A Novel Hybrid Aspirin-NO-Releasing Compound Inhibits TNF-alpha Release from LPS-Activated Human Monocytes and Macrophages
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-kappaB) 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 microM), their respective furazan NO-free counterparts (B16, B15; 10 microM), aspirin (10 microM), existing nitroaspirin (NCX4016; 10 microM), an NO donor (DEA/NO; 10M) or dexamethasone (1 microM), in the presence and absence of LPS (10ng/ml; 4h). Parallel experiments were conducted on undifferentiated fresh monocytes.
Supernatants were assessed by specific ELISA for TNFalpha 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 kappaB-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 TNFalpha 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 microM DEA/NO, B7, the furazans, aspirin or NCX4016.
LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic IkappaB-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.
Author: Catriona M Turnbull, Paolo Marcarino, Tara A Sheldrake, Loretta Lazzarato, Clara Cena, Roberta Fruttero, Alberto Gasco, Sarah Fox, Ian L Megson and Adriano G Rossi
Credits/Source: Journal of Inflammation 2008, 5:12
Published on: 2008-07-31
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
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