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In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats

This study examines the activity of the antioxidant N‐acetylcysteine on bleomycin‐induced pulmonary fibrosis in rats with emphasis on the early inflammatory phase. Rats receiving N‐acetylcysteine (300 mg kg−1 day−1, intraperitoneal) had less augmented lung wet weight, and lower levels of proteins, lactate dehydrogenase, neutrophil and macrophage counts in bronchoalveolar lavage fluid and lung myeloperoxidase activity with a betterment of histological score at 3 days postbleomycin. A diminished lung GSH/GSSG ratio and augmented lipid hydroperoxides were observed 3 days postbleomycin. These changes were attenuated by N‐acetylcysteine. Alveolar macrophages from bleomycin‐exposed rats released augmented amounts of superoxide anion and nitric oxide. N‐Acetylcysteine did not modify superoxide anion generation but reduced the increased production of nitric oxide. N‐Acetylcysteine suppressed the bleomycin‐induced increased activation of lung NF‐κB (shift assay and immunohistochemistry), and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor‐α, interleukin‐β, interleukin‐6 and macrophage inflammatory protein‐2 observed in bronchoalveolar lavage fluid at 1 and 3 days postbleomycin exposure. At 15 days postbleomycin, N‐acetylcysteine decreased collagen deposition in bleomycin‐exposed rats (hydroxyproline content: 6351±669 and 4626±288 μg per lung in drug vehicle‐ and N‐acetylcysteine‐treated rats, respectively; P<0.05). Semiquantitative histological assessment at this stage showed less collagen deposition in N‐acetylcysteine‐treated rats compared to those receiving bleomycin alone. These results indicate that N‐acetylcysteine reduces the primary inflammatory events, thus preventing cellular damage and the subsequent development of pulmonary fibrosis in the bleomycin rat model.

Ischemic preconditioning: A defense mechanism against the reactive oxygen species generated after hepatic ischemia reperfusion

Background. Preconditioning protects against both liver and lung damage after hepatic ischemia-reperfusion (I/R). Xanthine and xanthine oxidase (XOD) may contribute to the development of hepatic I/R. Objective. To evaluate whether preconditioning could modulate the injurious effects of xanthine/XOD on the liver and lung after hepatic I/R. Methods. Hepatic I/R or preconditioning previous to I/R was induced in rats. Xanthine and xanthine dehydrogenase/xanthine oxidase (XDH/XOD) in liver and plasma were measured. Hepatic injury and inflammatory response in the lung was evaluated. Results. Preconditioning reduced xanthine accumulation and conversion of XDH to XOD in liver during sustained ischemia. This could reduce the generation of reactive oxygen species (ROS) from XOD, and therefore, attenuate hepatic I/R injury. Inhibition of XOD prevented postischemic ROS generation and hepatic injury. Administration of xanthine and XOD to preconditioned rats led to hepatic MDA and transaminase levels similar to those found after hepatic I/R. Preconditioning, resulting in low circulating levels of xanthine and XOD activity, reduced neutrophil accumulation, oxidative stress, and microvascular disorders seen in lung after hepatic I/R. Inhibition of XOD attenuated the inflammatory damage in lung after hepatic I/R. Administration of xanthine and XOD abolished the benefits of preconditioning on lung damage. Conclusions. Preconditioning, by blocking the xanthine/XOD pathway for ROS generation, would confer protection against the liver and lung injuries induced by hepatic I/R.

