Cytosolic phospholipase A2 inhibition attenuates ischemia-reperfusion injury in an isolated rat lung model

BACKGROUND: Arachidonic acid metabolites and platelet-activating factor (PAF) are potentially involved in ischemia-reperfusion (IR) lung injury. A key enzyme regulating their metabolism is cytosolic phospholipase A2 (cPLA2). Arachidonyl trifluoromethyl ketone (AACOCF3) is reported to be a potent cPLA2 inhibitor. In the present study, we hypothesized that pharmacological inhibition of cPLA2 might ameliorate IR lung injury. METHODS: To test the hypothesis, we examined the effects of AACOCF3 in an isolated rat lung model. Three groups were defined (n=6, each): in the vehicle group, lungs were perfused for 2 hours without an ischemic period. In the ischemic groups, 20 mg/kg of AACOCF3 (AACOCF3 group) or saline (control group) was i.v. administered 15 min before lung harvest. Lungs were flushed with LPD solution, cold-stored 18 hours, and reperfused for 2 hours. RESULTS: IR increased cPLA2 activity mainly via alveolar macrophages, sPLA2 activity, thromboxane and leukotriene formation, and the expression of PAF receptor, whereas AACOCF3 treatment significantly reduced all of these. Compared to the vehicle group, the wet-to-dry ratio, proteins in BAL, and MPO activity increased significantly by twofold, fourfold, and threefold, respectively. Furthermore, the PO2 dropped from 615.7+/-31.2 to 452.1+/-30.9 mmHg at the end of reperfusion (P<0.001). AACOCF3 treatment maintained the PO2 at a level similar to the vehicle group throughout reperfusion and reduced significantly the alveolar-capillary leakage, edema formation, and neutrophil extravasation. CONCLUSION: Pharmacological inhibition of the cPLA2 cascade decreases bioactive lipid formation and attenuates IR-induced lung injury.     

Evidence for neutrophil-related acute lung injury after intestinal ischemia-reperfusion

Intestinal ischemia-reperfusion injury is a common and important clinical event associated with the activation of an endogenous inflammatory response. Some of the mediators of this response may be involved in the pathogenesis of multiple organ system failure. The purpose of this study was to determine whether remote organ dysfunction--specifically, acute lung injury--occurs after intestinal ischemia-reperfusion injury. After an ischemia-reperfusion event in rat intestine, whole lungs were obtained for measurement of tissue adenosine triphosphate (ATP) and myeloperoxidase values, and evaluation of histologic condition. In addition, lung microvascular permeability was assessed by determination of the rate at which iodine 125-labeled bovine serum albumin sequestration in the extravascular compartment occurred. Lung tissue ATP levels were no different in sham-operated animals than in those that had undergone 120 minutes of intestinal ischemia. Within 15 minutes of gut reperfusion, however, lung ATP decreased from 3.82 +/- 0.27 to 1.53 +/- 0.90 x 10(-7) moles/50 mg tissue, p less than 0.05. Neutrophil accumulation in the lungs, estimated by tissue myeloperoxidase determination, increased sevenfold (0.13 +/- 0.02 to 0.97 +/- 0.25 units/gm, p less than 0.05) after 120 minutes of ischemia and 15 minutes of reperfusion. Lung microvascular permeability increased threefold after 120 minutes of intestinal ischemia and 120 minutes of reperfusion (0.10 +/- 0.01 vs. 0.35 +/- 0.05 [lung/blood counts per minute], p less than 0.05). Intestinal ischemia followed by reperfusion is associated with acute lung injury characterized by increased microvascular permeability, histologic evidence of alveolar capillary endothelial cell injury, reduced lung tissue ATP levels, and the pulmonary sequestration of neutrophils. These data confirm an acute lung injury associated with intestinal ischemia-reperfusion and suggest a possible pathogenic role for the neutrophil.     

