Efeitos hepáticos de estatinas em modelo de sepse polimicrobiana em ratos

Orientadora : Profª Drª Alexandra AccoCo-Orientador : Prof. Dr. Aleksander Roberto ZampronioDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa: Curitiba, 14/02/2011Bibliografia: fls. 45-50Resumo: Contexto: Diversos dados sugerem beneficios do tratamento com estatinas durante a sepse, mas ainda nao existe um consenso a respeito disso na literatura. O objetivo principal deste trabalho foi investigar os efeitos do tratamento prolongado com estatinas em sepse, dando enfase em suas propriedades antioxidantes, antiinflamatorias e metabolicas. Desenho experimental: Ratos Wistar machos foram tratados com simvastatina (1,17 ou 5,85 mg/kg), atorvastatina (0,59 ou 2,95 mg/kg) ou veiculo uma vez ao dia, por gavagem. Apos 30 dias de tratamento, foi induzida a sepse pelo modelo de ligadura e puncao do ceco (CLP) nos grupos Controle, Simvastatina e Atorvastatina (n=6). O grupo Sham (n=6), que tambem recebeu veiculo, foi submetido apenas a laparotomia. Os grupos Simvastatina Basal e Atorvastatina Basal receberam 5,85 mg/Kg e 2,95 mg/kg, respectivamente (n=5), mas nao foram submetidos a nenhum procedimento cirurgico. Passadas 24 horas da CLP ou laparotomia, os animais foram anestesiados novamente para coleta de amostras (figado e sangue). Ensaios foram feitos para avaliar o estresse oxidativo hepatico (catalase, superoxido dismutase, glutationa-S-transferase, glutationa reduzida e peroxidacao lipidica), histologia hepatica, atividade de enzimas mitocondriais hepaticas, migracao leucocitaria, contagem de leucocitos no sangue, expressao genica de superoxido dismutase e TNFƒÑ no figado, bioquimica serica (aspartato aminotransferase, alanina aminotransferase, colesterol e triglicerideos) e nivel plasmatico de TNFƒÑ. Resultados: A maior parte dos parametros testados demonstrou alteracoes compativeis com a condicao de sepse. Neste estudo o unico parametro capaz de evidenciar algum beneficio proporcionado pelo tratamento com as estatinas em ratos submetidos ao modelo da CLP foi a atividade enzimatica mitocondrial. Nos demais parametros, a simvastatina e a atorvastatina nao protegeram o figado contra as injurias causadas pela inducao da sepse polimicrobiana. Conclusao: Ambas as estatinas quando usadas isoladamente por 30 dias antes da inducao de sepse pelo modelo de CLP parecem melhorar a atividade mitocondrial, mas esse resultado nao se repetiu em outros marcadores de funcao hepatica e migracao leucocitaria durante a sepse. Mais estudos devem ser realizados para avaliar a combinacao de estatinas com outras drogas utilizadas no tratamento dessa condicao patologica.Abstract: Background: Several data suggest benefical effects of treatment with statins during sepis, but there is not yet a consensus in scientific literature about that. The major objective of this study was to investigate the effects of long-term statins treatment on hepatic parameters during sepsis, focusing its antioxidant, antiinflammatory and metabolic properties. Experimental approach: Male Wistar rats were treated orally with simvastatin (1.17 or 5.85 mg/kg), atorvastatin (0.59 or 2.95 mg/kg) or vehicle once a day. After 30 days of treatment, sepsis was induced by CLP on Control, Simvastatin and Atorvastatin groups (n=6). Sham group (n=6), which also received vehicle, was submitted only to laparotomy. Basal Simvastatin and Basal Atorvastatin groups also received 5.85 mg/Kg and 2.95 mg/kg, respectively (n=5), but these animals were not submitted to any surgical procedure. After 24 hours of CLP or laparotomy, the animals were anaesthetized for sample collection (hepatic tissues and blood). Assays were performed to evaluate hepatic oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase, reduced glutathione and lipid peroxidation), liver histology, activity of hepatic mitochondria enzymes, leukocyte migration, leukocyte count on blood, gene expression of hepatic superoxide dismutase and TNFƒÑ, plasmatic biochemistry (aspartate aminotransferase, alanine aminotransferase, cholesterol and triglycerides) and plasmatic level of TNFƒÑ. Results: Most of the parameters tested showed alterations compatible with sepsis outcome. However, only liver mitochondrial enzymatic activity presented improvement with statins treatment in rat submitted to sepsis conditions. In other parameters, simvastatin and atorvastatin failed to protect liver against injuries of CLP polimicrobial sepsis model. Conclusion: The treatment with simvastatin and atorvastatin during 30 days as single drug before inducing CLP seems to improve liver enzymatic mitochondria activity, but this result was not reproducible in other biomarkers of liver function and leukocytes migration during sepsis. Future studies should be encouraged to evaluate the combination of statins with other drugs used in the therapy of sepsis