Differential activity of nitric oxide synthase in human nasal mucosa and polyps

Nitric oxide (NO) plays an important regulatory role in airway function and seems to be implicated in the pathophysiology of several airway diseases. To better understand the involvement of NO in the upper airways, we examined the presence of nitric oxide synthase (NOS) activity in human nasal mucosa and nasal polyp tissues. Nasal mucosa was obtained from seven patients undergoing septoplasty, and nasal polyps came from nine patients following polypectomy. NOS activity was quantified in tissue homogenates using the citrulline release assay and localized in tissue sections using reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. The results showed that nasal polyps (n = 9) contained higher levels of total NOS activity (mean +/- SD 5.94 +/- 5.71, range 1.29-18.0 pmol.min-1.mg protein) than nasal mucosa tissues (n = 7) (0.28 +/- 0.22, range 0.01-0.57 pmol.min-1.mg protein). In addition, nasal polyps mainly contained inducible NOS activity (4.67 +/- 4.57, range 1.23-15.5 pmol.min-1.mg protein) whereas in nasal mucosa all NOS activity detected was in constitutive form. In both cases, NOS activity was localized in the epithelial cells. Since NO synthase is induced in inflamed upper airways, we conclude that NO may be an important inflammatory mediator in the respiratory system and that the epithelium may be a source of NO production in the human upper airways.

Systemic prostacyclin in cirrhotic patients: Relationship with portal hypertension and changes after intestinal decontamination

The total body production of prostacyclin was shown to be increased in cirrhotic patients, suggesting that its synthesis by blood vessels of the systemic circulation is enhanced. However, the mechanism by which the synthesis of systemic prostacyclin is stimulated is not known. The present study investigated the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha, an index of total body prostacyclin synthesis, first, in cirrhotics with portal hypertension (n = 19) as compared with cirrhotics with reduced portal pressure after portacaval shunt surgery (n = 18) and with control noncirrhotic subjects (n = 11), and; second, in cirrhotics before and after intestinal decontamination by oral nonabsorbable antibiotics (n = 9 antibiotic treated patients, n = 10 control nontreated cirrhotics). Control noncirrhotic subjects showed lower urinary excretion of 2,3-dinor-6-keto-PGF1 alpha than both groups of cirrhotics (P less than 0.001). Interestingly, urinary excretion of 2,3-dinor-6-keto-PGF1 alpha was significantly higher in cirrhotics with portacaval shunt than in those with portal hypertension (P less than 0.01). The urinary excretion of 2,3-dinor-6-keto-PGF1 alpha decreased significantly after intestinal decontamination in the antibiotic-treated group (580.1 +/- 232.4 vs. 431.2 +/- 219.2 pg/mg creatinine; P less than 0.05) but not in nontreated patients (543.9 +/- 214.4 vs. 581.2 +/- 281.4 pg/mg creatinine; P = NS). These data suggest that the increased urinary excretion of 2,3-dinor-6-keto-PGF1 alpha observed in cirrhotics is not directly related to portal hypertension itself but to portal blood factors that bypass the liver. Some such factors may be of intestinal bacterial origin.

P-selectin upregulation in bleomycin induced lung injury in rats: effect of N-acetyl-L-cysteine

Background: A number of adhesion molecules are involved in the process of neutrophil infiltration into the lung. P-selectin is one of these neutrophil-endothelial cell adhesion molecules. A study was undertaken to examine the involvement of P-selectin in the development of bleomycin induced inflammation and the ability of N-acetyl-l-cysteine to reduce the potential expression of this selectin in rats. Methods:N-acetyl-l-cysteine (3 mmol/kg po) was administered daily for seven days prior to bleomycin administration (2.5 U/kg). The kinetics of P-selectin expression and the effect of N-acetyl-l-cysteine after bleomycin treatment were measured using radiolabelled antibodies. P-selectin localisation was evaluated by immunohistochemistry and neutrophil infiltration was assessed by myeloperoxidase activity. Results: Bleomycin administration resulted in an upregulation of P-selectin at 1 hour, returning to baseline at 3 hours. Myeloperoxidase activity showed a significant increase at 6 hours after bleomycin administration that lasted for 3 days. N-acetyl-l-cysteine treatment completely prevented these increases. Conclusion: Upregulation of P-selectin in the lung is associated with neutrophil recruitment in response to bleomycin. The beneficial effect of N-acetyl-l-cysteine on bleomycin induced lung injury may be explained in part by the prevention of neutrophil recruitment in the inflammatory stage of the disease.

