Thesis (Ph. D.)--Cornell University, 1900.

Thesis (Ph. D.)--Cornell University, 1900.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A pancreatite aguda (PA) Ã considerada uma situaÃÃo de emergÃncia abdominal, na forma grave da doenÃa os pacientes desenvolvem acentuada resposta inflamatÃria sistÃmica e SÃndrome de DisfunÃÃo de MÃltiplos ÃrgÃos (SDMO). Um terÃo das mortes relacionadas com PA acontecem antes da admissÃo hospitalar, e a maior parte dos casos estÃo relacionados com lesÃo pulmonar aguda (LPA) e sÃndrome do desconforto respiratÃrio agudo (SDRA). Objetivos: Avaliar as alteraÃÃes inflamatÃrias e funcionais do pulmÃo no curso da pancreatite aguda experimental induzida por ceruleÃna. MÃtodos: PA foi induzida em Ratos Wistar, machos pensando 100-150g, pela administraÃÃo de 4 doses de ceruleÃna (20Âg/kg) com intervalo de uma hora e os grupos controle receberam apenas soluÃÃo salina. ApÃs 24 horas, os animais foram sedados, analgesiados e traqueostomizados e anÃlise da funÃÃo pulmonar foi realizada atravÃs da espirometria, onde foram avaliados Fluxo, Volume Corrente (VC), FrequÃncia RespiratÃria (FR) e Volume Minuto (VM), e da mecÃnica pulmonar onde foram observados ElastÃncia DinÃmica (Edin), ComplacÃncia DinÃmica (Cdin), PressÃo de Pico, ResistÃncia (Res). Lavado bronco-alveolar (LBA) foi realizado para contagem total e diferencial de cÃlulas. Amostra de sangue arterial foi colhida para avaliaÃÃo dos parÃmetros gasomÃtricos. Em seguida os animais foram sacrificados e nÃveis sÃricos de amilase, lipase, EPO, TNF-α, GRO-KC, MIP-1, VEGF, IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-18 e de malondialdeÃdo (MDA) foram medidos. Atividade de mieloperoxidase (MPO) e avaliaÃÃo histolÃgica de pÃncreas e pulmÃo foram determinadas. AlÃm disso, amostras de sangue venoso foram colhidas para avaliaÃÃo de translocaÃÃo bacteriana. Resultados: NÃveis sÃricos de amilase, lipase, citocinas, MDA e atividade de MPO pancreÃtica e pulmonar estavam aumentados nos animais com PA; houve danos ao tecido pancreÃtico e pulmonar, revelados na histologia, nos animais que receberam ceruleÃna, quando comparados ao grupo controle. O LBA dos animais tratados com ceruleÃna demonstrou maior quantidade de cÃlulas, sendo predominantemente macrÃfagos. Gasometria arterial nÃo apresentou diferenÃa significativa entre os grupos. Fluxo, VC e VM se mostraram diminuÃdos nos animais com PA; FR permaneceu inalterada. Edin e PressÃo de Pico estavam maiores e Cdin estava menor nos animais com PA e nÃo houve alteraÃÃes na Res. Estudo da bacteremia foi negativo em ambos grupos. ConclusÃo: CeruleÃna induz PA em ratos com elevaÃÃo dos nÃveis de amilase e lipase pancreÃtica, com alteraÃÃes histopatolÃgicas no pÃncreas e no pulmÃo dependente do infiltrado neutrofÃlico, radicais livres e citocinas inflamatÃrias. PA induz alteraÃÃes espiromÃtricas e na mecÃnica pulmonar que nÃo sÃo dependentes de processo infeccioso. / Acute pancreatitis (AP) is considered an emergency abdominal, the severe form of the disease patients develop intense systemic inflammatory response and Multiple Organ Dysfunction Syndrome (MODS). About one-third of all deaths from acute pancreatitis has been reported to occur prior to admission to hospital, and in most cases, is associated with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Objectives: To assess the inflammatory and functional lung alterations in the course of AP induced by cerulein. Methods: Male Wistar rats (100-150g) were treated four times with one hour interval, intraperitoneally with cerulein (20 μg / kg, suspended in saline) or saline. Twenty-four hours after the first injection of cerulein, the animals were anesthetized, tracheostomized and placed in a spirometer for small animals and with following parameters evaluated: Flow, Volume(VC), Respiratory Frequency(RF) and Minute Volume(MV), and lung mechanics were