A neutrophil elastase inhibitor (ONO-5046) reduces neurologic damage after spinal cord injury in rats

In view of a cytoprotective effect of elastase inhibitor on chemokine-mediated tissue injury, we examined the neuroprotective effect of ONO-5046, a specific inhibitor of neutrophil elastase, in rats with spinal cord injury. Standardized spinal cord compression markedly increased cytokine-induced neutrophil chemo-attractant (CINC)-1 mRNA and protein. Their increases correlated with neurologic severity of injured rats. Immunohistochemically, CINC-1 protein was detected sequentially in vascular endothelial cells at 4 h, in perivascular neutrophils at 8 h, and in neutrophils infiltrating into cord substance at 12 h. Pretreatment with ONO-5046 (50 mg/kg) markedly ameliorated motor disturbance in injured rats, and reduced CINC-1 protein and mRNA expression. ONO-5046 also significantly reduced the increase of neutrophil accumulation or infiltration estimated by myeloperoxidase activity, and the extent of vascular permeability by Evans blue extravasation in the injured cord segment in comparison to control animals receiving vehicle. These results suggest that CINC-1 contributed to inflammation in rat spinal cord injury and ONO-5046 attenuated neurologic damage partly by blocking CINC-1 production of the chemoattractant, preventing neutrophil activation and vascular endothelial cell injury.     

Direct interaction of ONO-5046 with human neutrophil elastase through ¹H NMR and molecular docking

Human neutrophil elastase (HNE) has been implicated as a major contributor in the pathogenesis of diseases, such as pulmonary emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and other inflammatory diseases. Therefore, searching for appropriate and potential human neutrophil elastase inhibitors (HNEI) that would restore the balance between the free enzyme and the endogenous inhibitors would be of therapeutic interest. ONO-5046 is the first specific HNEI to improve respiratory function and protect lung tissues against various lung injuries. However, the mechanism of ONO-5046 to HNE is still unclear. In this study, the binding properties of ONO-5046 were investigated through (1)H NMR, molecular docking, and bioassay methods to understand the effect of ONO-5046 to HNE. The proton spin-lattice relaxation rate and molecular rotational correlation time results indicated that ONO-5046 has higher affinity with HNE. The molecular docking study showed that ONO-5046 is perfectly matched for the primary enzyme specificity pocket (S1 pocket), and is tightly bound to this pocket of HNE through hydrophobic and hydrogen bonding interactions. The results of both methods were validated through analysis of the HNE inhibitory activity bioassay of ONO-5046 with an IC(50) value of 87.05 nM. Our data suggested that ONO-5046 could bind to HNE through direct interaction, and that molecular docking and NMR methods are valid approaches to survey new HNEI.     

A novel potent inhibitor of inducible nitric oxide inhibitor, ONO-1714, reduces intestinal ischemia-reperfusion injury in rats.

The overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to the pathophysiology of intestinal injury induced by ischemia-reperfusion. The aim of the present study was to examine the effect of selective iNOS inhibition by a cyclic amidine analogue, ONO-1714, on reperfusion-induced small intestinal injury and inflammation in rats. Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion. The luminal nitrite concentration in the small intestine was measured by Griess reaction and the iNOS mRNA expression by RT-PCR. The severity of the intestinal mucosal injury and inflammation were evaluated by several biochemical markers and by the histological findings. The rats which were killed after ischemia-reperfusion had increased luminal concentrations of nitrite and iNOS mRNA expression, in addition to severe intestinal inflammation characterized by significant increases in myeloperoxidase activity, a marker of neutrophil infiltration, and by the mucosal content of CINC-1 cytokine, a neutrophil chemotactic cytokine. Administration with ONO-1714 significantly inhibited the luminal NO production. Reperfusion after 30-min ischemia resulted in an increase in luminal protein and hemoglobin concentrations, with levels reaching a maximum after 60 min of reperfusion. In contrast, pre-treatment with ONO-1714 2h before the ischemia inhibited the increases in luminal protein and hemoglobin concentration in a dose-dependent manner (0.001-0.1mg/kg). The contents of the thiobarbituric acid-reactive substances (a marker of oxidative lipid peroxidation) were significantly increased by ischemia-reperfusion, and this increase was reduced by ONO-1714. After reperfusion, the increase in tissue-associated myeloperoxidase activity, an index of neutrophil infiltration, was significantly inhibited by pre-treatment with ONO-1714. ONO-1714 also inhibited increases in intestinal CINC-1 protein and mRNA expression, as determined by ELISA and RT-PCR, respectively. In conclusion, the improvement of reperfusion-induced intestinal injury by ONO-1714 suggested that an excess of NO, produced by iNOS, may have contributed to the initiation/amplification of intestinal inflammatory injury by various mechanisms, including nitrosative and oxidative damage as well as the enhancement of inflammatory cytokine release.     

Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice.

Ischemia-reperfusion (I/R) lung injury is characterized by increased pulmonary endothelial permeability and edema, but the genetic basis for this injury is unknown. We utilized an in vivo mouse preparation of unilateral lung I/R to evaluate the genetic determinants of I/R lung injury. An index of pulmonary vascular protein permeability was measured by the ratio of left-to-right lung Evans blue dye of eight inbred mouse strains after 30 min of left lung ischemia and 150 min of reperfusion. The order of strain-specific sensitivity to I/R lung injury was BALB/c < SJL/J < CBA/J < C57BL/6J < 129/J < A/J < C3H/H3J < SWR/J. The reciprocal F1 offspring of the BALB/c and SWR/J progenitor strains had intermediate phenotypes but a differing variance. A similar pattern of right lung Evans blue dye content suggested the presence of contralateral injury because baseline vascular permeability was not different. Lung I/R injury was attenuated by NADPH oxidase inhibition, indicating a role for NADPH oxidase-derived reactive oxygen species (ROS). There was no strain-dependent difference in lung NADPH oxidase expression. Strain-related differences in zymosan-stimulated neutrophil ROS production did not correlate with I/R lung injury in that neutrophil ROS production in SWR/J mice was greater than C57BL/6J but not different from BALB/c mice. These data indicate the presence of a genetic sensitivity to lung I/R injury that involves multiple genes including a maternal-related factor. Although neutrophil-derived ROS production is also modulated by genetic factors, the pattern did not explain the genetic sensitivity to lung I/R injury.     

Role of neutrophils in xanthine/xanthine oxidase-induced oxidant injury in isolated rabbit lungs.

Reactive oxygen species have been shown to play an important role in the pathogenesis of lung injury. This study was designed to clarify the role of intrapulmonary neutrophils in the development of xanthine/xanthine oxidase (X/XO)-induced lung injury in isolated buffer-perfused rabbit lungs. We measured microvascular fluid filtration coefficient (K(f)) and wet-to-dry weight ratio to assess lung injury. X/XO induced a significant increase in K(f) and wet-to-dry weight ratio in neutrophil-replete lungs, whereas the lung injury was attenuated in neutrophil-depleted lungs. A neutrophil elastase inhibitor, ONO-5046, also attenuated the lung injury. In addition, X/XO induced a transient pulmonary arterial pressure (P(pa)) increase. The thromboxane inhibitor OKY-046 attenuated the P(pa) increase but did not alter the increase in permeability. Neutrophil depletion reduced the K(f) increase but had no effect on the P(pa) increase. These results suggest that intrapulmonary neutrophils activated by X/XO play a major role in development of the lung injury, that neutrophil elastase is involved in the injury, and that the X/XO-induced vasoconstriction is independent of intrapulmonary neutrophils.     

Estrogen prevents intestinal inflammation after trauma-hemorrhage via downregulation of angiotensin II and angiotensin II subtype I receptor

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.     