Efeitos da silmarina sobre a angiogênese e estresse oxidativo em camundongos normoglicêmicos e diabéticos

Orientador : Profª. Drª. Alexandra AccoCoorientadora : Profª. Drª. Cibele Campos CardosoTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa: Curitiba, 04/11/2016Inclui referências : f. 46-54;77-80;82-85Resumo: Contexto: Diversos dados sugerem benefícios do tratamento com estatinas durante a sepse, mas ainda não existe um consenso a respeito disso na literatura. O objetivo principal deste trabalho foi investigar os efeitos do tratamento prolongado com estatinas em sepse, dando ênfase em suas propriedades antioxidantes, antiinflamatórias e metabólicas. Desenho experimental: Ratos Wistar machos foram tratados com simvastatina (1,17 ou 5,85 mg/kg), atorvastatina (0,59 ou 2,95 mg/kg) ou veículo uma vez ao dia, por gavagem. Após 30 dias de tratamento, foi induzida a sepse pelo modelo de ligadura e punção do ceco (CLP) nos grupos Controle, Simvastatina e Atorvastatina (n=6). O grupo Sham (n=6), que também recebeu veículo, foi submetido apenas à laparotomia. Os grupos Simvastatina Basal e Atorvastatina Basal receberam 5,85 mg/Kg e 2,95 mg/kg, respectivamente (n=5), mas não foram submetidos a nenhum procedimento cirúrgico. Passadas 24 horas da CLP ou laparotomia, os animais foram anestesiados novamente para coleta de amostras (fígado e sangue). Ensaios foram feitos para avaliar o estresse oxidativo hepático (catalase, superóxido dismutase, glutationa-S-transferase, glutationa reduzida e peroxidação lipídica), histologia hepática, atividade de enzimas mitocondriais hepáticas, migração leucocitária, contagem de leucócitos no sangue, expressão gênica de superóxido dismutase e TNF? no fígado, bioquímica sérica (aspartato aminotransferase, alanina aminotransferase, colesterol e triglicerídeos) e nível plasmático de TNF?. Resultados: A maior parte dos parâmetros testados demonstrou alterações compatíveis com a condição de sepse. Neste estudo o único parâmetro capaz de evidenciar algum benefício proporcionado pelo tratamento com as estatinas em ratos submetidos ao modelo da CLP foi a atividade enzimática mitocondrial. Nos demais parâmetros, a simvastatina e a atorvastatina não protegeram o fígado contra as injúrias causadas pela indução da sepse polimicrobiana. Conclusão: Ambas as estatinas quando usadas isoladamente por 30 dias antes da indução de sepse pelo modelo de CLP parecem melhorar a atividade mitocondrial, mas esse resultado não se repetiu em outros marcadores de função hepática e migração leucocitária durante a sepse. Mais estudos devem ser realizados para avaliar a combinação de estatinas com outras drogas utilizadas no tratamento dessa condição patológica. Palavras-chave: sepse, fígado, estatinas, simvastatina e atorvastatina.Abstract: Background: Several data suggest benefical effects of treatment with statins during sepis, but there is not yet a consensus in scientific literature about that. The major objective of this study was to investigate the effects of long-term statins treatment on hepatic parameters during sepsis, focusing its antioxidant, antiinflammatory and metabolic properties. Experimental approach: Male Wistar rats were treated orally with simvastatin (1.17 or 5.85 mg/kg), atorvastatin (0.59 or 2.95 mg/kg) or vehicle once a day. After 30 days of treatment, sepsis was induced by CLP on Control, Simvastatin and Atorvastatin groups (n=6). Sham group (n=6), which also received vehicle, was submitted only to laparotomy. Basal Simvastatin and Basal Atorvastatin groups also received 5.85 mg/Kg and 2.95 mg/kg, respectively (n=5), but these animals were not submitted to any surgical procedure. After 24 hours of CLP or laparotomy, the animals were anaesthetized for sample collection (hepatic tissues and blood). Assays were performed to evaluate hepatic oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase, reduced glutathione and lipid peroxidation), liver histology, activity of hepatic mitochondria enzymes, leukocyte migration, leukocyte count on blood, gene expression of hepatic superoxide dismutase and TNF?, plasmatic biochemistry (aspartate aminotransferase, alanine aminotransferase, cholesterol and triglycerides) and plasmatic level of TNF?. Results: Most of the parameters tested showed alterations compatible with sepsis outcome. However, only liver mitochondrial enzymatic activity presented improvement with statins treatment in rat submitted to sepsis conditions. In other parameters, simvastatin and atorvastatin failed to protect liver against injuries of CLP polimicrobial sepsis model. Conclusion: The treatment with simvastatin and atorvastatin during 30 days as single drug before inducing CLP seems to improve liver enzymatic mitochondria activity, but this result was not reproducible in other biomarkers of liver function and leukocytes migration during sepsis. Future studies should be encouraged to evaluate the combination of statins with other drugs used in the therapy of sepsis. Key words: sepsis, liver, statins, simvastatin, atorvastati

