Reduction of inflammatory bowel disease-induced tumor development in IL-10 knockout mice with soluble epoxide hydrolase gene deficiency

Abstract

Soluble epoxide hydrolase (sEH) quickly inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs) by converting them to dihydroxyeicosatrienoic acids (DHETs). Inhibition of sEH has shown effects against inflammation, but little is studied about the role of sEH in inflammatory bowel disease (IBD) and its induced carcinogenesis. In the present study, the effect of sEH gene deficiency on the development of IBD-induced tumor development was determined in IL-10 knockout mice combined with sEH gene deficiency. Tumor development in the bowel was examined at the age of 25 wk for male mice and 35 wk for female mice. Compared to IL-10(-/-) mice, sEH (-/-)/IL-10(-/-) mice exhibited a significant decrease of tumor multiplicity (2 ± 0.9 tumors/mouse vs. 1 ± 0.3 tumors/mouse) and tumor size (344.55 ± 71.73 mm³ vs. 126.94 ± 23.18 mm³), as well as a marked decrease of precancerous dysplasia. The significantly lower inflammatory scores were further observed in the bowel in sEH(-/-)/IL-10(-/-) mice as compared to IL-10(-/-) mice, including parameters of inflammation-involved area (0.70 ± 0.16 vs. 1.4 ± 0.18), inflammation cell infiltration (1.55 ± 0.35 vs. 2.15 ± 0.18), and epithelial hyperplasia (0.95 ± 0.21 vs. 1.45 ± 0.18), as well as larger ulcer formation. qPCR and Western blotting assays demonstrated a significant downregulation of cytokines/chemokines (TNF-α, MCP-1, and IL-12, 17, and 23) and NF-κB signals. Eicosanoid acid metabolic profiling revealed a significant increase of ratios of EETs to DHETs and EpOMEs to DiOMEs. These results indicate that sEH plays an important role in IBD and its-induced carcinogenesis and could serve as a highly potential target of chemoprevention and treatment for IBD.

Keywords: IL-10; carcinogenesis; eicosanoid acid metabolic profiling; inflammatory bowel disease; soluble epoxide hydrolase.

Fig. 1. Morphology of colitis-induced adenocarcinoma and histograms of tumor number and size in IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice

(A) A polypoid well-differentiated adenocarcinoma was observed in the intestinal mucosa under low-power field (125X), and (B) high magnification (500X) of square area of (A), showing adenocarcinoma invading into laminar propria in the colonic mucosa in IL-10(−/−) mice. (C) low-power view of transmural invasion of mucinous carcinoma in the colon in IL-10(−/−) mice (125X), and (D) high magnification (250X) of square area of (C), showing well-differentiated mucinous carcinoma invading into muscularis propria. (E) Histogram of tumor multiplicity (number of tumor/mouse): showing a significant decrease of tumor multiplicity in sEH(−/−)/IL-10(−/−) mice (n=20) as compared to IL-10 (−/−) mice (n=19, P=0.01). (F) Distribution of tumor size: the tumor size in sEH(−/−)/IL-10(−/−) mice (n=20) was significantly smaller than that in IL-10(−/−) mice (n=19). Results were reported as mean ± SD. The statistical difference between sEH(−/−)/IL-10(−/−) and IL-10 (−/−) animals was calculated and labeled in the figure.

Fig. 2. Dysplastic lesions and cell proliferation labeled with Ki-67 immunohistochemistry

(A) Representative dysplastic lesion in IL-10(−/−) mice. (B) Representative dysplastic lesion in sEH(−/−)/IL-10(−/−) mice. (C) Dysplastic lesion, showing Ki-67-labeled proliferative cells diffusely distributed throughout the crypts to the luminal surface epithelium in the colon in IL-10(−/−) mice. (D) The proliferative cells labeled with Ki-67 were fewer and predominantly limited within the crypts and focally extend to the luminal surface of colon in sEH(−/−)/IL-10(−/−) mice. (E) Histogram of the number of dysplasia (counted throughout the entire colon): showing a marked decrease of the number of dysplasia in sEH(−/−)/IL-10(−/−) mice (n=19) as compared to that in IL-10 (−/−) (n=20); but not reached statistical significance (P>0.05). (F) Histogram of the average percentage of Ki67-labeld proliferative cells: showing a significant decrease of Ki67-labeld proliferative cells in sEH(−/−)/IL-10(−/−) mice (n=10) as compared to IL-10(−/−) mice (n=7) in lesions of inflammation (P=0.05), dysplasia (P=0.03), and adenocarcinoma (P=0.05). Results represented as mean ± SD. Percentage of positive stained Ki-67 cells in total cells counted in different lesions in the colon was analyzed by three randomly chosen areas of each lesion under 40X HPF.

