Pulmonary sepsis is the septic complication most frequently encountered in severely burn patients (1). Although the pathogenic mechanisms are not fully understood, it is clear that neutrophil accumulation in lung is involved (2) and oxygen-derived free radicals, cytokines, and nitric oxide (NO) are either signaling or effector molecules (3). Burn injury has been known to cause an enhanced migration of circulating neutrophils into the intestinal interstitial spaces in rats (4). Myeloperoxidase activity was shown to increase considerably in lung tissue at 2 h after burn injury. Arbak et al. have shown that neutrophil accumulation in lung is involved in pathogenesis of this distant organ after burn (2). Also, it has been shown that systemic burn results in early pulmonary vascular changes associated with leukocyte sequestration in burned rabbits (5). Furthermore, ultrastructural examination of the lung in thermal injured rats found disturbance of alveolar structure, intraalveolar hemorrhage, and prominent neutrophil infiltration, indicating the lung parenchymal injury (2).
NO was first introduced in 1980 as endothelium-derived relaxing factor (6). It is produced from L-arginine by NO synthase (NOS) in all mammalian cells. NO produced by the inducible isoform of NOS (iNOS) has been suggested to play a central role in the physiology and response to critical illness of the gastrointestinal tract (7). Part of these effects are mediated by the formation of highly toxic peroxynitrite when NO reacts with superoxide anions (8). Peroxynitrite is an extremely potent oxidant that can cause lipid peroxidation, DNA damage, and alterations of protein function in vitro (9). In addition, many of the actions of NO, including vasodilatation (10) and induction of vascular hyperpermeability (11), are mediated through the activation of soluble guanylate cyclase (sGC; Ref. 12). NO binds to the heme moiety of sGC and activates the enzyme, thereby increasing the cellular cyclic guanosine monophosphate (cGMP) level. Signal transduction via the NO/sGC pathway entails the activation of various cGMP-dependent protein kinases (13).
Studies have shown that NOS inhibition accelerated intestinal ischemia/reperfusion-induced capillary leak seen in acute lung injury and the effect is mediated by modulating the neutrophils (14). The activation of sGC has been known to contribute to the vascular hyporeactivity induced by endotoxin in vitro and in vivo. 1 H-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one (ODQ), a potent inhibitor of sGC has been shown to influence survival in a murine model of severe sepsis (8). Recently, ODQ has been used extensively as a specific inhibitor for sGC and a diagnostic tool for identifying a role for sGC in signal transduction events (15). In a previous study (16), we demonstrated that the thermal injury induced at least 2-fold increase in the lung neutrophil sequestration, plasma DHR oxidation and lung damage in rats. The application of selective iNOS inhibitor SMT significantly decreased the lung dysfunction syndrome, suggesting that the induction of NO production is closely related to the lung dysfunction in thermal injured rats. However, no study has yet determined whether these effects of NO are through a cGMP-dependent or a cGMP-independent (NO and/or peroxynitrite-dependent redox modification) pathway. Therefore, in this study, the role of cGMP and the effect of guanylate cyclase inhibitor on the lung neutrophil deposition and lung damage after burn were investigated.
MATERIALS AND METHODS
Both male and female specific pathogen-free Sprague–Dawley rats (200–275 g) used were obtained from the National Laboratory Breeding and Research Center (NLBRC, Taipei, Taiwan). Before the experiment, the animals were provided with standard rat chow and water ad libitum and maintained in a temperature-controlled room under a 12-h light/dark cycle for at least one week. All animal procedures were in compliance with regulations on animal used for experimental and other scientific purposes approved by the National Sun Yat-Sen University Animal Experiments Committee.
To evaluate the effect of guanylate cyclase inhibitor on lung damage after thermal injury, ODQ, a specific guanylate cyclase inhibitor (17–19), dissolved in sterile saline (0.5 mg/mL) was given (20 mg/kg, i.p.) to rats that underwent burn immediately after injury. The specific pathogen-free Sprague–Dawley rats were randomly divided into three groups: group I (control group, n = 6), received sham burn and saline injection (15 mL/kg, immediately post burn); group II (n = 6), received thermal injury and saline injection; and group III (n = 6), received thermal injury and ODQ injection. All animals were sacrificed at 8 h after burn and examined for the effect of ODQ on the lung neutrophil deposition, plasma DHR oxidation, and histology. In another experiment, the lung permeability of the animals with the same quantity and treatment was measured in anesthetized animals.