异丙酚对大鼠肠缺血再灌注后肺损伤的影响

目的探讨异丙酚对大鼠肠缺血再灌注后肺损伤的影响。方法32只成年SD大鼠,随机分为4组（n=8）,缺血再灌注组（I/R组）缺血1 h,再灌注2 h;异丙酚1组（P1组）在缺血前10 min、异丙酚2组（P2组）在再灌注前10min静脉注射异丙酚10mg,kg,然后以10mg·kg^-1·h^-1持续输注,余处理同I/R组;假手术组（C组）不行缺血再灌注及异丙酚输注。所有大鼠在再灌注120 min时处死。光镜下观察肺组织形态学及细胞凋亡;测定肺组织超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量及含水量。结果I/R组光镜下可见大量肺泡塌陷、实变,肺实质水肿及中性粒细胞浸润聚集。与C组比较,I/R组及P2组肺组织细胞凋亡计数增加,I/R组肺组织SOD活性降低,MDA含量升高,含水量升高（P＜0．05或0．01）;与I/R组比较,P1组SOD活性升高,MDA含量降低（P＜0．01）。结论细胞凋亡参与了大鼠肠缺血再灌注后肺损伤的发生,肠缺血前给予异丙酚可明显减轻肠缺血再灌注后肺损伤。     

Pretreatment with simvastatin reduces lung injury related to intestinal ischemia-reperfusion in rats

In this rat model study we evaluated whether pretreatment with simvastatin affects the severity of acute lung injury caused by intestinal ischemia-reperfusion (I/R). Twenty-four animals were randomly allocated to three equal groups (sham, control, simvastatin). The simvastatin group was pretreated with simvastatin 10 mg x kg(-1) x day(-1) for 3 days, whereas the other groups received placebo. The simvastatin and control groups underwent 60 min of superior mesenteric artery occlusion and 90 min of reperfusion. Compared with the simvastatin group, the control group exhibited significantly more severe intestinal I/R-induced acute lung injury, as indicated by lower Pao2 and oxygen saturation (P = 0.01 and P = 0.005, respectively) and higher mean values for neutrophil infiltration of the lungs (P = 0.003), total lung histopathologic injury score (P = 0.003), lung wet-to-dry weight ratio (P = 0.009), and lung-tissue malondialdehyde levels (P = 0.016). The control and simvastatin groups had similar serum levels and similar bronchoalveolar lavage fluid levels of cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) and P-selectin at all measurements, except for a significantly higher level of bronchoalveolar lavage fluid P-selectin in the control group (P = 0.006). Pretreatment with simvastatin reduces the severity of acute lung injury induced by intestinal I/R in rats.     

Phospholipase A2 inhibition decouples lung injury from gut ischemia-reperfusion.

BACKGROUND. Phospholipase A2 (PLA2) has recently been implicated as a key enzyme of local inflammation after gut ischemia-reperfusion (I/R). The hypothesis of this study is that PLA2 inhibition decouples remote organ injury from gut I/R. METHODS. Sprague-Dawley rats were pretreated with a PLA2 inhibitor, quinacrine (10 mg/kg, intravenously), before the induction of gut ischemia (45 minutes of superior mesenteric artery occlusion) followed by 6 hours of reperfusion. 125I-labeled albumin leak was employed as a marker of pulmonary endothelial permeability and myeloperoxidase as a monitor of neutrophil (PMN) traffic in the gut and lung. To further characterize the impact of PLA2 inhibition, PMNs were harvested at 6 hours of reperfusion and superoxide production was measured in the presence or absence of an activating stimulus, N-formyl-methionyl-leucyl-phenylalanine. RESULTS. Gut I/R increased gut PLA2 activity, elicited gut PMN influx, and produced lung leak; these events were prevented by PLA2 blockade. Gut I/R also markedly enhanced PMN superoxide production with N-formyl-methionyl-leucyl-phenylalanine, and this priming was ablated by PLA2 inhibition. CONCLUSION. These data suggest that PLA2 activation is a proximal step in the pathogenesis of distant organ injury after splanchnic hypoperfusion, a process that appears to involve PMN priming in the gut bed.     

