Inhibition of soluble epoxide hydrolase enhances the anti-inflammatory effects of aspirin and 5-lipoxygenase activation protein inhibitor in a murine model

Abstract

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.

Conflict of interest statement

Conflict of interest statement BDH is the founder of Arête Therapeutics. This company is moving sEHI through clinical trials for treating hypertension, pain, metabolic disease, inflammation, and other disorders. However, this study is independent from the company.

Published by Elsevier Inc.

Figures

Fig. 1

(A) A simplified schematic of the arachidonic acid cascade showing the target metabolite oxylipins that were simultaneously measured. The effects of inhibition of COX, FLAP, and/or sEH on the AA metabolism in LPS-challenged murine model are present in Figures 2, 3 and 4, respectively. (B) Structures of inhibitors used. Aspirin is a non-selective COX inhibitor. 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid (MK 886) is a FLAP inhibitor, and trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) is a sEH inhibitor.

Fig. 2

Selective modulation of three branches of the AA cascade in a murine model of inflammation reveals unexpected interactions. The COX and LOX branches were blocked by aspirin and MK 886, respectively. The CYP450 branch was impacted by preventing the degradation of EETs using a sEHI t-AUCB. (A) LPS administration led to a net increase in DHETs (black bars, sum of 5,6-, 8,9-, 11,12- and 14,15-DHETs) levels and to slight changes in EETs (gray bars, sum of 8,9-, 11,12- and 14,15-EETs) levels. A high dose of aspirin and t-AUCB decreased DHET levels. MK886 dramatically increased DHETs while displaying small changes in EETs levels. The data for 5,6-EET are excluded because of lactone formation and hydrolysis during sample preparation. (B) LPS administration led to a net increase in TXB2 (black bars) and PGE2 (gray bars) levels. A low dose of t-AUCB led to a decrease in TXB2 levels but not PGE2 levels. Aspirin and MK886 decreased the levels of both TXB2 and PGE2. (C) LPS administration led to a net increase in both 5-HETE (black bars) and 15-HETE (gray bars). High dose of aspirin, t-AUCB, and MK886 all led to decreases in 5-HETE and 15-HETE levels, while the lower dose of aspirin decreased only15-HETE levels significantly. The data in Figures 2-5 represent average ± SD (n = 4) unless otherwise noted. Animals were sacrificed 6 hours after treatments. * Significantly different from LPS control (P > 0.05) determined by ANOVA followed by Tukey’s posthoc comparison test.

Fig. 3

Dual inhibition of two branches of the AA acid cascade is synergistically effective in decreasing inflammatory mediators. (A) LPS administration led to significant increases in both PGE2 (gray bar) and TXB2 (black bar). t-AUCB is ineffective in significantly reducing PGE2 by itself but when co-administered with a low dose of aspirin led to a synergistic reduction in PGE2 (gray bar) levels. TXB2 (black bar) levels were also reduced by t-AUCB, aspirin, and co-administration of t-AUCB with a lower dose of aspirin. Aspirin decreased the production of TXB2 and PGE2 in a dose-related manner. (B) LPS administration led to significant increases in both 5-HETE (black bar) and 15-HETE (gray bar). t-AUCB, effective by itself, however, when co-administered with MK 886, led to a synergistic decrease in the production of both HETEs. MK 886 decreased the production of 5-HETE 15-HETE in a dose-related manner. These data indicate that co-administration of a low dose of t-AUCB with a therapeutic dose of MK 886 can further reduce the levels of proinflammatory molecules 5-HETE and 15-HETE. The data are depicted as percentage of control mice receiving vehicle without LPS. Control values are TXB2, 4.4 ± 1.4, and PGE2, 0.5 ± 0.1 nM, 5-HETE, 3.4 ± 0.3 nM; 15-HETE, 6.6 ± 1.2 nM. * Significantly different (P > 0.05) determined by ANOVA followed by Tukey’s or Games-Howell’s posthoc comparison test.

Fig.4

Disruption of the sEH gene in mice demonstrates an inflammatory mediator profile similar to that of a chemical knockout using a sEHI. LPS was administered to Ephx2-null mice (gray bar) and their wildtype conspecifics (black bar) and plasma eicosanoid profiles 6 h post-LPS were determined. (A) The plasma levels of PGD2 in Ephx2-null mice were not different than wild-type counterparts upon LPS administration. However whereas aspirin reduced PGD2 significantly in wild-type mice, a further synergistic reduction of PGD2 was detected in Ephx2-null mice receiving aspirin. (B) The plasma levels of PGE2 in Ephx2-null mice were significantly lower than wild-type mice upon inflammation, pointing out lower COX activity in the Ephx2-null animals. However whereas aspirin reduced PGE2 significantly in wild-type mice, a further reduction of PGE2 was detected in Ephx2-null mice receiving aspirin. (C) The plasma levels of 5-HETE in both Ephx2-null (n=4) and wild-type (n = 6) mice significantly increased after LPS administration. MK886 administration reduced 5-HETE levels in both strains. However, the Ephx2-null mice had still lower levels of 5-HETE after MK 886 administration. † denotes significant difference, * significant difference from wild mice with same treatment (P > 0.05) determined by ANOVA followed by Tukey’s posthoc comparison test.

Fig. 5

Selective modulation of branches of the AA cascade during a murine model of inflammation results in unexpected anti-hypotensive effects. Animals were treated as detailed in Fig.2 and systolic blood pressure was monitored 4 h after LPS administration. (A) LPS administration led to a profound decrease in blood pressure that was lower than the detection limit (40 mm Hg) of the instrument used (dashed line). Both doses of t-AUCB, and aspirin significantly reversed LPS-induced hypotension but MK 886 was effective only at a high dose. However, co-administration of the low doses of t-AUCB and MK 886 led to a synergistic reversal of blood pressure. Similarly, co-administration of t-AUCB and aspirin resulted in stronger reversal of hypotension. *Significantly different from its lower dose alone (P > 0.05) and † significantly different from the individual inhibitors alone as determined by ANOVA followed by Tukey’s posthoc comparison test. (B) The baseline systolic blood pressure of wild-type (black bar, n = 6) and Ephx2-null (gray bar, n = 4) mice were not different and LPS administration led to a similar decrease in both strains of mice. Aspirin or MK886 administration both led to reversal of hypotension only in the Ephx2-null mice but not in wild-type conspecifics. * Significantly different from Ephx2 wild mice control (P > 0.05) determined by by ANOVA followed by Tukey’s posthoc comparison test.