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Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats
Injury, Int. J. Care Injured (2007) 38, 53—59
www.elsevier.com/locate/injury
Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats Zhong-yuan Xia a, Xiao-yuan Wang a, Xiangdong Chen a,b,*, Zhengyuan Xia a,c a
Anesthesiology Research Laboratory, Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China b Department of Pharmacology, University of Virginia, Charlottesville, VA 22904, USA c Centre for Anesthesia and Analgesia, Department of Pharmacology and Therapeutics, the University of British Columbia,Vancouver, Canada V6T 1Z3 Accepted 27 September 2006
KEYWORDS Inducible haeme oxygenase; Inducible nitric oxide synthase; Acute lung injury; Lipopolysaccharide; Sodium nitroprusside
Summary Nitric oxide (NO) donor-sodium nitroprusside (SNP) mitigates acute lung injury (ALI), but the mechanism of this protection is incompletely known. We investigated the effect of SNP on lipopolysaccharide (LPS)-induced ALI in rats. Forty-eight male Wistar rats were randomly assigned into six groups: the sham-operation group (S group), the LPS instillation group (LPS group), the haemin, a haeme oxygenase-1 (HO-1) inducer, pretreatment group (HM group), the haemin pretreatment plus LPS instillation group (HM + LPS group), the SNP alone and SNP plus LPS treatment groups. Macroscopic and histopathological examinations and immunohistochemistry analysis were performed for the lung specimens 8 h after LPS instillation. Intratracheal administration of LPS induced significant expressions of the inducible isoform of NO synthase (iNOS) and HO-1, while both haemin pretreatment and SNP treatment increased the expression of HO-1 and prevented the expression of iNOS. In the LPS group, the wet—dry weight ratio (W/D), bronchoalveolar lavage fluid (BALF) protein, and lung malondialdehyde (MDA) content were significantly higher than those in the sham-operation group, which were reversed by the pretreatment with haemin or administration of SNP. These results suggest that HO-1 plays a protective role against LPS-induced acute lung injury, which may be achieved at least in part, via inactivating the iNOS/NO system that is involved in the pathophysiological process of LPS-induced acute lung injury. The nitric oxide (NO) donor-SNP ameliorates LPS-induced ALI, which may be related to the induction of HO-1 and the subsequent inhibition of iNOS. # 2006 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China. Tel.: +86 27 88041919x2272; fax: +86 27 88042292. E-mail address: [email protected] (X. Chen). 0020–1383/$ — see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2006.09.021
54
Z-y. Xia et al.
Introduction
Animal model and experimental protocols
The pathogenic and genetic basis of acute lung injury (ALI) remains incompletely understood. Over the past few years, numerous researches have revealed that nitric oxide (NO), a free radical produced by the inducible isoform of NO synthase (iNOS), plays a critical role in the pathogenesis of oxidative stressinduced ALI. In addition, the importance of haeme oxygenase-1 (HO-1) as a cytoprotective defense mechanism against oxidative insults in ALI has also been recognised2,19,20. Only recently has it become known that it is akin to NO, the simple gaseous substance carbon monoxide (CO) is an intracellular signaling molecule that plays a functional role in the pathogenesis of a wide spectrum of biological and pathological processes.19 In vivo, there are intricate metabolic loops between NO and CO. For example, NO can up-regulate HO-1 expression, leading to the formation of endogenous CO production. On the other hand, CO, a negative regulatory modulator of iNOS, can inhibit iNOS-derived NO overproduction.17 The interaction between HO-1/CO and iNOS/NO systems has recently seen an explosion of research interest due to its potential role in the injury/anti-injury balance under oxidative stress.3,4,15 Lipopolysaccharide (LPS) is an important predisposing factor in causing ALI. Despite years of intensive research, no specific therapy has been proven to be effective in preventing ALI. Recent studies showed that the NO donor-sodium nitroprusside (SNP) reduced lung injury under cardiopulmonary bypass,1 but the exact mechanism of this action is unknown. We hypothesized that the interplay between HO-1/ CO and iNOS/NO plays a role in SNP mediated protection against LPS-induced ALI. To test this hypothesis, we employed an animal model of ALI induced by intratracheal instillation of LPS10 and examined the effects of haemin, an HO-1 inducer, and SNP on the lung injury and the expressions of iNOS and HO-1.
All rats were randomly assigned into six groups of eight animals each: the sham-operation group (S group), the LPS intratracheal instillation group (LPS group), the haemin alone group (HM group), the SNP alone intratracheal administration group (SNP group), the haemin pretreatment plus LPS intratracheal instillation group (HM + LPS group), and the SNP treatment plus LPS intratracheal instillation group (SNP + LPS group). Prior to experimentation, animals were fasted overnight, but had free access to water. On the day of the experiment, the rats were weighed and anaesthetised, with 7% chloral hydrate (5 ml kg 1) by intraperitoneal injection. LPS was dissolved in 0.9% saline. An anterior midline incision was made to expose the trachea, and a 24-gauge needle attached to a 1-ml syringe containing 300 ml of either 750 mg kg 1 of LPS in the LPS group or drug vehicle (0.9% saline) in the S group was inserted into the trachea.10 The fluid was injected. Then, the incision was closed and the rats were allowed to recover from the procedure. In the HM + LPS or HM group, animals were injected intraperitoneally with haemin at a dose of 40 mmol kg 1, 12 h prior to LPS instillation (HM + LPS group) or without LPS instillation (HM group), respectively, as described by Morisaki and colleagues.13 Haemin was prepared immediately before use by dissolving in 0.1N NaOH, adjusted to pH 7.4 with HCl. In the SNP or SNP + LPS group, SNP (25 mg kg 1) were instilled intratracheally, with or without equal volumes (300 ml) of LPS (750 mg kg 1) at the same time. We did not observe any tissue damage by SNP intratracheal instillation in this dose in our SNP control group. Our pilot study and previously reported investigations have shown that the maximal effect of LPS occurs at 8 h following intratracheal administration.11 8 h after LPS or vehicle instillation, arterial blood was collected for blood gases analysis under sterile condition8 and then rats were anaesthetised and killed by carotid exsanguination. The trachea was exposed and incised, and the pedicle of the right lung was occluded with a microvascular clip. Two aliquots of 4 ml of normal physiological saline at 4 8C were injected into and withdrawn from the left lung. The bronchoalveolar lavage (BAL) fluid was centrifuged at 1000 g for 10 min at 4 8C and the supernatant was stored at 70 8C until assayed for protein content. The upper, middle and lower lobes of the right lung tissues were chosen randomly to be harvested for the determination of wet—dry weight ratio (W/D), lung malondialdehyde (MDA) content and to be fixed in 10% formalin for immunohistochemistry assay.
