Gastroprotective activity of ethyl-4-[(3,5-di-tert-butyl-2-hydroxybenzylidene) amino]benzoate against ethanol-induced gastric mucosal ulcer in rats

Abstract

Background: The study was carried out to determine the cytotoxic, antioxidant and gastro-protective effect of ethyl-4-[(3,5-di-tert-butyl-2-hydroxybenzylid ene)amino] benzoate (ETHAB) in rats.

Methodology/principal findings: The cytotoxic effect of ETHAB was assessed using a MTT cleavage assay on a WRL68 cell line, while its antioxidant activity was evaluated in vitro. In the anti-ulcer study, rats were divided into six groups. Group 1 and group 2 received 10% Tween 20 (vehicle). Group 3 received 20 mg/kg Omeprazole. Groups 4, 5 and 6 received ETHAB at doses of 5, 10, and 20 mg/kg, respectively. After an hour, group 1 received the vehicle. Groups 2-6 received absolute ethanol to induce gastric mucosal lesions. In the WRL68 cell line, an IC50 of more than 100 µg/mL was observed. ETHAB results showed antioxidant activity in the DPPH, FRAP, nitric oxide and metal chelating assays. There was no acute toxicity even at the highest dosage (1000 mg/kg). Microscopy showed that rats pretreated with ETHAB revealed protection of gastric mucosa as ascertained by significant increases in superoxide dismutase (SOD), pH level, mucus secretion, reduced gastric lesions, malondialdehyde (MDA) level and remarkable flattened gastric mucosa. Histologically, pretreatment with ETHAB resulted in comparatively better gastric protection, due to reduction of submucosal edema with leucocyte infiltration. PAS staining showed increased intensity in uptake of Alcian blue. In terms of immunohistochemistry, ETHAB showed down-expression of Bax proteins and over-expression of Hsp70 proteins.

Conclusion/significance: The gastroprotective effect of ETHAB may be attributed to antioxidant activity, increased gastric wall mucus, pH level of gastric contents, SOD activity, decrease in MDA level, ulcer area, flattening of gastric mucosa, reduction of edema and leucocyte infiltration of the submucosal layer, increased PAS staining, up-regulation of Hsp70 protein and suppressed expression of Bax.

Figure 1. Chemical structure of Schiff base ETHAB (ethyl-4-[(3,5-di-tert-butyl-2-hydroxybenzylidene)amino]benzoate).

Figure 2. Histological study of acute toxicity in mice liver: (a) Mice orally administrated with 10% Tween 20 (normal group).

(b) Mice orally administrated with low dose (500 mg/kg) ETHAB. (c) Mice orally administrated with high dose (1000 mg/kg) ETHAB. Mice kidney: (d) Mice orally administrated with 10% Tween 20 (normal group). (e) Mice orally administrated with low dose (500 mg/kg) ETHAB. (f) Mice orally administrated with high dose (1000 mg/kg) ETHAB. In all groups no lesions appear and no significant change compared to the normal control. (H&E staining, 20×).

Figure 3. (a) Effect of pretreatment with ETHAB on gastric contents pH. (b) Effect of pretreatment with ETHAB on mucus weight.

(c) Effect of treatment with ETHAB on the ulcer area (mm²) and ulcer inhibition (%); number rat 6/group; p<0.05. All results are presented as average mean ± SEM. *The mean difference is significant at the 0.05 level (p<0.05) compared to PC. ^#The mean difference is significant at the 0.05 level (p<0.05) compared to UC.

Figure 4. Gross lesion of the gastric mucosa in rats.

(a) In rats pretreated with 10%Tween 20 without Eth-induced ulcer (normal control) no injury to the gastric mucosa appears. (b) Rats pretreated with 10% Tween 20 (ulcer control) followed with Eth-induced ulcer, have severe hemorrhage injuries in the gastric mucosa. (c) Rats pretreated with Omeprazole (20 mg/kg) have less injury to the gastric mucosa as compared to the ulcer control rats. (d) Rats pretreated with ETHAB (5 mg/kg) have moderate injuries in the gastric mucosa. The compound reduces the formation of gastric lesions induced by absolute ethanol. (e) Rats pretreated with ETHAB (10 mg/kg) have mild to moderate injuries in the gastric mucosa. (f) Rats pretreated with ETHAB (20 mg/kg) have mild lesions but significantly restored gastric mucosa with visible flattening of the mucosa.

Figure 5. Histological study of ethanol-induced gastric mucosal damage in rats.

(a) In rats pretreated with 10% Tween 20 (negative control) normal tissue has intact surface mucosal epithelium. (b). Rats pre-treated with 10% Tween 20 (ulcer control) have severe disruption of the surface epithelium and necrotic lesions that penetrate deeply into the mucosa (c) Rats pretreated with Omeprazole (20 mg/kg) have mild disruption of the surface epithelium mucosa, and there is a reduction in submucosal edema and leucocyte infiltration. (d) Rats pre-treated with ETHAB (5 mg/kg) have moderate disruption of the surface epithelium with edema and leucocyte infiltration of the submucosal layer. (e) Rats pretreated with ETHAB (10 mg/kg) have a mild to moderate disruption of the surface epithelium and a reduction in submucosal edema. (f) Rats pre-treated with ETHAB (20 mg/kg) have mild disruption of the surface epithelium (H&E staining; 10×).

Figure 6. Periodic Acid Schiff (PAS) staining of mucosal glycoproteins.

The observed intense magenta color in the apical epithelial cells in the groups pretreated with ETHAB (d, e and f) and Omeprazole (c) compared with the ulcer control group (b) and normal group (a)(Magnification 10x).

Figure 7. Immunohistochemistry staining of Hsp70 proteins.

The analysis revealed an over expression of Hsp 70 protein in the stomach of rats pretreated with ETHAB(d, e and f) and Omeprazole (c) compared with the ulcer control group (b) and normal group (a)(Magnification 20x).

Figure 8. Immunohistochemistry staining of Bax proteins.

The analysis revealed a down-expression of Bax protein in the stomach of rats pretreated with ETHAB (d, e and f) and Omeprazole (c) compared with the ulcer control group (b) and normal group (a)(Magnification 20x).