To examine the ODQ effect on lung cGMP production, the rats were divided into three groups: group I (sham group), n = 4, received sham burn treatment and vehicle injection; group II (sodium nitroprusside [SNP] group), n = 4, received sham burn treatment, sodium nitroprusside (SNP, 2 mM, i.p.), and saline injection; group III (SNP + ODQ group), n = 4, received sham burn treatment, sodium nitroprusside (SNP, 2 mM, i.p.), and ODQ (20 mg/kg, i.p.) injection; group IV (SNP + methylene blue group), n = 4, received sham burn treatment, sodium nitroprusside (SNP, 2 mM, i.p.), and methylene blue (100 μM, i.p.) injection. SNP has been known to dose-dependently elevate intracellular cGMP levels (20,21) and elevate intracellular cGMP 10-fold in spleen cells (22). Methylene blue has been known as an inhibitor of GC (22). The animals were sacrificed 4 h after injection and lung tissues were harvested for iNOS mRNA study.
The thermal injury procedures were as described previously (16). Briefly, animals were anesthetized intraperitoneally with sodium pentobarbital (35 mg/kg), and a marked area (130 ± 15 cm2) of the shaved dorsal skin was exposed from a wooden template and immersed in 95°C water for 10 s. This procedure produced a 30–35% total burn surface area full-thickness burn of the rats. All animals received sterile saline (15 mL/kg) intraperitoneally for fluid resuscitation right after the scald burn or sham treatment. The sham control animals were anesthetized, shaved and maintained in identical settings except that room temperature water was used for immersion. The burn injury caused 8% mortality within the first 24 h after burn and nonsurviving animals were excluded from the subsequent study.
Determination of lung MPO activity
Lung content of MPO, an index of neutrophil accumulation (20), was determined to assess the degree of pulmonary neutrophil sequestration. Rats were anesthetized and the thorax was opened with median sternotomy. The bilateral lungs and heart were harvested together and the pulmonary vasculature was cleared of blood by gentle injection of 10 mL of sterile saline into the right ventricle. The lungs were then blotted dry of surface blood and weighed.
Lung tissues were placed in 50 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethylammonium bromide and homogenized. The homogenate was sonicated on ice and centrifuged for 30 min at 3,000 g, 4°C. An aliquot (0.1 mL) of supernatant was added to 2.9 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.167 mg/mL of O-dianisidine and 0.0005% hydrogen peroxide (23). The rate of change in absorbance at 460 nm was measured over 3 min. One unit of MPO activity was defined as the amount of enzyme that reduces 1 mmol of peroxide per min and the data were expressed as units per gram of lung tissue (U/g tissue).
Quantification of pulmonary injury
Thermal injury-induced pulmonary microvascular dysfunction was quantified by measuring the concentration of Evans blue dye within the lung after intravenous injection of dye. Evans blue dye binds avidly to albumin and has been used as a marker of protein extravasation in models of inflammatory tissue injury (24). Evans blue dye (30 mg/kg) was injected to the right femoral vein slowly, the bilateral lungs and heart were harvested 30 min after infusion. The pulmonary vasculature was cleared of blood and the lungs were weighed and placed in 5 mL of formamide at 37°C for 16 h. The absorbance of Evans blue dye in eluate was measured at 620 nm. The concentration of Evans blue dye extracted from lungs was calculated against a standard curve and expressed as nanograms dye per milligram lung tissue (ng/mg tissue).