Materials and methods Animals and reagents Forty-eight male Wistar rats, weighing 200 10 g, were fed a standard diet and water ad libitum. E. coli LPS (serotype 0111:B4) and haemin, a specific inducer of HO-1, were purchased from Sigma. All experiments were conducted in accordance with the approved protocols of the Experimental Animal Centre Review Board of Wuhan University, Wuhan, China. Rabbit polyclonal anti HO-1 and iNOS antibodies and other chemicals were purchased from Boster Biotechnology Co. (Wuhan, China).
Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats
HO-1 and iNOS immunohistochemistry Inflation-fixed lung tissues were paraffin-embedded and cut into 4-mm sections. Immunohistochemical staining for HO-1 and iNOS were performed by using the streptomyces avidin-biotin-peroxidase method (SABC kit, Boster Biotechnology Co., Wuhan, China). Sections were deparaffinised in xylene and hydrated in graded alcohol solutions, and endogenous peroxidase activity was quenched. After incubation overnight at 4 8C with rabbit polyclonal antibodies (Boster Biotechnology Co., Wuhan, China) against HO-1 or iNOS, sections were treated with biotinylated anti-rabbit IgG antibody. After incubation with the SABC reagent, sections were developed with diaminobensidine (DAB) solution and counterstained with heamatoxylin. Some sections were incubated with nonspecific immunoglobins (IgG) and served as negative controls. The protein expressions of HO-1 and iNOS were measured by optical density (OD), through HIPAS-2000 image analysis system, semiquantitatively, under OLYMPUS400 microscope.
Lung malondialdehyde (MDA) content and BAL fluid protein content One of the right lung lobe (100 mg) was harvested and immediately, homogenised on ice in 1 ml of cold (4 8C) normal saline (0.9% NaCl). The homogenate was centrifuged at 1000 g for 10 min at 4 8C. The content of MDA in the supernatant was measured by thiobarbituric acid colorimetric method (MDA assay kit, Jiancheng Co. Nanjing, China). The BAL fluid protein content was detected by Coomassie Brilliant Blue method.
55
(ANOVA) and Student—Newman—Keuls-q test were used for statistical evaluation. A P value less than 0.05, was considered statistically significant.
Results The changes of arterial oxygen tension and W/D of lung and BAL fluid protein content Eight hours after intratracheal LPS instillation, acute lung injury was confirmed by arterial blood gas analysis and macroscopic and histopathological examinations. Fig. 1 shows that 8 h intratracheal administration of LPS induced a 20% decrease in PaO2 (from 105 9 to 84 6 mmHg, P < 0.01 8 h after treatment vs. before treatment). Haemin or SNP alone did not affect the PaO2, while protected against the LPS-induced decrease in PaO2 (P < 0.05, HM + LPS or SNP + LPS vs. LPS group). As shown in Table 1, intratracheal administration of LPS resulted in a significant increase in W/D and BAL fluid protein contents (P < 0.01). Pretreatment with haemin or intratracheal administration of SNP significantly attenuated the increases in the lung W/D and BAL fluid protein seen in the LPS group (P < 0.01). The lung W/D and BAL fluid protein content did not differ among HM, SNP, HM + LPS, SNP + LPS and S groups (P > 0.05).
The changes of lung MDA content In the LPS group, the lung MDA content was significantly higher than that in the S group (P < 0.01). Haemin or SNP alone did not have an effect on MDA
Wet-to-dry weight ratio (W/D) of the lung Another lobe of the right lung was harvested, flushed with saline, weighed immediately, dried (at 80 8C for 72 h in oven until weights were unchanged), and reweighed. The W/D was calculated.
Histological observation Inflation-fixed third lobe of the right lung was paraffin embedded and cut into 4-mm sections. The sections were stained with haematoxylin and eosin (HE), and lung histopathological alteration was detected under light microscopy.
Statistical analysis All data were expressed as mean S.D. Commercial SPSS 11.5 software for Windows was employed to analyse all data. One-way analysis of variance
Figure 1 The effects of LPS on arterial oxygen tension (PaO2). 8 h after intratracheal administration of LPS a 20% decrease in PaO2 was induced (#P < 0.01 vs. before treatment, n = 8). Co-treatment of haemin or SNP with LPS ameliorated the reduction in PaO2 (*P < 0.05 vs. LPS group, n = 8, respectively) and there is no difference between control (before LPS treatment) and HM or SNP groups in PaO2 (P > 0.05 vs. control, n = 8, respectively).
