Volume 147, Issue 8 p. 966-974
Free Access

Nitric oxide-releasing flurbiprofen reduces formation of proinflammatory hydrogen sulfide in lipopolysaccharide-treated rat

Farhana Anuar

Farhana Anuar

Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD2, 18 Medical Drive, Singapore 117597, Singapore

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Matthew Whiteman

Matthew Whiteman

Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD7, 18 Medical Drive, Singapore 117597, Singapore

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Jia Ling Siau

Jia Ling Siau

Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD7, 18 Medical Drive, Singapore 117597, Singapore

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Shing Erl Kwong

Shing Erl Kwong

Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD7, 18 Medical Drive, Singapore 117597, Singapore

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Madhav Bhatia

Madhav Bhatia

Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD2, 18 Medical Drive, Singapore 117597, Singapore

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Philip K Moore

Corresponding Author

Philip K Moore

Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD2, 18 Medical Drive, Singapore 117597, Singapore

Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD2, 18 Medical Drive, Singapore 117597, Singapore. E-mail: [email protected]Search for more papers by this author
First published: 29 January 2009
Citations: 48

Abstract

  • The biosynthesis of both nitric oxide (NO) and hydrogen sulfide (H2S) is increased in lipopolysaccharide (LPS)-injected mice and rats but their interaction in these models is not known. In this study we examined the effect of the NO donor, nitroflurbiprofen (and the parent molecule flurbiprofen) on NO and H2S metabolism in tissues from LPS-pretreated rats.

  • Administration of LPS (10 mg kg−1, i.p.; 6 h) resulted in an increase (P<0.05) in plasma TNF-α, IL-1β and nitrate/nitrite (NOx) concentrations, liver H2S synthesis (from added cysteine), CSE mRNA, inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO) activity (marker for neutrophil infiltration) and nuclear factor-kappa B (NF-κB) activation.

  • Nitroflurbiprofen (3–30 mg kg−1, i.p.) administration resulted in a dose-dependent inhibition of the LPS-mediated increase in plasma TNF-α, IL-1β and NOx concentration, liver H2S synthesis (55.00±0.95 nmole mg protein−1, c.f. 62.38±0.47 nmole mg protein−1, n=5, P<0.05), CSE mRNA, iNOS, MPO activity and NF-κB activation.

  • Flurbiprofen (21 mg kg−1, i.p.) was without effect.

  • These results show for the first time that nitroflurbiprofen downregulates the biosynthesis of proinflammatory H2S and suggest that such an effect may contribute to the augmented anti-inflammatory activity of this compound.

  • These data also highlight the existence of ‘crosstalk’ between NO and H2S in this model of endotoxic shock.

British Journal of Pharmacology (2006) 147, 966–974. doi:10.1038/sj.bjp.0706696

Abbreviations:

  • CBS
  • cystathionine β synthetase
  • CSE
  • cystathionine γ lyase
  • FLU
  • flurbiprofen
  • H2S
  • hydrogen sulfide
  • IL-1β
  • interleukin-1β
  • LPS
  • lipopolysaccharide
  • MPO
  • myeloperoxidase
  • NO
  • nitric oxide
  • NOF
  • nitroflurbiprofen
  • NO-NSAID
  • NO-releasing nonsteroidal anti-inflammatory drug
  • NOx
  • nitrate/nitrite
  • TNF-α
  • tumour necrosis factor-α
  • VEH
  • vehicle
  • Introduction

    Until relatively recently hydrogen sulphide (H2S) was considered as a toxic environmental pollutant of little or no physiological significance. However, it is now clear that H2S is synthesised naturally in a range of mammalian and non-mammalian tissues by two pyridoxal phosphate-dependent enzymes (cystathionine γ lyase (CSE) and cystathione β synthetase (CBS)) acting on cysteine as substrate and most likely plays a part in physiological and pathophysiological processes (Moore et al., 2003).