Resveratrol ameliorates subacute intestinal ischemia-reperfusion injury

Background

Resveratrol has been shown to attenuate reactive oxygen species formation and protect against ischemia-reperfusion (I/R) injury. However, the effects of resveratrol against subacute intestinal I/R injury are not clearly elucidated. Therefore, this study was designed to investigate the effects and possible protective mechanisms of resveratrol on subacute intestinal I/R injury in mice.

Methods

BALB/c mice were subjected to 1 h ischemia by occluding the superior mesenteric artery and 24 h reperfusion. Histologic injury; myeloperoxidase, superoxide dismutase, and glutathione peroxidase activity; malondialdehyde level; inducible nitric oxide synthase (iNOS), Ac-NF-κBp65, and sirtuin 1 (SIRT1) expression; NF-κB translocation; and nitric oxide (NO) production were examined in treated with or without resveratrol in the absence or presence of pharmacologic inhibitors.

Results

Resveratrol significantly ameliorated subacute intestinal I/R injury accompanied with the decrease of NO production as well as iNOS expression. In addition, resveratrol obviously upregulated the expression of SIRT1 and inhibited the activity of NF-κB. After application of iNOS inhibitor S-methylisothiourea and NF-κB inhibitor pyrrolidine dithiocarbamate, the protective effect of resveratrol was significantly augmented by attenuating iNOS and NO production, indicating that resveratrol exerted its protective effect on intestinal I/R injury via NF-κB-mediated iNOS pathway. Furthermore, the protective effect of resveratrol was correlated with SIRT1, because application of SIRT1 inhibitor nicotinamide strikingly weakened the protective effect of resveratrol.

Conclusions

Taken together, our findings showed that resveratrol protects intestinal subacute I/R injury via the SIRT1-NF-κB pathway in an iNOS-NO-dependent manner. Therefore, resveratrol has a potential clinical prospect for further development of anti-injury therapy.     

Role of integrins and intracellular adhesion molecule-1 in lung injury after intestinal ischemia-reperfusion

BACKGROUND: We tested the hypothesis that lung injury after intestinal ischemia-reperfusion (IR) requires the activation of CD11/CD18 glycoprotein complex and its ligand, intracellular adhesion molecule-1 (ICAM-1), on pulmonary endothelial surface. METHODS: Rats were assigned to one of six groups including sham operation, intestinal IR (60/120 min) and IR plus treatment with one of the following monoclonal antibodies against CD11a, CD11b, CD18, and ICAM-1. Pulmonary microvascular permeability, neutrophil accumulation, and expression of adhesion molecules were evaluated. RESULTS: Intestinal IR resulted in lung injury characterized by a marked increase in microvascular permeability, neutrophil accumulation and upregulated expression of leukocyte integrins and ICAM-1. The increase in pulmonary microvascular permability and neutrophil accumulation elicited by intestinal reperfusion was effectively prevented by administration of blocking antibodies against ICAM-1, CD11, and CD18. CONCLUSIONS: These results indicate that adhesion molecules contribute to the lung injury after intestinal IR. Immunoneutralization of certain of these adhesion molecules may prevent intestinal IR-induced lung injury.     

Neutrophil and nonneutrophil-mediated injury in intestinal ischemia-reperfusion.