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Table 1 Changes of wet-to-dry weight ratio (W/D), BAL fluid (BALF) protein and lung malondialdehyde (MDA) content in lung tissue in rats (n = 8/group, mean S.D.) Group
W/D
BALF (g/L)
MDA (nmol/mg protein)
S group HM group SNP group LPS group HM + LPS group SNP + LPS group
3.60 0.47 3.67 0.52 3.48 0.31 5.48 0.82 b 4.65 0.72b, c 4.35 0.43a, c
0.39 0.10 0.37 0.15 0.34 0.09 1.18 0.21 b 0.56 0.17 c 0.44 0.16 c
1.59 0.39 1.62 0.44 1.43 0.22 4.87 0.93 b 3.00 0.27b, c 2.28 0.73 c
a b c
P < 0.05 vs. S group. P < 0.01 vs. S group. P < 0.01 vs. LPS group.
content as compared with S group (P > 0.05, HM or SNP vs. S group, Table 1), however, pretreatment with haemin or administration of SNP concomitantly with LPS prevented the increase of MDA seen in the LPS group (P < 0.01).
lung damage in the HM + LPS and SNP + LPS groups were reduced as compared to that in the LPS group. There are no significant morphological changes in S group, HM alone and SNP alone groups (data not shown).
Morphological changes
The expressions of HO-1 and iNOS in lung
Morphological evidences for lung oedema, haemorrhage, numerous inflammatory cells sequestration, neutrophil infiltration with cell damage, and compensatory emphysema were manifested 8 h after LPS intratracheal instillation. Under light microscopy,
As shown in Fig. 2, staining of HO-1 was not detectable in the alveolar region in the S group. Eight hours after LPS instillation, inflammatory cells had accumulated in the lung and some of these cells stained positive for HO-1 and, so, did some alveolar
Figure 2 Immunohistochemical analysis for HO-1 expression in rat lung (SABC and haematoxylin and eosin (HE), original magnification 400). (A) sham-operation group, no staining of HO-1 was detected in the alveolar region. (B) LPS group, some of the inflammatory cells and alveolar epithelia were stained positive for HO-1. In the HM + LPS (C) and SNP + LPS group (D), intense positive staining for HO-1 was seen in quite a number of inflammatory cells and alveolar epitheliums. All figures are representative of at least three experiments performed on different days, respectively.
Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats Table 2 Comparing of the average OD of HO-1, iNOS protein in each group (n = 8/group, mean S.D.) Group
INOS
HO-1
S group HM group SNP group LPS group HM + LPS group SNP + LPS group
0.0883 0.0139 0.0836 0.0117 0.0897 0.0176 0.1826 0.0246 a 0.1563 0.0207a, d
0.0854 0.0164 0.1167 0.0114 b 0.1034 0.0157 b 0.1751 0.0220 a 0.2172 0.0291a, d
0.1489 0.0253a, c
0.2014 0.0160a, c
a b c d
P < 0.01 P < 0.05 P < 0.05 P < 0.01
vs. vs. vs. vs.
S group. S group. LPS group. LPS group.
epithelium cells (Fig. 2, slide B). The HO-1 protein expressions were significantly increased in LPS group compared to the S group (P < 0.01, Fig. 2. and Table 2). In the HM + LPS and SNP + LPS groups, the level of HO-1 expression was significantly higher than that in the LPS and S groups (P < 0.01 or P < 0.05, Table 2), and there was intense staining for HO-1 in inflammatory cells and alveolar epithelium cells in the HM + LPS (Fig. 2, slide C) and SNP + LPS groups (Fig. 2, slide D).
57
As shown in Fig. 3, iNOS was barely detectable in the alveolar region in the S group (Fig. 3 slide A). In the LPS group, it showed intense staining of iNOS in macrophages and some inflammatory cells and alveolar epithelium cells (Fig. 3, slide B). The expressions of iNOS in the lung, macrophages, inflammatory cells, and alveolar epithelium cells were significantly higher in the LPS group than those seen in the HM + LPS (Fig. 3, slide C) and SNP + LPS (Fig. 3, slide D) groups (P < 0.01, Table 2).
Discussion In the present in vivo study, we demonstrated that acute lung injury induced by lipopolysaccharide intratracheal instillation was associated with the induction of iNOS and HO-1. Pretreatment with haemin or intratracheal administration of NO donor SNP induced an increase of HO-1 and a decrease of the iNOS expression, as well as attenuation of LPSinduced lung injury in rats. It has been shown that SNP reduces lung injury under cardiopulmonary bypass.1 In the present study, we used an in vivo rat model of endotoxininduced ALI to investigate the possible beneficial effects of intratracheal administration of SNP on
Figure 3 Immunohistochemical analysis for iNOS expression in rat lung (SABC and haematoxylin and eosin (HE), original magnification 400). (A) In the sham-operation group iNOS expression was barely detectable in the alveolar region. (B) In the LPS group intensive staining of iNOS was found in macrophages, some inflammatory cells, and alveolar epitheliums. There was light staining for iNOS in some inflammatory cells and alveolar epitheliums in the haemin + LPS group (C) and the SNP + LPS group (D). All figures are representative of at least three experiments performed on different days, respectively.
