Repercussão do diabete moderado no estresse oxidativo e hormônios pancreáticos ao longo da vida de ratas
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Data
2018-03-01
Autores
Souza, Franciane Quintanilha Gallego
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Universidade Estadual Paulista (Unesp)
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Introdução: Durante o período neonatal de ratos, o pâncreas endócrino sofre remodelamento, sendo considerado uma janela crítica de desenvolvimento, que determina a fisiologia do pâncreas no organismo e possíveis doenças na vida adulta, dentre elas o diabete. Na gravidez, o pâncreas sofre adaptações. Por isso, é relevante entender os mecanismos envolvidos nas adaptações das ilhotas pancreáticas frente à associação gravidez e diabete, a qual pode estar relacionada ao quadro de estresse oxidativo e alterações no desempenho reprodutivo materno. Objetivo: Analisar a morfologia das ilhotas pancreáticas e o quadro de estresse oxidativo durante a janela crítica de desenvolvimento pós-natal e na prenhez de ratas com diabete. Materiais e método: Para a indução do diabete, os recém-nascidos fêmeas receberam droga beta (β)-citotóxica (streptozotocin – STZ, 100 mg/kg de peso corporal via subcutânea) no primeiro dia de vida. As fêmeas controle receberam apenas o tampão de citrato. O critério de inclusão para o grupo diabético foi glicemia ≥ 400 mg/dL no dia 5 de vida (D5) e, para o grupo controle, glicemia < 120mg/dL em D5. Os animais de ambos os grupos foram mortos em momentos diferentes: D5, D10, D15, D30 e no dia 18 de prenhez. Foi realizado a coleta de sangue (dosagem sérica de insulina e glucagon) e do pâncreas (análise morfológica). Os anticorpos utilizados para análise imunoistoquímica foram: anti-Pdx-1, anti-Ki-67, anti-insulina, anti-glucagon, anti-somatostatina, anti-superóxido dismutase 1, anti-glutationa peroxidase, anti-malondialdeído. O útero gravídico foi pesado e dissecado para análise das porcentagens de perdas embrionárias e fetais. Para todas as comparações foi considerado o limite mínimo de significância de p<0,05. Resultados: Em todos os momentos estudados (D5, D10, D15, D30 e prenhez) houve diminuição da porcentagem de células imunomarcadas para insulina e aumento da porcentagem de somatostatina e glucagon nas ilhotas pancreáticas das ratas diabéticas. O grupo diabético apresentou menor intensidade de marcação para SOD1 no D5, D10, D15 e D30, menor intensidade de marcação para GSH-Px no D10, D30 e na prenhez, aumento da intensidade da marcação de MDA na prenhez, menor porcentagem de células imunomarcadas para Pdx-1 e maior para Ki-67 quando comparadas às das ratas controle. Houve aumento da porcentagem de perdas pré e pós-implantação nas ratas prenhes com diabete moderado. Conclusão: A hiperglicemia afetou a composição celular das ilhotas pancreáticas durante toda vida, incluindo a prenhez, contribuindo para a diminuição da intensidade das enzimas antioxidantes e aumento do marcador de lipoperoxidação nos animais diabéticos, comprovando o quadro de estresse oxidativo pancreático. Isso influenciou negativamente a ativação do Pdx-1, diminuindo o estímulo para a síntese de insulina, levando a alterações glicêmicas mediante a um estímulo maior de glicose. Este desbalanço na homeostase da glicose sobrecarrega o pâncreas e levando ao aparecimento do diabete. Sendo assim, estas alterações morfológicas pancreáticas e o quadro anormal de estresse oxidativo decorrentes do diabete durante a janela crítica do desenvolvimento e na prenhez podem explicar as alterações existentes para o aparecimento de doenças na vida adulta. Desta forma, mais estudos precisam ser realizados visando medidas terapêuticas que possam prevenir ou minimizar as repercussões diabéticas a longo prazo.
