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

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-alpha, IL-1beta and nitrate/nitrite (NO(x)) 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-kappaB) activation. Nitroflurbiprofen (3-30 mg kg(-1), i.p.) administration resulted in a dose-dependent inhibition of the LPS-mediated increase in plasma TNF-alpha, IL-1beta and NO(x) 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-kappaB 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.

Figure 1

Plasma concentration of (a) TNF-α, (b) IL-1β, (c) NO_x 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).

Figure 2

(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.

Figure 3

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).

Figure 4

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).

Figure 5

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).

Figure 6

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).