Supplementation with γ-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell responses in rats

Neutrophil-mediated tissue injury is a shared pathogenesis of both chronic pulmonary diseases and acute responses to pathogens, allergens, and airborne pollutants. Interventions to minimize toxic effects of neutrophil-derived oxidants and proteases are usually limited to corticosteroids, which can have adverse side effects. We used a rodent model of endotoxin-induced lung injury to test the hypothesis that the dietary supplement γ-tocopherol (γT), a natural form of vitamin E with antioxidant and novel anti-inflammatory properties, will protect from adverse nasal and pulmonary inflammatory responses induced by endotoxin (lipopolysaccharide; LPS). Male Fisher F344 rats were intranasally (i.n.) instilled with LPS for 2 consecutive days. Beginning 2 days before i.n. LPS, the rats were gavaged daily with 30 mg/kg γT. Twenty-four hours after the last i.n. LPS, bronchoalveolar lavage fluid (BALF) was collected, and pulmonary and nasal tissues were analyzed for gene expression and morphometric analyses of neutrophils and intraepithelial mucosubstances (IM). LPS caused increased BALF total cells (70% increase), neutrophils (300%), protein (35%), PGE₂ (500%), and secreted mucins (75%). Robust increases in neutrophils and IM were detected in conducting airways. Pulmonary expression of MUC5AC, MIP-2, CINC-1, and MCP-1 was elevated three- to eightfold by LPS. Treatment with γT inhibited LPS-induced increases in BALF total cells, neutrophils, protein, PGE₂, and secreted mucins, as well as IM and tissue neutrophil influx. Furthermore γT induced the expression of the regulatory cytokines IL-10 and IFN-γ while decreasing MUC5AC, MIP-2, CINC-1, and MCP-1. These data demonstrate novel therapeutic effects of the dietary vitamin E γT promoting anti-inflammatory pathways to protect from neutrophil-mediated lung injury.     

Time course of lung ischemia-reperfusion-induced ICAM-1 expression and its role in ischemia-reperfusion lung injury.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression is an important mechanism underlying ischemia-reperfusion (I/R) induced neutrophil activation and tissue injury in other organs. However, I/R of the lungs has not been shown to upregulate ICAM-1 expression. We determined the time course profile of lung I/R-induced ICAM-1 expression and assessed the role of ICAM-1 in mediating neutrophil sequestration, transmigration, and I/R injury in the isolated blood-perfused rat lungs. I/R had a biphasic effect on ICAM-1 expression, an early downregulation and a late-phase upregulation. Superoxide dismutase and neutrophil depletion prevented the early ICAM-1 downregulation. The late-phase ICAM-1 upregulation coincided with the I/R-induced increase in pulmonary microvascular leakage index. ICAM-1 monoclonal antibody (MAb) reversed the I/R-induced increase in pulmonary microvascular leakage index, with control antibody being ineffective. Neither I/R nor ICAM-1 MAb affected lung MPO activity and circulating neutrophil count. Lung I/R significantly increased bronchoalveolar lavage fluid neutrophil count and the GSSG-to-(GSSG+GSH) ratio. ICAM-1 MAb blocked the I/R-induced increase in GSSG-to-(GSSG+GSH) ratio but had no effect on bronchoalveolar lavage fluid neutrophil count. Our results demonstrated that lung I/R up- and downregulates ICAM-1 expression depending on the duration of reperfusion. ICAM-1 upregulation is an important mechanism of I/R-induced pulmonary endothelial injury.     

Edaravone protects against lung injury induced by intestinal ischemia/reperfusion in rat

Intestinal ischemia/reperfusion (I/R) is a critical and triggering event in the development of distal organ dysfunction, frequently involving the lungs. Respiratory failure is a common cause of death and complications after intestinal I/R. In this study we investigated the effects of edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) on the prevention of lung injury induced by intestinal I/R in rats. Edaravone has been used for protection against I/R injury in patients with cerebral infarction. When rats were subjected to 180 min of intestinal ischemia, a high incidence of mortality was observed within 24 h. In this situation, intravenous administration of edaravone just before the start of reperfusion reduced the mortality in a dose-dependent manner. To examine the efficacy of edaravone on the lung injury induced by intestinal I/R in more detail, we performed 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone treatment decreased the neutrophil infiltration, the lipid membrane peroxidation, and the expression of proinflammatory cytokine interleukin-6 mRNA in the lungs after intestinal I/R compared to the I/R-treated rat lungs without edaravone treatment. Histopathological analysis also indicated the effectiveness of edaravone. In conclusion, edaravone ameliorated the lung injury induced by intestinal I/R, resulting in a reduction in mortality.     

