EP4 agonist alleviates indomethacin-induced gastric lesions and promotes chronic gastric ulcer healing

Abstract

Aim: To investigate EP4-selective agonist effect on indomethacin-induced gastric lesions and on the spontaneous healing of chronic gastric ulcers.

Methods: In a mouse model of gastric bleeding with high dose of indomethacin (20 mg/kg), an EP4-selective agonist was administered orally. Stomach lesions and gastric mucous regeneration were monitored. In a mouse model of chronic gastric ulcer induced by acetic acid, EP4 agonist effect on the healing of chronic gastric ulcer was evaluated in the presence or absence of low dose indomethacin (3 mg/kg). In cultured human gastric mucous cells, EP4 agonist effect on indomethacin-induced apoptosis was assessed by flow cytometry.

Results: The EP4-selective agonist reduced high dose indomethacin-induced acute hemorrhagic damage and promoted mucous epithelial regeneration. Low-dose indomethacin aggravated ulcer bleeding and inflammation, and delayed the healing of the established chronic gastric ulcer. The EP4 agonist, when applied locally, not only offset indomethacin-induced gastric bleeding and inflammation, but also accelerated ulcer healing. In the absence of indomethacin, the EP4 agonist even accelerated chronic gastric ulcer healing and suppressed inflammatory cell infiltration in the granulation tissue. In vitro, the EP4 agonist protected human gastric mucous cells from indomethacin-induced apoptosis.

Conclusion: EP4-selective agonist may prevent indomethacin-induced gastric lesions and promote healing of existing and indomethacin-aggravated gastric ulcers, via promoting proliferation and survival of mucous epithelial cells.

Figure 1

EP4 agonist effect on indomethacin-induced apoptosis in human gastric mucus epithelial cells (AGS). AGS cells were pre-treated with EP4 agonist for 30 min followed by addition of indomethacin for 24 h and sorted by flow cytometry after PI staining. Sub-2N population was quantified as apoptotic cells. A: indomethacin dose-dependently induced apoptosis, ^bP < 0.0001 vs the rest, n = 4; B: shown are values minus vehicle controls, ^aP < 0.005, n = 5-9; indomethacin dose was 400 μmol/L.

Figure 2

EP4 agonist effect on indomethacin-induced gastric lesion in mice. EP4 agonist was orally administered 24 h and 30 min prior to indomethacin dosing at 20 mg/kg. The stomachs were assessed for mucus lesions 24 h after indomethacin dosing. A: HE and BrdU immunohistochemistry staining of stomachs (× 200). Superficial mucosal cells had sloughed off gastric mucus with infiltration of inflammatory cells in vehicle-treated group (arrow points to one lesion site). The mucus of EP4 agonist-treated stomachs was almost normal, except for a sparse focal defect of superficial mucous cells without inflammatory cells (arrow points to one lesion site). BrdU labeling showed robust mucous epithelial regeneration and migration in EP4 agonist-treated rats compared with that of vehicle treatment; B: Quantification of gross lesion under dissection microscopy (× 16) and percentage of BrdU-positive cells among mucus cells. Shown are lesion scores, ^bP < 0.0001, n = 10; and BrdU percentage, ^dP < 0.01, n = 10, respectively.

Figure 3

Low dose indomethacin effect on chronic gastric ulcer of mice. Chronic gastric ulcers were created by 40% acetic acid as detailed in “Methods”. Then the animals were given indomethacin from day 3 to day 6 at 3 mg/kg per day and the ulcers were assessed after blood withdrawal on day 7 post-surgery. A: Ulcer sizes were measured under dissection microscopy with 16 × magnification; ^bP = 0.006, n = 18; B: Hematology analysis of ulcerated animals on day 7, ^aP < 0.05, n = 6; RBC: Red blood cell; HGB: Hemoglobin; HCT: Hematocrit; C: Hematology of ulcerated animals on day 7, ^aP < 0.05, n = 6; WBC: White blood cells; LYM: Lymphocytes; NEU: Neutrophils; MONO: Monocytes.

Figure 4

EP4 agonist alleviated indomethacin effect on gastric ulcer in mice. Mice with gastric ulcer were given low-dose indomethacin (3 mg/kg per day) with or without EP4 agonist from day 3 to day 6 post-surgery. The blood was withdrawn on day 7 for hematology analysis. A and B are results of hematology analysis, ^aP < 0.05, n = 20.

Figure 5

EP4 agonist promoted gastric ulcer healing in mice. Mice with gastric ulcers were treated with vehicle or EP4 agonist from day 3 to day 10 post-surgery. Gastric ulcers were assessed on day 7 or 11, respectively. A is quantification of ulcer sizes under dissection microscopy with 16 × magnification, ^bP = 0.003, n = 16; B is representative images of gastric ulcers; the macroscopy images are at original magnification, × 16; the microscopy images are at original magnification, × 40 from slide sections.