RESUMO
We have shown previously that rats subjected to tourniquet shock develop an acute form of remote organ injury of the liver that is both Kupffer cell (KC) and polymorphonuclear (PMN) leukocyte dependent. Circulating plasma xanthine oxidase (XO) has been shown to be responsible for the development of endothelial dysfunction and for remote organ injury of the lung and intestine after ischemia-reperfusion protocols. We now hypothesize that XO is released from rat hind limbs upon reperfusion and that it is responsible for KC and PMN leukocyte activation in this shock model. Our results show that about 30% of rat gastrocnemius muscle xanthine dehydrogenase (XD) is converted to XO during the 5-h tourniquet period and that it is released into the femoral vein within 10 min of reperfusion. Total muscle xanthine oxidoreductase activity (XO + XD) decreases within 30 min of reperfusion and is paralleled by a corresponding increase in femoral vein lactic dehydrogenase. In addition, liver tissue XO increases significantly within 30 min of reperfusion without a corresponding conversion of endogenous XD. Conversion of hepatic XD becomes evident 60 min after reperfusion is initiated, as does XO, and alanine aminotransferase (ALT) release into the hepatic vein, presumably from damaged hepatocytes as a consequence of oxidative stress. Tissue myeloperoxidase activity also increases significantly after the 60-min reperfusion period. That XO mediates KC and PMN activation is supported by the following observations: a) the close relationships between plasma XO and the time courses of tumor necrosis factor-alpha TNFalpha release into the hepatic vein and colloidal carbon clearance by KCs; b) that colloidal carbon clearance, TNFalpha and ALT release, loss of tissue free thiols, lipid peroxidation (TBARS), and liver infiltration by PMN neutrophils can also be induced by the administration of exogenous XO to normal rats; and c) pretreatment of rats with allopurinol inhibits KC activation and liver leukocyte infiltration. These results suggest that XO, released from the ischemic limb on reperfusion, is taken up by the liver were it mediates KC and PMN neutrophil activation and thus contributes to the development of multiple system organ failure after hind limb reperfusion.
Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Células de Kupffer/fisiologia , Fígado/fisiopatologia , Neutrófilos/fisiologia , Estresse Oxidativo/fisiologia , Choque/fisiopatologia , Xantina Oxidase/metabolismo , Alanina Transaminase/sangue , Animais , Feminino , L-Lactato Desidrogenase/sangue , Fígado/fisiologia , Ativação de Macrófagos , Músculo Esquelético/fisiopatologia , Ratos , Ratos Sprague-Dawley , Reperfusão , Fatores de Tempo , Torniquetes , Xantina Desidrogenase/metabolismoRESUMO
Küpffer cells (KCs) have been implicated in leukocyte recruitment and microvascular dysfunction associated with liver inflammation. The overall objective of this study was to assess the role of KCs and polymorphonuclear (PMN) leukocytes on the oxidative stress elicited in the liver as a consequence of hind limb reperfusion in rats subjected to tourniquet shock, a shock model that differs from other models in that hepatic injury is a consequence of remote organ damage. Colloidal carbon clearance from blood and its incorporation into KCs demonstrate that these cells are activated after the 2 h hind limb reperfusion period and that they are responsible for the observed oxidative stress and for PMN leukocyte recruitment and activation. Liver oxidative stress in this model is evidenced by increased liver tissue GSSG/GSH ratio, thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, myeloperoxidase (MPO) activity, an index of tissue-associated neutrophil accumulation, and a significant loss in total tissue superoxide dismutase (SOD) activity. Mean arterial blood pressure (MAP), as well as plasma levels of alanine aminotransferase (ALT), an index of hepatic tissue injury, total SOD activity, plasma levels of alpha-tocopherol and beta-carotene, and total plasma nitrite are also affected as a consequence of KC activation after the 2 h hind limb reperfusion period. Inhibition of KC activity by gadolinium chloride (GdCl3) reverted most of the above alterations to values that do no differ from those found in control animals. These results support the hypothesis that hepatic and systemic oxidative stress elicited by hind limb reperfusion in rats subjected to tourniquet shock is both KC and PMN leukocyte dependent.