Fig. 3. Morphological analysis and semi-quantification of inflammatory activity and ulcer formation in IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice

(A) The colon of IL-10(−/−) mouse exhibits a large ulcer with adjacent hyperplastic epithelial changes and extensive inflammatory cell infiltration. (B) The colon of sEH(−/−)/IL-10(−/−) mouse showed a small healed ulcer with mild hyperplastic epithelium and less inflammatory cell infiltration. (C) Immunohistochemistry of myeloperoxidase (MPO), a square area in photo A shows intense MPO-positive neutrophil infiltration; and D) MPO Immunohistochemistry, a square area in photo B displays much significant less MPO-positive neutrophil infiltration. E) Histogram of inflammatory severity in IBD: showing a semi-quantification of inflammatory severity, ulcer, hyperplasia, and lesion area involved in sEH(−/−)/IL-10(−/−) mice (n=20) and in IL-10(−/−) mice (n=19). The statistically significant difference was observed between sEH(−/−)/IL-10(−/−) and IL-10(−/−) mice in inflammatory severity (P=0.03), hyperplasia (P=0.04), extent of that involved (P=0.007), and total inflammatory index (P=0.003). Results were reported as mean ± SE.

Fig. 4. Quantitative real-time PCR analysis of TNF-α, IFN- γ, MCP-1, IL-12, IL-17 and IL-23 mRNA expressions in the colon in wild type, sEH(−/−). IL-10(−/−), sEH(−/−)/IL-10(−/−) mice

The statistically significant difference of these cytokines and chemokines was observed between IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice. (n=6/group, mucosa specimens from 3 males and 3 females chosen randomly from each group). Results were reported as mean ± SE.

Fig. 5. Western blot and densitometry analysis for NF-κB p65, phosphorylated NF-κB p65 (S276), IKK-α, and VCAM-1 for freshly collected colon mucosa from IL-10(−/−) and sEH(−/−)/IL-10(−/−) mice

(A) Western blot assay, and (B) Histogram of densitometry analysis. Specimens from male and female animals were pooled together within each group (n=6/group, 3 males and 3 females chosen randomly from each group). The significant decreases of NF-κB p65, phosphorylated NF-κB p65 (S276), IKK-α, and VCAM-1 were seen in sEH(−/−)/IL-10(−/−) mice as compared with IL-10(−/−) mice. β-actin was used as internal control.

Fig. 6. Metabolites of eicosanoid profile in plasma specimens analyzed using a LC/MS-MS method

The levels of epoxygenase-dependent metabolites in plasma (A) EETs (including 11(12)-, 14(15)-EET), (B) DHETs (including 11(12)-DHET, and 14(15)-DHET), and (C) the ratio of total EETs to DHETs. (D) EpOMEs (including 9(10)-, 11(12)-EpOME), (E) DiHOMEs (including 9(10)-DiHOME, and 11(12)-DiHOME), and (F) the ratio of total EpOME to DiHOME. The statistically significant difference was labeld in the figures in comparison between wild type mice and sEH (−/−) mice and between sEH(−/−)/IL-10(−/−) mice and IL-10(−/−) mice. Especially, both EETs and EpOMEs were significantly increased, while DHETs and DiHOME were significantly decreased in sEH(−/−)/IL-10(−/−) mice compared to IL-10(−/−) mice (n=20 per group), therefore, the ratios of EETs/DHETs and EpOMEs/DiHOME were much more significantly increased. Results were reported as mean ± SE.

Fig. 7. Representative metabolites of eicosanoid profile in COX and LOX pathways analyzed using a LC/MS-MS method

The levels of COX-dependent metabolites in plasma (A) PGE2, and PGD2, and (B) TXB2. The levels of LOX-dependent metabolites in plasma (C) LTB4 and (D) 5-HETE. Results were reported as mean ± SE. The statistically significant difference was labeled in the figure.