Inhibition of Rho kinase by fasudil hydrochloride attenuates lung injury induced by intestinal ischemia and reperfusion

AimThe aim of this study is to evaluate the role of Rho-kinase in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R) and the preconditioning effects of fasudil hydrochloride. The novel therapeutic approach of using Rho-kinase inhibitors in the treatment of intestinal I/R is introduced.MethodsSprague–Dawley (SD) rats were divided into 4 groups: intestinal I/R group, two fasudil pretreatment groups (7.5 mg/kg and 15 mg/kg), and controls. Intestinal and lung histopathology was evaluated; myeloperoxidase (MPO) and superoxide dismutase (SOD) levels in lung parenchyma were determined. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. eNOS and P-ERM expression were measured by Western Blot.ResultsLung and intestinal injury were induced by intestinal I/R, characterized by histological damage and a significant increase in BALF protein. Compared to controls, serum TNF-α, IL-6, and lung MPO activity increased significantly in the I/R group, while SOD activity decreased. A strongly positive P-ERM expression was observed, while eNOS expression was weak. After fasudil administration, injury was ameliorated. Serum TNF-α, IL-6, lung MPO and P-ERM expression decreased significantly as compared to the I/R group, while SOD activity and eNOS expression increased significantly.SignificanceRho-kinase plays a key role in the pathogenesis of lung injury induced by intestinal I/R. The inhibition of the Rho-kinase pathway by fasudil hydrochloride may prevent lung injury.     

Protective effect of hesperidin against lung injury induced by intestinal ischemia/reperfusion in adult albino rats: Histological,immunohistochemical and biochemical study

Hesperidin is a naturally common flavonoid. It is an abundant and cheap by-product of citrus cultivation. It is reported to have antioxidative, anti-inflammatory and anticarcinogenic effects. This work was performed to investigate the possible protective role of hesperidin in ameliorating the effect of experimentally induced intestinal ischemia/reperfusion injury (I/R) on lung tissue, histologically, immunohistochemically and biochemically. Thirty male Wistar adult albino rats were randomized into three groups named: group I (control group); group II (I/R); and group III (I/R with hesperidin). Intestinal I/R was induced by occluding the superior mesenteric artery for 60 min, followed by 120 min of reperfusion period. Animals were given hesperidin orally 1 h before the onset of ischemia. At the end of the reperfusion period the lung tissues were extracted for histopathological examination and immunohistochemical detection of the distribution of inducible nitric oxide synthase (iNOS). Pulmonary edema was evaluated by lung tissue wet/dry weight ratios. The levels of malondialdehyde (MDA, a biomarker of oxidative damage), myeloperoxidase (MPO, an index of the degree of neutrophil accumulation) and glutathione (GSH, a biomarker of protective oxidative injury) were also determined in all dissected tissues. Pretreatment with hesperidin (in group III) alleviated lung morphological changes noticed in I/R group and the levels of MDA and MPO were significantly decreased whereas those of GSH were significantly increased. Immunohistochemical study revealed a significant decrease in the iNOS. Hesperidin also significantly alleviated the formation of pulmonary edema as evidenced by the decreased organ wet/dry weight ratios. Hesperidin exerts a protective effect against lung damage induced by intestinal I/R injury in rats by reducing oxidative stress.     

Mechanism for the increased permeability in endothelial monolayers induced by elastase

The aim of this study was to investigate the mechanism for the increase in endothelial permeability induced by human neutrophil elastase (HNE). Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with HNE(0-30 mug/ml) for 1 h produced a concentration dependent increase in (125)I-albumin clearance. The effect was reversible and was not due to cytolysis. Pretreatment of BPAEC with sodium tungstate, which depletes xanthine oxidase, or with oxypurinol, did not prevent HNE induced increased permeability. Heparin, which neutralizes the cationic charge of HNE, also had no protective effect. Pretreatment with heat inactivated HNE, which still had positive charge sites, did not result in increased endothelial permeability. Also, ONO-5046, a novel specific inhibitor of HNE, did prevent increased permeability. These results suggest that elastase increases endothelial permeability mainly through its proteolytic effects.     

