Attenuation of iNOS in an LPS-stimulated macrophage model by omega-3 fatty acids is independent of COX-2 derived PGE2.
BACKGROUND
Omega-3 fatty acids (n-3 FA) demonstrate significant anti-inflammatory properties thought to occur through three principal mechanisms; (1) displacement of arachidonic acid from the cellular membrane, (2) differential prostaglandin E2 (PGE2) and LTB4 production, and (3) molecular level alterations such as diminished nuclear factor kappa B and AP-1 activation. Recently, n-3 FA have been demonstrated to significantly decrease nitric oxide (NO) production in a lipopolysaccharide (LPS)-stimulated M Phi model. We hypothesized that decreased NO production by n-3 FA occurs through inhibition of cyclooxygenase-2 (COX-2) derived PGE2 and that repletion of the system with PGE2 would obliterate these effects. Selective COX-2 inhibitor (L-748,731) experiments and separate PGE2 repletion studies were used to test this hypothesis.


METHODS
NO production was assessed following 24 h with or without LPS/PGE2 in the presence of n-3 FA, L-748,731 (a selective COX-2 inhibitor), or combination (n-3 FA + L-748,731) treatment. Western blots were used to assess inducible NO synthase protein expression.


RESULTS
Independently or in the presence of LPS, treatment with a COX-2 inhibitor significantly increased NO production compared with control, n-3 FA, and combination treatment. NO production in combination treatment is slightly increased compared to n-3 FA treatment. In control cells treated with LPS, PGE2 repletion resulted in a significant decrease in NO. All other treatment groups repleted with PGE2 demonstrated no significant alterations in NO production. Inducible NO synthase protein expression levels were similar to NO production across all treatments.


CONCLUSIONS
These experiments disproved our original hypothesis that the decrease in NO production associated with n-3 FA treatment occurs through a COX-2 derived PGE2 dependent mechanism. Eliminating COX-2 derived PGE2 by a selective inhibitor actually increased NO production. Exogenous PGE2 repletion did not restore the system. Therefore, mechanisms other than n-3 FA associated alterations in COX-2 derived PGE2 are likely involved in decreasing NO production in LPS stimulated M Phi.
Razzak
Anthony
A
Department of Surgery, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Aldrich
Chris
C
Babcock
Tricia A
TA
Saied
Abdul
A
Espat
N Joseph
NJ
D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism*; D004347:Drug Interactions; D015525:Fatty Acids, Omega-3 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*
D016861:Cyclooxygenase Inhibitors; D015525:Fatty Acids, Omega-3; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D015232:Dinoprostone