    Recently, attention has been focused on the possible role of H2S in inflammation. Interestingly, the ability of inhaled H2S (following accidental environmental exposure) to trigger pulmonary inflammation (e.g. damage to pulmonary epithelial cells, alveolar swelling, oedema etc.) was noted several decades ago and is one of the major factors in the toxic response to this gas (Milby & Baselt, 1999; Knight & Presnell, 2005). Rats exposed (4 h) to an atmosphere of H2S (615 mg m−3) also showed histological evidence of pulmonary oedema and fibrinocellular alveolitis (Lopez et al., 1988). In stark contrast, exposure to H2S baths (balnaeotherapy) has been widely employed in some parts of the world as a treatment for arthritis (Grabski et al., 2004) although the mechanism(s) involved have not been determined. Over the last year, work in this and other laboratories has provided evidence of a role for H2S as a proinflammatory mediator in various animal models of haemorrhagic (Mok et al., 2004) and endotoxic (Collin et al., 2005; Li et al., 2005) shock as well as in caerulein-induced pancreatitis (Bhatia et al., 2005b) and carrageenan-induced hindpaw oedema in the rat (Bhatia et al., 2005a). In each of these disease models, tissue CSE expression is upregulated leading to enhanced H2S biosynthesis and plasma levels while CSE inhibitors (e.g. DL-propargylglycine) have been shown to exhibit pronounced anti-inflammatory activity. In addition, H2S administration to rats reportedly causes neurogenic inflammation in the airways which is mediated by activation of vanilloid 1 receptors (Trevisani et al., 2005).

    Numerous analogies exist between H2S and nitric oxide (NO). Both are cell permeable gases under physiological conditions and are synthesised in a range of mammalian cell types and tissues from simple amino acids (cysteine and arginine, respectively). In addition, both gases occur naturally in the bloodstream (H2S concentration in human plasma, approximately, 50 μM Richardson et al., 2000; NO detected as its stable breakdown products, nitrate and nitrite). Furthermore H2S and NO exhibit vasodilator activity in vitro and in vivo albeit by different mechanisms (for H2S most probably opening of smooth muscle K-ATP channels (Zhao et al., 2001) and for NO activation of soluble guanylate cyclase) and the cellular synthesis of each mediator is enhanced in a variety of inflammatory disorders most probably following the induction of CSE and inducible NO synthase (iNOS) in inflammatory and other cells (Evans, 1995). While induction of iNOS most probably occurs by several mechanisms notably activation of the intracellular nuclear factor-kappa B (NF-κB) transduction pathway, the mechanism(s) by which CSE expression is upregulated either in vitro or in vivo is not known.

    Intriguingly, recent studies have suggested that NO may work in concert (i.e. ‘crosstalk’) with H2S. A number of potential ‘crossover’ points between H2S and NO have been reported. For example, H2S stabilises NO in solution (Sorensen et al., 1980) and augments NO release from S-nitrosothiols (Roediger & Babidge, 2000). Furthermore, NO acts on cultured smooth muscle cells to increase both H2S production and CSE expression (Zhao et al., 2001). Both NO and H2S bind to, and are consequently quenched by, haemoglobin (Searcy & Lee, 1998). In addition, H2S scavenges peroxynitrite (Whiteman et al., 2004). Finally, the vasorelaxant effect of NO is variously augmented or inhibited (Zhao et al., 2003) by the presence of H2S. While the precise nature of this ‘crosstalk’ remains unclear it seems reasonable to suggest that the overall biological effect of H2S on smooth muscle (and other systems) in vivo may well be inextricably linked with the synthesis/activity of NO.

    With these observations in mind we decided to investigate the interaction between H2S and NO in an animal model of systemic inflammation (Escherichia coli lipopolysaccharide (LPS)-induced endotoxic shock). For these experiments, the NO-releasing nonsteroidal anti-inflammatory drug (NO-NSAID), nitroflurbiprofen was used.

    Methods

    LPS-induced endotoxic shock model

    The ‘Principles of laboratory animal care’ (NIH publication no. 85-23, revised 1985; http://grants1.nih.gov/grants/olaw/references/phspol.htm) were followed in these experiments which were approved by the animal ethics committee of National University of Singapore.

    Conscious rats (male, Sprague–Dawley, 250–280 g) were injected i.p. with either saline (2.5 ml kg−1, sham), E. coli LPS (10 mg kg−1, LPS treated) or LPS pretreatment with either equivalent volume (2 ml kg−1, i.p.) of vehicle (ethanol : olive oil, 5:95 v v−1), flurbiprofen (21 mg kg−1, i.p., the mol for mol of the highest dose of nitroflurbiprofen used in this study) or nitroflurbiprofen (3–30 mg kg−1, i.p.). After 6 h, rats were terminally anaesthetized (1 ml kg−1, i.p.) with a mixture of hypnorm-containing fentanyl, fluanisone and 1 ml of midazolam and blood (approx. 5 ml, 50 U ml−1 heparin) was obtained by cardiac puncture prior to assay for plasma nitrate/nitrite (NOx), TNF-α and IL-1β. Tissues (liver and kidney) were also removed and kept in cryotubes that were frozen in liquid nitrogen and stored at −80°C. They were later assayed for protein iNOS expression, H2S synthesizing ability, CSE mRNA expression, myeloperoxidase (MPO) activity and NF-κB activation.

