Pharmacological inhibition of soluble epoxide hydrolase or genetic deletion reduces diclofenac-induced gastric ulcers

Abstract

Aims: This research was conducted to evaluate the hypothesis that gastric ulcers caused by the NSAID diclofenac sodium (DCF) can be prevented by the soluble epoxide hydrolase inhibitor TPPU.

Main methods: Mice were administered a single dose of 10, 30 or 100mg/kg of DCF. Once an ulcerative dose of DCF was chosen, mice were pretreated with TPPU for 7days at 0.1mg/kg to evaluate anti-ulcer effects of the sEH inhibitor on anatomy, histopathology, pH, inflammatory markers and epithelial apoptosis of stomachs.

Key findings: Diclofenac caused ulceration of the stomach at a dose of 100mg/kg and a time post dose of 6h. Ulcers generated under these conditions were associated with a significant increase in the levels of TNF-α and IL-6 in serum and increased apoptosis compared to control mice. Pretreatment with TPPU resulted in a decrease of ulceration in mice treated with DCF with a significant decrease in the level of apoptosis, TNF-α and IL-6 in the serum in comparison to diclofenac-treated mice. TPPU did not affect the pH of the stomach, whereas omeprazole elevated the pH of the stomach as expected. A similar anti-ulcer effect was observed in sEH gene knockout mice treated with DCF.

Significance: The sEH inhibitor TPPU decreases the NSAID-induced stomach ulcers.

Keywords: Apoptosis; Diclofenac; IL-6; Soluble epoxide hydrolase inhibitor TPPU; Stomach ulcer; TNF-α.

Fig. 1. Inhibition of sEH or genetic deletion decreases diclofenac-induced ulcers in stomachs of mice

(A) Among the 3 doses tested for the ulcerative effect of diclofenac (DCF), the 100 mg/kg dose exhibited ulcers in Swiss Webster mice (*** p < 0.001 vs 10 mg/kg dose). The severity of the ulcers was greater at 6 h post diclofenac dose than at 18 h after diclofenac treatment. (B) 100 mg/kg of diclofenac-induced stomach ulcers significantly in Swiss Webster mice (*** p < 0.001, DCF treated animals vs vehicle control animals; * p < 0.01, DCF + OME vs vehicle-treated animals). Severity of diclofenac-induced ulcers decreased significantly with TPPU or OME treatment (^### p < 0.001, DCF vs DCF + TPPU, DCF vs DCF + OME). OME-treated animals exposed to DCF had higher (^% p < 0.01) incidence of ulceration than TPPU + DCF treated animals. (C) DCF significantly induced stomach ulcers in C57BL6 mice (*** p < 0.001, DCF vs vehicle). The sEH gene deletion (KO) also decreased ulcerative effects of diclofenac in stomach (^### p < 0.001). The vehicle control animals used for this figure are C57BL6 mice. Values are presented as the mean ± standard deviation, n=4. (D) Anatomical observation at 7X under a dissecting microscope of stomachs of Swiss Webster mice administered with different treatments. Ulceration is pointed with an arrow. Stomachs from mice exposed to DCF exhibited ulcers and hemorrhagic streaks. Pre-treatment with the sEHI TPPU, omeprazole (OME), or (F) sEH knock out (KO) decreased stomach ulceration as shown at 7X under a dissecting microscope. (E) Histopathological observation of tissue section complemented anatomical observation. Morphology of gastric mucosa of vehicle treated Swiss Webster mice looked normal while ulceration was observed on stomachs of DCF treated mice. Luminal surface necrosis tissue debris is shown as highlighted area and it’s beneath granulation tissue formation is noticed. Ulceration was minimal in mice treated with DCF + TPPU. Minimal glandular cystic injury/erosion on surface of the mucosal layer in TPPU pre-treated mice is shown with an arrow. Omeprazole also minimized gastric erosion due to DCF. Minimal erosion on surface of mucosal layer is pointed by arrow. (G) Gastric mucosa of C57BL6 mice was normal but ulceration in mucosa (highlighted area) was observed in DCF treated mice. Mucosa with minimal injury/erosion and predominant reactive epithelial changes is seen in stomachs of sEH knockout mice. Doses of TPPU and OME were 0.1 mg/kg and 50 mg/kg respectively.