Efeitos hepáticos de estatinas em modelo de sepse polimicrobiana em ratos

Orientadora : Profª Drª Alexandra AccoCo-Orientador : Prof. Dr. Aleksander Roberto ZampronioDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa: Curitiba, 14/02/2011Bibliografia: fls. 45-50Resumo: Contexto: Diversos dados sugerem beneficios do tratamento com estatinas durante a sepse, mas ainda nao existe um consenso a respeito disso na literatura. O objetivo principal deste trabalho foi investigar os efeitos do tratamento prolongado com estatinas em sepse, dando enfase em suas propriedades antioxidantes, antiinflamatorias e metabolicas. Desenho experimental: Ratos Wistar machos foram tratados com simvastatina (1,17 ou 5,85 mg/kg), atorvastatina (0,59 ou 2,95 mg/kg) ou veiculo uma vez ao dia, por gavagem. Apos 30 dias de tratamento, foi induzida a sepse pelo modelo de ligadura e puncao do ceco (CLP) nos grupos Controle, Simvastatina e Atorvastatina (n=6). O grupo Sham (n=6), que tambem recebeu veiculo, foi submetido apenas a laparotomia. Os grupos Simvastatina Basal e Atorvastatina Basal receberam 5,85 mg/Kg e 2,95 mg/kg, respectivamente (n=5), mas nao foram submetidos a nenhum procedimento cirurgico. Passadas 24 horas da CLP ou laparotomia, os animais foram anestesiados novamente para coleta de amostras (figado e sangue). Ensaios foram feitos para avaliar o estresse oxidativo hepatico (catalase, superoxido dismutase, glutationa-S-transferase, glutationa reduzida e peroxidacao lipidica), histologia hepatica, atividade de enzimas mitocondriais hepaticas, migracao leucocitaria, contagem de leucocitos no sangue, expressao genica de superoxido dismutase e TNFƒÑ no figado, bioquimica serica (aspartato aminotransferase, alanina aminotransferase, colesterol e triglicerideos) e nivel plasmatico de TNFƒÑ. Resultados: A maior parte dos parametros testados demonstrou alteracoes compativeis com a condicao de sepse. Neste estudo o unico parametro capaz de evidenciar algum beneficio proporcionado pelo tratamento com as estatinas em ratos submetidos ao modelo da CLP foi a atividade enzimatica mitocondrial. Nos demais parametros, a simvastatina e a atorvastatina nao protegeram o figado contra as injurias causadas pela inducao da sepse polimicrobiana. Conclusao: Ambas as estatinas quando usadas isoladamente por 30 dias antes da inducao de sepse pelo modelo de CLP parecem melhorar a atividade mitocondrial, mas esse resultado nao se repetiu em outros marcadores de funcao hepatica e migracao leucocitaria durante a sepse. Mais estudos devem ser realizados para avaliar a combinacao de estatinas com outras drogas utilizadas no tratamento dessa condicao patologica.Abstract: Background: Several data suggest benefical effects of treatment with statins during sepis, but there is not yet a consensus in scientific literature about that. The major objective of this study was to investigate the effects of long-term statins treatment on hepatic parameters during sepsis, focusing its antioxidant, antiinflammatory and metabolic properties. Experimental approach: Male Wistar rats were treated orally with simvastatin (1.17 or 5.85 mg/kg), atorvastatin (0.59 or 2.95 mg/kg) or vehicle once a day. After 30 days of