Heparin mobilizes xanthine oxidase and induces lung inflammation in acute pancreatitis

ObjectiveTo evaluate the effect of low molecular weight heparin on plasma xanthine oxidase concentrations and lung inflammatory response during acute pancreatitis. DesignRandomized, controlled trial. SettingExperimental laboratory. SubjectsMale Wistar rats. InterventionsAcute pancreatitis was induced by intraductal administration of 5% sodium taurocholate. Low molecular weight heparin (0, 30, 90, or 300 units/kg) was administered immediately after induction of pancreatitis. Measurements and Main ResultsLipase and xanthine oxidase plasma concentrations were measured 3 hrs after pancreatitis induction. Expression of P-selectin messenger RNA and myeloperoxidase activity as a marker of neutrophil infiltration were determined in the lung. An increase in xanthine oxidase plasma concentrations was observed during pancreatitis. Administration of heparin also increased plasma xanthine oxidase activity in both control and pancreatitis animals. Measures of xanthine oxidase present in the endothelial surface indicate that during pancreatitis, the enzyme is released from the gastrointestinal endothelium. By contrast, heparin mobilizes xanthine oxidase from almost all organs evaluated. Neutrophil infiltration was increased in the lung during pancreatitis. Heparin administration further increased, in a dose-dependent manner, myeloperoxidase activity and P-selectin expression in the lung in animals with pancreatitis. By contrast, in control animals, heparin had no effect on myeloperoxidase activity and did not induce P-selectin up-regulation. ConclusionDuring acute pancreatitis, heparin administration might mobilize xanthine oxidase attached to endothelial cells, originating a free radical-generating system in the circulation that would trigger an inflammatory response in the lung.

Oxidised lipids present in ascitic fluid interfere with the regulation of the macrophages during acute pancreatitis, promoting an exacerbation of the inflammatory response

Background: Pancreatitis-associated ascitic fluid (PAAF) plays a critical role in the pathogenesis of acute pancreatitis. Taking into consideration that damaged pancreas exudes high concentrations of lipolytic enzymes in the peritoneal cavity, large amounts of lipid metabolism derived products could occur in PAAF. In this study, we have examined the involvement of the lipid fraction of PAAF generated in the early stages of experimental acute pancreatitis. Methods: Pancreatitis was induced in rats by intraductal administration of 5% sodium taurocholate. After 3 h, PAAF was collected and its lipid fraction was obtained. Lipid composition and levels of lipid peroxidation were measured. Toxicity was evaluated by measuring the effects of the PAAF lipid fraction on cell viability of hepatic and macrophage cell lines. In vivo effects on the liver were also evaluated. Effects on the inflammatory response were determined by measuring the levels of nuclear factor kappa B (NFκB) activation, the effect on the inhibitory activity of 15-deoxy-PGJ2 and the possible interference on PPARγ activation. Results: High concentrations of oxidised free fatty acids were detected in PAAF. Besides the direct cell toxicity, the PAAF-derived lipid extract interfered with the anti-inflammatory pathway mediated by PPARγ. Addition of this lipid extract to macrophage cell cultures had no direct effect on the activation of NFκB, but abolished the inhibitory activity of endogenous PPARγ agonists such as 15-deoxy-PGJ2. Conclusions: Oxidised free fatty acids present in PAAF interfere with the endogenous regulatory mechanism of the inflammatory response, thus promoting an exacerbation of macrophage activation in acute pancreatitis.

Strategies to modulate the deleterious effects of endothelin in hepatic ischemia-reperfusion.

Background. This study evaluates whether bosentan (endothelin [ET] receptor antagonist) or preconditioning (mechanism that inhibits the postischemic ET release) could reduce the microvascular disorders and the injurious effects of tumor necrosis factor (TNF) associated with hepatic ischemia-reperfusion (I/R). Methods. Hepatic I/R was induced in rats and the effects of bosentan or preconditioning on the deleterious effects of ET in hepatic I/R were evaluated. Transaminase and TNF levels in plasma; edema, vascular permeability, lactate, ET, and TNF levels in liver; and edema and myeloperoxidase activity levels in lung were measured after hepatic reperfusion. Results. The administration of bosentan or the induction of preconditioning previous to I/R attenuated the increase in vascular permeability, edema and lactate levels observed in liver after I/R. However, the addition of ET before preconditioning abolished its benefits. Preconditioning prevented both the increase in hepatic TNF and its release from the liver into the systemic circulation. This resulted in an attenuation of liver and lung damage. Addition of ET or TNF to the preconditioned group abolished the benefits of preconditioning, whereas the previous inhibition of TNF release with GdCl3 in the preconditioned group pretreated with ET did not modify the effects of preconditioning. The inhibition of ET with bosentan prevented the increase of both hepatic and plasma TNF, thus attenuating the liver and lung injury, whereas TNF addition abolished the benefits of bosentan. Conclusions. These findings suggest that both bosentan and preconditioning, by inhibition of ET could attenuate the microvascular disorders and the deleterious effect of TNF on the liver and lung elicited by hepatic I/R.

