Furthermore, DHA pretreatment significantly abro gated the TNF dependent increase in mRNA levels of both proinflammatory markers I��B and TNF. As chemical information expected from the mRNA results, DHA pretreatment also significantly re duced TNF protein production and thus the translational inflammatory response. These findings ensure that DHA exhibits anti inflammatory properties in the hypothalamic rHypoE 7 cell model. The anti inflammatory effects of are AKT and ERK independent To determine if the anti inflammatory actions of DHA is mediated through the phosphoinositide 3 kinase AKT or mitogen activated Inhibitors,Modulators,Libraries protein kinase extracellular signal regulated kinase pathways, rHypoE 7 cells were co treated with DHA and their respective inhibitors prior to TNF exposure.

Pretreatment of rHypoE 7 cells with the PI3K inhibitor, Wortmannin significantly reduced AKT phosphorylation levels in response to DHA but failed to prevent the anti inflammatory effects of DHA as shown from the analysis of I��B mRNA and TNF mRNA. Treatment of the rHypoE 7 cells with the PKC inhibitor, Staurosporine algyone reduced Inhibitors,Modulators,Libraries ERK phosphorylation Inhibitors,Modulators,Libraries indicating pERK formation upon DHA exposure is PKC dependent. Pretreatment with Stauro abolished or reduced the enhancement of I��B or TNF respectively, indicating that PKC itself participates in the induction of the transcriptional inflammatory re sponse to TNF in the rHypoE 7 cell line. Relative to DHA pretreat ment alone, DHA and Stauro co pretreatment prior to TNF exposure did not cause a further reduc tion in I��B mRNA levels but led to a further decrease in TNF mRNA levels.

These results indicate that the ability of DHA to activate PKC activity plays a role in its anti inflammatory actions. These results suggest that although DHA acti vates PI3K and PKC, neither signaling cascade plays significant roles in mediating the anti inflammatory actions of this omega 3 FA. GPR120 mediates the anti inflammatory Inhibitors,Modulators,Libraries effect of DHA in rHypoE 7 cells The Inhibitors,Modulators,Libraries transcriptional anti inflammatory actions of DHA in rHypoE 7 cells was abolished by the presence of BSA as shown upon quantifying mRNA levels of I��B TNFhistone after TNF ex posure. As the addition of BSA and the forma tion of DHA BSA complexes abolished the ability of DHA to inhibit the transcriptional inflammatory response, it is clear that DHA must exist in its free form to mediate its acute anti inflammatory actions and may interact with free FA receptors at the cell surface.

The free FA receptor, GPR120, was recently identified to mediate the anti inflammatory actions of omega 3 FAs in macrophages, and to determine if GPR120 may be involved in the anti different inflammatory actions in the hypo thalamic rHypoE 7 cell model, we employed the GPR120 synthetic agonist GW9508. It must be noted here that GW can also activate GPR40, but as GPR40 is not expressed in the rHypoE 7 cell model, this agonist is considered a GPR120 specific in our studies.