58 LPS-induced ALI. In this study we chose intratracheal LPS administration rather than intraperitoneal or intravenous LPS injection in an attempt to focus on lung injury without causing systemic inflammation and multi-organ failure. Intratracheal LPS administration resulted in increased expression of iNOS and HO-1 and apparent alveolar inflammation, characterised by pulmonary oedema, neutrophil infiltration with cell damage and tissue injury. Intratracheal LPS administration also caused a significant increase of BAL fluid protein contents and MDA contents, a result that is consistent with previous studies.19,20 SNP administered intratracheally at the early stage of acute lung injury ameliorated LPS-induced ALI, prevented the LPS-induced decrease in PaO2 and attenuated the increases in lung W/D and BAL fluid protein induced by LPS. Data from this study, together with previously published results,1 support the notion that SNP may be useful for the treatment of ALI or acute respiratory distress syndrome (ARDS). However, only the effects of pretreatment with SNP on LPS-induced ALI have been investigated in the current study. Whether posttreatment with SNP could confer similar effects is yet to be determined. The therapeutic effect of SNP may be attributable to multiple mechanisms. In the current study, both pretreatment with haemin and intratracheal administration of SNP led to an increased expression of HO-1 and an attenuation of the LPS induced expression of iNOS and lung injury. The parallel results induced by haemin and SNP suggest that SNP may mainly share similar mechanisms with haemin in protecting LPS-induced lung injury. The beneficial effect of haemin treatment on the lung injury may be attributable to the down regulation of LPS induced expression of iNOS. Currently, the specific mechanisms of the pathogenesis of ALI are poorly elucidated. A general understanding is that oxidants that are generated in excess of antioxidant defences can result in ALI.21 Therefore, the balance of oxidant-antioxidant defence mechanisms has been extremely important in our understanding of ALI/ARDS. Numerous researches have demonstrated that endotoxin (LPS) exposure leads to the induction of iNOS protein expression in macrophages, neutrophil, and endothelial cells accompanied by generation of sustained high quantities of NO,7,22 and that the expression of iNOS and the subsequent production of supraphysiologic amounts of NO could have played a key role in the pathogenesis of LPS-induced ALI.6 In the LPS-induced ALI, the regulatory cross-talk between HO-1 and iNOS may be operative. It has been shown that NO is a determinant in the induction of the HO-1.5,9 Further, study has demonstrated
Z-y. Xia et al. that there are regulatory loops between NO and HO-1: NO can up-regulate the HO-1 expression, leading to the formation of endogenous CO. On the other hand, CO, a negative feedback modulator of iNOS, can inhibit the overproduction of iNOSderived NO.14 Therefore, our results suggest that the interaction between HO-1/CO and iNOS/NO system may be attributable, at least in part, to the protective action of NO donor SNP on lung injury. However, further studies incorporating an HO-1 blocker would be helpful to address the mechanism(s). In addition, the direct modulation of iNOS by SNP may represent one of the mechanisms through which SNP induces its therapeutic effects. It has been shown that NO can directly down-regulate iNOS expression, by an inhibitory action on cytokine-induced NF-kB activation, due to its direct blockade on phosphorylation and subsequent degradation of IkB-a via the cGMP-independent pathway.12 Furthermore, SNP may attenuate the lung injury by inducing polymorphonuclear leukocyte apoptosis and attenuating the accumulation of polymorphonuclear leukocyte in the lung.18 Finally, the amelioration of LPS induced pulmonary hypertension by SNP may contribute to the protective action of SNP on lung injury. However, it has been shown that the vasodilatory mechanism was not attributable to the pulmonary protection in ischaemiareperfusion injury exerted by SNP.16 In summary, the results of our study suggest an interaction between HO-1 and iNOS systems in LPSinduced ALI. Our studies also suggest that the NO donor-SNP mediated amelioration of LPS-induced ALI may be related to the increase of HO-1 and inhibition of iNOS. On the other hand, the protective effect of HO-1/CO system may be mediated, at least in part, by preventing the activation of iNOS/NO system that is involved in the pathophysiological process of LPS-induced lung injury. These results provide evidence that SNP may be a therapy in the prevention and treatment of ALI or ARDS.
Acknowledgement This work was supported, in part, by the National Natural Science Foundation of China (30471059 to Z.X., 30400422 to X.C.).
References 1. Cakir O, Oruc A, Eren S, et al. Does sodium nitroprusside reduce lung injury under cardiopulmonary bypass? Eur J Cardiothorac Surg 2003;23:1040—5.
Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats 2. Choi AM, Alam J. Haeme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidantinduced lung injury. Am J Respir Cell Mol Biol 1996;15:9—19. 3. Datta PK, Koukouritaki SB, Hopp KA, et al. Haeme oxygenase1 induction attenuates inducible nitric oxide synthase expression and proteinuria in glomerulonephritis. J Am SOC Nephrol 1999;10:2540—50. 4. Demple B. Protection from the dark side of NO: signaling and cellular defenses against nitric oxide toxicity. IUBMB Life 2004;56:59—64. 5. Foresti R, Hoque M, Bains S, et al. Haem and nitric oxide: synergism in the modulation of the endothelial haem oxygenase-1 pathway. Biochem J 2003;372:381—90. 6. Gu Z, Ling Y, Cong B, et al. Peroxynitrite mediated acute lung injury induced by lipopolysaccharides in rats. Natl Med J China 2000;80:58—61. 7. Gutierrez HH, Pitt BR, Schwarz M, et al. Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators. Am J Physiol 1995;268:L501—8. 8. Hartog A, Gommers D, Haitsma JJ, Lachmann B. Improvement of lung mechanics by exogenous surfactant: effect of prior application of high positive end-expiratory pressure. Br J Anaesth 2000;85:752—6. 9. Hartsfield CL, Alam J, Cook JL, Choi AM. Regulation of haeme oxygenase-1 gene expression vascular smooth muscle cells by nitric oxide. Am J Physiol 1997;237:L980—8. 10. Honda K, Kobayashi H, Hataishi R, et al. Inhaled nitric oxide reduces tyrosine nitration after lipopolysaccharide instillation into lungs of rats. Am J Respir Crit Care Med 1999;160(2): 678—88. 11. Jeyaseelan S, Chu H-W, Young SK, Worthen GS. Transcriptional profiling of lipopolysaccharide-induced acute lung injury. Infect Immun 2004;72:7247—56. 12. Katsuyama K, Shichiri M, Marumo F, et al. NO inhibits cytokine-induced iNOS expression and NF-kB activation by
