Made available in DSpace on 2014-12-17T14:03:33Z (GMT). No. of bitstreams: 1 JannisonKCR_DISSERT.pdf: 3798069 bytes, checksum: 8a7cf649e9d79113b8221c30286c11d0 (MD5) Previous issue date: 2010-05-07 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Seeds from legumes including the Glycine max are known to be a rich source of protease inhibitors. The soybean Kunitz trypsin inhibitor (SKTI) has been well characterised and has been found to exhibit many biological activities. However its effects on inflammatory diseases have not been studied to date. In this study, SKTI was purified from a commercial soy fraction, enriched with this inhibitor, using anion exchange chromatography Resource Q column. The purified protein was able to inhibit human neutrophil elastase (HNE) and bovine trypsin. . Purified SKTI inhibited HNE with an IC50 value of 8 ?g (0.3 nM). At this concentration SKTI showed neither cytotoxic nor haemolytic effects on human blood cell populations. SKTI showed no deleterious effects on organs, blood cells or the hepatic enzymes alanine amine transferase (ALT) and aspartate amino transferase (AST) in mice model of acute systemic toxicity. Human neutrophils incubated with SKTI released less HNE than control neutrophils when stimulated with PAF or fMLP (83.1% and 70% respectively). These results showed that SKTI affected both pathways of elastase release by PAF and fMLP stimuli, suggesting that SKTI is an antagonist of PAF/fMLP receptors. In an in vivo mouse model of acute lung injury, induced by LPS from E. coli, SKTI significantly suppressed the inflammatory effects caused by elastase in a dose dependent manner. Histological sections stained by hematoxylin/eosin confirmed this reduction in inflammation process. These results showed that SKTI could be used as a potential pharmacological agent for the therapy of many inflammatory diseases / Sementes de leguminosas s?o conhecidas como uma rica fonte de inibidores de proteinases, destacando-se dentre estes o inibidor de tripsina da soja (SKTI) que ? uma prote?na amplamente estudada e caracterizada para muitas propriedades biol?gicas. Entretanto seus efeitos aplicados a desordens inflamat?rias ainda s?o pouco conhecidos. SKTI foi purificado ? partir de uma fra??o comercial de soja atrav?s de cromatografia de troca ani?nica em Resource Q. A prote?na purificada foi capaz de inibir a elastase de neutr?filos humanos (ENH) e a tripsina bovina. O valor da sua IC50 foi de 8 μg.mL-1 (0.3 nM) e nessa concentra??o o SKTI n?o foi capaz de provocar efeitos hemol?ticos ou citot?xicos sobre as popula??es celulares sangu?neas humanas. Por meio do modelo de toxicidade sist?mica aguda, utilizando camundongos, tamb?m n?o foram observados efeitos delet?rios sobre ?rg?os, c?lulas sangu?neas e altera??es nos n?veis das enzimas hep?ticas aspartato amino transferase (AST) e alanina amino transferase (ALT). Neutr?filos humanos incubados com SKTI na concentra??o de 0.3 nM apresentaram uma diminui??o da libera??o de ENH quando estimulados pelos ativadores PAF e fMLP (83,1% e 70 %, respectivamente). Estes resultados mostram que o SKTI foi capaz de afetar ambas as vias PAF/fMLP de libera??o de ENH, sugerindo esta prote?na como um poss?vel antagonista dos receptores PAF/fMLP. Modelos in vivo de inj?ria pulmonar aguda mediante estimula??o por LPS de Escherichia. coli demonstraram uma supress?o significativa dos eventos inflamat?rios atribu?dos ? atividade elast?sica de forma dose dependente. Cortes histol?gicos corados por hematoxilina e eosina confirmaram a diminui??o da inflama??o tecidual. Estes resultados sugerem que o SKTI pode ser indicado como um potencial agente farmacol?gico na terapia de muitas doen?as inflamat?rias

Made available in DSpace on 2014-12-17T14:03:37Z (GMT). No. of bitstreams: 1 NorbertoKVM_DISSERT.pdf: 2522986 bytes, checksum: 6298d49730e0d9c9d5418ad46a9b33f5 (MD5) Previous issue date: 2011-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer / Estudos indicam que v?rios componentes medicinais foram isolados de vegetais, os quais apresentam atividades antibacterianas, antif?ngicas, antitumorais e anti-inflamat?rias. Sepse ? caracterizada por uma inflama??o sist?mica que tem como conseq??ncia a produ??o exarcebada de mediadores inflamat?rios, pela excessiva ativa??o de c?lulas inflamat?rias e coagula??o intravascular disseminada (CIVD), na qual a elastase neutrof?lica humana exerce um papel importante na sua patog?nese. Diversos estudos epidemiol?gicos sugerem que componentes de vegetais, especialmente de leguminosas, podem desempenhar um papel ben?fico na redu??o da incid?ncia de diferentes tipos de c?ncer. Um inibidor de quimotripsina do tipo Kunitz (Varela, 2010) foi purificado de sementes de Erythrina velutina (Mulungu) por fracionamento com sulfato de am?nio, cromatografias de afinidade em Tripsina-Sepharose e Quimotripsina-Sepharose e cromatografia de troca i?nica em Resource Q 1 mL (GE Healthcare), em sistema FPLC/AKTA. O inibidor, denominado EvCI, apresentou uma massa molecular de 17 kDa, determinada por SDS-PAGE. A prote?na purificada foi capaz de inibir a elastase de neutr?filos humanos (ENH), apresentando um IC50 de 3,12 nM. O EvCI foi capaz de inibir ambas as vias de libera??o de ENH estimuladas por PAF e fMLP (75,6% e 65%, respectivamente). O inibidor tamb?m inibiu a migra??o leucocit?ria em camundongos s?pticos em cerca de 87% e prolongou o tempo de coagula??o com inibi??o do fator Xa. EvCI n?o apresentou atividade hemol?tica nem citot?xica. EvCI apresentou um efeito antiproliferativo seletivo para linhagens de c?lulas HepG2 com IC50 de 0,5 μg /mL. Estes resultados sugerem o EvCI como uma mol?cula antagonista dos receptores PAF/fMLP e um potencial emprego no combate a dist?rbios relacionados a inflama??o, coagula??o intravascular disseminada (CIVD) e cancer