AICAR Attenuates Organ Injury and Inflammatory Response after Intestinal Ischemia and Reperfusion

Intestinal ischemia and reperfusion (I/R) is encountered in various clinical conditions and contributes to multiorgan failure and mortality as high as 60% to 80%. Intestinal I/R not only injures the intestine, but affects remote organs such as the lung leading to acute lung injury. The development of novel and effective therapies for intestinal I/R are critical for the improvement of patient outcome. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) is a cell-permeable compound that has been shown to possess antiinflammatory effects. The objective is to determine that treatment with AICAR attenuates intestinal I/R injury and subsequent acute lung injury (ALI). Male Sprague Dawley rats (275 to 325 g) underwent intestinal I/R injury with blockage of the superior mesenteric artery for 90 min and subsequent reperfusion. At the initiation of reperfusion, vehicle or AICAR (30 mg/kg BW) was given intravenously (IV) for 30 min. At 4 h after reperfusion, blood and tissues were collected for further analyses. Treatment with AICAR significantly decreased the gut damage score and the water content, indicating improvement in histological integrity. The treatment also attenuated tissue injury and proinflammatory cytokines, and reduced bacterial translocation to the gut. AICAR administration after intestinal I/R maintained lung integrity, attenuated neutrophil chemotaxis and infiltration to the lungs and decreased lung levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Inflammatory mediators, lung-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, were decreased in the lungs and lung apoptosis was significantly reduced after AICAR treatment. These data indicate that AICAR could be developed as an effective and novel therapeutic for intestinal I/R and subsequent ALI.     

Nitric oxide decreases lung injury after intestinal ischemia

Terada, Lance S., Nancy N. Mahr, and Eugene D. Jacobson.Nitric oxide decreases lung injury after intestinal ischemia. J. Appl. Physiol. 81(6):2456-2460, 1996.After injury to a primary organ, mediators arereleased into the circulation and may initiate inflammation of remoteorgans. We hypothesized that the local production of nitric oxide (NO)may act to limit the spread of inflammation to secondarily targetedorgans. In anesthetized rats, 30 min of intestinal ischemia followed by2 h of reperfusion (I/R) did not increase lung albumin leak. However,after treatment with N^G-nitro-L-arginine methyl ester(L-NAME), intestinal I/R led to increased lung leak, suggesting a protective effect of endogenous NO.The site of action of NO appeared to be the lung and not the gutbecause 1) after treatment withL-NAME, local delivery of NO tothe lung by inhalation abolished the increase in intestinal I/R-inducedlung leak; 2)L-NAME had no effect onepithelial permeability (⁵¹Cr-labeled EDTA clearance) ofreperfused small bowel; and 3) after treatment with L-NAME, localdelivery of NO to the gut by luminal perfusion did not improveepithelial permeability of reperfused intestines. Furthermore,L-NAME increased, and inhaled NOde- creased, the density of lung neutrophils in rats subjected to intestinal I/R, and treatment with the selectin antagonist fucoidan abolished L-NAME-induced lungleak in rats subjected to intestinal I/R. We conclude thatendogenous lung NO limits secondary lung injury after intestinal I/R bydecreasing pulmonary neutrophil retention.

     

The α2AR/Caveolin-1/p38MAPK/NF-κB axis explains dexmedetomidine protection against lung injury following intestinal ischaemia-reperfusion

Intestinal ischaemia-reperfusion (I/R) injury can result in acute lung injury due to ischaemia and hypoxia. Dexmedetomidine (Dex), a highly selective alpha2-noradrenergic receptor (α2AR) agonist used in anaesthesia, is reported to regulate inflammation in organs. This study aimed to investigate the role and mechanism of Dex in lung injury caused by intestinal I/R. After establishing a rat model of intestinal I/R, we measured the wet-to-dry specific gravity of rat lungs upon treatments with Dex, SB239063 and the α2AR antagonist Atipamezole. Moreover, injury scoring and histopathological studies of lung tissues were performed, followed by ELISA detection on tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 expression. Correlation of Caveolin-1 (Cav-1) protein expression with p38, p-p38, p-p65 and p65 in rat lung tissues was analysed, and the degree of cell apoptosis in lung tissues after intestinal I/R injury was detected by TUNEL assay. The lung injury induced by intestinal I/R was a dynamic process. Moreover, Dex had protective effects against lung injury by mediating the expression of Cal-1 and α_2A-AR. Specifically, Dex promoted Cav-1 expression via α_2A-AR activation and mitigated intestinal I/R-induced lung injury, even in the presence of Atipamezole. The protective effect of Dex on intestinal I/R-induced lung injury was also closely related to α_2A-AR/p38 mitogen-activated protein kinases/nuclear factor-kappaB (MAPK/NF-κB) pathway. Dex can alleviate pulmonary inflammation after in intestinal I/R by promoting Cav-1 to inhibit the activation of p38 and NF-κB. In conclusion, Dex can reduce pulmonary inflammatory response even after receiving threats from both intestinal I/R injury and Atipamezole.     

