The human sEH is encoded by the EPHX-2 gene. The EPHX-2 gene-encoded product has an N-terminal domain that contains lipid phosphatase activity [30
] and a carboxy-terminal domain with epoxide hydrolase activity. The bifunctional nature of the EPHX-2 encoded protein allows for inhibition of one domain with no effect on the enzymatic activity of the other domain. The role of the c-terminal epoxide hydrolase in inflammation has been preliminarily assessed in rodent models.[2
] sEH hydrolyzes EETs to their much less biologically active DHETs, dramatically reducing anti-inflammatory activity of EETs.[1
] Isoprenoid pyro- and monophosphates are substrates for the N-terminal phosphatase domain[31
] and these lipid phosphates are the metabolic precursors of cholesterol biosynthesis, and most importantly they are use for isoprenylation, a protein post-translational lipid modification process that is involved in the process of inflammation.[33
] In the present study, using an advanced approach of the IBD model in IL-10(−/−) mice combined with sEH/EPHX-2 gene deficiency or sEH inhibition, we demonstrated that sEH gene deficiency or inhibition significantly ameliorated chronic active inflammation in the bowel, particularly reduced active ulcer formation, trnasmural inflammation and inflammatory cell infiltration.
The neutrophil crytitis and crypt abscesses are key pathogenic events in the disease activity in IBD. The intense infiltration of leukocytes along with the overproduction of reactive oxygen and nitric oxide free radicals, inflammatory cytokines/chemokines and arachidonic acid metabolites are the key contributory factors to inflammatory activity and inflammatory injury in the bowel. It has been demonstrated that physiologic concentrations of EETs decrease the expression of endothelial cell adhesion molecules such as VCAM-1 (induced by TNF-α via NF-κB) and thus prevent subsequent leukocyte adhesion to the vascular wall and reduce inflammatory cell infiltration into the inflamed tissues.[2
] sEH, as a pro-inflammatory enzyme, converts EETs to DHETs that inactivate anti-inflammatory function of EETs. In this study, we demonstrated a significant reduction in the intensity of MPO-labeled neutrophils and lymphoplasmacytosis in IBD in sEH(−/−)/IL-10(−/−)mice, as well as in IL-10(−/−) mice treated with the sEH inhibitor t
-AUCB. These results indicate that the inhibition of inflammatory activity, particularly neutrophil infiltration, is a major anti-inflammatory event by sEH inhibition.
The infiltration of inflammatory cells is mediated in part via pro-inflammatory cytokines and chemokines. TNF-α, a central inflammatory cytokine, involves in both systemic inflammation and the stimulation of the acute phase reaction. TNF-α promotes the inflammatory response and causes many of the clinical problems associated with idiopathic inflammatory disorders such as IBD. TNF-α together with other cytokines and chemokines including interferon-γ (IFN-γ) and monocyte chemotactic protein-1 (MCP-1) plays crucial role in stimulating a proinflammatory cytokine produced by Th1 cells [36
], inducing the expression of integrins and VCAM-1 required for chemotaxis, and acting as a potent attractant for inflammatory cells (monocytes and lymphocytes) [37
]. Particularly, VCAM-1 induced by TNF-α via NF-κB is important for leukocyte adhesion to the vascular wall and infiltrating into inflamed tissues [2
]. Our results revealed that the levels of TNF-α, IFN-γ, and MCP-1 as well as VCAM-1 in the inflamed colonic tissues were significantly decreased in sEH(−/−)/IL-10(−/−) mice and in IL-10(−/−) mice treated with t
-AUCB. Furthermore, Ikk-α and phosphorylated NF-κB were significantly down-regulated in sEH(−/−)/IL-10(−/−) mice. These findings suggest that the suppression of these inflammatory cytokines and chemokines as well as NF-κB signaling by sEH gene deficiency or inhibition are key mechanism in suppressing inflammatory activity in IBD.
