Abstract
Purpose :
Epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP) are endogenous cytochrome P450-derived epoxygenated fatty acids that decrease retinal expression, secretion, and propagation of pro-inflammatory cytokines under diabetic conditions, providing a potential endogenous system for correcting diabetic retinopathy (DR)-related retinal inflammation. This study tested the hypothesis that increasing levels of EET and EDP, by administration of exogenous epoxide or use of soluble epoxide hydrolase (sEH) inhibitor GSK2256294, attenuates Müller cell inflammation in response to diabetes-relevant stimuli (DRS).
Methods :
Primary human Müller cells (hMC) were treated in serum-depleted media with BSA-bound palmitic acid (PA, 250μM) for 24hrs or IL-1β (1ng/ml) for 8hrs to stimulate DR inflammation (n=3-4). PA- or IL-1β-treated hMC were administered sEH inhibitor GSK2256294 (0.1-10nM), 11,12-EET (0.5μM) or 19,20- EDP (0.5μM). Relative expression of inflammatory mediators was analyzed via qRT-PCR and ANOVA with Tukey’s multiple comparisons test was used to evaluate significant differences among treatment groups. hMC were transfected with luciferase constructs and treated with PA or IL-1β in the presence or absence of epoxide (n=6). NFκB activity was determined via the ratio of firefly-to-Renilla luciferase.
Results :
Addition of exogenous epoxide reduced both PA- and IL-1β-induced hMC expression of IL-6, IL-8, and IL-1β (p<0.04, PA+IL-8: ns). GSK2256294 also significantly reduced PA- and IL-1β-stimulated hMC cytokine expression (p<0.002) and showed no significant change in efficacy between treatment concentrations. An NFκB promoter assay showed significant decreases in luciferase activity in response to epoxide administration (p<0.01), suggesting a mechanism of action by which EET/EDP exert anti-inflammatory activity.
Conclusions :
Consistent with our hypothesis, two strategies aimed at elevating hMC epoxide levels were shown to decrease cellular cytokine expression in response to DRS. Findings support the notion that epoxide elevation works by reducing hMC NFκB activity. Therefore elevation of retinal epoxide levels, by administration of exogenous epoxide or inhibition of epoxide hydrolysis, may provide a rational therapeutic strategy for inhibition of early retinal inflammation in DR.
This is a 2020 ARVO Annual Meeting abstract.