OBJECTIVE: The role of polymorphonuclear neutrophils (PMN) was examined in local and remote organ injury after intestinal ischemia-reperfusion. SUMMARY BACKGROUND DATA: PMN have been found to mediate the local injury in low flow intestinal ischemia-reperfusion. However, in complete intestinal ischemia-reperfusion, prevention of PMN adhesion by monoclonal antibodies to CD11b and CD18 reduces remote but not local intestinal injury. The role of PMN was further investigated in this setting. METHODS: In a rat model of 1-hour complete intestinal ischemia and 4-hour reperfusion. PMN were manipulated in the following four ways: (1) inhibition of oxygen-free radicals using manganese superoxide dismutase and catalase (SOD/CAT), (2) antagonism of PMN elastase using secretory leukocyte protease inhibitor (SLPI), (3) neutropenia using PMN antisera, and (4) inhibition of activation and adhesion using interleukin-1 receptor antagonist (IL-1ra) and tumor necrosis factor binding protein (TNFbp). Lung injury was quantified by the pulmonary permeability index, which is the ratio of bronchoalveolar lavage to blood concentration of radiolabeled bovine serum albumin, and PMN sequestration by myeloperoxidase (MPO) activity. Liver injury was estimated by PMN counts using quantitative histologic examination and by serum glutamic pyruvic transaminase (SGPT). Local injury was quantified by MPO activity and histologic grading. RESULTS: Neutropenia reduced the pulmonary permeability 80% from 11.0 +/- 0.5 x 10(-3) with saline treatment to 5.50 +/- 0.12 x 10(-3); IL-1ra, to 5.62 +/- 0.44 x 10(-3); and TNFbp, to 4.32 +/- 0.18 x 10(-3) (all p < 0.05). Pulmonary MPO rose from 0.03 +/- 0.01 U/g to 0.25 +/- 0.03 U/g (p < 0.05). This was reduced by neutropenia, 0.01 +/- 0.003 U/g, but not by inhibition of oxygen-free radicals or PMN elastase. IL-1ra inhibited PMN sequestration, reducing MPO to 0.12 +/- 0.01 (p < 0.05). Liver injury was 60% dependent on PMN. Ischemia-reperfusion increased SGPT from 20.3 +/- 0.7 IU/L in the sham-treated animals to 97.0 +/- 3.1 IU/L in the experimental animals. Neutropenia reduced this to 48.1 +/- 3.9 IU/L; IL-1ra, to 44.7 +/- 3.7 IU/L; SOD/CAT, to 64.0 +/- 3.38 IU/L; and SLPI, to 57.1 +/- 3.4 IU/L (p < 0.05 in all cases). Local injury was severe and unaffected by manipulation of the PMN. CONCLUSIONS: These data suggest that PMN and their products mediate most of the lung, part of the liver, and none of the local gut injury after intestinal ischemia-reperfusion.     

Acute lung injury in a rat model of intestinal ischemia-reperfusion: the potential time depended role of phospholipases A(2)

BACKGROUND: A pivotal role of phospholipase A(2) (PLA(2)) and platelet-activating factor-acetylhydrolase (PAF-AcH) as enzymes involved in lung inflammation has recently been suggested. The objective of this study was to elucidate the role and the time dependence of PLA(2) and PAF-AcH fluctuations in the lung relative to the evolution of intestinal ischemia-reperfusion (IIR). MATERIALS AND METHODS: Rats were randomly allocated to five groups of IIR induced by occlusion of the superior mesenteric artery for 45 min followed by 1 min, 2, 4, and 8 h of reperfusion (expGroups) and five corresponding sham groups (sGroups). Bronchoalveolar lavage fluid was obtained from the right lung and its biochemical (protein, PLA(2), PAF-AcH) and cytological characteristics were determined. Plasma malonyldialdehyde was measured as a marker of lipid peroxidation. The 4 and 8 h reperfusion expGroups had significantly (P < 0.05) elevated alveolar-arterial O(2) gradient values compared with the corresponding controls. Total protein, PLA(2) and PAF-AcH levels significantly (P < 0.05) increased in expGroups compared with the corresponding shams after 4 h of reperfusion. Total bronchoalveolar lavage fluid cells and plasma malonyldialdehyde were significantly (P < 0.05) elevated in expGroups compared with the sGroups after 2 h of reperfusion. CONCLUSIONS: PLA(2) could act synergistically or parallel with the reactive oxygen species produced during IIR, resulting in the induction or even in the exacerbation of the inflammatory reaction in acute respiratory distress syndrome. PAF-AcH could play an anti-inflammatory role by reducing the concentration of PAF.     