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interfering with phosphorylation and degradation of IkB-a. Arterioscler Thromb Vasc Biol 1998;18:1796—802. Morisaki H, Katayama T, Kotake Y, et al. Roles of carbon monoxide in leukocyte and platelet dynamics in rat mesenteric during sevoflurane anesthesia. Anesthesiology 2001;95: 192—9. Otterbein LE, Bach FH, Alam J, et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422—42. Sarady JK, Zuckerbraun BS, Bilban M, et al. Carbon monoxide protection against endotoxic shock in involves reciprocal effects on iNOS in the lung and liver. FASEB J 2004;18: 854—6. Schutte H, Lockinger A, Seeger W, Grimminger F. Aerosolized PGE1, PGI2 and nitroprusside protect against vascular leakage in lung ischaemia-reperfusion. Eur Respir J 2001;18:15— 22. Slebos DJ, Rvter SW, Choie AM. Haeme oxygenase-1 and carbon monoxide in pulmonary medicine. Respir Res 2003;4:7. Sookhai S, Wang JJ, McCourt M, et al. A novel therapeutic strategy for attenuating neutrophil-mediated lung injury in vivo. Ann Surg 2002;235:285—91. Tavaf-Motamen H, Miner TJ, Starnes BW, Shea-Donohue T. Nitric oxide mediates acute lung injury by modulation of inflammation. J Surg Res 1998;78:137—42. Tsuburai T, Kaneko T, Nagashima Y, et al. Pseudomonas aeruginose-induced neutrophilic lung inflammation is attenuated by adenovirus-mediated transfer of the 9 oxygenase-1 cDNA in mice. Hum Gene Ther 2004;15:273—85. Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000;342:1334—49. Wright CD, Mulsch A, Busse R, Osswald H. Generation of nitric oxide by human neutrophils. Biochem Biophys Res Commun 1989;160:813—9.
www.elsevier.com/locate/injury
Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats Zhong-yuan Xia a, Xiao-yuan Wang a, Xiangdong Chen a,b,*, Zhengyuan Xia a,c a
Anesthesiology Research Laboratory, Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China b Department of Pharmacology, University of Virginia, Charlottesville, VA 22904, USA c Centre for Anesthesia and Analgesia, Department of Pharmacology and Therapeutics, the University of British Columbia,Vancouver, Canada V6T 1Z3 Accepted 27 September 2006
KEYWORDS Inducible haeme oxygenase; Inducible nitric oxide synthase; Acute lung injury; Lipopolysaccharide; Sodium nitroprusside
Summary Nitric oxide (NO) donor-sodium nitroprusside (SNP) mitigates acute lung injury (ALI), but the mechanism of this protection is incompletely known. We investigated the effect of SNP on lipopolysaccharide (LPS)-induced ALI in rats. Forty-eight male Wistar rats were randomly assigned into six groups: the sham-operation group (S group), the LPS instillation group (LPS group), the haemin, a haeme oxygenase-1 (HO-1) inducer, pretreatment group (HM group), the haemin pretreatment plus LPS instillation group (HM + LPS group), the SNP alone and SNP plus LPS treatment groups. Macroscopic and histopathological examinations and immunohistochemistry analysis were performed for the lung specimens 8 h after LPS instillation. Intratracheal administration of LPS induced significant expressions of the inducible isoform of NO synthase (iNOS) and HO-1, while both haemin pretreatment and SNP treatment increased the expression of HO-1 and prevented the expression of iNOS. In the LPS group, the wet—dry weight ratio (W/D), bronchoalveolar lavage fluid (BALF) protein, and lung malondialdehyde (MDA) content were significantly higher than those in the sham-operation group, which were reversed by the pretreatment with haemin or administration of SNP. These results suggest that HO-1 plays a protective role against LPS-induced acute lung injury, which may be achieved at least in part, via inactivating the iNOS/NO system that is involved in the pathophysiological process of LPS-induced acute lung injury. The nitric oxide (NO) donor-SNP ameliorates LPS-induced ALI, which may be related to the induction of HO-1 and the subsequent inhibition of iNOS. # 2006 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China. Tel.: +86 27 88041919x2272; fax: +86 27 88042292. E-mail address: [email protected] (X. Chen). 0020–1383/$ — see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2006.09.021
54
Z-y. Xia et al.
Introduction
Animal model and experimental protocols
The pathogenic and genetic basis of acute lung injury (ALI) remains incompletely understood. Over the past few years, numerous researches have revealed that nitric oxide (NO), a free radical produced by the inducible isoform of NO synthase (iNOS), plays a critical role in the pathogenesis of oxidative stressinduced ALI. In addition, the importance of haeme oxygenase-1 (HO-1) as a cytoprotective defense mechanism against oxidative insults in ALI has also been recognised2,19,20. Only recently has it become known that it is akin to NO, the simple gaseous substance carbon monoxide (CO) is an intracellular signaling molecule that plays a functional role in the pathogenesis of a wide spectrum of biological and pathological processes.19 In vivo, there are intricate metabolic loops between NO and CO. For example, NO can up-regulate HO-1 expression, leading to the formation of endogenous CO production. On the other hand, CO, a negative regulatory modulator of iNOS, can inhibit iNOS-derived NO overproduction.17 The interaction between HO-1/CO and iNOS/NO systems has recently seen an explosion of research interest due to its potential role in the injury/anti-injury balance under oxidative stress.3,4,15 Lipopolysaccharide (LPS) is an important predisposing factor in causing ALI. Despite years of intensive research, no specific therapy has been proven to be effective in preventing ALI. Recent studies showed that the NO donor-sodium nitroprusside (SNP) reduced lung injury under cardiopulmonary bypass,1 but the exact mechanism of this action is unknown. We hypothesized that the interplay between HO-1/ CO and iNOS/NO plays a role in SNP mediated protection against LPS-induced ALI. To test this hypothesis, we employed an animal model of ALI induced by intratracheal instillation of LPS10 and examined the effects of haemin, an HO-1 inducer, and SNP on the lung injury and the expressions of iNOS and HO-1.