Effect of NADPH oxidase inhibition on cardiopulmonary bypass-induced lung injury

Cardiopulmonary bypass (CPB) causes acute lung injury. Reactive oxygen species (ROS) from NADPH oxidase may contribute to this injury. To determine the role of NADPH oxidase, we pretreated pigs with structurally dissimilar NADPH oxidase inhibitors. Low-dose apocynin (4-hydroxy-3-methoxy-acetophenone; 200 mg/kg, n = 6), high-dose apocynin (400 mg/kg, n = 6), or diphenyleneiodonium (DPI; 8 mg/kg) was compared with diluent (n = 8). An additional group was treated with indomethacin (10 mg/kg, n = 3). CPB was performed for 2 h with deflated lungs, complete pulmonary artery occlusion, and bronchial artery ligation to maximize lung injury. Parameters of pulmonary function were evaluated for 25 min following CPB. Blood chemiluminescence indicated neutrophil ROS production. Electron paramagnetic resonance determined the effect of apocynin and DPI on in vitro pulmonary endothelial ROS production following hypoxia-reoxygenation. Both apocynin and DPI attenuated blood chemiluminescence and post-CPB hypoxemia. At 25 min post-CPB with Fi(O(2)) = 1, arterial Po(2) (Pa(o(2))) averaged 52 +/- 5, 162 +/- 54, 335 +/- 88, and 329 +/- 119 mmHg in control, low-dose apocynin, high-dose apocynin, and DPI-treated groups, respectively (P < 0.01). Indomethacin had no effect. Pa(O(2)) correlated with blood chemiluminescence measured after drug administration before CPB (R = -0.60, P < 0.005). Neither apocynin nor DPI prevented the increased tracheal pressure, plasma cytokine concentrations (tumor necrosis factor-alpha and IL-6), extravascular lung water, and pulmonary vascular protein permeability observed in control pigs. NADPH oxidase inhibition, but not xanthine oxidase inhibition, significantly blocked endothelial ROS generation following hypoxia-reoxygenation (P < 0.05). NADPH oxidase-derived ROS contribute to the severe hypoxemia but not to the increased cytokine generation and pulmonary vascular protein permeability, which occur following CPB.     

Murine model of gastrointestinal ischemia associated with complement-dependent injury.

Gastrointestinal ischemia-reperfusion (I/R) injury is often associated with remote tissue injury. Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal I/R. We developed a new murine model of gastrointestinal I/R that has complement-dependent local and remote tissue injury. Twenty, but not thirty, minutes of gastrointestinal ischemia followed by 3 h of reperfusion induced a significant loss of intestinal lactate dehydrogenase that was significantly prevented by a murine anti-murine C5 monoclonal antibody. Anti-C5 also significantly decreased neutrophil infiltration into the gut and lung. Gastrointestinal I/R significantly increased pulmonary intercellular adhesion molecule-1 mRNA and protein expression that was significantly inhibited by anti-C5. Pulmonary macrophage inflammatory protein-2 mRNA was significantly induced by gastrointestinal I/R and inhibited by anti-C5 treatment. These data demonstrate that brief periods of murine gastrointestinal I/R activate complement, leading to tissue injury and neutrophil accumulation. Anti-C5 treatment attenuates tissue injury, neutrophil recruitment, and leukocyte adherence molecule and chemokine expression in the mouse. This model will be well suited to investigate the role of complement-mediated tissue injury and gene expression after gastrointestinal I/R.     

