Gallic Acid Enriched Fraction of Phyllanthus emblica Potentiates Indomethacin-Induced Gastric Ulcer Healing via e-NOS-Dependent Pathway

Abstract

The healing activity of gallic acid enriched ethanolic extract (GAE) of Phyllanthus emblica fruits (amla) against the indomethacin-induced gastric ulceration in mice was investigated. The activity was correlated with the ability of GAE to alter the cyclooxygenase- (COX-) dependent healing pathways. Histology of the stomach tissues revealed maximum ulceration on the 3rd day after indomethacin (18 mg/kg, single dose) administration that was associated with significant increase in inflammatory factors, namely, mucosal myeloperoxidase (MPO) activity and inducible nitric oxide synthase (i-NOS) expression. Proangiogenic parameters such as the levels of prostaglandin (PG) E(2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), von Willebrand Factor VIII, and endothelial NOS (e-NOS) were downregulated by indomethacin. Treatment with GAE (5 mg/kg/day) and omeprazole (3 mg/kg/day) for 3 days led to effective healing of the acute ulceration, while GAE could reverse the indomethacin-induced proinflammatory changes of the designated biochemical parameters. The ulcer healing activity of GAE was, however, compromised by coadministration of the nonspecific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), but not the i-NOS-specific inhibitor, L-N6-(1-iminoethyl) lysine hydrochloride (L-NIL). Taken together, these results suggested that the GAE treatment accelerates ulcer healing by inducing PGE(2) synthesis and augmenting e-NOS/i-NOS ratio.

Figure 1

Healing capacities of GAE under various treatment regimes against indomethacin-induced acute gastric mucosal injury in mice. Ulceration in the mice was induced by indomethacin (18 mg /kg, single dose, p.o.). Treatment was carried out with GAE (3, 4, 5, 6, 7 mg/kg, single dose daily up to 7 days, p. o.) after indomethacin administration. The section of mice stomachs were dissected on the 1st, 3rd, and 7th days of ulceration, 4 h after the last dose of the test sample, and the damage scores of different mice groups were measured. The values are mean ± S.E. of four independent experiments, each with 8 mice/group. *P < 0.001, compared to 1st day ulcerated mice; **P < 0.01, ***P < 0.001, compared to untreated mice of the same day.

Figure 2

Healing capacities of GAE, gallic acid and omeprazole under the optimized treatment regime. (a) ulcer indices; (b) histology. Ulceration in the mice was induced by indomethacin (18 mg/kg, single dose, p.o.). Treatment was carried out with GAE (5 mg/kg daily, p.o.), gallic acid (3 mg/kg daily, p.o.), and omeprazole (3 mg/kg daily, p.o.) for 3 days, starting the first dose 6 h postulcer induction. The sections of mice stomachs were processed for capturing the images. Representative histology of gastric tissue sections are shown at 10x magnification. (i) normal, (ii) Ulcerated untreated, (iii) Ulcerated + GAE treated, (iv) Ulcerated + Gallic acid treated, (v) Ulcerated + Omeprazole treated, mucosal, and submucosal layers are shown by blue and green arrows, respectively. The ulcer indiceswere calculated from the damage scores.The values are mean ± S.E. of four independent experiments, each with 8 mice/group. *P < 0.001, compared to normal mice; **P < 0.01,***P < 0.001, compared to untreated mice.

Figure 3

Reduction of the mucosal MPO activity in ulcerated mice by GAE. (a) under various treatment regimes; (b) optimized treatment regime. Ulceration in the mice was induced by indomethacin (18 mg/kg, single dose, p.o.). Treatment was carried out with different doses of GAE upto 7 days after indomethacin (18 mg/kg, single dose, p.o.) administration. The section of mice stomachs were dissected on the 1st, 3rd, and 7th days of ulceration, 4 h after the last dose of the test sample, and the MPO activities of different groups of mice were measured. Omeprazole (3 mg/kg × 3 days, p. o.) was used as the positive control. The values are mean ± S.E. of four independent experiments, each with 8 mice/group. *P < 0.001, compared to 1st day ulcerated mice; **P < 0.01, ***P < 0.001, compared to untreated mice of the same day. ^# P < 0.001, compared to 3rd day normal mice; ^$ P < 0.001, compared to 3rd day untreated mice.

Figure 4

Effect of GAE on mucosal PGE₂ synthesis in indomethacin-induced ulcerated mice. Ulceration in the mice was induced by indomethacin (18 mg/kg, single dose, p.o.). Treatment was carried out for 3 days with GAE (5 mg/kg, daily, p. o.) or omeprazole (3 mg/kg, p. o.) after ulcer induction. The mucosal PGE₂ levels of the ulcerated untreated and treated mice were measured. The values are mean ± S.E. of four independent experiments, each with 8 mice/group. *P < 0.001, compared to normal mice; **P < 0.001, compared to untreated mice.

Figure 5

The e-NOS and i-NOS expressions in normal, ulcerated and GAE (5 mg/kg, single dose daily × 3 days, p. o.) or omeprazole (3 mg/kg, p. o.) treated gastric tissues of mice, and their quantifications. Western blots of the expressions of the enzymes (A). Ratios of the intensities of i-NOS (B) and e-NOS (C) bands to that of the respective β-actin bands as quantified from the western blot images, using Kodak Gelquant software. The values (arbitrary unit, mean ± S.E.M.) are the density scanning results of three independent experiments, considering that of normal mice as 1.

Figure 6

Effect of GAE on mucosal NO level in indomethacin-induced ulcerated mice. Ulceration in the mice was induced by indomethacin (18 mg/kg, single dose, p. o.). Treatment was carried out for 3 days with GAE (5 mg/kg, single dose daily × 3 days, p. o.) or omeprazole (3 mg/kg, p. o.) after ulcer induction. The mucosal NO levels of the ulcerated untreated and treated mice were measured. The values are mean ± S.E. of four independent experiments, each with 8 mice/group. *P < 0.001, compared to normal mice; **P < 0.001, compared to untreated mice.

Figure 7

Effect of GAE on different angiogenic parameters in indomethacin-induced ulcerated mice. (a) von Willebrand Factor VIII; (b) mucosal VEGF level; (c) mucosal HGF level. Ulceration in the mice was induced by indomethacin (18 mg /kg, single dose, p. o.). Treatment was carried out for 3 days with GAE (5 mg/kg, single dose daily × 3 days, p. o.) or omeprazole (3 mg/kg, p. o.) after ulcer induction. The mucosal von Willebrand Factor VIII (expressed as number of microvessels/mm²) and growth factors (expressed as ng/ml tissue extract) were measured by immunohistochemistry and colorimetry, respectively. *P < 0.05, **P < 0.01, compared to normal mice; ^# P < 0.01, ***P < 0.001, compared to untreated mice.

Figure 8

Effect of NOS inhibitors on the healing activity of GAE in indomethacin-induced ulcerated mice. (a) damage score, (b) MPO activity, (c) von Willebrand Factor VIII, (d) NO level. Ulceration in the mice was induced by indomethacin (18 mg /kg, single dose, p.o.). After ulcer induction, treatment was carried out with GAE (5 mg/kg, single dose daily, p. o.) alone or in conjunction with L-NAME (15 mg/kg, once daily) or L-NIL (3 mg/kg, twice daily) for 3 days. The parameters of the ulcerated untreated and treated mice were measured. *P < 0.001 compared to normal mice; **P < 0.01, compared to GAE-treatment.

Figure 9

Schematic representation of the plausible mechanism of the ulcer healing by GAE.