All rats were randomly assigned into six groups of eight animals each: the sham-operation group (S group), the LPS intratracheal instillation group (LPS group), the haemin alone group (HM group), the SNP alone intratracheal administration group (SNP group), the haemin pretreatment plus LPS intratracheal instillation group (HM + LPS group), and the SNP treatment plus LPS intratracheal instillation group (SNP + LPS group). Prior to experimentation, animals were fasted overnight, but had free access to water. On the day of the experiment, the rats were weighed and anaesthetised, with 7% chloral hydrate (5 ml kg 1) by intraperitoneal injection. LPS was dissolved in 0.9% saline. An anterior midline incision was made to expose the trachea, and a 24-gauge needle attached to a 1-ml syringe containing 300 ml of either 750 mg kg 1 of LPS in the LPS group or drug vehicle (0.9% saline) in the S group was inserted into the trachea.10 The fluid was injected. Then, the incision was closed and the rats were allowed to recover from the procedure. In the HM + LPS or HM group, animals were injected intraperitoneally with haemin at a dose of 40 mmol kg 1, 12 h prior to LPS instillation (HM + LPS group) or without LPS instillation (HM group), respectively, as described by Morisaki and colleagues.13 Haemin was prepared immediately before use by dissolving in 0.1N NaOH, adjusted to pH 7.4 with HCl. In the SNP or SNP + LPS group, SNP (25 mg kg 1) were instilled intratracheally, with or without equal volumes (300 ml) of LPS (750 mg kg 1) at the same time. We did not observe any tissue damage by SNP intratracheal instillation in this dose in our SNP control group. Our pilot study and previously reported investigations have shown that the maximal effect of LPS occurs at 8 h following intratracheal administration.11 8 h after LPS or vehicle instillation, arterial blood was collected for blood gases analysis under sterile condition8 and then rats were anaesthetised and killed by carotid exsanguination. The trachea was exposed and incised, and the pedicle of the right lung was occluded with a microvascular clip. Two aliquots of 4 ml of normal physiological saline at 4 8C were injected into and withdrawn from the left lung. The bronchoalveolar lavage (BAL) fluid was centrifuged at 1000 g for 10 min at 4 8C and the supernatant was stored at 70 8C until assayed for protein content. The upper, middle and lower lobes of the right lung tissues were chosen randomly to be harvested for the determination of wet—dry weight ratio (W/D), lung malondialdehyde (MDA) content and to be fixed in 10% formalin for immunohistochemistry assay.
Materials and methods Animals and reagents Forty-eight male Wistar rats, weighing 200 10 g, were fed a standard diet and water ad libitum. E. coli LPS (serotype 0111:B4) and haemin, a specific inducer of HO-1, were purchased from Sigma. All experiments were conducted in accordance with the approved protocols of the Experimental Animal Centre Review Board of Wuhan University, Wuhan, China. Rabbit polyclonal anti HO-1 and iNOS antibodies and other chemicals were purchased from Boster Biotechnology Co. (Wuhan, China).
Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats
HO-1 and iNOS immunohistochemistry Inflation-fixed lung tissues were paraffin-embedded and cut into 4-mm sections. Immunohistochemical staining for HO-1 and iNOS were performed by using the streptomyces avidin-biotin-peroxidase method (SABC kit, Boster Biotechnology Co., Wuhan, China). Sections were deparaffinised in xylene and hydrated in graded alcohol solutions, and endogenous peroxidase activity was quenched. After incubation overnight at 4 8C with rabbit polyclonal antibodies (Boster Biotechnology Co., Wuhan, China) against HO-1 or iNOS, sections were treated with biotinylated anti-rabbit IgG antibody. After incubation with the SABC reagent, sections were developed with diaminobensidine (DAB) solution and counterstained with heamatoxylin. Some sections were incubated with nonspecific immunoglobins (IgG) and served as negative controls. The protein expressions of HO-1 and iNOS were measured by optical density (OD), through HIPAS-2000 image analysis system, semiquantitatively, under OLYMPUS400 microscope.
Lung malondialdehyde (MDA) content and BAL fluid protein content One of the right lung lobe (100 mg) was harvested and immediately, homogenised on ice in 1 ml of cold (4 8C) normal saline (0.9% NaCl). The homogenate was centrifuged at 1000 g for 10 min at 4 8C. The content of MDA in the supernatant was measured by thiobarbituric acid colorimetric method (MDA assay kit, Jiancheng Co. Nanjing, China). The BAL fluid protein content was detected by Coomassie Brilliant Blue method.
55
(ANOVA) and Student—Newman—Keuls-q test were used for statistical evaluation. A P value less than 0.05, was considered statistically significant.