Measurement of dihydrorhodamine 123 oxidation in plasma
The formation of peroxynitrite in plasma was assayed by the peroxynitrite-dependent oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine as described by Kooy et al. (25). Animals were injected with DHR 123 (2 μmol/kg in 0.3 mL of saline i.v.) and sacrificed 30 min after the injection. Plasma samples were taken for the measurement of rhodamine fluorescence using a fluorometer with the excitation wavelength of 500 nm and emission wavelength of 536 nm. The rate of rhodamine formation, an index of peroxynitrite production, was calculated against a standard curve obtained with authentic rhodamine prepared in plasma from untreated rats.
RNA isolation and reverse transcription of RNA
Total RNA was isolated from lung using TRIZOL reagent (Invitrogen, Carlsbad, CA). Tissue samples were added TRIZOL reagent (50–100 mg/mL) and homogenized. The homogenized samples were incubated at 24°C for 8 min to permit the complete dissociation of nucleoprotein complexes. Chloroform (0.2 mL per ml of TRIZOL reagent) was added and shaken vigorously for 15 s. The samples were centrifuged at 13,000 g for 15 min at 4°C and the colorless upper aqueous phase was transferred into a fresh tube. RNA was precipitated with isopropyl alcohol (0.5 mL per milliliter of TRIZOL reagent). Samples were incubated at 24°C for 10 min and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was removed and the RNA pellet was washed once with 70% ethanol (1 mL per milliliter of TRIZOL reagent). At the end, samples were centrifuged at 7500 g for 5 min at 4°C and the RNA pellets were briefly dried. RNA dissolved in DEPC treated water was quantified by UV absorption at 260 nm and stored at −80°C. As cDNA synthesis, 2.5 μg of total RNA was incubated with 500 ng of oligo-(dT)18 at 70°C for 10 min and quickly chilled on ice. The mixture was added to obtain a 20-μL reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each of dNTP, 1 μL of RNase inhibitor (40 Units, Promega, Madison, WI), and 1 μL (10 U) of SUPERSCRIPT II RNase H-Reverse Transcriptase (Invitrogen, Carlsbad, CA). Then the contents were incubated at 42°C for 50 to 60 min and heating at 70°C for 10 min to stop the reaction. The solution was stored at −20°C.
Polymerase chain reaction (PCR) and quantification of PCR products
One set of iNOS primer was designed according to the iNOS gene documented in GenBank. The sequences are 5´-GCAGCTGTGCTCCATAGTTT3´ (sense) and 5´-GAAGGTATGCCCGAGTTCTT-3´ (antisense). Meanwhile, we designed one pair of primer: 5´-CAGCCCAGAACATCATCCC-3´ (sense) and 5´-TGAGGTCTACCACCCTGTT-3´ (antisense) for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene as a control. The PCR products of iNOS and GADPH were 600 bp and 378 bp in length, respectively.
To a sterile 0.2-mL tube, 5 μL of 10X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl and 15 mM MgCl2), 2 μL of DMSO, 4 μL of 2.5 mM dNTP mixture, 0.5 μL each of the sense and antisense primers (0.5 mg/mL), 25 ng to 100 ng of the cDNA template, and an appropriate amount of water to make a total volume of 50 μL was added. After adding 0.5 μL of Taq polymerase (5 units/μL), the tube was heated for 2 min at 95°C in a thermocycler (Touchgene, TECHNE, Duxford, Cambridge, UK). The PCR cycles for iNOS and GAPDH were 40 s at 95°C, 1 min at 58°C, and 1 min at 72°C. The PCR products were electrophoresed in 1.0% agarose gel and stained with ethidium bromide. The approximate length of the PCR product was obtained by comparing the marker (100-bp ladder, Biolabs, Beverly, MA). We recorded the electrophoresis by CCD camera and compared the band concentration by Kodak Digital Science TM ID Image Analysis Software (Eastman Kodak Company, Rochester, NY).