Reducing ischemia-reperfusion injury in clinical lung transplantation

OBJECTIVE: Acute graft dysfunction secondary to ischemia-reperfusion injury (IRI) continues to be the most common cause of early mortality after lung transplantation. The perioperative management with aprotinin could decrease the incidence of severe IRI. METHODS: A retrospective analysis was conducted of the data from 180 patients who underwent either single lung (56%) or bilateral sequential lung transplantation for similar end-stage lung disease between 1997 and 2005. The most recent 68 patients were managed perioperatively with the high-dose aprotinin infusion regimen (aprotinin group). The ISHLT grade III injury score was used for the diagnosis of severe IRI, which is based on a Pao(2)-FIo(2) ratio of less than 200 mmHg. RESULTS: Grade III injury was observed in 18% of the patients who were not managed with aprotinin (control group, 152 grafts, 64% single transplants, 68% male, 54 +/- 8 years of age). Early ECMO support was required in 25% of these patients. The associated mortality rate was 40%. Despite significantly longer cold ischemic times (290 +/- 14 minutes vs 231 +/- 14 minutes), older donors (42 +/- 12 years of age), and more frequently observed severely elevated systolic PAP of greater than 60 mmHg (60% vs 48%) as well as more frequently required extracorporeal circulatory support (24%* vs 12%) in the aprotinin group, the incidence of severe IRI (8%) and associated mortality (9%) was markedly reduced. CONCLUSIONS: The use of aprotinin in LTX surgery, which had strong beneficial effects on patient outcomes, significantly decreased the incidence of severe posttransplant IRI.     

Beta-chemokine function in experimental lung ischemia-reperfusion injury

BACKGROUND: Although chemokines are functionally important in models of ischemia-reperfusion injury, little is known about their role in lung ischemia-reperfusion injury (LIRI). This study examined the role of the beta-chemokines, macrophage inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T cells expressed and secreted (RANTES) in LIRI. METHODS: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received anti-MIP-1alpha, anti-MCP-1, or anti-RANTES antibodies before reperfusion. Changes in lung vascular permeability were measured with iodine 125-labeled bovine serum albumin. Neutrophil accumulation in the lung parenchyma was determined by myeloperoxidase activity, and bronchoalveolar lavage was performed to measure leukocyte cell counts. Western blots, Northern blots, and ribonuclease protection assays assessed beta-chemokine messenger RNA and protein levels. RESULTS: Animals receiving anti-MIP-1alpha demonstrated reduced vascular permeability compared with controls (p < 0.001). Attenuation of permeability was less dramatic in animals treated with anti-MCP-1 and anti-RANTES antibody, which demonstrated permeability decreases of 15% and 16%, respectively (p < 0.02). Lung neutrophil accumulation was reduced in animals receiving anti-MIP-1alpha antibody (p < 0.005) but was unchanged in animals receiving either anti-MCP-1 or anti-RANTES. Bronchoalveolar lavage leukocyte content was also reduced by treatment with anti-MIP-1alpha (p < 0.003) and was unchanged in anti-MCP-1-treated and anti-RANTES-treated animals. MIP-1alpha treatment decreased tumor necrosis factor-alpha messenger RNA in injured left lungs. CONCLUSIONS: MIP-1alpha is functionally significant in the development of LIRI. It likely exerts its effects in part by mediating the expression of proinflammatory and antiinflammatory cytokines and influencing tissue neutrophil recruitment. MCP-1 and RANTES seem to play relatively minor roles in the development of direct LIRI.     

A Mac-1 antibody reduces liver and lung injury but not neutrophil sequestration after intestinal ischemia-reperfusion.

