The document describes developing a model to assess the anti-inflammatory potential of 5,6-EET-EA. 5,6-EET-EA is a selective CB2 receptor agonist formed by oxidation of anandamide. Since 5,6-EET-EA activates the CB2 receptor in the endocannabinoid system, which is being researched for its anti-inflammatory effects, the authors hypothesized it would inhibit pro-inflammatory molecules IL-6, TNF-α, and IFN-γ. Experiments showed 5,6-EET-EA decreased IL-6 levels in splenocytes stimulated with LPS, similarly to anandamide, demonstrating its potential as an anti-inflammatory compound. Further

1. Development of a model to assess the anti-inflammatory potential of 5,6-EET-EA
William Parker, John Stokes and Barbara L. F. Kaplan
The compound 5,6- epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA) is a highly
selective cannabinoid receptor 2 (CB2) agonist. 5,6-EET-EA is formed by oxidation of
anandamide by cytochrome P450. Since 5,6-EET-EA is a CB2 agonist, it works in the
endocannabinoid system. The endocannabinoid system consists of lipids known as
endocannabinoids, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and
their receptors. Two of these receptors are G-protein coupled receptors expressed in the
central nervous system (CB1) and on immune system cells (CB2). The endocannabinoid
system is under research to help ameliorate symptoms of inflammation. In early stages of
inflammation, the infected tissue is infiltrated with macrophages and neutrophils.
Macrophages are activated by the molecule interferon-γ (IFN-γ). Two other molecules,
tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6), are produced by macrophages
and known to induce inflammation. The molecule CD11b, is one of the protein subunits
that make up the molecule macrophage-1 antigen (Mac-1). Upregulation of mac-1 can
prolong immune cell life. Since 5,6-EET-EA is a CB2 receptor agonist, it is hypothesized
that 5,6-EET-EA will inhibit IL-6, TNF-α, and IFN-γ. To test this, we isolated
splenocytes and stimulated them in vitro with lipopolysaccharide (LPS), a part of gram
negative bacterial cell walls. Then we used antibodies conjugated to fluorophores
directed against IL-6, TNF-α, IFN-γ, and flow cytometry was used to detect these
proteins. The results showed that after a 4-hour stimulation with LPS, IL-6 and TNF-α
were detected, but not IFN-γ. 5,6-EET-EA decreased IL-6 at certain concentrations. In
order to compare the effectiveness of 5,6-EET-EA, we used anandamide also and saw
that IL-6 was also decreased at certain concentrations. This decreased concentration of
IL-6 shows that 5,6-EET-EA is a strong candidate for anti-inflammatory uses. More
studies with longer incubations of 5,6-EET-EA and LPS need to be conducted in order to
confirm 5,6-EET-EA’s anti-inflammatory properties.