    Measurement of plasma NOx, TNF-α and IL-1β

    Aliquots (80 μl) of plasma were incubated (37°C, 30 minNOx, final incubation volume of 200 μl) with nitrite reductase (10 mU) in the presence of NADPH (100 μmol l−1) to reduce nitrate to nitrite and then centrifuged (14,000 r.p.m., 25 min, 40°C). The resulting supernatant as well as sodium nitrite standard (100 μl; 5–35 μmol l−1) were added, in duplicate, to 96-well microtitre plates. Thereafter, Greiss reagent (0.2% w v−1 N-1-napthyl ethylenediamine dihydrochloride and 2% w v−1 sulfanilamide in 5% v v−1 H3PO4) was added to the above mixture in a ratio of 1 : 1 (v v−1) and incubated for 10 min at room temperature after which absorbance was determined at 550 nm in a 96-well microplate reader (Tecan Systems, Inc., San Jose, CA, U.S.A.). The concentration of nitrite (indicative of NOx in the original samples) was calculated from a standard curve of NaNO2 (5–35 μmol l−1) and expressed as μmol l−1 nitrite. TNF-α and IL-1β were measured in plasma aliquots (75 and 50 μl, respectively) by ELISA using commercially available kits according to the manufacturer's instructions. Results are shown as pg ml−1 of each cytokine in plasma.

    Detection of liver tissue iNOS expression by Western blot analysis

    Tissues (0.15 g) were homogenized in 4 ml of ice cold homogenization buffer (250 mM sucrose, 20 mM Tris, 1 mM DTT). The homogenates were centrifuged at 4°C, 9000 × g for 30 min. Supernatants were collected and centrifuged at 4°C, 14,000 × g for 30 min. Protease Inhibitor Cocktail (F. Hoffmann-La Roche Ltd, Grenzacherstrasse, Basel, Switzerland) was added to the final supernatants. Western blotting conditions were conducted essentially as described (Whiteman et al., 2004; 2005) using an enhanced chemiluminescence detection kit (Amersham Biosciences, Amersham, Buckinghamshire, U.K.) followed by analysis using a Kodak Image Analyser (IS440CF, NEN Life Science, Boston, MA, U.S.A.) and captured images analysed using Kodak digital science 1D image analysis software. Protein concentration was determined using a commercial kit (Dc protein assay, Bio-Rad Ltd, Hercules, CA, U.S.A.) and 30 μg total protein analysed for each assays.

    Assay of tissue H2S synthesis

    Liver and kidney tissues from animals treated as above were thawed and homogenized in 100 mM ice-cold potassium phosphate buffer (pH 7.4). Optimal w v−1 ratios of 1 : 20 v v−1 were determined from preliminary experiments. The reaction mixture (total volume, 500 μl) contained L-cysteine (10 mM, 20 μl), pyridoxal 5′-phosphate (2 mM, 20 μl), saline (30 μl) and tissue homogenate (430 μl). The reaction was performed in parafilmed eppendorf tubes and initiated by transferring the tubes from ice to a water bath at 37°C. In some experiments, the enzymatic reactions were stopped immediately by addition of trichloroacetic acid (10% w v−1, 250 μl) to denature protein prior to addition of cysteine. After incubation for 30 min, zinc acetate (1% w v−1, 250 μl) was added to trap evolved H2S, followed by tricholoroacetic acid. Subsequently, N,N-dimethyl-p-phenylenediamine sulphate (20 μM, 133 μl) in 7.2 M HCl and FeCl3 (30 μM, 133 μl) in 1.2 M HCl were added and the absorbance of the resulting solution (670 nm) measured 15 min thereafter, using a 96-well microplate reader (Tecan Systems Inc., San Jose, CA, U.S.A.). The basal concentration of H2S was determined in incubates in which trichloroacetic acid was added at zero time (T=0) prior to addition of cysteine and incubation (37°C, 30 min). At the end of this period, trichloroacetic acid was added and H2S generated assayed spectrophotometrically as described above. All samples were assayed in duplicate. The H2S concentration of each sample was calculated against a calibration curve of sodium hydrosulphide (NaHS; 3.12–250 μM) and results are expressed as nmol H2S formed mg−1 protein (determined using the Bradford assay, Bio-Rad Ltd, Hercules, CA, U.S.A.).