Fig. 2. Inhibition of sEH or gene deletion does not affect gastric pH

Diclofenac (DCF, 100 mg/kg, single dose) itself and diclofenac along with TPPU (0.1 mg/kg, 7 days) did not alter the pH of stomachs when compared to mice treated with vehicle. Similarly, DCF did not alter the pH in stomachs of sEH knockout mice. However, omeprazole treatment (50 mg/kg, 5 days) elevated the pH of stomachs and the difference was statistically significant when compared with vehicle (^## p < 0.01), DCF (*** p < 0.001), DCF + TPPU (^$ p < 0.05) and effect of DCF on gastric pH of sEH knockout animals (^%% p < 0.01). One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. Data are presented as the mean ± standard deviation of 4 readings.

Fig. 3. Inhibition of sEH or knock out reverses diclofenac-induced increases in the levels of TNF-α and IL-6 in the serum of mice

(A) Diclofenac (DCF) (100 mg/kg, p.o., single dose) treatment increased level of TNF-α significantly (* p < 0.01; DCF vs vehicle) while pretreatment of TPPU (0.1 mg/kg) (^$ p < 0.01; DCF vs DCF+TPPU), omeprazole (OME) (50 mg/kg) (^% p < 0.01; DCF vs DCF + OME) or sEH knock out mice (^# p < 0.01; DCF vs DCF + sEH KO) decreased level of TNF-α significantly. (B) Diclofenac (DCF) treatment increased levels of IL-6 significantly (* p < 0.01; DCF vs vehicle) while pre-treatment of TPPU (^$ p < 0.01; DCF vs DCF+TPPU), omeprazole (OME) (^% p < 0.01; DCF vs DCF + OME) and sEH knocked out (^# p < 0.01; DCF vs DCF + sEH KO) decreased level of IL-6 significantly. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. The assays were run in duplicate. Data are presented as mean ± standard deviation of 3–4 animals.

Fig 4. Inhibition of sEH or gene knockout, and pre-treatment of Omeprazole decrease diclofenac-induced apoptosis in epithelial cells of stomachs. (A)

Apoptosis of stomach epithelial cells was detected by TUNEL positive cells with brown nuclei. DCF (100 mg/kg) treatment increased apoptosis significantly (* p < 0.001; DCF vs vehicle) while pre-treatment with TPPU (0.1 mg/kg) (^$ p < 0.001; DCF vs DCF+TPPU), OME (50 mg/kg) (^% p < 0.01; DCF vs DCF + OME) or sEH knockout (^# p < 0.001; DCF vs DCF + sEH KO) decreased apoptosis significantly. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. Data are presented as the mean ± standard deviation of 3–4 observation (B) Stomach epithelial cells seen under a microscope at 200X magnification after TUNEL assay.

Fig 5. Effect of treatment on activities of sEH and COX. (A)

DCF (100 mg/kg) did not increase or decrease the activity of sEH in vitro. Negligible sEH activity was observed in the stomach of sEH knockout mice. The sEH activity in sEH KO mice was significantly less (p < 0.001) in comparison to vehicle (*), DCF (@), DCF + TPPU (#), DCF + OME ($). (B) Levels of PGE₂ were quantified in stomachs of treated animals (n=4) as a marker of COX activity. Diclofenac alone and in combination with TPPU and OME inhibited COX as expected. (C) Levels of TxB2/PGE₂ were quantified in the plasma of animals. Diclofenac treatment significantly decreased the levels of TxB2/PGE₂. DCF + OME (* p < 0.05) decreased the level of TxB2/PGE₂ to a lesser extent than DCF (** p < 0.01) and DCF + TPPU (** p < 0.01) in comparison to vehicle. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. The assay was run in triplicate. Data are presented as the mean ± standard deviation of 3–5 animals.