Fig 1.
Name and structure for GSK1910364 and GSK2256294.
IUPAC names and molecular structures (ChemDraw) are given for the two sEHi compounds used in the experiments described in this publication.
Table 1.
DAI scoring system for DSS model.
Table 2.
Histopathology scoring system for DSS model.
Histopathology Scoring Pattern (Scale 0–40).
Fig 2.
DSS-induced colitis mouse model.
DSS-induced colitis is a common in vivo model for IBD. Our study employed acute dosing of DSS from Day 0 to Day 5. Compound treatments were from Day 0 to Day 9. Experimental groups were: Sham = vehicle control (no DSS or other treatment, n = 6), DSS = DSS-only (n = 12), CsA = 10 mg/kg i.p. treatment (n = 6), EPHX2i = GSK1910364A 50 mg/kg orally, twice daily (n = 6). (A) Mean DAI is plotted versus time; error bars reflect standard errors. (B) AUC values for each group were calculated at experiment conclusion. Sample sizes for each group are the same as in (A). Both CsA and EPHX2i significantly decreased the AUC compared to DSS-only control (ANOVA P-value < 0.05). (C) MPO protein expression is used as a surrogate biomarker for inflammation. MPO was nearly absent in the sham control mice, but levels were significantly increased in the DSS-treated animals. Both cyclosporine and EPHX2i treatment significantly decrease MPO expression, with EPHX2i showing a return to near normal level. (D) The histopathology scoring system is given in Table 2. DSS-treatment significantly increased the score, compared to vehicle control group. Both CsA and EPHX2i treatment significantly decrease the histopathology score by similar amount. (E) RT-PCR measurement of selected marker gene mRNA expression. All expression values were normalized to the levels in the vehicle control group. For most marker genes, DSS-treatment caused large increases/decreases in mRNA levels. Both CsA and EPHX2i treatment tended to restore expression levels back toward baseline (Il1b, Ccr6, Sstr2, Lgr5) or to near baseline levels (Tnf, Ltb, Rela). (F) H&E staining of selected colonic tissue sections from DSS-mice. 10x magnification of representative colon sections from each of the four treatments groups. (i) the sham control shows normal histology. (ii) the DSS-control shows widespread damage. (iii) CsA and (iv) EPHX2i treatment both show substantial protection from DSS-induced damage.
Fig 3.
Western blot analysis for EPHX2 protein level in human colon.
Human colon samples were from healthy donors or UC donors. Each UC donor had matched uninflamed (unin) and inflamed (infl) biopsies. (A) Representative western blot results. (B) Normalized mean EPHX2 intensity vs. control actin from combined donors for each group. Data derived from duplicate western blot analyses with the same set of samples. Error bars represent standard deviations. Differences between groups were not statistically significant. (C) EPHX2/actin ratio for individual donors from each sample group. Differences between groups were not statistically significant.
Fig 4.
Hematoxylin and eosin (H&E; A-C) and EPHX2 immunohistochemical (IHC; D-I) staining in human normal, CD and UC biopsy samples. Minimal cytoplasmic EPHX2 IHC staining is present in the colonocytes of the normal colon sample (D & G) with no EPHX2 IHC staining evident in the lamina propria leukocytes. Increased EPHX2 immunoreactivity was observed in the CD and UC samples. Marked cytoplasmic colonocyte and minimal to mild lamina propria leukocyte (arrow) EPHX2 IHC staining is present in the CD sample (E & H); whereas, mild cytoplasmic colonocyte and minimal lamina propria leukocyte (arrow) EPHX2H IHC staining is present in the UC sample (F & I).
Table 3.
Qualitative histopathology using the GHAS scoring system for IHC scoring.
Fig 5.
Effect of GSK2256294 on cytokine release from UC and CD colon explants measured by MSD.
UC or CD tissue samples were incubated for 18 h with varying doses of GSK2256294 (GSK294), or prednisolone (positive control). GSK2256294 doses are in nM. Cytokine release in the cell-free supernatant was determined using MSD assay as described in Materials and Methods and were normalized to DMSO (negative control). Samples from N = 6 UC and N = 10 CD donors were used in the cytokine analysis. Error bars represent standard errors. * denotes p <0.05 vs DMSO control.
Fig 6.
Effect of GSK2256294 on cytokine release from UC and CD colon explants measured by ELISA.
UC or CD tissue samples were incubated for 18 h with varying doses of GSK2256294 (294), or prednisolone (positive control). GSK2256294 doses are in nM. Cytokine release in the cell-free supernatant was determined by ELISA as described in Material and Methods and were normalized to DMSO (negative control). Samples from N = 6 UC and N = 10 CD donors were used in the cytokine analysis. Error bars represent standard errors. * denotes p<0.05 vs DMSO control.
Fig 7.
Summary of EPHX2i and knockout in mouse models and human disease explants.
Similar protective effects on histology and inflammation were observed whether EPHX2 was decreased using chemical inhibitors or due to genetic deficiency. This was the case for both DSS-induced colitis and IL10 knockout mouse models. In colon explants from human patients, EPHX2i decreased production of inflammatory cytokines, compared to DMSO controls. This effect was seen in both UC and CD patient tissue.