Pulmonary histological studies
The lung tissue specimens harvested from different groups of animals were immersed in 4% formaldehyde solution at 8 h after thermal injury. The tissue was embedded in paraffin wax, serially sectioned, and stained with hematoxylin-eosin. Pulmonary morphological characteristics were evaluated under light microscope. A pathologist blinded for treatment evaluated the lungs histopathological findings. A scoring system to grade the degree of lung injury was devised based on the following histologic features. 1) septal edema, 2) congestion, 3) degree of inflammatory cells infiltration, and 4) alveolar edema. A scale from 0 to 4 for each feature was used (0 = normal, 4 = most severe). A total score was obtained by adding values for each feature form every animal. Mean scores and standard deviation were calculated for each group of animals.
All values in the figures and text are expressed as mean ± standard deviation of the mean, and P < 0.05 was considered to be statistically significant. The plasma DHR 123 oxidation level, lung MPO activity, and lung permeability between groups were assessed with one-way analysis of variance, followed by Scheffe's F test.
There was a marked increase in neutrophil recruitment in lung after thermal injury as demonstrated by lung MPO activity assay (Fig. 1). The MPO activity in lung of the control group was 30.70 ± 1.19 U/g tissue and it was increased significantly to 92.55 ± 2.64 U/g tissue at 8 h after injury. Similarly, the level of plasma DHR 123 oxidation (Fig. 2) was increased by 303% as compared with the sham group and the lung damage increased 2.5-fold as demonstrated by extravasation of Evans blue dye (Fig. 3) at 8 h after injury. These results demonstrated that thermal injury induced significant increases in lung neutrophil accumulation, plasma DHR oxidation and lung damage and also in agreement with our previous studies (16) reporting that the MPO activity and plasma DHR 123 oxidation peaked at 8 h after burn but the lung permeability increased to a maximum as early at 4 h after burn.
Effect of ODQ on lung MPO activity
Rats received sterile saline after burn (group II) showed a significant 201% increase in lung MPO activity in comparison with sham group (Fig. 1). Although rats injected with ODQ after burn (group III) had an increase in lung MPO activity (34%) in comparison with group I, ODQ administration significantly decreased 55% of the neutrophil accumulation in lung in comparison with saline injection group (group II).
Effect of ODQ on plasma rhodamine level
Rats receiving sterile saline after burn had a significant increase in plasma DHR 123 oxidation level in comparison with group I (Fig. 2). Injection of ODQ (group III) caused a significant 66% decrease in comparison with saline injection group, and the DHR 123 oxidation level of group III showed no significant difference with the control group (group I).
Effect of ODQ on lung permeability
The lung permeability in rats of group II increased 149% in comparison with that of group I (Fig. 3). Although the injection of ODQ (group III) after thermal injury significantly decreased (15.04 ± 1.26 vs. 32.07 ± 0.95 ng/mg) the lung permeability up to 53% in comparison with the saline injection group, lung permeability in Group III after burn still showed a significant increase (17%) in comparison with group I.
Effect of ODQ or methylene blue on iNOS expression
As shown in Figure 4, iNOS strongly expressed in burn rat at 4 h but not at 8 h after thermal injury. Both of the selective sGC inhibitor ODQ and methylene blue significantly reduced the expression of iNOS at 4 h after burn, suggesting the involvement of the sGC/cGMP pathway in the expression of iNOS induced by burn in rats. SNP (2 mM) significant increased the iNOS mRNA expression in lungs of sham treated animals, suggesting the involvement of the cGMP in the induction of iNOS in the lung. ODQ significantly reduced the expression of iNOS at 4 h after SNP treatment.
The lungs in the saline injection group showed a marked interstitial edema and increased perivascular and interstitial inflammatory cells infiltration in comparison with control (Fig. 5). The inflammatory infiltrate was composed of predominantly of lymphocytes and polymorphonuclear leukocytes and the infiltrate was markedly attenuated in ODQ group. The lung injury score was presented in Table 1.