treatment, sepsis was induced by CLP on Control, Simvastatin and Atorvastatin groups (n=6). Sham group (n=6), which also received vehicle, was submitted only to laparotomy. Basal Simvastatin and Basal Atorvastatin groups also received 5.85 mg/Kg and 2.95 mg/kg, respectively (n=5), but these animals were not submitted to any surgical procedure. After 24 hours of CLP or laparotomy, the animals were anaesthetized for sample collection (hepatic tissues and blood). Assays were performed to evaluate hepatic oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase, reduced glutathione and lipid peroxidation), liver histology, activity of hepatic mitochondria enzymes, leukocyte migration, leukocyte count on blood, gene expression of hepatic superoxide dismutase and TNFƒÑ, plasmatic biochemistry (aspartate aminotransferase, alanine aminotransferase, cholesterol and triglycerides) and plasmatic level of TNFƒÑ. Results: Most of the parameters tested showed alterations compatible with sepsis outcome. However, only liver mitochondrial enzymatic activity presented improvement with statins treatment in rat submitted to sepsis conditions. In other parameters, simvastatin and atorvastatin failed to protect liver against injuries of CLP polimicrobial sepsis model. Conclusion: The treatment with simvastatin and atorvastatin during 30 days as single drug before inducing CLP seems to improve liver enzymatic mitochondria activity, but this result was not reproducible in other biomarkers of liver function and leukocytes migration during sepsis. Future studies should be encouraged to evaluate the combination of statins with other drugs used in the therapy of sepsis

Characterization of an alcoholic hepatic steatosis model induced by ethanol and high-fat diet in rats

Alcoholic liver disease is characterized by a wide spectrum of liver damage, which increases when ethanol is associated with high-fat diets (HFD). This work aimed to establish a model of alcoholic hepatic steatosis (AHS) by using a combination of 10% ethanol and sunflower seeds as the source of HFD. Male rats received water or 10% ethanol and regular chow diet and/or HFD, which consisted of sunflower seeds. The food consumption, liquid intake and body weight of the rats were monitored for 30 days. After this period, blood was collected for biochemical evaluation, and liver samples were collected for histological, mitochondrial enzyme activity and oxidative stress analyses. Our results indicated that the combination of 10% ethanol and HFD induced micro- and macrosteatosis and hepatocyte tumefaction, decreased the levels of reduced glutathione and glutathione S-transferase activity and increased the level of lipoperoxidation and superoxide dismutase activity. The mitochondrial oxidation of NADH and succinate were partially inhibited. Complexes I and II were the main inhibition sites. Hepatic steatosis was successfully induced after 4 weeks of the diet, and the liver function was modified. The combination of 10% ethanol and sunflower seeds as an HFD produced an inexpensive model to study AHS in rats

Sesquiterpene lactones of Moquiniastrum polymorphum subsp. floccosum have antineoplastic effects in Walker-256 tumor-bearing rats