    Measurement of liver and kidney tissue CSE mRNA

    Tissue (100 mg) was homogenized in 1 ml ice-cold TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) and thereafter incubated for 10 min at room temperature. Samples were mixed with chloroform (0.2 ml), vigorously shaken and incubated at room temperature for 3 min, followed by centrifugation (12,000 × g, 4°C, 15 min). The top aqueous phase was transferred to an eppendorf tube and isopropanol (0.5 ml) was added. After further incubation at room temperature (10 min), samples were centrifuged (12,000 × g, 4°C, 10 min) and the resulting RNA pellet was washed with 75% v v−1 ethanol (1.5 ml) and centrifuged again (7500 × g, 4°C, 5 min). Supernatants were discarded and the RNA pellets were air dried (5–10 min), dissolved in diethyl pyrocarbonate (DEPC)-treated water (20–80 μl) and incubated (55–60°C) for 10 min. The concentration of isolated nucleic acids was determined spectrophotometrically by measuring the absorbance at 260 nm. All samples were stored at −80°C until required. One-step RT–PCR method was employed in this study (QIAGEN® one-step RT–PCR kit, Qiagen Inc., Valencia, CA, U.S.A.). Total RNA template (1 μg) was mixed with 5 × RT–PCR buffer (4 μl), dNTP mix (400 μM, 0.8 μl), 1.2 μl of each primer (0.6 μM), enzyme mix (0.8 μl, a mixture of omniscript, sensiscript reverse transcriptases and HotStar Taq DNA polymerase) and DEPC-treated water. The final volume was 20 μl. For the detection of CSE mRNA, the forward primer sequence used was 5′-CATGGATGAAGTGTATGGAGGC-3′, and the reverse primer sequence was 5′-CGGCAGCAGAGGTAACAATCG-3′. The PCR product size for CSE was 445 bp. RT–PCR was performed at 50°C for 30 min and at 95°C for 15 min for reverse transcription, followed by 36 cycles of PCR reaction consisting of 94°C (30 s) for denaturation, 58.5°C (30 s) for primer-specific annealing, and 72°C (30 s) for extension. The reaction without RNA template (none template control) was also performed as a negative control. PCR products were analysed by 1.5% w v−1 agarose ethidium bromide gel electrophoresis and imaged under ultraviolet light by the MultiGenius Bioimaging system (Syngene, Cambridge, U.K.). The band intensity was semiquantified by densitometry using Gel analysis software (Syngene, Cambridge, U.K.).

    Measurement of MPO activity

    Neutrophil sequestration in liver and kidney was quantified by measuring tissue MPO activity. Tissue samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), centrifuged (10,000 × g, 10 min, 4°C) and the resulting pellet resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5% w v−1 hexadecyltrimethylammonium bromide (Sigma-Aldrich Corp., St Louis, MO, U.S.A.). The suspension was subjected to four cycles of freezing and thawing and further disrupted by sonication (40 s). The sample was then centrifuged (10,000 × g, 5 min, 4°C) and the supernatant used for the MPO assay. The reaction mixture consisted of the supernatant (50 μl), 1.6 mM tetramethylbenzidine (Sigma-Aldrich Corp., St Louis, MO, U.S.A.), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide (reagent volume: 50 μl). This mixture was incubated at 37°C for 110 s, the reaction terminated with 50 μl of 0.18 M H2SO4 and the absorbance measured at 450 nm using a 96-well microplate reader (Tecan Systems Inc., San Jose, CA, U.S.A.). This absorbance was then corrected for the DNA content of the tissue sample and results are expressed as enzyme activity (fold increase over control).