NO has been identified as a versatile and ubiquitous mediator in mammalians. Excessive production of NO by iNOS has been implicated as an important factor that contributes to many manifestations of septic shock, including vasodilatation (26), diminished myocardial contractility, hepatic damage, microvascular hyperpermeability, and intestinal barrier dysfunction (27). Binding of NO activates sGC to catalyze the conversion of GTP to cGMP. The sGC is a key enzyme in the signal transduction pathway leading to NO-induced vasodilatation. Therefore, inhibition of sGC has been recognized as a potential way to block the NO overproduction and its cytopathic effects in septic shock or endotoxemia. Two agents, 4 H-8-bromo-[1,2,4] oxidazolo (3,4-d) benz (b) (1,4) oxazin-1-one (NS 2028) and 1 H-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one (ODQ), have been identified as highly selective sGC inhibitors. Zingarelli et al. have demonstrated that ODQ application increased the survival of mice in lipopolysaccharide (LPS)-induced sepsis without influencing NO- or peroxynitrite-induced cytotoxicity (8). Inhibition of sGC has been shown to attenuate IL-1β/TNFα-elicited iNOS induction in human mesangial cells (28). Our data also show sGC inhibition by ODQ or methylene blue reduces SNP-induced iNOS expression in lung tissues. This further demonstrates the positive feedback mechanism in which iNOS-induced cGMP will upregulate iNOS.
In many pathologic conditions and inflammation, simultaneous cellular production of superoxide anion (O2) and NO may occur, potentially leading to the continuous formation of peroxynitrite. Recently, nitrogen-derived oxidants were found in human acute lung injury, suggesting the possibility of an important role for peroxynitrite in inflammatory lung disease (29). In rodent models, enhanced formation of NO and peroxynitrite have been implicated in the pathogenesis of various forms of shock (30). Since peroxynitrite has a half-life of less than 1 s at pH 7.4, the measurement of DHR 123 oxidation has been taken as a marker of peroxynitrite production in plasma. This method is sensitive and specific, because while authentic peroxynitrite oxidizes DHR 123, neither NO nor superoxide causes DHR 123 oxidation (25). Previously, our studies showed that there is a significant increase of intestinal mucosal iNOS activity and a substantial increase in the level of nitrotyrosine immunostaining within the base of intestinal villi following thermal injury (31), suggesting that peroxynitrite plays a vital role in thermal injury-induced intestine damage. As shown in Figure 2, the plasma DHR 123 oxidation level in injured rats increased dramatically to 4-fold of control rats at 8 h post burn, demonstrating that thermal injury induced the peroxynitrite formation in plasma and use of guanylate cyclase inhibitor ODQ reduced the production of peroxynitrite and lung damage. NO released in small quantities may be beneficial by increasing protein synthesis and DNA-repair enzymes (24). However, overproduction of NO may contribute to the tissue injury after the activation of iNOS under the influence of inflammatory mediators (25). NO has been known to neutralize the prooxidative effect of peroxynitrite (26). As demonstrated by RT-PCR of iNOS, the expression of iNOS was barely detected at 8 h after burn but peaked at 4 h after burn. This observation suggests that induction of iNOS and excess production of NO directly result in the significantly increase in lung permeability in injured rats, and the increased lung neutrophil recruitment and plasma DHR oxidation level are likely caused by the indirect action of NO derived from iNOS. In addition, the effect of GC inhibitor ODQ on blood DHR 123 oxidation may be related to the inhibition of iNOS expression by ODQ (Fig. 4), subsequently decreasing the overproduction of NO by iNOS and the level of plasma DHR oxidation.
It has been shown that excess NO produced by iNOS during systemic endotoxemia causes a vascular leak in the lung (32) and play a critical role in the pathogenesis of LPS-induced acute lung injury and smoke inhalation injury (29). L-NAME, a nonselective NO inhibitor, has been shown to increase neutrophil adhesion in the lung, while decrease that in the peritoneum in peritonitis rats (33). Aminoguanidine, a relatively selective inhibitor of iNOS, has been demonstrated to block NO production, decrease neutrophil chemotaxis and sequestration in the lung, and attenuate lung inflammation (34). Our previous report demonstrated that a potent iNOS inhibitor significantly decreased the intestinal mucosal iNOS activity and iNOS/tNOS ratio on day 2 after burn (31). Recently, sGC inhibitor is preferred to a selective iNOS inhibitor in blocking the overproduction of NO and its downstream effect, because sGC inhibitors have been known not to interfere with the cGMP-independent actions of NO. sGC inhibitors would block both normal endothelium-dependent NO-mediated signaling as well as pathologic vasodilatation induced by LPS. Our data show that the lung neutrophil deposition and lung damage as demonstrated by lung permeability increase were induced by burn in rats. Using ODQ, a highly selective sGC inhibitor (8), we show that cGMP released by GC plays an important role in the pathogenesis of thermal injury-induced lung damage and GC inhibition reduces thermal injury-induced DHR123 oxidation in blood, lung neutrophil sequestration and damage. These results suggest that GC inhibitors may have potential in the treatment of thermal injury or splanchnic ischemia and reperfusion-induced acute respiratory distress syndrome.