Background and aim: This study aimed to evaluate the in vivo antitumor actions and toxicity of the dichloromethane fraction (F1B) of Moquiniastrum polymorphum subsp. floccosum (formerly Gochnatia polymorpha ssp. floccosa), composed of sesquiterpene lactones, against Walker-256 carcinosarcoma in rats. Methods: Male Wistar rats received 100 mg kg-1 F1B per day orally for 16 days after subcutaneous inoculation of Walker-256 cells in the pelvic limb. The tumor progression was monitored, and after treatment, tumor weight, oxidative stress, plasma biochemistry, inflammatory parameters, gene expression and histology of tumor and/or liver were evaluated. The toxicity of F1B was analyzed through the relative weight of organs. Additionally, an LD50 test was performed in mice. Results: F1B treatment significantly reduced tumor volume and weight. There was no difference in oxidative stress in tumor tissue after treatment. F1B treatment modified hepatic glutathione and superoxide dismutase, and normalized plasma glucose, alkaline phosphatase, and amylase. F1B did not affect the activity of myeloperoxidase and N-acetylglucosaminidase or the nitric oxide levels in tumor tissue. However, F1B decreased the tumor necrosis factor (TNF)-α levels. Additionally, F1B increased apoptosis in the tumor, mediated by up-regulation of the p53 and Bax gene expression. No clinical signs of toxicity or death were observed in the rats treated with F1B. The LD50 calculated for mice was 1209 mg kg-1. Conclusions: F1B, which is rich in sesquiterpene lactones, showed antitumor activity against Walker-256 carcinosarcoma. This effect may be, at least in part, related to the induction of apoptosis and inhibition of TNF-α signaling

The FXR agonist 6ECDCA reduces hepatic steatosis and oxidative stress induced by ethanol and low-protein diet in mice

Excessive ethanol consumption can lead to development of hepatic steatosis. Since the FXR receptor regulates adipose cell function and liver lipid metabolism, the aim of this work was to examine the effects of the FXR agonist 6ECDCA on alcoholic liver steatosis development and on oxidative stress induced by ethanol consumption. Swiss mice (n=24) received a low-protein diet (6%) and a liquid diet containing 10% ethanol or water for 6weeks. In the last 15days mice received oral treatment with 6ECDCA (3mgkg(-1)) or 1% tween (vehicle). The experimental groups (n=6) were: water+tween, water+6ECDCA, ethanol+tween and ethanol+6ECDCA. Moreover, as a diet control, we used a basal group (n=6), fed by a normal-proteic diet (23%) and water. After the treatment period, the animals were anesthetized for sample collection to perform plasma biochemistry assays, hepatic oxidative stress assays, hepatic cholesterol and triglycerides measurements, liver histology and hepatic gene expression. Ethanol associated with low-protein diet induced hepatic oxidative stress, increased plasma transaminases and induced hepatic lipid accumulation. Many of these parameters were reversed by the administration of 6ECDCA, including amelioration of lipid accumulation and lipoperoxidation, and reduction of reactive oxygen species. These effects were possibly mediated by regulation of Srebpf1 and FAS gene expression, both reduced by the FXR agonist. Our data demonstrated that 6ECDCA reverses the accumulation of lipids in the liver and decreases the oxidative stress induced by ethanol and low-protein diet. This FXR agonist is promising as a potential therapy for alcoholic liver steatosi

Catalase expression in liver (A) and tumour (B), and SOD-1 expression in liver (C) and tumour (D), as measured by Western blot.

<p>The densitometry graphs are displayed beside each picture. Group abbreviations: CO: Control, BR: BHE extract, BU: BuOH fraction, CH: CHCl₃ fraction.</p

HPLC results.

<p>(A) Chromatogram of the BuOH fraction of <i>U. tomentosa</i> showing phenolic compounds, including proanthocyanidins in the region between 50 and 60 minutes. (B) Chromatogram of the CHCl₃ fraction of <i>U. tomentosa</i>. The following compounds were tentatively identified as 1- speciophylline; 2- uncarine F; 3- mitraphyline; 4- isomitraphyline; 5- pteropodine and 6- isopteropodine by comparison with data from the literature.</p