Introduction: During the neonatal period of rats, the endocrine pancreas undergoes remodeling, being considered a critical window of development, which determines the physiology of the pancreas in the body and possible diseases in adult life, among them diabetes. In pregnancy, the pancreas undergoes adaptations. Therefore, it is relevant to understand the mechanisms involved in the adaptation of pancreatic islets to the pregnancy and diabetes association, which may be related to oxidative stress and changes in maternal reproductive performance. Objective: To analyze the morphology of pancreatic islets and oxidative stress during the critical window of postnatal development and in the pregnancy of rats with diabetes. Materials and methods: For the induction of diabetes, female newborns received beta (β) -cytotoxic (streptozotocin - STZ, 100 mg / kg body weight subcutaneously) on the first day of life. Control females received only the citrate buffer. The inclusion criterion for the diabetic group was glycemia ≥ 400 mg / dL at day 5 of life (D5) and, for the control group, glycemia <120mg / dL at D5. The animals of both groups were killed at different times: D5, D10, D15, D30 and on day 18 of pregnancy. Blood was collected (serum insulin and glucagon) and pancreas (morphological analysis). Anti-Pdx-1, anti-Ki-67, anti-insulin, anti-glucagon, anti-somatostatin, anti-superoxide dismutase 1, anti-glutathione peroxidase, anti-malondialdehyde were used for immunohistochemical analysis. The gravid uterus was weighed and dissected to analyze the percentages of embryonic and fetal losses. For all comparisons, the minimum significance level of p <0.05 was considered. Results: At all the time points studied (D5, D10, D15, D30 and pregnancy) there was a decrease in the percentage of cells immunolabelled for insulin and an increase in the percentage of somatostatin and glucagon in the pancreatic islets of diabetic rats. The diabetic group presented lower intensity of SOD1 in D5, D10, D15 and D30, lower GSH-Px marking intensity in D10, D30 and in pregnancy, increased MDA marking intensity in pregnancy, lower percentage of immunolabelled cells for Pdx-1 and higher for Ki-67 when compared to the control rats. There was an increase in the percentage of pre and post implantation losses in pregnant rats with moderate diabetes. Conclusion: Hyperglycemia affected the cellular composition of pancreatic islets throughout life, including pregnancy, contributing to the reduction of the intensity of antioxidant enzymes and increase of the marker of lipoperoxidation in diabetic animals, proving the pancreatic oxidative stress. This negatively influenced the activation of Pdx-1, decreasing the stimulus for insulin synthesis, leading to glycemic changes through a greater glucose stimulus. This imbalance in glucose homeostasis overwhelms the pancreas and leads to the onset of diabetes. Thus, these pancreatic morphological alterations and the abnormality of oxidative stress due to diabetes during the critical window of development and pregnancy may explain the existing alterations to the onset of diseases in adult life. Thus, more studies need to be performed aiming at therapeutic measures that may prevent or minimize diabetic repercussions in the long term.
Introduction: During the neonatal period of rats, the endocrine pancreas undergoes remodeling, being considered a critical window of development, which determines the physiology of the pancreas in the body and possible diseases in adult life, among them diabetes. In pregnancy, the pancreas undergoes adaptations. Therefore, it is relevant to understand the mechanisms involved in the adaptation of pancreatic islets to the pregnancy and diabetes association, which may be related to oxidative stress and changes in maternal reproductive performance. Objective: To analyze the morphology of pancreatic islets and oxidative stress during the critical window of postnatal development and in the pregnancy of rats with diabetes. Materials and methods: For the induction of diabetes, female newborns received beta (β) -cytotoxic (streptozotocin - STZ, 100 mg / kg body weight subcutaneously) on the first day of life. Control females received only the citrate buffer. The inclusion criterion for the diabetic group was glycemia ≥ 400 mg / dL at day 5 of life (D5) and, for the control group, glycemia <120mg / dL at D5. The animals of both groups were killed at different times: D5, D10, D15, D30 and on day 18 of pregnancy. Blood was collected (serum insulin and glucagon) and pancreas (morphological analysis). Anti-Pdx-1, anti-Ki-67, anti-insulin, anti-glucagon, anti-somatostatin, anti-superoxide dismutase 1, anti-glutathione peroxidase, anti-malondialdehyde were used for immunohistochemical analysis. The gravid uterus was weighed and dissected to analyze the percentages of embryonic and fetal losses. For all comparisons, the minimum significance level of p <0.05 was considered. Results: At all the time points studied (D5, D10, D15, D30 and pregnancy) there was a decrease in the percentage of cells immunolabelled for insulin and an increase in the percentage of somatostatin and glucagon in the pancreatic islets of diabetic rats. The diabetic group presented lower intensity of SOD1 in D5, D10, D15 and D30, lower GSH-Px marking intensity in D10, D30 and in pregnancy, increased MDA marking intensity in pregnancy, lower percentage of immunolabelled cells for Pdx-1 and higher for Ki-67 when compared to the control rats. There was an increase in the percentage of pre and post implantation losses in pregnant rats with moderate diabetes. Conclusion: Hyperglycemia affected the cellular composition of pancreatic islets throughout life, including pregnancy, contributing to the reduction of the intensity of antioxidant enzymes and increase of the marker of lipoperoxidation in diabetic animals, proving the pancreatic oxidative stress. This negatively influenced the activation of Pdx-1, decreasing the stimulus for insulin synthesis, leading to glycemic changes through a greater glucose stimulus. This imbalance in glucose homeostasis overwhelms the pancreas and leads to the onset of diabetes. Thus, these pancreatic morphological alterations and the abnormality of oxidative stress due to diabetes during the critical window of development and pregnancy may explain the existing alterations to the onset of diseases in adult life. Thus, more studies need to be performed aiming at therapeutic measures that may prevent or minimize diabetic repercussions in the long term.
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Palavras-chave
ilhotas pancreáticas, estresse oxidativo, diabete, prenhez, ratas