It is known that severe burns result in early pulmonary vascular changes associated with leukocyte sequestration in burned rabbits (5). Arbak et al. have shown that the accumulation of neutrophil in lung is involved in the pathogenesis of this distant organ after burn injury (2). In our data, the lung permeability and the MPO activity in lung increased up to a plateau at 4 h and 8 h respectively after thermal injury. Although iNOS-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia (35), peroxynitrite has been known to be an effective priming agent for neutrophils by enhancing O2 − production on subsequent activation with N-formyl-methionine-leucine-phenylalanine (36). As shown in Figures 1 and 2, the inhibition of GC decreases the blood DHR 123 oxidation level and the lung neutrophil deposition. Peroxynitrite-induced neutrophil priming and migration may represent the mechanism through which GC inhibition decreases the lung neutrophil deposition. Our findings suggest that intestinal ischemia and reperfusion injury-induced lung damage might be closely related to plasma DHR oxidation and lung neutrophil sequestration. Overproduction of NO and the following cGMP formation might play a key role in thermal injury-induced or intestinal ischemia/reperfusion injury-induced lung damage.
We have shown that the lung permeability increased significantly as early as 4 h after burn in injured rats but the lung neutrophil accumulation and plasma DHR oxidation did not increase significantly until 8 h after burn. Also, the thermal injury-induced lung damage is reduced by use of SMT, a specific inhibitor of iNOS (16). Inhibition of iNOS by SMT decreased the blood DHR 123 oxidation and lung neutrophil deposition, suggesting that peroxynitrite-induced neutrophil priming and migration may represent the mechanism for the effect of SMT on lung deposition. However, SMT decreased the lung neutrophil deposition is only partial (∼41%) but completely prevented the lung damage. Thus, factors other than neutrophil deposition, NO overproduction and peroxynitrite might play a key role in thermal injury-induced lung damage. In this study, we observed that the beneficial effect of ODQ administration is comparable to the use of SMT, meaning that the blockade of NO production or inhibition of NO-dependent signaling pathway via cGMP have the same effect on thermal-injury induced lung damage. In addition, burn caused a significant increase in iNOS mRNA as determined by semi-quantitative RT-PCR. The expression peaked at 4 h but decreased to barely detectable level at 8 h after burn. Therefore, NO is important in initiating burn-induced increase in lung permeability (most likely to be peroxynitrite). Once NO has exerted its effects and even when iNOS expression is minimal as seen at 8 h after burn, the long-lasting effects on lung permeability and neutrophil accumulation appear to depend on a complex NO-dependent downstream cGMP signal pathway.
In summary, specific inhibition of GC immediately after thermal injury decreases the plasma DHR oxidation, the neutrophil deposition in lung, and attenuates the lung damage, suggesting that thermal injury-induced plasma DHR oxidation, lung neutrophil deposition, and lung damage are mediated by the NO/cGMP system since the lung dysfunction syndromes are abolished by administration of specific iNOS inhibitor SMT or a selective inhibitor of guanylate cyclase ODQ. Also, the observed protective effects of ODQ are attributed to the blockade of cGMP signaling pathway and the inhibition of iNOS expression. The mechanism of this inhibition clearly requires further work.
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Guanylate cyclase; iNOS; neutrophil; ODQ; peroxynitrite