Soluble epoxide hydrolase gene deficiency or inhibition attenuates chronic active inflammatory bowel disease in IL-10(-/-) mice

Abstract

Background: Soluble epoxide hydrolase (sEH) metabolizes anti-inflammatory epoxyeicosatrienoic acids (EETs) into their much less active dihydroxy derivatives dihydroxyeicosatrienoic acids. Thus, targeting sEH would be important for inflammation.

Aims: To determine whether knockout or inhibition of sEH would attenuate the development of inflammatory bowel disease (IBD) in a mouse model of IBD in IL-10(-/-) mice.

Methods: Either the small molecule sEH inhibitor trans/-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) or sEH knockout mice were used in combination with IL-10(-/-) mice. t-AUCB was administered to mice in drinking fluid. Extensive histopathologic, immunochemical, and biochemical analyses were performed to evaluate effect of sEH inhibition or deficiency on chronic active inflammation and related mechanism in the bowel.

Results: Compared to IL-10 (-/-) mice, sEH inhibition or sEH deficiency in IL-10(-/-) mice resulted in significantly lower incidence of active ulcer formation and transmural inflammation, along with a significant decrease in myeloperoxidase-labeled neutrophil infiltration in the inflamed bowel. The levels of IFN-γ, TNF-α, and MCP-1, as well VCAM-1 and NF-kB/IKK-α signals were significantly decreased as compared to control animals. Moreover, an eicosanoid profile analysis revealed a significant increase in the ratio of EETs/DHET and EpOME/DiOME, and a slightly down-regulation of inflammatory mediators LTB(4) and 5-HETE.

Conclusion: These results indicate that sEH gene deficiency or inhibition reduces inflammatory activities in the IL-10 (-/-) mouse model of IBD, and that sEH inhibitor could be a highly potential in the treatment of IBD.

Fig.1. Histopathologic analysis of inflammatory activity in the bowel in IL-10(−/−) and sEH (−/−)/IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB

A) active ulcer in the colon in IL-10(−/−) mice; B and C) a healed ulcer with hyperplasic/regenerative epithelium in sEH (−/−)/IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB, respectively; D) active transmural inflammation and epithelial hyperplasic change in IL-10(−/−) mice; E and F) mild lymphocyte and plasma cells in lamina propria and hyperplastic epithelial change but no or minimal transmural inflammation in sEH (−/−)/IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB, respectively. G and H) Histogram of active ulcer formation and transmural inflammation scores in IBD in IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice as well as in IL-10(−/−) mice treated with t-AUCB. The statistically significant difference between IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice as well between IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB were marked in the histogram.

Fig.2. Immunohistochemical staining of myeloperoxidase(MPO)-labeled neutrophils

A-C) MPO-positive cells in the inflamed colonic mucosa in IL-10(−/−) mice, sEH(−/−)/IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB, respectively. D-E) Histogram of semi-quantitative analysis of MPO-positive cells in the inflamed mucosa: the number of MPO-positive cells was expressed as Mean±SD. F-G) Histogram of semi-quantitative analysis of lymphoplasmacytosis in the inflamed colonic mucosa in IBD mice. Statistically significant difference between IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice as well between IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB were marked in the histograms.

Fig. 3. Immunohistochemical staining of PCNA-labeled cell proliferation

A-D) PCNA-labeled proliferative cells in morphologically normal colonic mucosa in wild type mice (A), sEH(−/−) mice (B), IL-10(−/−) mice (C), and sEH(−/−)/IL-10(−/−) mice (D); E-F) PCNA-labeled proliferative cells in hyperplasic mucosa adjacent to ulcer in IL-10(−/−) mice (E) and in sEH(−/−)/IL-10(−/−) mice (F). G) Histogram of PCNA proliferation index in the inflamed colonic mucosa in IBD in IL-10(−/−) mice compared to sEH(−/−)/IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB. Statistically significant difference between IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice as well between IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB were marked in the histograms.

Fig.4. Analysis of IFN-γ, TNF-α, and MCP-1 as well VCAM-1 mRNA expressions using quantitative real-time PCR and assay of NF-κB signaling using western blot approach

IFN-γ, TNF-α, and MCP-1 as well VCAM-1 mRNA expressions in wild type, sEH(−/−), IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice (A); and in wild type mice, IL-10(−/−) mice and IL-10(−/−) mice treated with t-AUCB (B). Western blot assay for phosphorylated NF-kB p65 (S276) and Ikk-α as well VCAM-1 in sEH(−/−)/IL-10(−/−), IL-10(−/−), sEH(−/−) and wild type mice (C), and quantitative densitometry analysis of phosphorylated NF-kB p65 (S276) and Ikk-α as well VCAM-1 (D-F). Statistically significant difference of these cytokines and NF-κB signals either in IL-10(−/−) mice compared to sEH(−/−)/IL-10(−/−) mice or in IL-10(−/−) mice compared to IL-10(−/−) mice treated with t-AUCB was marked in the histogram figures.

Fig.5. Analysis of plasma levels of epoxygenase-dependent metabolites using a LC/MS/MS assay

The difference of the ratios of total EpOME to DiHOME and EETs to DHETs, and the concentration (nM, Mean ± SD) of EpOMEs (including 9(10) - and 12(13)-EpOME), DiHOMEs (including 9(11)-DHOME and 12(13)-DHOME), 14(15)-EET, and 14(15)-DHET among IL-10(−/−), sEH(−/−)/IL-10(−/−), sEH(−/−), and wild type (wt) mice (A) as well among wild type (wt) mice, IL-10(−/−) mice, and IL-10(−/−) mice treated with t-AUCB (B). Statistically significant difference of these metabolites either in sEH(−/−)/IL-10(−/−) mice compared to IL-10(−/−) mice (A, left two panels of histograms) or in IL-10(−/−) mice compared to IL-10(−/−) mice treated with t-AUCB (B, right two panels of histograms) was marked in the figures.

Fig. 6. Analysis of plasma levels of COX-2 and 5-LOX-dependent metabolites using a LC/MS-MS assay

The concentration (nM, Mean ± SD) of PGE₂, LTB₄, and 5-HETE in wild type (wt) mice, sEH(−/−), IL-10(−/−), and sEH(−/−)/IL-10(−/−) mice (A), and in wild type (wt) mice, IL-10(−/−) mice, and IL-10(−/−) mice treated with t-AUCB (B).