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We investigated the effect of hydroxytyrosol (HT), a phenolic compound from virgin olive oil, on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in J774 murine macrophages stimulated with lipopolysaccharide (LPS). Incubation of cells with LPS caused an increase in iNOS and COX-2 mRNA and protein level as well as ROS generation, which was prevented by HT. In addition, HT blocked the activation of nuclear factor-kappaB (NF-kappaB), signal transducer and activator of transcription-1alpha (STAT-1alpha) and interferon regulatory factor-1 (IRF-1). These results, showing that HT down-regulates iNOS and COX-2 gene expression by preventing NF-kappaB, STAT-1alpha and IRF-1 activation mediated through LPS-induced ROS generation, suggest that it may represent a non-toxic agent for the control of pro-inflammatory genes.

In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type-, but not mutant-decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-kappaB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, exhibited a similar profile of activity of decoy ODN. Our results indicate that decoy ODN, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions.

Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amounts of prostaglandins (PGs) generated by the inducible isoform of cyclooxygenase (COX-2). Nitric oxide (NO), a pleiotropic free radical, has been demonstrated to modulate the release of a broad range of inflammatory mediators, amongst these PGs. In the present study we investigated the molecular mechanism by which NO affects cyclooxygenase pathway. Incubation of J774 cells with LPS caused an increase of prostaglandin E2 production and COX-2 protein expression which was prevented in a concentration-dependent fashion by pre-incubating cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating agents. Electrophoretic mobility shift assay indicated that both NO-generating agents blocked LPS-induced activation of nuclear factor-kappaB (NF-kappaB) by increasing IkappaB-alpha protein expression and blocking nuclear translocation of NF-kappaB subunits p50 and p65. SNP and GSNO also inhibited nuclear factor-interleukin-6 (NF-IL6) activation. These results show for the first time that SNP and GSNO down-regulate LPS-induced COX-2 expression by inhibiting NF-kappaB and NF-IL6 activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged PGs production in pathological events.