Results The changes of arterial oxygen tension and W/D of lung and BAL fluid protein content Eight hours after intratracheal LPS instillation, acute lung injury was confirmed by arterial blood gas analysis and macroscopic and histopathological examinations. Fig. 1 shows that 8 h intratracheal administration of LPS induced a 20% decrease in PaO2 (from 105 9 to 84 6 mmHg, P < 0.01 8 h after treatment vs. before treatment). Haemin or SNP alone did not affect the PaO2, while protected against the LPS-induced decrease in PaO2 (P < 0.05, HM + LPS or SNP + LPS vs. LPS group). As shown in Table 1, intratracheal administration of LPS resulted in a significant increase in W/D and BAL fluid protein contents (P < 0.01). Pretreatment with haemin or intratracheal administration of SNP significantly attenuated the increases in the lung W/D and BAL fluid protein seen in the LPS group (P < 0.01). The lung W/D and BAL fluid protein content did not differ among HM, SNP, HM + LPS, SNP + LPS and S groups (P > 0.05).
The changes of lung MDA content In the LPS group, the lung MDA content was significantly higher than that in the S group (P < 0.01). Haemin or SNP alone did not have an effect on MDA
Wet-to-dry weight ratio (W/D) of the lung Another lobe of the right lung was harvested, flushed with saline, weighed immediately, dried (at 80 8C for 72 h in oven until weights were unchanged), and reweighed. The W/D was calculated.
Histological observation Inflation-fixed third lobe of the right lung was paraffin embedded and cut into 4-mm sections. The sections were stained with haematoxylin and eosin (HE), and lung histopathological alteration was detected under light microscopy.
Statistical analysis All data were expressed as mean S.D. Commercial SPSS 11.5 software for Windows was employed to analyse all data. One-way analysis of variance
Figure 1 The effects of LPS on arterial oxygen tension (PaO2). 8 h after intratracheal administration of LPS a 20% decrease in PaO2 was induced (#P < 0.01 vs. before treatment, n = 8). Co-treatment of haemin or SNP with LPS ameliorated the reduction in PaO2 (*P < 0.05 vs. LPS group, n = 8, respectively) and there is no difference between control (before LPS treatment) and HM or SNP groups in PaO2 (P > 0.05 vs. control, n = 8, respectively).
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Z-y. Xia et al.
Table 1 Changes of wet-to-dry weight ratio (W/D), BAL fluid (BALF) protein and lung malondialdehyde (MDA) content in lung tissue in rats (n = 8/group, mean S.D.) Group
W/D
BALF (g/L)
MDA (nmol/mg protein)
S group HM group SNP group LPS group HM + LPS group SNP + LPS group
3.60 0.47 3.67 0.52 3.48 0.31 5.48 0.82 b 4.65 0.72b, c 4.35 0.43a, c
0.39 0.10 0.37 0.15 0.34 0.09 1.18 0.21 b 0.56 0.17 c 0.44 0.16 c
1.59 0.39 1.62 0.44 1.43 0.22 4.87 0.93 b 3.00 0.27b, c 2.28 0.73 c
a b c
P < 0.05 vs. S group. P < 0.01 vs. S group. P < 0.01 vs. LPS group.
content as compared with S group (P > 0.05, HM or SNP vs. S group, Table 1), however, pretreatment with haemin or administration of SNP concomitantly with LPS prevented the increase of MDA seen in the LPS group (P < 0.01).
lung damage in the HM + LPS and SNP + LPS groups were reduced as compared to that in the LPS group. There are no significant morphological changes in S group, HM alone and SNP alone groups (data not shown).
Morphological changes
The expressions of HO-1 and iNOS in lung
Morphological evidences for lung oedema, haemorrhage, numerous inflammatory cells sequestration, neutrophil infiltration with cell damage, and compensatory emphysema were manifested 8 h after LPS intratracheal instillation. Under light microscopy,
As shown in Fig. 2, staining of HO-1 was not detectable in the alveolar region in the S group. Eight hours after LPS instillation, inflammatory cells had accumulated in the lung and some of these cells stained positive for HO-1 and, so, did some alveolar
Figure 2 Immunohistochemical analysis for HO-1 expression in rat lung (SABC and haematoxylin and eosin (HE), original magnification 400). (A) sham-operation group, no staining of HO-1 was detected in the alveolar region. (B) LPS group, some of the inflammatory cells and alveolar epithelia were stained positive for HO-1. In the HM + LPS (C) and SNP + LPS group (D), intense positive staining for HO-1 was seen in quite a number of inflammatory cells and alveolar epitheliums. All figures are representative of at least three experiments performed on different days, respectively.
Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats Table 2 Comparing of the average OD of HO-1, iNOS protein in each group (n = 8/group, mean S.D.) Group
INOS
HO-1
S group HM group SNP group LPS group HM + LPS group SNP + LPS group
0.0883 0.0139 0.0836 0.0117 0.0897 0.0176 0.1826 0.0246 a 0.1563 0.0207a, d
0.0854 0.0164 0.1167 0.0114 b 0.1034 0.0157 b 0.1751 0.0220 a 0.2172 0.0291a, d
0.1489 0.0253a, c
0.2014 0.0160a, c
a b c d
P < 0.01 P < 0.05 P < 0.05 P < 0.01
vs. vs. vs. vs.
S group. S group. LPS group. LPS group.
epithelium cells (Fig. 2, slide B). The HO-1 protein expressions were significantly increased in LPS group compared to the S group (P < 0.01, Fig. 2. and Table 2). In the HM + LPS and SNP + LPS groups, the level of HO-1 expression was significantly higher than that in the LPS and S groups (P < 0.01 or P < 0.05, Table 2), and there was intense staining for HO-1 in inflammatory cells and alveolar epithelium cells in the HM + LPS (Fig. 2, slide C) and SNP + LPS groups (Fig. 2, slide D).