    Preparation of nuclear extracts and determination of NF-κB activation

    Nuclear extracts of livers (100 mg) were prepared by using the Compartmental Protein Extraction Kit as described by the manufacturer (CHEMICON International Inc., Temecula, CA, U.S.A.). Protein concentrations in nuclear extracts were determined using the Bradford assay (Bio-Rad Ltd, Hercules, CA, U.S.A.). To monitor NF-κB activation in liver tissues, we used a TransAM NF-κB p65 Transcription Factor Assay Kit (Active Motif, Tokyo, Japan). The kit consists of a 96-well plate into which oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′) is bound. The active form of NF-κB in the nuclear extract specifically binds to this consensus site and is recognised by a primary antibody. A horseradish peroxidase-conjugated secondary antibody provides the basis for the colorimetric quantification. The absorbance of the resulting solution was measured 5 min thereafter (450 nm with a reference wavelength of 655 nm), using a 96-well microplate reader (Tecan Systems Inc., San Jose, CA, U.S.A.). The wild-type consensus oligonucleotide is provided as a competitor for NF-κB binding in order to monitor the specificity of the assay. The oligonucleotide will prevent NF-κB binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on NF-κB binding. To allow for optimum competition, the oligonucleotide was added to the well prior to the addition of nuclear extract. Results were expressed as percentages of the positive control (TNF-α-stimulated HeLa whole cell extract) provided by the manufacturer.

    Statistical analysis

    Data show mean±s.e.m. Statistical analysis of data was by one-way ANOVA followed by post hoc Tukey's test. A P-value of <0.05 was taken to indicate a statistically significant difference.

    Results

    Effect of flurbiprofen and nitroflurbiprofen on plasma NOx, TNF-α and IL-1β in LPS rats

    Low concentrations of TNF-α, IL-1β, and nitrite/nitrate (NOx) (Figure 1a–c) were detected in plasma of rats at 6 h after injection with saline. Conversely, LPS-induced septic shock was associated with significantly increased plasma concentrations of TNF-α, IL-1β and NOx (Figure 1a–c). Treatment of LPS rats with flurbiprofen did not affect the resulting rise in plasma TNF-α, IL-1β and NOx concentrations (Figure 1a–c). In contrast, nitroflurbiprofen administration resulted in a significant (P<0.05) dose-dependent inhibition of LPS-mediated increase in plasma TNF-α and IL-1β (c.f. LPS rats) (Figure 1a and b). Treatment with nitroflurbiprofen also resulted in a significant (P<0.05) inhibition of plasma NOx concentrations at 6 h after LPS-induced septic shock (Figure 1c).

    Details are in the caption following the image

    Plasma concentration of (a) TNF-α, (b) IL-1β, (c) NOx of rats killed 6 h after injection of either saline (sham), E. coli LPS (LPS) or LPS pretreatment with either equivalent volume of vehicle (2 ml kg−1, i.p., VEH), flurbiprofen (21 mg kg−1, i.p., FLU) or nitroflurbiprofen (3–30 mg kg−1, i.p., NOF). Results show plasma concentration and are mean±s.e.m., n=5, *P<0.05 c.f. Sham, +P<0.05 c.f. LPS, #P<0.05 c.f. LPS+NOF (30 mg kg−1).

    Effect of flurbiprofen and nitroflurbiprofen on liver iNOS expression in LPS rats

    Blots were reacted with antibodies against iNOS, a protein whose expression is known to be increased by LPS. Rats injected with LPS and killed 6 h thereafter showed significant induction in the expression of iNOS in the liver (Figure 2a and b). However iNOS expression was not detected in tissues from rats injected with saline in the liver (Figure 2a and b). Treatment of LPS rats with flurbiprofen did not affect the resulting increase in the expression of iNOS in the liver (Figure 2a and b). In contrast, nitroflurbiprofen administration resulted in a significant (P<0.05) inhibition of LPS-mediated increase in iNOS expression (Figure 2a and b).

    Details are in the caption following the image

    (a) Representative blot from one liver showing the presence of iNOS (∼130 kDa). (b) Quantitation of liver (shown in (a)) iNOS. Homogenates were prepared from liver removed from rats killed 6 h after injection of either saline (sham), E. coli LPS (LPS) or LPS pretreatment with either equivalent volume of vehicle (2 ml kg−1, i.p., VEH), flurbiprofen (21 mg kg−1, i.p., FLU) or nitroflurbiprofen (30 mg kg−1, i.p., NOF). Results indicate the relative intensities in arbitrary units, and are mean±s.e.m., n=5, *P<0.05 c.f. sham, +P<0.05 c.f. LPS.

