and CCh determined in the proximal colon did not differ among C, DSS, FBu and DSS+FBu groups. In distal colon of DSS-mice, Emax values of ACh and CCh were reduced 19 and 47%, respectively of control values. After treatment of DSS-mice with BuF (0,3 g/kg), the Emax and EC50 values of ACh and CCh did not differ from those determined C group. Concentration-response curves constructed to nicotine (0,3 -100 µM) in colon preparations from C group showed relaxation effect related to the agonist concentration. The maximum relaxation produced by nicotine xvi was greater by 5-fold in distal colon than the proximal colon. Nicotine-induced relaxation effects in the proximal colon did not differ among C, DSS, BuF or DSS+BuF groups. In distal colon, nicotine-induced maximal relaxation was reduced in both DSS and BuF groups, and both effects were summed up in DSS+BuF group. These observations indicate that nicotine-induced colon relaxation in mice is mediated by other mechanisms in addition to nitric oxide (NO) release. The results showed that BuF was more effective than AE in reducing the macroscopic effects and histopathological alterations of the colon induced by acute DSS-colitis. Treatment of DSS-mice with BuF resulted in the increase of the maximal contractile effects of muscarinic agonists on colon preparations to control values, while it did not influence the altered nicotine-induced muscle relaxation. Together, the data indicate that the effects of Buf on DSS-colitis appear to be related mainly to a local action exerted on the colon, possibly related to the anti-inflammatory and antioxidant activities of its major constituents, the flavonoids and procyanidins.
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BECHARA, Fabiana Moreira Nogueira. Ações do extrato quimicamente padronizado de C. glaziovii Snethl. na inflamação intestinal induzida em camundongos: efeitos na neurotransmissão colinérgica. 2016. Tese (Doutorado em Farmacologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2016.