Assuntos
Células de Kupffer/metabolismo , Fígado/metabolismo , Neutrófilos/metabolismo , Estresse Oxidativo , Choque/fisiopatologia , Alanina Transaminase/metabolismo , Animais , Carbono/metabolismo , Feminino , Gadolínio/farmacologia , Glutationa/metabolismo , Malondialdeído/metabolismo , Ativação de Neutrófilo , Nitritos/sangue , Peroxidase/metabolismo , Pressão , Ratos , Ratos Sprague-Dawley , Reperfusão , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Torniquetes , Vitamina E/sangue , beta Caroteno/sangueRESUMO
The role of nitric oxide (NO) on liver oxidative stress and tissue injury in rats subjected to tourniquet shock was investigated. This shock model differs from others in that injury is a consequence of remote organ damage. Liver oxidative stress becomes evident after hind limb reperfusion, as evidenced by the loss of total tissue thiols; by increases in tissue oxidized glutathione (GSSG), lipid peroxidation (LPO), plasma aminotransferases (alanine aminotransferase (ALT) and (aspartate aminotransferase (AST)), and plasma nitrites; and by a 36% loss in total superoxide dismutase (SOD) activity. Portal blood flow is reduced by 54.1% after 2 h of hind limb reperfusion. Inhibition of NO synthesis with Nomega-nitro-L-arginine methyl ester or L-arginine methyl ester increased mean arterial blood pressure; further reduced portal blood flow; and aggravated liver injury as assessed by further loss in total thiols, increased LPO and GSSG content, and further increases in plasma ALT and AST. Total plasma nitrites were lower than in control animals, and total tissue SOD activity decreased by more than 80%. Treatment with the NO donor sodium nitroprusside reverted the decrease in portal blood flow and also reverted tissue thiol loss, LPO, and GSSG increases, as well as the loss of ALT and AST to plasma and of SOD activity to levels comparable to untreated control shock animals. As expected, plasma nitrites were greater than in tourniquet control animals. These data support the hypothesis that endogenous NO formation protects the rat liver from the consequences of oxidative stress elicited by hind limb reperfusion in rats subjected to tourniquet shock.
Assuntos
Glutationa/metabolismo , Fígado/fisiopatologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Estresse Oxidativo , Choque Traumático/fisiopatologia , Superóxido Dismutase/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Pressão Sanguínea , Cisteína/metabolismo , Feminino , Dissulfeto de Glutationa/metabolismo , Peroxidação de Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Malondialdeído/análise , Nitroprussiato/farmacologia , Ratos , Ratos Sprague-Dawley , Reperfusão , Choque Traumático/metabolismo , Superóxido Dismutase/antagonistas & inibidores , TorniquetesRESUMO
Rats subjected to tourniquet shock suffer a severe form of circulatory shock, tissue and organ oxidative stress, and final multiple system organ failure (MSOF) and death of the animals within 24 h of tourniquet release. The oxidative damage observed in hind-limb muscle tissue after reperfusion does not by itself account for the final systemic and lethal MSOF. We have postulated that organ failure has its genesis in a primary perfusion abnormality, e.g. the hind limbs, which is followed by secondary hypoperfusion of other organs, such as the liver, as has been shown to be the case in several septic shock models. It has also been shown that injured or necrotic tissue can activate neutrophils, Küpffer cells, platelets, and both the complement and coagulation cascades. In turn, complement activation also leads to neutrophil and Küpffer cell activation as assessed by their capacity to generate oxyradicals. Herein we have evaluated the potential protective effect of ibuprofen on hepatic oxygen-derived free radical production, as well as its effects on both polymorphonuclear leucocyte (PMN) activation and liver infiltration. The protective effect of ibuprofen on hepatic oxidative injury was assessed by determining total thiol groups (SH), thiobarbituric acid-reactive substances (TBARS), and by the release of aspartic acid (AsT) and alanine (AIT) aminotransferases in control animals, in animals subjected to 5 h of tourniquets, and in animals after 2 h of hind-limb reperfusion. Liver infiltration by PMNs was determined by histology after staining with eosin-hematoxylin, and PMN activation by their capacity to reduce nitro blue tetrazolium (NBT).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ibuprofeno/uso terapêutico , Hepatopatias/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Choque Traumático/metabolismo , Alanina Transaminase/sangue , Animais , Doença Hepática Induzida por Substâncias e Drogas , Estudos de Avaliação como Assunto , Radicais Livres , Hepatopatias/metabolismo , Masculino , Ativação de Neutrófilo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/metabolismo , Choque Traumático/complicações , Compostos de Sulfidrila/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , TorniquetesRESUMO
