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 MΦ 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.

Conclusion

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 MΦ.

Key Words: omega-3 fatty acids, nitric oxide, selective NSAIDS