BACKGROUND. Intestinal ischemia and reperfusion (I/R) result in leukosequestration and injury to the liver and lungs. The adherence-dependent oxidative burst of neutrophils requires cell adhesion through the Mac-1 integrin. Neutrophil-mediated tissue injury may depend on this specific cell adhesion event. This study tests the effect of a Mac-1 (CD 11b) monoclonal antibody (MAb) (R17) on liver and lung injury after intestinal I/R. METHODS. After collaterals were ligated in anesthetized rats, the superior mesenteric artery was clamped for 60 minutes followed by 3 hours of reperfusion. Animals were treated with saline solution, R17, or nonspecific immunoglobulin M. Another nonischemic group of rats were sham controls. Lung and intestinal polymorphonuclear leukocyte sequestration was assessed by measurement of myeloperoxidase and lung permeability by bronchoalveolar lavage blood concentration ratio of 125I-labeled bovine serum albumin. RESULTS. At 3 hours of reperfusion lung and intestinal myeloperoxidase and lung permeability were increased. Treatment with R17 MAb did not reduce intestinal or lung myeloperoxidase but prevented increased lung permeability. Similarly, after treatment with saline solution, liver polymorphonuclear leukocyte sequestration increased after 3 hours of reperfusion, and serum alanine aminotransferase level rose eightfold. R17 MAb did not significantly reduce liver neutrophil sequestration; however, it reduced alanine aminotransferase level more than 50% when compared to saline solution controls. At 3 hours of reperfusion there was a leukocytosis (white blood cell count, 14.9 +/- 1.0 x 10(3)/mm3 vs 6.0 +/- 0.8 in sham [p less than 0.05]). The white blood cell count was unaffected by R17 MAb. CONCLUSIONS. These data indicate that a MAb to the neutrophil Mac-1 integrin reduces lung and liver injury after intestinal I/R but does not reduce lung or intestinal leukosequestration.     

Hepatocellular oxidant stress following intestinal ischemia-reperfusion injury

Reperfusion of ischemic intestine results in acute liver dysfunction characterized by hepatocellular enzyme release into plasma, reduction in bile flow rate, and neutrophil sequestration within the liver. The pathophysiology underlying this acute hepatic injury is unknown. This study was undertaken to determine whether oxidants are associated with the hepatic injury and to determine the relative value of several indirect methods of assessing oxidant exposure in vivo. Rats were subjected to a standardized intestinal ischemia-reperfusion injury. Hepatic tissue was assayed for lipid peroxidation products and oxidized and reduced glutathione. There was no change in hepatic tissue total glutathione following intestinal ischemia-reperfusion injury. Oxidized glutathione (GSSG) increased significantly following 30 and 60 min of reperfusion. There was no increase in any of the products of lipid peroxidation associated with this injury. An increase in GSSG within hepatic tissue during intestinal reperfusion suggests exposure of hepatocytes to an oxidant stress. The lack of a significant increase in products of lipid peroxidation suggests that the oxidant stress is of insufficient magnitude to result in irreversible injury to hepatocyte cell membranes. These data also suggest that the measurement of tissue GSSG may be a more sensitive indicator of oxidant stress than measurement of products of lipid peroxidation.     

肠黏膜屏障缺血/再灌注损伤研究进展

肠黏膜缺血／再灌注损伤是肠黏膜的主要损伤因素之一。各种原因引起肠道的缺血缺氧,导致肠黏膜的缺血／再灌注损伤,影响肠黏膜屏障功能。与肠黏膜缺血／再灌注损伤有关的主要因素包括：氧自由基、细胞因子、核转录因子-kB、细胞凋亡等。本文就肠黏膜屏障缺血／再灌注损伤相关因素的研究进展予以综述。     

腺病毒介导bcl-2基因转染预防肺再灌注损伤

目的 探讨bcl-2基因对体外肺缺血再灌注损伤的保护作用.方法 按肺移植供肺获取和保存方法,对家兔的肺进行获取、保存,然后采用体外循环装置,建立体外肺缺血再灌注损伤模型.共分为3组,分别在手术前48 h从耳背静脉注入生理盐水、含标记基因Laz的腺病毒载体（Ad/LacZ）、重组人bcl-2正义腺病毒载体（Ad/s-bcl-2）.结果 Ad/s-bcl-2可使肺组织中bcl-2 Mrna表达增加,使肺组织中超氧化物歧化酶（SOD）含量升高,丙二醛（MDA）含量降低;使肺动脉压（PAP）降低,肺组织湿干重比降低;降低肺组织中的凋亡细胞数和改善肺组织病理变化.Ad/s-bcl-2组的上述指标均优于生理盐水组和Ad/LacZ组.结论 bcl-2对肺缺血再灌注损伤有明显的保护作用.