Hydrogen-rich saline reduces lung injury induced by intestinal ischemia/reperfusion in rats

Objective. Hydrogen has been reported to selectively reduce the hydroxyl radical, the most cytotoxic of reactive oxygen species. In this study we investigated the effects of hydrogen-rich saline on the prevention of lung injury induced by intestinal ischemia/reperfusion (I/R) in rats. Methods. Male Sprague-Dawley rats (n = 30, 200-220 g) were divided randomly into three experimental groups: sham operated, intestinal I/R plus saline treatment (5 ml/kg, i.v.), and intestinal I/R plus hydrogen-rich saline treatment (5 ml/kg, i.v.) groups. Intestinal I/R was produced by 90 min of intestinal ischemia followed by a 4 h of reperfusion. Results. Hydrogen-rich saline treatment decreased the neutrophil infiltration, the lipid membrane peroxidation, NF-κB activation and the pro-inflammatory cytokine interleukin IL-1β and TNF-α in the lung tissues compared with those in saline-treated rat. Conclusion. Hydrogen-rich saline attenuates lung injury induced by intestinal I/R.     

Acute alcohol intoxication increases interleukin-18-mediated neutrophil infiltration and lung inflammation following burn injury in rats

In this study, we examined whether IL-18 plays a role in lung inflammation following alcohol (EtOH) and burn injury. Male rats ( approximately 250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dl before burn or sham injury ( approximately 12.5% total body surface area). Immediately after injury, rats were treated with vehicle, caspase-1 inhibitor AC-YVAD-CHO to block IL-18 production or with IL-18 neutralizing anti-IL-18 antibodies. In another group, rats were treated with anti-neutrophil antiserum approximately 16 h before injury to deplete neutrophils. On day 1 after injury, lung tissue IL-18, neutrophil chemokines (CINC-1/CINC-3), ICAM-1, neutrophil infiltration, MPO activity, and water content (i.e., edema) were significantly increased in rats receiving a combined insult of EtOH and burn injury compared with rats receiving either EtOH intoxication or burn injury alone. Treatment of rats with caspase-1 inhibitor prevented the increase in lung tissue IL-18, CINC-1, CINC-3, ICAM-1, MPO activity, and edema following EtOH and burn injury. The increase in lung IL-18, MPO, and edema was also prevented in rats treated with anti-IL-18 antibodies. Furthermore, administration of anti-neutrophil antiserum also attenuated the increase in lung MPO activity and edema, but did not prevent the increase in IL-18 levels following EtOH and burn injury. These findings suggest that acute EtOH intoxication before burn injury upregulates IL-18, which in turn contributes to increased neutrophil infiltration. Furthermore, the presence of neutrophils appears to be critical for IL-18-meditaed increased lung tissue edema following a combined insult of EtOH and burn injury.     

Proteasome inhibition attenuates lung injury induced by intestinal ischemia reperfusion in rats

The aim of this study is to investigate the role of proteasome in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R) by examining the effect of the proteasome inhibitor lactacystin on neutrophil infiltration, intracellular adhesion molecule-1 (ICAM-1) expression and nuclear factor kappa B (NF-κB) activation. Thirty-two Wistar rats were divided into (1) control, (2) intestinal I/R, (3) 0.2 mg/kg lactacystin pretreated, and (4) 0.6 mg/kg lactacystin pretreated groups (n = 8). Injuries in lung and intestine were induced by intestinal I/R, and were characterized by histological edema, hemorrhage and infiltration of inflammatory cells. The results showed a significant increase in serum creatine kinase B (CK-B) and lung water content in intestine and lung injuries. As compared with the control group, the myeloperoxidase (MPO) activity in intestine and lung as well as the serum TNF-α level increased significantly in intestinal I/R group. Simultaneously, expression of ICAM-1 and NF-κB p65 was also observed in the I/R group. Pre-treatment with lactacystin markedly reduced 20S proteasome activity in circulating white blood cells and ameliorated intestine and lung injuries. These results demonstrated that the proteasome participates in the pathogenesis of lung injury induced by intestinal I/R. Lactacystin as a proteasome inhibitor can prevent this kind of injury by decreasing ICAM-1 and TNF-α production via the inhibition of NF-κB activation.