observed where Dynamic Elastance (Edyn), Dynamic Compliance (Cdyn), Peak Pressure, Resistance (Raw). Bronchoalveolar lavage (BAL) was performed to count and differential cell. Arterial blood sample was drawn for assessment of pulmonary gas exchange parameters. Then the animals were sacrificed and serum amylase, lipase, EPO, TNF-α, GRO-KC, MIP-1, VEGF, IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-18 and malondialdehyde (MDA) were measured. Myeloperoxidase activity (MPO) and histological evaluation of pancreas and lung were determined. In addition, venous blood samples were collected for evaluation of bacterial translocation. Results: Serum levels of amylase, lipase, cytokines, MDA and MPO activity of pancreatic and lung were increased in animals with PA, there was damage to pancreatic tissue and lung histology revealed, in animals that received cerulein compared to the control group. There was an increase in the number of BAL cells, predominantly macrophages. Arterial blood gas analysis showed no significant difference between groups. Flow, and MV proved lower in animals with PA; FR remained unchanged. Edyn and pressure peak were larger and Cdyn was lower in animals with PA and no changes in Res. There was no translocation in any groups. Conclusion: Cerulein induced AP in rats with elevated serum amylase and pancreatic lipase, with histopathological changes in the pancreas and lung dependent neutrophilic infiltrate, free radicals and inflammatory cytokines. PA induces spirometric and lung mechanics alterations that are not dependent on bacterial translocation

Inflammation caused by oxidative stress (ROS) demonstrates an essential mechanism in the pathogenesis of acute pancreatitis (AP). Important sources for ROS comprise the reactive compound methylglyoxal (MGO) itself and the MGO-derived formation of advanced glycation end-products (AGEs). AGEs bind to the transmembrane receptor RAGE and activate NF-κB, and lead to the production of pro-inflammatory cytokines. MGO is detoxified by glyoxalase-I (Glo-I). The importance of Glo-I was shown in different models of inflammation and carcinogenesis. Nevertheless, the role of Glo-I and MGO in AP has not been evaluated so far. This study analyzed Glo-I in cerulein-(CN)-induced AP and determined the effects of Glo-I knockdown, overexpression and pharmacological modulation. Methods: AP was induced in C57BL6/J mice by i.p. injection of CN. Glo-I was analyzed in explanted pancreata by Western Blot, qRT-PCR and immunohistochemistry. AR42J cells were differentiated by dexamethasone and stimulated with 100 nM of CN. Cells were simultaneously treated with ethyl pyruvate (EP) or S-p-bromobenzylglutathione-cyclopentyl-diester (BrBz), two Glo-I modulators. Knockdown and overexpression of Glo-I was achieved by transient transfection with Glo-I siRNA and pEGFP-N1-Glo-I-Vector. Amylase secretion, TNF-α production (ELISA) and expression of Glo-I, RAGE and NF-κB were measured. Results: Glo-I was significantly upregulated on protein and mRNA levels in CN-treated mice and AR42J cells. Dexamethasone-induced differentiation of AR42J cells increased the expression of Glo-I and RAGE. Treatment of AR42J cells with CN and EP or BrBz resulted in a significant reduction of CN-induced amylase secretion, NF-κB, RAGE and TNF-α. Overexpression of Glo-I led to a significant reduction of CN-induced amylase levels, NF-κB expression and TNF-α, whereas Glo-I knockdown revealed only slight alterations. Measurements of specific Glo-I activity and MGO levels indicated a complex regulation in the model of CN-induced AP. Conclusion: Glo-I is overexpressed in a model of CN-induced AP. Pharmacological modulation and overexpression of Glo-I reduced amylase secretion and the release of pro-inflammatory cytokines in AP in vitro. Targeting Glo-I in AP seems to be an interesting approach for future in vivo studies of AP.