One of the pivotal molecules in inflammation is arachidonic acid, which simplistically has three potential metabolic fates, including cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 epoxygenases mediated metabolic pathways to generate biologically active mediators involved in the inflammatory cascade. A LC/MS-MS method is highly efficient approach to analyze endogenous lipid mediators [38
] and facilitates the quantitative analysis of over 120 eicosanoid metabolites using an ABI linear trap instrument in MRM mode [39
]. This “omic” approach provides much more insight than can be obtained from monitoring a single analyte such as EET or DHET and provide an opportunity on understanding the interactions among three arachidonic acid metabolic pathways via targeting the key enzyme/s. Similar to the previous studies using sEH inhibitors[10
], a simple biomarker for monitoring the effect of sEH inhibition is the ratio of lipid epoxides to diols. As expected, sEH gene deficiency or sEH inhibition resulted in a significant increase in the ratios of EETs/DHETs and EpOMEs/DiHOMEs, confirming the known function of sEH in metabolizing epoxide products, particularly converting 14(15) EET to 14(15) DHET. Analysis of the eicosanoid profile further revealed that sEH gene deficiency displayed a marked reduction of the LTB4 and 5-HETE metabolites when compared to IL-10(−/−) mice. These results simply imply that sEH gene deficiency shifts the eicosanoid profile from propagation of inflammation to its resolution in which LTB4
and HETEs stimulate inflammatory cell activities and PGE2
participates in regeneration and ulcer healing. But, t
-AUCB, sEH inhibitor, appears not as strong as sEH gene deficiency in modulation of these metabolic pathways.
The clinical use of nonsteroidal anti-inflammatory drugs (NSAIDs) exacerbates inflammatory bowel disease (IBD).[41
] NSAIDs and COX-2-specific inhibitors (e.g.,
celecoxib) have been shown to be harmful in animal models of IBD as well.[42
] These findings may be explained by the idea that 1) COX2 and PGE2
inhibition leads to inhibition of cell proliferation and a delay in ulcer healing, and 2) leads to shunting of arachidonic acid substrates to other pathways, particularly to the 5-LOX pathway.[44
] Here, sEH gene deficiency or inhibition results in slightly decreased level of LTB4
and 5-HETE and no significant change on PGE2, indicating that direct targeting of sEH would be different from NSAIDs and may even overcome the toxicity of NSAIDs to IBD. In particular, shifting the eicosanoid profile from propagation of inflammation (LTB4 and HETEs) to EETs and the PGE2-led regenerative healing process could prove beneficial for the treatment of IBD.
The results of the eicosanoid profiling also correlate well with the morphological inflammatory activities in the bowels. LTB4
and 5-HETE are well known pro-inflammatory mediators that are able to induce inflammatory cell infiltration by inducing adhesion and activation of leukocytes on the endothelium, allowing them to bind to and cross it into the inflamed tissue [48
]. Down-regulation of the levels of LTB4
and 5-HETE is also a partial mechanism of reduction of MPO-labeled inflammatory cell infiltration in sEH(−/−)/IL-10(−/−) mice in addition to lowering inflammatory cytokines and chemokines. Whether or not the modulation of the eicosanoid profile by sEH gene deficiency or inhibition is involved in regulation of inflammatory cytokines need to be investigated further.
t-AUCB is a potent sEH inhibitor with IC50 of sEH inhibition 1.5 ± 0.2 nM. LC/MS-MS based analytical procedures allow to detect picomolar concentrations of the sEH inhibitors including t-AUCB from ~5 μl of blood, and to perform the rapid ADME studies. Herein, we have demonstrated that administration of 8mg/liter t-AUCB to mice in drinking fluid reached to 2.63 ± 2.34 nM plasma levels which was within the range of IC50 sEH inhibition and showed the inhibitory effect on inflammation in the bowel. Thus, t-AUCB will be highly potential for treating IBD in human in future.
To conclude, the sEH gene deficiency or inhibition results in a significant reduction in inflammatory cell infiltration and active ulcer formation in IBD in IL-10(−/−) mice, as well as decreased inflammatory cytokine expression and shifting of the eicosanoid profile from propagation of inflammation to its resolution. These findings indicate that sEH involves in the process of inflammation in IBD and thus is a potential target for the treatment of IBD.