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Consultar: Programa de Ps-Graduao em Cincias Biolgicas (Biologia Celular)

Incio > Dissertaes e Teses > Cincias Biolgicas > Biologia Geral > Programa de Ps-Graduao em Cincias Biolgicas (Biologia Celular)

Ttulo [PT]: Aes do metil jasmonato sobre a inflamao e o estresse oxidativo sistmicos em ratos com artrite induzida por adjuvante
Ttulo [EN]: Anti-inflammatory action of methyl jasmonate is associated with oxidative modifications in the liver of arthritic rats
Autor(es): Jamil Soni Neto
Palavras-chave [PT]:

Artrite reumatide. Produo de ROS mitocondrial. Metil jasmonato (MeJA). Atividades anti-inflamatrias. Atividade da enzima glicoquinase. Brasil.
Palavras-chave [EN]:
Rheumatoid arthritis. Mitochondrial ROS production. Glucokinase activity. Brazil.
rea de concentrao: Biologia Celular e Molecular
Titulao: Mestre em Cincias Biolgicas
Banca:
Jurandir Fernando Comar [Orientador] - UEM
Monique Cristine de Oliveira - UNING
Maria Raquel Maral Natali - DCM/PBC/UEM
Cristiane Vizioli de Castro Ghizoni - UEM
Resumo:
Resumo: O metil jasmonato (MeJA) uma ciclopentanona natural derivada de cidos graxos que compartilha semelhanas estruturais com as prostaglandinas e, por isso, muitos estudos vm sendo realizados no sentido de avaliar suas aes em clulas de mamferos, principalmente as potenciais atividades anti-inflamatrias e anticancergenas. O MeJA inibiu a proliferao de diversas linhagens celulares de cncer em murinos e humanos. As mitocndrias parecem ser o alvo principal do MeJA, onde estimula a produo de espcies reativas de oxignio (ROS), dissocia a hexocinase ligada a mitocndria, estimula a depleo de ATP e causa morte celular. Em relao ao anti-inflamatria, o MeJA foi capaz de inibir a produo mediada por NFκB de xido ntrico, prostaglandina E, TNF-alfa, IL-1 e IL-6 em macrfagos de murinos ativados por lipopolissacardeo (LPS) (RAW 264.7). Entretanto, estes efeitos foram demonstrados apenas em clulas isoladas e nenhum estudo at a presente data de acordo com nosso conhecimento, avaliou se MeJA capaz de inibir in vivo a inflamao. O presente estudo, portanto, investigou a ao do MeJA sobre a inflamao e o estado oxidativo sistmicos em ratos com artrite induzida por adjuvante, uma imunopatologia em ratos que compartilha muitas caractersticas da artrite reumatide humana. Esses animais apresentam inflamao sistmica que afetam outros rgos alm das articulaes, como o fgado, que apresenta marcadas alteraes metablicas associadas a um pronunciado estresse oxidativo. O presente estudo tambm avaliou as aes do MeJA sobre produo de ROS e atividade respiratria em mitocndrias isoladas de fgado. Por fim, foi investigado tambm o efeito do MeJA sobre a atividade da glicoquinase heptica, a enzima que catalisa o primeiro passo limitante da gliclise, uma via que reportada estar mais acelerada no fgado artrtico. A induo de artrite foi realizada em ratos Holtzman com adjuvante de Freund. Os animais foram distribudos em sete grupos: controles (saudveis), os quais receberam leo de milho; controles tratados com MeJA 300 mgKg-1; ratos artrticos, os quais receberam leo de milho; ratos artrticos tratados com MeJA nas doses de 75, 150 e 300 mgKg-1; e artrticos tratados com ibuprofeno 30 mgKg-1. O tratamento foi feito por via oral (gavagem) uma vez ao dia durante 5 dias antes da induo da artrite e 18 dias depois. O volume das patas foi medido por pletismografia e as leses secundrias foram monitoradas entre os dias 10 e 18. Os leuccitos circulantes e os leuccitos recrutados na cavidade da articulao femorotibial foram adicionalmente quantificados nos ratos artrticos. No dia 19, a cavidade peritoneal dos ratos anestesiados foi exposta, o sangue coletado da veia cava e o fgado removido para preparar o homogenato e isolar mitocndrias. Os nveis de protenas carboniladas, TBARS e ROS foram medidos no homogenato para avaliar o estresse oxidativo heptico. O contedo de glutationa oxidada (GSSG) e reduzida (GSH) e as atividades das enzimas catalase, SOD, mieloperoxidase (MPO) e glicoquinase foram medidas no sobrenadante do homogenato. A capacidade antioxidante do plasma (FRAP), grupos tiol, albumina, protenas carboniladas e atividade das enzimas MPO, AST, ALT e fosfatase alcalina foram medidas no plasma. As mitocndrias foram isoladas por centrifugao diferencial e utilizadas para investigar a produo de ROS e atividade respiratria. A respirao mitocondrial