Soluble epoxide hydrolase: Sex differences and role in estrogen-mediated neuroprotection

INTRODUCTION: In experimental models of stroke, females sustain smaller brain damage than males, in part due to the neuroprotective effects of estrogen. The precise mechanism of estrogen-mediated neuroprotection remains unknown. We previously identified P450 eicosanoids termed eopoxyeicosatrienoic acid (EETs) as important mediators of ischemic preconditioning and tolerance. We subsequently demonstrated that inhibition or gene deletion of EETs-metabolizing enzyme soluble epoxide hydrolase (sEH) reduces infarct size after middle cerebral artery occlusion (MCAO) in mice. In peripheral tissue, sEH expression is sexually dimorphic and regulated by sex hormones. Therefore, we tested the hypothesis that the protective effect of sEH inactivation is sex-specific and modulated by estrogen. METHODS: Adult male and female C57/Bl6 wild type (WT) and sEH knockout (sEHKO) mice were subjected to 2-hour MCAO, and infarct size was measured at 22 hours of reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC). Male and female WT mice were also treated with sEH inhibitor 12-(3-adamantan-1-yl-ureido)- dodecanoic acid n-butyl ester (AUDA-BE, 10 mg/kg i.p.) or vehicle at reperfusion. Body temperature was kept within normal range, and cortical laser-Doppler perfusion was monitored to confirm vascular occlusion and reperfusion. The level of sEH expression was determined by Western blotting, and sEH activity was determined by measuring EETs metabolite 14,15-dihydroxyeicosatrienoic acids (14,15-DHETs), specifically produced by sEH, in plasma using ELISA. To determine the effect of estrogen on sEH expression, ovariectomized female rats (OVX) were implanted subcutaneously with pellets containing 17beta-estradiol (E2, 25 μg, 21-day release) for 7 days. RESULTS: The level of sEH immunoreactivity (IR) in brain was higher in WT male compared to female mice (0.45·0.02 vs 0.34·0.03 relative to beta-actin, n=4 each, p=0.007), and was undetectable in male and female sEHKO mice (n=4 each). Strong sEH-IR was consistently detected in brains from OVX rats. Replacement with E2 reduced sEH protein by 38% (n=4 each, p=0.029). Plasma 14,15-DHET was higher in male vs. female mice [3908·458 (n=14) vs. 710·257 (n=11) pg/ml, p<0.001], and was reduced by AUDA-BE (n=10) in male mice by approximately 35% (p=0.04). The level of 14,15-DHET in male sEHKO mice was significantly lower than WT males [115·73 (n=10) vs 4179·482 (n=12), p<0.001]. Female WT mice sustained smaller infarcts after MCAO compared to male mice (23.9·7.9%, n=5 vs. 35.9·2.5%, n=5, p<0.05). The sex difference, however, was absent in sEHKO mice and mice treated with AUDA-BE. Infarct size was significantly smaller in sEHKO compared to WT male (16.0·6.4% vs. 35.9·2.5%, n=5 each, p<0.05), but not female mice (17.0·7.7% compared to 23.9·7.9, n=5 each, p>0.05). Infarct size was also smaller in AUDA- compared to vehicle-treated male (15.7·5.6% vs. 33.1·2.3%, n=5 each, p<0.05), but not female mice (28.1·6.0 % vs. 25.4·5.8 %, n=5 each, p>0.05). CONCLUSION: We conclude that sEH is expressed at a lower level in young adult female vs male brain, which serves a beneficial role due to reduced breakdown of protective 14,15-EET, and may contribute to the protection against cerebral ischemia observed in females, and to the neuroprotective effect of estrogen.