The placement of rubber band tourniquets upon rat hind-limbs for 5 h followed by reperfusion of the extremities results in a severe form of circulatory shock characterized by hypotension and death within 24 h of tourniquet release. Oxidative damage to muscle tissue is an early consequence of hind-limb reperfusion on tourniquet release, yet this local damage does not explain the lethal hypotensive shock state which evolves within the next 24 h. Multiple system organ failure (MSOF), of as of yet unknown causes, is usually described in relation to several shock states. It has been suggested that injured or necrotic tissue may activate neutrophils, platelets, and the coagulation system leading to embolization in remote tissues. Effective decreases in hepatic blood flow have been observed in several forms of sepsis which precedes the biochemical evidence consistent with an ischemic insult of the liver. In support of our original hypothesis, that organ failure has its genesis in a primary perfusion abnormality with secondary ischemic organ injury, herein we have assessed the possibility that oxygen-derived free radicals are generated in the liver of rats after reperfusion of their hind-limbs on release of the tourniquets. We report on the protective effects of allopurinol (ALLO) and a mixture of superoxide dismutase (SOD) catalase (CAT) and dimethylsulfoxide (DMSO) on liver free sulfhydryl content (SH), thiobarbituric acid-reactive substances (TBARS), and on the release of aspartic acid (AsT) and alanine aminotransferase (AlT) activities, and of alkaline phosphatase during a 5 h tourniquet period and after 2 h of reperfusion of the hind-limbs. During the hind-limb ischemic period hepatis tissue SH levels remained essentially constant during the first hour (6.02 +/- 0.36 to 5.65 +/- 0.20 mumoles/g wet tissue), and decreased significantly, over and above the normal circadian decrease of liver glutathione levels, to 4.02 +/- 0.69 mumoles/g wet tissue after the third hour and remained lowered until tourniquet release. A further significant decrease (3.11 +/- 0.49 mumoles/g wet tissue) was observed after 2h of reperfusion. TBARS production remained constant during the 5 h hind-limb ischemic period (168.4 +/- 37.3 mumoles/g wet tissue) and rose by 55% to 261.7 +/- 55.8 mumoles/g wet tissue after 2 h of tourniquet release. ALLO, but not the SOD-CAT-DMSO combination, protected hepatic SH loss during the hind-limb ischemic insult, yet both offered protection after 2 h of tourniquet release.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Hepatopatias/etiologia , Oxigênio/metabolismo , Traumatismo por Reperfusão/complicações , Choque/complicações , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Feminino , Radicais Livres , Membro Posterior/irrigação sanguínea , Isquemia , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/metabolismo , Tiobarbitúricos , TorniquetesRESUMO
To assess the possible role of oxygen-derived free radical in the pathogenesis of cutaneous necrotizing vasculitis (Thomas reaction) induced by epinephrine in endotoxin-primed rabbits, animals were treated with superoxide dismutase (intraperitoneal or local), catalase, allopurinol, or ibuprofen 2 h before inducing the reaction. This is done by an earlobe intravenous injection (10 micrograms) of endotoxin followed by an intradermal injection (100 micrograms) of epinephrine in the shaved abdomen; 18-24 h later, a hemorrhagic-necrotic lesion can be observed at the site of the epinephrine injection. The reaction was completely inhibited by superoxide dismutase, catalase, and ibuprofen, while allopurinol afforded only partial protection. Thus, oxyradicals seem to participate in the pathogenesis of this reaction.
Assuntos
Endotoxinas/toxicidade , Epinefrina/toxicidade , Escherichia coli , Oxigênio/metabolismo , Vasculite/etiologia , Animais , Catalase/farmacologia , Feminino , Radicais Livres , Lipopolissacarídeos/toxicidade , Masculino , Necrose , Coelhos , Pele/patologia , Superóxido Dismutase/farmacologiaRESUMO
1. Three zymogens have been isolated from human gastric mucosae and two from the stomachs of the toad Caudiverbera caudiverbera. 2. Human zymogens I and III were immunologically related and cross-reacted with antisera prepared against porcine pepsinogen. The third, (II), showed no cross-reactivity. 3. Human zymogens I and III and toad zymogen ZII gave rise to two human pepsins and to a pepsin-like enzyme, respectively. 4. Human zymogen II (gastricsinogen) and toad zymogen ZI gave rise to human gastricsin and to a gastricsin-like enzyme respectively. 5. The toad enzymes showed much greater stability at neutral and alkaline pH values than the human enzymes.