foi medida na presena (estado III) e ausncia (basal e estado IV) de ADP e as atividades das enzimas NADH e succinato oxidases foram medidas em mitocndrias rompidas. A produo de ROS e a atividade respiratria foram medidas em mitocndrias hepticas incubadas com MeJA na faixa de at 10 mM e tambm nas mitocndrias isoladas de animais tratados com MeJA. Os ratos artrticos desenvolveram uma intensa resposta inflamatria ao adjuvante nas patas injetada e contralateral. Em adio, estes animais apresentaram menor ganho de peso corporal e manifestaes inflamatrias sistmicas, conforme foi evidenciado pela maior atividade da MPO plasmtica e heptica, pelos baixos nveis de albumina plasmtica, surgimento e persistncia de leses secundrias artrite e maior nmero de leuccitos no sangue e tambm recrutados para as cavidades articulares. O estresse oxidativo tambm foi pronunciado no plasma e no fgado de ratos artrticos, conforme evidenciado pelos maiores nveis de ROS, TBARS e protenas carboniladas no fgado, diminuio da atividade da catalase e da razo GSH/GSSG no fgado, pelos maiores nveis de protenas carboniladas no plasma e diminuio do FRAP e tiis no plasma. Os volumes das patas injetada e no injetada foram respectivamente 55% menores e no alterados nos ratos artrticos tratados com MeJA 300 mgKg-1, grupo no qual tambm houve uma diminuio do nmero de leuccitos no sangue e recrutados para as articulaes. No entanto, o escore de leses secundrias e os nveis de albumina plasmtica em ratos artrticos no foram modificados pelo tratamento com MeJA. As atividades da MPO plasmtica e heptica foram respectivamente 40 e 27% menores em ratos artrticos tratados com MeJA 300 mgKg-1. O tratamento de ratos controles e artrticos com MEJA no modificou as atividades da AST, ALT e fosfatase alcalina no plasma. O MeJA (150 e 300 mgKg-1) aboliu o aumento das protenas carboniladas no plasma e no fgado, o teor de ROS no fgado e diminuiu (apenas 300 mgKg-1) a razo GSH/GSSG e atividade da catalase no fgado de ratos artrticos. Alm disso, o MeJA aumentou os grupos tiis e FRAP plasmticos. O MeJA adicionado exogenamente (1,25-10 μM) estimulou a produo de ROS e inibiu a respirao em mitocndrias isoladas hepticas de ratos controles e artrticos, mas apenas a produo de ROS mitocondrial foi estimulada nos ratos tratados com MeJA (300 mgKg-1). As atividades da NADH e succinato oxidases foram inibidas em mitocndrias isoladas hepticas incubadas com MeJA e tambm em ratos artrticos e controles tratados com MeJA 300 mgKg-1. A atividade da glicoquinase heptica foi 60% maior em ratos artrticos (comparada com controles) e o tratamento de ratos controles e artrticos (todas as doses) com MeJA inibiu a atividade da glicoquinase em aproximadamente 60%.Os resultados foram comparados com tratamento controle positivo de ibuprofeno 30 mg Kg-1. O MeJA foi efetivo como anti-inflamatrio, em particular na dose de 300 mgKg-1, o qual foi capaz de diminuir o edema de pata, leucocitose e o recrutamento de leuccitos para as articulaes, aes que foram semelhantes ao do ibuprofeno. O mecanismo de ao do MeJA parece estar associado inibio da expresso de citocinas pr-inflamatrias induzidas por NFκB . O acentuado estresse oxidativo plasmtico em ratos artrticos ocorre como consequncia dos baixos nveis de albumina e tiis. Em relao ao fgado, o estresse oxidativo acentuado como consequncia de um impedido sistema antioxidante associado aumentada produo de ROS, ambos induzidos por citocinas pr-inflamatrias de forma direta e tambm indireta ao estimular o metabolismo oxidativo heptico. Em adio, a gliclise acelerada no fgado artrtico aumenta o fluxo de equivalentes redutores do citosol para as mitocndrias, tornando o citosol mais oxidado. O MeJA foi capaz de diminuir o estresse oxidativo no plasma e no fgado de ratos artrticos, um efeito que parece ser a consequncia da supresso do sistema pr-oxidante induzido por citocinas e de um aumento da atividade antioxidante, em particular a razo GSH/GSSG. Embora o MeJA tenha estimulado a produo mitocondrial de ROS em ratos artrticos e controles, o estresse oxidativo no se modificou no fgado, um fenmeno que pode estar associado a uma diminuio do fluxo atravs da gliclise devido inibio da glicoquinase. As doses efetivas de MeJA no presente estudo podem ser ainda relativamente altas, mas no tiveram efeitos txicos, o que faz do MeJA um ponto de partida importante para desenvolvimento de drogas anti-inflamatrias.

