Ulcer Prevention Effect Of 3,4,5-Tihydroxy-N0-[(2-Methyl-1H-Indol-3yl)Methylidene]Benzohydrazide In HCl/Ethanol-Induced Gastric Mucosal Damage In Rats

Abstract

The newly synthesized, 3,4,5-Trihydroxy-N 0-[(2-methyl-1H-indol-3-yl)-methylidene] benzohydrazide (TIBH), is an indole and gallic acid derivative. The aim of this research investigation was to evaluate the acute toxicity and the ulcer prevention potential of TIBH in HCl/Ethanol-induced gastric ulcer rat model. Six groups of rats were orally received 5ml/kg of vehicle (1 % Carboxy methyl cellulose) for the normal and ulcer control groups each, Omeprazole (20mg/kg) for positive control, 50 mg/kg, 100 mg/kg and 200 mg/kg of TIBH for experimental groups, respectively. After one hour, instead of rats in the normal group which received 5ml/kg of 1% CMC, other groups received 5ml/kg of HCl/Ethanol. All rats were sacrificed after one additional hour. Gastric juice, gastric mucosa, morphologies of gastric ulcers and protein expressions of both control and treatment groups were evaluated. TIBH showed a ulcer prevention potential by increase of the mucus secretion, decrease of the gastric acidity, up-regulation of HSP70 protein, down-regulation of Bax protein, decrease of the lipid peroxidation and the increase of the Superoxide dismutase (SOD) activity in gastric tissue homogenate. Acute toxicity assay exposed valuable information on the safety of this compound. TIBH had a dose dependent ulcer prevention potential against HCl/Ethanol-triggered gastric ulcer.

Keywords: Acute toxicity; Gastric ulcer; Gastro-protective activity; Immunostaining..

Figure 1

Renal and liver sections of rats in acute toxicity study (H&E staining, 20x). a) Normal rat renal tissue; b) 300 mg/kg TIBH treated rat renal tissue, c) 2000 mg/kg TIBH treated rat renal tissue; d) Normal rat hepatic tissue; e) 300 mg/kg TIBH treated rat hepatic tissue; f) 2000 mg/kg TIBH treated rat hepatic tissue; No significant differences was observed in the analysed kidney and liver tissues between TIBH pre-treated and control groups (20× magnifications).

Figure 2

Effect of TIBH on macroscopic morphology of the rat's stomach in the HCl/ethanol-induced ulcer in the experimental groups (n = 6). a) Normal control group did not display any lesions in the gastric mucus membrane; b) in ulcer control group, strong ulceration was generated in the stomach; c) Positive control group (Omeprazole, 20 mg/kg) displayed minor gastric damages; d) low dose group pre-treated with 50 mg/kg of the TIBH also displayed moderate gastric damages; e) Medium dose and f)High dose pretreated groups with respectively 100 and 200 mg/kg of the TIBH revealed decrease of the gastric damages in comparison with the ulcer control. White arrows show the example of ulceration.

Figure 3

A. Histological analysis of gastric tissues obtained from TIBH pre-treated groups and control groups using H&E staining 10x. Histopathology of gastric tissue of 3a) Normal control group; 3b) Ulcer control group; 3c) Positive control (Omeprazole); 3d, 3e, 3f) TIBH low dose, medium dose and high dose pre-treated groups, respectively. B. Histological analysis of gastric tissues obtained from TIBH pre-treated groups and control groups using PAS staining 10×. Histopathology of gastric tissue of 3a) Normal control group; 3b) Ulcer control group; 3c) Positive control (Omeprazole); 3d, 3e, 3f) TIBH low dose, medium dose and high dose pre-treated groups, respectively. Medium (3e) and high dose (3f) TIBH pre-treated groups showed increment of magenta color (white arrow) compared to ulcer control groups (3b) which indicated the increase of the secreted mucus by gastric glands due to TIBH pre-treatment.

Figure 3

A. Histological analysis of gastric tissues obtained from TIBH pre-treated groups and control groups using H&E staining 10x. Histopathology of gastric tissue of 3a) Normal control group; 3b) Ulcer control group; 3c) Positive control (Omeprazole); 3d, 3e, 3f) TIBH low dose, medium dose and high dose pre-treated groups, respectively. B. Histological analysis of gastric tissues obtained from TIBH pre-treated groups and control groups using PAS staining 10×. Histopathology of gastric tissue of 3a) Normal control group; 3b) Ulcer control group; 3c) Positive control (Omeprazole); 3d, 3e, 3f) TIBH low dose, medium dose and high dose pre-treated groups, respectively. Medium (3e) and high dose (3f) TIBH pre-treated groups showed increment of magenta color (white arrow) compared to ulcer control groups (3b) which indicated the increase of the secreted mucus by gastric glands due to TIBH pre-treatment.

Figure 4

A. Expression of HSP70 proteins in the gastric tissue obtained from TIBH pre-treated groups and control groups. 4a) Normal control group; 4b) Ulcer control group; 4c) Positive control group (Omeprazole); 4d) Low dose TIBH pre-treated group; 4e) Medium dose TIBH pre-treated group; 4f) High dose TIBH pre-treated group. Positive control, TIBH high dose and medium dose pre-treated rats show the over-expression of HSP70 protein (HSP70 stain 20x). B. Expression of Bax proteins in the gastric tissue obtained from TIBH pre-treated groups and control groups. 4a) Normal control group; 4b) Ulcer control group; 4c) Positive control group (Omeprazole); 4d) Low dose TIBH pre-treated group; 4e) Medium dose TIBH pre-treated group; 4f) High dose TIBH pre-treated group. Ulcer control group animals show Bax over-expression (brown color) while in positive control, TIBH high dose and medium dose pretreated rats, Bax expression decreased (Bax stain 20x).

Figure 4

A. Expression of HSP70 proteins in the gastric tissue obtained from TIBH pre-treated groups and control groups. 4a) Normal control group; 4b) Ulcer control group; 4c) Positive control group (Omeprazole); 4d) Low dose TIBH pre-treated group; 4e) Medium dose TIBH pre-treated group; 4f) High dose TIBH pre-treated group. Positive control, TIBH high dose and medium dose pre-treated rats show the over-expression of HSP70 protein (HSP70 stain 20x). B. Expression of Bax proteins in the gastric tissue obtained from TIBH pre-treated groups and control groups. 4a) Normal control group; 4b) Ulcer control group; 4c) Positive control group (Omeprazole); 4d) Low dose TIBH pre-treated group; 4e) Medium dose TIBH pre-treated group; 4f) High dose TIBH pre-treated group. Ulcer control group animals show Bax over-expression (brown color) while in positive control, TIBH high dose and medium dose pretreated rats, Bax expression decreased (Bax stain 20x).