Bestandteil dieser Arbeit sind zwei Publikationen, welche sich mit einer möglichen Bedeutung der Glyoxalase-I (GLO-I) bei der akuten Pankreatitis (AP) auseinander-setzen. Die erste Publikation „Pitfalls in AR42J - model of cerulein-induced acute pancreatitis“ befasst sich mit der Gestalt eines in-vitro Modells der durch Cerulein (CN) induzierten akuten Pankreatitis. Darauf aufbauend setzt sich die zweite Publi-kation „Glyoxalase-I is upregulated in acute cerulein-induced pancreatitis: A new mechanism in pancreatic inflammation?“ mit der Fragestellung zur Rolle der GLO-I bei der CN-induzierten AP als Modell der AP auseinander. Als Basis zur fachlichen Einordnung der in dieser Arbeit inkludierten Publikationen wird zu Beginn ein fokussierter Überblick über das Pankreas und die AP gegeben. Es folgt eine Darstellung des Modells der CN-induzierten AP als Modell der AP. Da-rauf aufbauend werden inflammatorische Zusammenhänge im Modell betrachtet, welche die explorative Forschung zur GLO-I begründen.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Os triterpenos pentacÃclicos sÃo compostos naturais com atividade antiinflamatÃria e citoprotetora e sÃo relativamente atÃxicos. A pancreatite aguda, uma inflamaÃÃo aguda do pÃncreas, pode levar à sÃndrome da resposta inflamatÃria sistÃmica (SRIS) e à sÃndrome da disfunÃÃo mÃltipla de ÃrgÃos (MODS), condiÃÃes que podem levar o paciente ao Ãbito. Neste trabalho, a mistura de triterpenos pentacÃclicos alfa,β-amirina, isolada do Protium hepthaphyllum, foi investigada quanto aos seus efeitos nos modelos de pancreatite aguda induzida por L-arginina em ratos e por ceruleÃna em camundongos. No modelo de pancreatite aguda induzida por L-arginina, ratos Wistar machos foram tratados com a mistura de alfa,β-amirina (10, 30 e 100 mg/kg, v.o.) ou com o veÃculo (2% de Tween 80 em Ãgua destilada, 10ml/kg) 48, 24 e 1,5h antes da administraÃÃo de L-arginina (2 x 2,5 g/kg; 1 h de intervalo) ou com metilprednisolona (30 mg/kg, i.m.) 30 min antes da administraÃÃo de L-arginina. Na pancreatite induzida por ceruleÃna, camundongos Swiss machos foram tratados com a mistura de alfa,β-amirina (10, 30 e 100 mg/kg, v.o.) ou com o veÃculo (2% de Tween 80 em Ãgua destilada, 10ml/kg) 48, 24 e 1,5h antes da administraÃÃo de ceruleÃna (5 x 50 μg/kg; 1 h de intervalo) ou com talidomida (200 mg/kg, v.o.) 1h antes da administraÃÃo de ceruleÃna. Animais tratados apenas com salina (0,9%, NaCl) foram incluÃdos nos dois modelos. Foram analisados o edema pancreÃtico, nÃveis sÃricos de amilase, lipase e citocinas (TNF alfa, IL-6), mieloperoxidase, substÃncias reativas ao Ãcido tiobarbitÃrico (TBARS), histologia e imunohistoquÃmica (TNF-alfa, iNOS e nitrotirosina) pancreÃtica. L-arginina e ceruleÃna aumentaram significativamente o edema pancreÃtico e os nÃveis sÃricos de amilase, lipase, TNF alfa, IL-6. A avaliaÃÃo histopatolÃgica do pÃncreas revelou a presenÃa de edema, infiltraÃÃo neutrofÃlica, hemorragia, vacuolizaÃÃo e necrose acinar. Foi observado um aumento acentuado na expressÃo de TNF alfa, iNOS e nitrotirosina na avaliaÃÃo por imunohistoquÃmica. O prÃ-tratamento com alfa e beta-amirina (10, 30 e 100 mg/kg, v.o.), metilprednisolona (30 mg/kg, i.m.) ou talidomida (200 mg/kg, v.o.) atenuaram significativamente a severidade da pancreatite aguda induzida tanto por L-arginina, quanto por ceruleÃna, evidenciado pela reduÃÃo do edema pancreÃtico, amilase, lipase e citocinas sÃricas, mieloperoxidase e TBARS pancreÃtico. AlÃm disso, o tratamento com alfa,β-amirina e com as drogas de referÃncia suprimiram as alteraÃÃes histopatolÃgicas e a expressÃo de citocinas e nitrotirosina pancreÃticas. Em conjunto, esses resultados indicam que alfa,β-amirina melhora a severidade da pancreatite aguda induzida por L-arginina ou ceruleÃna por agir como antiinflamatÃrio e antioxidante. / Triterpenes are natural compounds with anti-inflammatory and cytoprotective effects relatively non-toxic. Acute pancreatitis, an inflammation of the pancreas, may lead to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), conditions that can lead the patient to death. In this study, a mixture of triterpenes isolated from Protium hepthaphyllum was investigated for their effects in models of acute pancreatitis. In the model of acute pancreatitis induced by L-arginine, male Wistar rats were treated with a mixture of ,β-amyrin (10, 30 and 100 mg/kg, p.o.) or with vehicle (2% Tween 80 in distilled water, 10 ml/kg) 48, 24 and 1.5 h before the administration of L-arginine (2 x 2.5 g/kg, 1 h apart) or with metilprednisolone (30 mg/kg, i.m.) 30 min before the administration of L-arginine. In cerulein-induced pancreatitis, male Swiss mice were treated with a mixture of ,β-amyrin (10, 30 and 100 mg/kg, p.o.) or with vehicle (2% Tween 80 in distilled water, 10 ml/kg) 48, 24 and 1.5 h before administration of cerulein (5 x 50 mg/kg, 1 h apart) or with thalidomide (200 mg/kg, p.o.) 1h before administration of cerulein. Animals treated with saline (0.9% NaCl) were included in both models. We analyzed the pancreatic edema, serum amylase, lipase and cytokines (TNF-, IL-6), myeloperoxidase, reactive substances to thiobarbituric acid (TBARS), histology and immunohistochemistry (TNF-, iNOS and nitrotyrosine) pancreatic. L-arginine and cerulein significantly increased pancreatic edema and serum levels of amylase, lipase, TNF- and IL-6. Histopathologic evaluation of pancreas revealed the presence of edema, neutrophil infiltration, hemorrhage, acinar vacuolization and necrosis. We observed a marked increase in the expression of TNF-, iNOS and nitrotyrosine in the evaluation by immunohistochemistry. The pre-treatment with alpha and beta-amyrin (10, 30 and 100 mg/kg, p.o.), metilprednisolone (30 mg/kg, i.m.) or thalidomide (200 mg/kg, p.o.) significantly attenuated the severity of acute pancreatitis induced by either L-arginine, and by cerulein, as evidenced by the reduction of pancreatic edema, amylase, lipase and serum cytokines, myeloperoxidase and pancreatic TBARS. Furthermore, treatment with ,β-amyrin and the reference drugs suppressed the histopathological changes and expression of cytokines and nitrotyrosine pancreatic. Together, these results indicate that the mixture of ,-amyrin reduces the severity of acute pancreatitis induced by L-arginine or cerulein acting as anti-inflammatory and antioxidant agent.