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Three Arachidonoylamide Derivatives Inhibit Pro-Inflammatory Genes Expression by Modulating NF-κB and AP1 Activities

[ Vol. 12 , Issue. 7 ]

Author(s):

Alex Gregorelli, Anna Sgarbossa, Shahbaz Khan, Annunziata Soriente, Margherita De Rosa, Carmela Saturnino and Marta Menegazzi   Pages 662 - 673 ( 12 )

Abstract:


Background: Since the anti-inflammatory activity of arachidonic acid derivatives was previously reported, we synthesized three new amide derivatives of arachidonic acid (AA-Ds) and tested their anti-inflammatory effects on an in vitro skin inflammation model. Aim of our study was to find derivatives of natural compounds able to down regulate inflammatory signal transduction pathway.

Methods: Human keratinocytes cell line (HaCaT) was cultured and induced by cytokines in the presence of AA-Ds. Cytokines administration elicited an inflammatory response mediated by NF-κB and STAT-1 activation that induced proinflammatory genes expression.

Results: By real time PCR we found that 24 hours after induction all AA-Ds significantly inhibit inducible Nitric Oxide Synthase (iNOS), TNFα, Inhibitor α of NF-κB, chemokine (C-X-C motif) ligand 9 and 10 genes expression. We analyzed their molecular effects in particular on the iNOS gene expression. Since iNOS transcript half-life did not change with AA-Ds treatment, we excluded a prominent role of post-transcriptional regulation for this gene and focused our attention on its transcriptional regulation. Starting three-five hours after cytokines induction, HaCaT cells, pretreated with each compound, showed inhibition of both NF-κB DNA-binding and NF-κB p65-Ser536 phosphorylation. STAT1 activation was inhibited only by AA-D4 derivative. To explain why the inhibition of iNOS expression began late after induction we analyzed activities of others key transcription factors. AA-Ds treatment elicited early increases of AP1 DNA binding as well as c-Jun, c-Fos and Fra-1 mRNA levels. Our data agree with the repressing effects of AP1 on human iNOS promoter previously described in others cell systems (Kleinert et al.).

Conclusion: AA-Ds shown to be good candidates as inhibitors of several pro-inflammatory genes induction and our study provides indications for their possible use as new antiinflammatory drugs.

Keywords:

Arachidonoylamide derivatives, NF- B, AP-1, STAT1, iNOS, inflammatory cytokines, Inflammation.

Affiliation:

Department of Pharmacy, University of Salerno, 84084 Fisciano (SA), Italy., Department of Neurosciences, Biomedicine and Movement Sciences, Biochemistry Section, University of Verona, Strada Le Grazie, 8, 37134 Verona, Italy.

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