    Effect of flurbiprofen and nitroflurbiprofen on liver and kidney H2S biosynthesis in LPS rats

    Incubation of liver and kidney homogenates (30 min at 37°C) with cysteine (10 mM) and pyridoxal 5′-phosphate (2 mM) resulted in the formation of significant amounts of H2S as determined spectrophotometrically (Figure 3). Interestingly, the formation of H2S by liver and kidney homogenates from rats injected with LPS was significantly greater (P<0.05) than in homogenates prepared from rats injected with saline (Figure 3). Treatment of LPS rats with flurbiprofen did not affect the resulting rise in formation of significant amounts of H2S both in the liver and kidney (Figure 3). In contrast, nitroflurbiprofen administration resulted in a significant (P<0.05) dose-dependent inhibition of LPS-mediated increase in H2S formation both in the liver and kidney (c.f. LPS rats) (Figure 3).

    Details are in the caption following the image

    Formation of H2S from cysteine (10 mM) in the presence of pyridoxal 5′-phosphate (1 mM) following incubation (37°C, 30 min). Homogenates were prepared from liver or kidney removed from rats killed 6 h after injection of either saline (sham), E. coli LPS (LPS) or LPS pretreatment with either equivalent volume of vehicle (2 ml kg−1, i.p., VEH), flurbiprofen (21 mg kg−1, i.p., FLU) or nitroflurbiprofen (3–30 mg kg−1, i.p., NOF). Results show H2S formation as nmol formed mg protein−1, and are mean±s.e.m., n=5, *P<0.05 c.f. sham, +P<0.05 c.f. LPS, #P<0.05 c.f. LPS+NOF (30 mg kg−1).

    Effect of flurbiprofen and nitroflurbiprofen on liver and kidney CSE mRNA in LPS rats

    Rat liver and kidney homogenates from sham animals contained detectable amounts of mRNA for CSE (Figure 4). Rats injected with LPS and killed 6 h thereafter showed significant (P<0.05) increase in CSE mRNA expression in liver and kidney (c.f. sham rats) (Figure 4). Treatment with flurbiprofen did not affect the CSE mRNA expression for both liver and kidney in LPS-injected animals (Figure 4). Conversely, nitroflurbiprofen administration resulted in a significant (P<0.05) dose-dependent inhibition of LPS-mediated increase in CSE mRNA expression for both liver and kidney (c.f. LPS rats) (Figure 4).

    Details are in the caption following the image

    Quantitation of liver and kidney CSE. Homogenates were prepared from liver or kidney removed from rats killed 6 h after injection of either saline (sham), E. coli LPS (LPS) or LPS pretreatment with either equivalent volume of vehicle (2 ml kg−1, i.p., VEH), flurbiprofen (21 mg kg−1, i.p., FLU) or nitroflurbiprofen (3–30 mg kg−1, i.p., NOF). Data indicate the relative intensities in arbitrary units, and are mean±s.e.m., n=5, *P<0.05 c.f. sham, +P<0.05 c.f. LPS, #P<0.05 c.f. LPS+NOF (30 mg kg−1).

    Effect of flurbiprofen and nitroflurbiprofen on liver and kidney injury in LPS rats

    Evidence of liver and kidney injury in rats injected with LPS and killed 6 h thereafter was indicated by a significant (P<0.05) increase in liver and kidney MPO as a measure of neutrophil infiltration (c.f. sham rats) (Figure 5). Treatment with flurbiprofen did not inhibit MPO activity either in the liver or kidney (Figure 5). However, nitroflurbiprofen administration in LPS injected rats resulted in a significant (P<0.05) dose-dependent inhibition of MPO activity in the liver but not in the kidney (Figure 5).

    Details are in the caption following the image

    Effect of LPS injection on MPO activity. Homogenates were prepared from liver or kidney removed from rats killed 6 h after injection of either saline (sham), E. coli LPS (LPS) or LPS pretreatment with either equivalent volume of vehicle (2 ml kg−1, i.p., VEH), flurbiprofen (21 mg kg−1, i.p., FLU) or nitroflurbiprofen (3–30 mg kg−1, i.p., NOF). Results indicate the relative MPO activity fold increase over control in arbitrary units, and are mean±s.e.m., n=5, *P<0.05 c.f. sham, +P<0.05 c.f. LPS, #P<0.05 c.f. LPS+NOF (30 mg kg−1).

