A novel hybrid aspirin-NO-releasing compound inhibits TNFalpha release from LPS-activated human monocytes and macrophages

Background: The cytoprotective nature of nitric oxide (NO) led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNF alpha release from lipopolysaccharide (LPS)-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-kappa B) activation.

Methods: Peripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 mu M), their respective furazan NO-free counterparts (B16, B15; 10 mu M), aspirin (10 mu M), existing nitroaspirin (NCX4016; 10 mu M), an NO donor (DEA/NO; 10 mu M) or dexamethasone (1 mu M), in the presence and absence of LPS (10 ng/ml; 4 h). Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNF alpha release and by lactate dehydrogenase (LDH) assay for cell necrosis. To assess NF-kappa B activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-kappa B, I kappa B alpha (assessed by western blotting) and nuclear localisation (assessed by immunofluorescence) of the p65 subunit of NF-kappa B were determined.

Results: B8 significantly reduced TNF alpha release from LPS-treated macrophages to 36 +/- 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNF alpha release to 28 +/- 5, and 49 +/- 9% of control, respectively. The B8 effect was equivalent in magnitude to that of dexamethasone, but was not shared by 10 mu M DEA/NO, B7, the furazans, aspirin or NCX4016. LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic I kappa B alpha and nuclear translocation of the p65 NF-kappa B subunit was inhibited by the active NO-furoxans.

Conclusion: Here we show that furoxan-aspirin, B8, significantly reduces TNF alpha release from both monocytes and macrophages and suggest that inhibition of NF-kappa B activation is a likely mechanism for the effect. This anti-inflammatory action highlights a further therapeutic potential of drugs of this class.