Effect of Subcutaneous Administration of Octreotide on Endogenous Vasoactive Systems and Renal Function in Cirrhotic Patients with Ascites

Splanchnic and systemic arteriolar vasodilationplays an important role in ascites formation incirrhosis. Octreotide produces splanchnicvasoconstriction, but the effects on systemichemodynamics and renal function are controversial. This studyevaluated the effect of subcutaneous octreotideadministration on systemic hemodynamics, endogenousvasoactive systems, and renal function in cirrhoticpatients with ascites. Twenty patients were included: 10received octreotide 250 μg/12 hr subcutaneously (forfive days), and 10 did not. No statistically significantchanges were found in mean arterial pressure and cardiac rate. Octreotide induced astatistically significant decrease in plasma reninactivity (P < 0.01), plasma aldosterone (P = 0.01)and plasma glucagon (P < 0.05). No significantvariations were observed in other systemic vasoactivesubstances (nitric oxide and prostacyclin). Renalfunction was not modified in either group. Inconclusion, in cirrhotic patients with ascites,subcutaneous octreotide administration decreases plasma glucagon, reninactivity, and aldosterone without changing in systemichemodynamics or renal function.

Mobilization of xanthine oxidase from the gastrointestinal tract in acute pancreatitis

BackgroundXanthine oxidoreductase has been proposed to play a role in the development of local and systemic effects of acute pancreatitis. Under physiologic conditions, the enzyme exists mainly as xanthine dehydrogenase (XDH) but can be converted by proteolytic cleavage to its superoxide-generating form xanthine oxidase (XOD). In addition to its intracellular location XDH/XOD is also associated to the polysaccharide chains of proteoglycans on the external endothelial cell membrane.In the early stages of acute pancreatitis, this enzyme seems to be arising from its mobilization from the gastrointestinal endothelial cell surface. Taking into account the ability of α-amylase to hydrolyze the internal α-1,4 linkages of polysaccharides, we wanted to elucidate the involvement of α-amylase in XDH/XOD mobilization from the gastrointestinal endothelial cell surface and the relevance of the ascitic fluid (AF) as the source of α-amylase in experimental acute pancreatitis.MethodsAcute pancreatitis was induced in male Wistar rats by intraductal administration of 5% sodium taurocholate. In another experimental group 3000 U/Kg α-amylase was i.v. administered. The concentrations of XDH, XOD and α-amylase in plasma and AF and myeloperoxidase (MPO) in lung have been evaluated. In additional experiments, the effect of peritoneal lavage and the absorption of α-amylase present in the AF by an isolated intestine have been determined.ResultsSimilar increase in XDH+XOD activity in plasma was observed after induction of acute pancreatitis and after i.v. administration of α-amylase. Nevertheless, the conversion from XDH to XOD was only observed in the pancreatitis group. Lung inflammation measured as MPO activity was observed only in the pancreatitis group. In addition peritoneal lavage prevented the increase in α-amylase and XDH+XOD in plasma after induction of pancreatitis. Finally, it was observed that α-amylase is absorbed from the AF by the intestine.ConclusionsDuring the early stages of acute pancreatitis, α-amylase absorbed from AF through the gastrointestinal tract could interfere with the binding of XDH/XOD attached to glycoproteins of the endothelial cells. Proteolytic enzymes convert XDH into its oxidase form promoting an increase in circulating XOD that has been reported to be one of the mechanisms involved in the triggering of the systemic inflammatory process.