57
As shown in Fig. 3, iNOS was barely detectable in the alveolar region in the S group (Fig. 3 slide A). In the LPS group, it showed intense staining of iNOS in macrophages and some inflammatory cells and alveolar epithelium cells (Fig. 3, slide B). The expressions of iNOS in the lung, macrophages, inflammatory cells, and alveolar epithelium cells were significantly higher in the LPS group than those seen in the HM + LPS (Fig. 3, slide C) and SNP + LPS (Fig. 3, slide D) groups (P < 0.01, Table 2).
Discussion In the present in vivo study, we demonstrated that acute lung injury induced by lipopolysaccharide intratracheal instillation was associated with the induction of iNOS and HO-1. Pretreatment with haemin or intratracheal administration of NO donor SNP induced an increase of HO-1 and a decrease of the iNOS expression, as well as attenuation of LPSinduced lung injury in rats. It has been shown that SNP reduces lung injury under cardiopulmonary bypass.1 In the present study, we used an in vivo rat model of endotoxininduced ALI to investigate the possible beneficial effects of intratracheal administration of SNP on
Figure 3 Immunohistochemical analysis for iNOS expression in rat lung (SABC and haematoxylin and eosin (HE), original magnification 400). (A) In the sham-operation group iNOS expression was barely detectable in the alveolar region. (B) In the LPS group intensive staining of iNOS was found in macrophages, some inflammatory cells, and alveolar epitheliums. There was light staining for iNOS in some inflammatory cells and alveolar epitheliums in the haemin + LPS group (C) and the SNP + LPS group (D). All figures are representative of at least three experiments performed on different days, respectively.
58 LPS-induced ALI. In this study we chose intratracheal LPS administration rather than intraperitoneal or intravenous LPS injection in an attempt to focus on lung injury without causing systemic inflammation and multi-organ failure. Intratracheal LPS administration resulted in increased expression of iNOS and HO-1 and apparent alveolar inflammation, characterised by pulmonary oedema, neutrophil infiltration with cell damage and tissue injury. Intratracheal LPS administration also caused a significant increase of BAL fluid protein contents and MDA contents, a result that is consistent with previous studies.19,20 SNP administered intratracheally at the early stage of acute lung injury ameliorated LPS-induced ALI, prevented the LPS-induced decrease in PaO2 and attenuated the increases in lung W/D and BAL fluid protein induced by LPS. Data from this study, together with previously published results,1 support the notion that SNP may be useful for the treatment of ALI or acute respiratory distress syndrome (ARDS). However, only the effects of pretreatment with SNP on LPS-induced ALI have been investigated in the current study. Whether posttreatment with SNP could confer similar effects is yet to be determined. The therapeutic effect of SNP may be attributable to multiple mechanisms. In the current study, both pretreatment with haemin and intratracheal administration of SNP led to an increased expression of HO-1 and an attenuation of the LPS induced expression of iNOS and lung injury. The parallel results induced by haemin and SNP suggest that SNP may mainly share similar mechanisms with haemin in protecting LPS-induced lung injury. The beneficial effect of haemin treatment on the lung injury may be attributable to the down regulation of LPS induced expression of iNOS. Currently, the specific mechanisms of the pathogenesis of ALI are poorly elucidated. A general understanding is that oxidants that are generated in excess of antioxidant defences can result in ALI.21 Therefore, the balance of oxidant-antioxidant defence mechanisms has been extremely important in our understanding of ALI/ARDS. Numerous researches have demonstrated that endotoxin (LPS) exposure leads to the induction of iNOS protein expression in macrophages, neutrophil, and endothelial cells accompanied by generation of sustained high quantities of NO,7,22 and that the expression of iNOS and the subsequent production of supraphysiologic amounts of NO could have played a key role in the pathogenesis of LPS-induced ALI.6 In the LPS-induced ALI, the regulatory cross-talk between HO-1 and iNOS may be operative. It has been shown that NO is a determinant in the induction of the HO-1.5,9 Further, study has demonstrated
Z-y. Xia et al. that there are regulatory loops between NO and HO-1: NO can up-regulate the HO-1 expression, leading to the formation of endogenous CO. On the other hand, CO, a negative feedback modulator of iNOS, can inhibit the overproduction of iNOSderived NO.14 Therefore, our results suggest that the interaction between HO-1/CO and iNOS/NO system may be attributable, at least in part, to the protective action of NO donor SNP on lung injury. However, further studies incorporating an HO-1 blocker would be helpful to address the mechanism(s). In addition, the direct modulation of iNOS by SNP may represent one of the mechanisms through which SNP induces its therapeutic effects. It has been shown that NO can directly down-regulate iNOS expression, by an inhibitory action on cytokine-induced NF-kB activation, due to its direct blockade on phosphorylation and subsequent degradation of IkB-a via the cGMP-independent pathway.12 Furthermore, SNP may attenuate the lung injury by inducing polymorphonuclear leukocyte apoptosis and attenuating the accumulation of polymorphonuclear leukocyte in the lung.18 Finally, the amelioration of LPS induced pulmonary hypertension by SNP may contribute to the protective action of SNP on lung injury. However, it has been shown that the vasodilatory mechanism was not attributable to the pulmonary protection in ischaemiareperfusion injury exerted by SNP.16 In summary, the results of our study suggest an interaction between HO-1 and iNOS systems in LPSinduced ALI. Our studies also suggest that the NO donor-SNP mediated amelioration of LPS-induced ALI may be related to the increase of HO-1 and inhibition of iNOS. On the other hand, the protective effect of HO-1/CO system may be mediated, at least in part, by preventing the activation of iNOS/NO system that is involved in the pathophysiological process of LPS-induced lung injury. These results provide evidence that SNP may be a therapy in the prevention and treatment of ALI or ARDS.
Acknowledgement This work was supported, in part, by the National Natural Science Foundation of China (30471059 to Z.X., 30400422 to X.C.).
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