Abstract: Methyl jasmonate (MeJA) is a natural fatty acid-derived cyclopentanone which shares structural similarities with prostaglandins and therefore many studies have been carried out to evaluate their actions on mammal cells, specially the potential anti-inflammatory and anti-can-cer activities. MeJA was able to inhibit the proliferation in various murine and human cancer cell lines and the mitochondria seem to be the main target of MeJA action, which stimulates the production of reactive oxygen species (ROS), bind to and detach the mitochondria-bound hexo-kinase, ATP depletion and finally cell death. Regarding the anti-inflammatory action MeJA was able to inhibit NFκB -mediated production of nitric oxide, prostaglandin E, TNF-α, IL-1 and IL-6 in LPS-activated murine macrophages (RAW264.7). However, these effects have been demonstrated only in isolated cells and no study has until now evaluated if MeJA is able to inhibit in vivo the inflammation. The present study therefore investigated the action of MeJA on the systemic inflammation and oxidative status in rats with adjuvant-induced arthritis, an experimental pathology in rats that shares many features with the human rheumatoid arthritis. Arthritic animals present systemic inflammation and in addition to articular sites other organs are affected, such as liver, which presents marked metabolic alterations associated to a pro-nounced oxidative stress. The present study has also evaluated the actions of MeJA on ROS production and respiratory activity of hepatic isolated mitochondria. Finally, this study also investigated the effect of MeJA on hepatic hexokinase (glucokinase) activity, enzyme that cat-alyzes the first rate-limiting step of glycolysis, which is reported to be accelerated in the arthritic liver. The arthritis induction was performed in Holtzman rats with Freund's adjuvant. Animals were distributed into seven groups: controls (healthy), which received corn oil; controls treated with 300 mgKg-1 MeJA; arthritic rats, which received corn oil; arthritic rats treated with 75, 150 and 300 mgKg-1 MeJA; and arthritic rats treated with 30 mgKg-1 ibuprofen. The treatment was made orally (gavage) once a day for 5 days prior to arthritis induction and by additional 18 days after. Paw volume was measured by plethysmography and the score of secondary lesions was assessed from the 10th to 18th day. Circulating leukocytes and those recruited into the fem-orotibial joint cavities were further quantified in the arthritic rats. At day 19th, peritoneal cavity of anesthetized rats was exposed, the blood was collected from the cava vein and the liver was removed to perform mitochondria isolation and homogenate preparation. Protein carbonyl groups, TBARS and ROS were measured in the homogenate to evaluate the liver oxidative stress. Oxidized (GSSG) and reduced (GSH) glutathione contents and activity of the enzymes catalase, SOD, myeloperoxidase (MPO) and glucokinase were measured in the homogenate supernatant. The ferric reducing ability of plasma (FRAP), thiol groups, albumin, protein car-bonyl groups and the activities of MPO, AST, ALT and alkaline phosphatase were measured in the plasma. Mitochondria were isolated by differential centrifugation and used to measure real time ROS production and respiratory activity. Mitochondrial respiration was measured in the presence (state III) and absence (basal and state IV) of ADP and activities of the NADH and succinate oxidases were measured in disrupted mitochondria. ROS production and respiratory activity were measured in isolated hepatic mitochondria incubated with MeJA exogenously added in the range up to 10 mM and also in mitochondria isolated from animals treated with MeJA. Arthritic rats developed an intense inflammatory response to adjuvant in both injected and contralateral paws. Animals furthermore presented very low body weight gain and systemic inflammatory manifestations, as evidenced by higher plasma and liver MPO activity, low levels of plasma albumin, severe secondary lesions to arthritis and higher number of total leukocytes in the blood and also recruited into the femorotibial joint cavities. Oxidative stress was also pronounced in the plasma and liver of arthritic rats, as evidenced