    Effect of flurbiprofen and nitroflurbiprofen on liver NF-κB activation in LPS rats

    Rats injected with LPS and killed 6 h thereafter showed significant (P<0.05) increase in NF-κB activation in the liver (c.f. sham rats) (Figure 6). Treatment with flurbiprofen did not affect the NF-κB activation in the liver of LPS-injected animals (Figure 6). On the other hand, nitroflurbiprofen administration resulted in a significant (P<0.05) dose-dependent inhibition of LPS-mediated increase in NF-κB activation in the liver (c.f. LPS rats) (Figure 6).

    Details are in the caption following the image

    Effect of flurbiprofen (21 mg kg−1, i.p., FLU) and nitroflurbiprofen (3–30 mg kg−1, i.p., NOF) on NF-κB activation in the liver of rats at 6 h after LPS injection. Results indicate the relative percentage of NF-κB activation over the positive control provided by the manufacturer, and are mean±s.e.m., n=5, *P<0.05 c.f. sham, +P<0.05 c.f. LPS, #P<0.05 c.f. LPS+NOF (30 mg kg−1).

    Discussion

    We have previously reported that LPS administration in the mouse results in increased plasma H2S concentration coupled with upregulated liver and kidney CSE mRNA expression (Li et al., 2005) and have further shown that DL-propargylglycine (an inhibitor of CSE enzyme activity) abrogates endotoxic shock in the rat as evidenced by a reduction in plasma markers of liver (aspartate aminotransferase), pancreas (lipase) and skeletal muscle (creatinine kinase) damage (Collin et al., 2005). Furthermore, H2S administration (by injection of NaHS – a water-soluble H2S donor drug) resulted in augmented liver and kidney MPO activity as well as microscopic changes in the lung, liver and kidney typical of an inflammatory insult (Li et al., 2005). Interestingly, similar airways inflammation has been noted in man following accidental environmental exposure to H2S (for review, see Knight & Presnell, 2005). Based on these studies we have proposed that H2S acts as a proinflammatory mediator in endotoxic shock. A similar proinflammatory effect of H2S has been reported in other inflammatory conditions including caerulein-induced pancreatitis (Bhatia et al., 2005b) and carrageenan-induced hindpaw oedema (Bhatia et al., 2005a).

    The aetiology of LPS-evoked endotoxic shock is complex and undoubtedly involves the overproduction of a number of different mediators other than H2S. For example, it has long been recognised that the excessive formation of NO, as a consequence of upregulation of iNOS, plays an important role in the cardiovascular depression and organ damage associated with endotoxic and septic shock (Stein et al., 1996). Furthermore, several recent reports have suggested that H2S and NO interact together in such a way as to modify the biological activity of each mediator (see Introduction for references). However, whether such an interaction takes place in pathological states such as endotoxic shock, or indeed in other examples of inflammatory disease, is not known. As such, it seemed of interest to determine whether exogenously provided NO affected the upregulated biosynthesis of H2S which we have reported in LPS-injected animals. Nitroflurbiprofen was used as the NO donor in the present series of experiments. This particular NO donor was chosen since LPS-induced endotoxic shock in the rat becomes evident over a relatively long time period (up to 6 h) and as such administration of classical NO donors such as sodium nitroprusside which produce a rapid, but transient (min), release of NO is inappropriate. It is now well established in the literature that the administration of nitroflurbiprofen (or indeed other NO-NSAID) to animals (Aldini et al., 2002) or man (Zacharowski et al., 2004) results in esteratic cleavage of the parent molecule and leads to the slow release (maximum plasma concentrations at 2–4 h) of both component molecules, that is, NO and flurbiprofen. An added advantage of the use of nitroflurbiprofen in these experiments is that it does not alter blood pressure in the rat (Keeble et al., 2001) most probably due to the slow time course of NO release. Such a vasodepressor effect would likely confound the interpretation of the present results.

    We demonstrate here that nitroflurbiprofen, produces a dose-related inhibition of LPS-mediated endotoxic shock in the rat as evidenced by reduced plasma TNF-α and IL-1β concentration and liver and kidney MPO activity (indicative of neutrophil infiltration in these organs) coupled with a significant fall in plasma NOx concentration and liver iNOS protein expression. The decrease in plasma NOx concentration was not as great as the decline in plasma TNF-α and IL-1β levels which presumably reflects the ability of other cytokines to uprgeulate iNOS expression. Flurbiprofen pretreatment was without effect on all parameters studied indicating that NO, released following cleavage of nitroflurbiprofen, was the molecule responsible for the changes observed. These data suggest that at least part of the mechanism of action of nitroflurbiprofen may be attributed to an NO-mediated downregulation of iNOS with consequent diminished biosynthesis of proinflammatory and vasodilator NO. In support of this possibility, we have previously shown that nitroflurbiprofen inhibits LPS-induced generation of TNF-α and IL-1β in whole human blood (Marshall & Moore, 2004) while others have observed a similar inhibitory effect of this molecule in cultured macrophages and neutrophils exposed to LPS (Mariotto et al., 1995a, 1995b).