by higher levels of ROS, TBARS and protein carbonyl groups, as well as decreased activity of catalase and GSH/GSSG ratio in the liver, whereas higher levels of protein carbonyl groups and decreased FRAP and thiols were verified in the plasma. Injected and non-injected paw volumes were respectively 55% lower and not altered in arthritic rats treated with 300 mgKg-1 MeJA, which also decreased the number of total leukocytes in the blood and recruited into the femorotibial joints. However, score of secondary lesions and plasma albumin levels in arthritic rats were not modified by MeJA treatment. MPO activities in the plasma and livers were respectively 40 and 27% lower in arthritic rats treated with 300 mgKg-1 MeJA. Treatment of control and arthritic rats with MEJA did not modify the AST, ALT and phosphatase alkaline activities in the plasma. MeJA (150 and 300 mgKg-1) abolished the increase of carbonylated proteins in the plasma and liver, and ROS content in the liver, and decreased (only 300 mgKg-1) the hepatic GSH/GSSG ratio and catalase activity in arthritic rats. In addition, MeJA increased FRAP and thiols groups in the plasma. MeJA exogenously added (1.25-10 mM) stimulated ROS production and inhibited respiration in isolated hepatic mitochondria of control and arthritic rats, but only mitochondrial ROS production was stimulated when rats were treated with MeJA (300 mgKg-1). The activi-ties of NADH and succinate oxidases were inhibited in isolated hepatic mitochondria by MeJA exogenously added and also in arthritic and control rats treated with 300 mgKg-1 MeJA. He-patic glucokinase activity was 60% higher in arthritic rats (compared to the controls) and treat-ment of control (only 300 mgKg-1) and arthritic rats with MeJA (all doses) inhibited the glu-cokinase activity by approximately 60%. MeJA was effective as an anti-inflammatory agent, particularly at the dose of 300 mgKg-1, which was able to decrease the paw edema, leukocytosis and leukocytes recruitment into the femorotibial joints, actions that were similar to ibuprofen, a classical anti-inflammatory drug. The mechanism of MeJA action seems to be associated with inhibition of NFκB -induced ex-pression and release of cytokines. Pronounced oxidative stress in the plasma of arthritic rats occurs as consequence of low levels of albumin and thiol groups. Only this last was improved by the remission of inflammation, but it was enough to decrease the plasma oxidative stress. Regarding the liver, oxidative stress is more pronounced as consequence of an impaired ROS scavenging system associated with increased production of ROS, which are induced by proin-flammatory cytokines directly and indirectly by stimulating the liver oxidative metabolism. In fact, the accelerated glycolysis in the liver of arthritic rats lead to an increased flux of reducing equivalents from the cytosol to mitochondria, making the cytosol more oxidized. MeJA was able to diminish the oxidative stress in the liver of arthritic rats, an effect that seems to be the consequence of both a suppression of the cytokine-induced pro-oxidant system and an improve-ment of the antioxidant defense, particularly the GSH/GSSG ratio. Although MeJA has also stimulated production of ROS in the liver mitochondria of arthritic rats, oxidative stress was not modified in the organ, a phenomenon that could be associated to a diminished flux through the glycolysis due to glucokinase inhibition. The effective doses of MeJA in the present study may be still relatively high, but they had no toxic effects and make this compound a potentially important starting point for anti-inflammatory and anti-rheumatic drugs development.
Data da defesa: 11/08/2017
Cdigo: vtls000227092
Informaes adicionais:
Idioma: mul
Data de Publicao: 2017
Local de Publicao: Maring, PR
Orientador: Prof. Dr. Jurandir Fernando Comar
Co-Orientador: Prof. Dr. Anacharis Babeto de S Nakanishi
Instituio: Universidade Estadual de Maring . Centro de Cincias Biolgicas
Nvel: Dissertao (mestrado em Cincias Biolgicas)
UEM: Programa de Ps-Graduao em Cincias Biolgicas

Responsavel: edson
Categoria: Aplicao
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Arquivo: Dissertao Jamil 2c.pdf
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