    Of even greater significance, we also provide evidence that nitroflurbiprofen administration affects the metabolism of endogenous H2S. Animals administered nitroflurbiprofen exhibited reduced liver and kidney H2S synthesising activity (measured ex vivo) associated with decreased liver and kidney CSE mRNA expression. Again, flurbiprofen was devoid of such activity indicating that NO, released from nitroflurbiprofen in vivo, was responsible for the changes in H2S metabolism observed. These data suggest that exogenous NO (provided by nitroflurbiprofen) downregulates not only the biosynthesis of NO but also that of H2S. Whether endogenous NO exerts a similar effect on H2S metabolism in either physiological or pathophysiological states warrants further study.

    The molecular mechanism by which NO (released from nitroflurbiprofen) downregulates iNOS protein and CSE mRNA expression has also been investigated. We observed that pretreatment of LPS-injected animals with nitroflurbiprofen (but not flurbiprofen) caused a dose-related inhibition of liver NF-κB activation. There are several reports in the literature that NO modulates NF-κB activity either by interfering with the phosphorylation of IκB (Peng et al., 1995) and/or by inhibition of NF-κB-DNA-binding affinity by nitrosation of the p50 subunit (Matthews et al., 1996). Interestingly, nitroaspirin (a structural analogue of nitroflurbiprofen) has previously been reported to reduce transduction via the NF-κB pathway in cultured macrophages (Cui et al., 2001) as has nitroflurbiprofen in cultured L929 cells (Fratelli et al., 2003). The present data may additionally be taken to suggest that NO also inhibits the expression of CSE mRNA (and consequent ability to synthesise H2S) at least in the liver and kidney of LPS-injected rats. While we, and other researchers, have reported changes in CSE mRNA expression and/or H2S synthesising activity in a number of animal models of disease including hypertension (Zhong et al., 2003), diabetes mellitus (Yusuf et al., 2005) and pancreatitis (Bhatia et al., 2005b), the molecular basis underlying this effect has not been examined. The present data suggesting that both iNOS and CSE mRNA expression may be regulated by exogenous NO is therefore of potential significance in a range of different disease states.

    Notwithstanding the interaction between NO and H2S identified in this work the present experiments also offer an opportunity to probe further the complex anti-inflammatory effect of nitroflurbiprofen (for review, see Scatena, 2004). On a mol for mol basis, nitroflurbiprofen has been reported to exhibit greater anti-inflammatory activity in animal models of Alzheimer's disease (injection of preaggregated Aβ 1–42 into the nucleus basalis in the rat) (Prosperi et al, 2004) and experimental autoimmune encephalomyelitis (Furlan et al., 2004). Nitroflurbiprofen also reduced brain inflammation resulting form chronic infusion of LPS into the fourth ventricle in the rat (Wenk et al., 2002). Since the brain contains high levels of H2S (approx. 100–150 μM) and is an excellent source of cystathionine β synthetase (which, like CSE, acts on cysteine to form H2S) it is tempting to suggest that the augmented anti-inflammatory effect of nitroflurbiprofen (c.f. flurbiprofen) noted in the brain may also be due, at least in part, to inhibition of H2S biosynthesis.

    In conclusion, we show here that NO (provided by nitroflurbiprofen) abrogates the upregulation of both NO and H2S biosynthesis following LPS injection in the rat. The mechanism involved may be by inhibition of transduction via the NF-κB pathway. The data suggests, (i) that NO and H2S interact together to promote inflammatory changes in endotoxic shock and (ii) lend further support to our proposal that inhibition of H2S formation or action may represent a novel approach to the treatment of this and perhaps other inflammatory disorders.

    Acknowledgments

    We thank the Agency for Science, Technology and Research (A*STAR) for the award of a Graduate Scholarship to FA and the Office of Life Science (OLS) of the National University of Singapore for financial support (Grant no. R-184-000-074-712). We also thank NicOx SA, Sophia-Antipolis, France for the generous gift of nitroflurbiprofen.