Acute toxicity and gastroprotective role of M. pruriens in ethanol-induced gastric mucosal injuries in rats

Abstract

The investigation was to evaluate gastroprotective effects of ethanolic extract of M. pruriens leaves on ethanol-induced gastric mucosal injuries in rats. Forty-eight rats were divided into 8 groups: negative control, extract control, ulcer control, reference control, and four experimental groups. As a pretreatment, the negative control and the ulcer control groups were orally administered carboxymethylcellulose (CMC). The reference control was administered omeprazole orally (20 mg/kg). The ethanolic extract of M. pruriens leaves was given orally to the extract control group (500 mg/kg) and the experimental groups (62.5, 125, 250, and 500 mg/kg). After 1 h, CMC was given orally to the negative and the extract control groups. The other groups received absolute ethanol. The rats were sacrificed after 1 h. The ulcer control group exhibited significant mucosal injuries with decreased gastric wall mucus and severe damage to the gastric mucosa. The extract caused upregulation of Hsp70 protein, downregulation of Bax protein, and intense periodic acid schiff uptake of glandular portion of stomach. Gastric mucosal homogenate showed significant antioxidant properties with increase in synthesis of PGE2, while MDA was significantly decreased. The ethanolic extract of M. pruriens leaves was nontoxic (<5 g/kg) and could enhance defensive mechanisms against hemorrhagic mucosal lesions.

Figure 1

Effects of M. pruriens extract on ulcer area (mm²) and inhibition percentage (a) and alcian-blue-binding capacity (b). Ulcer area (mm²) and inhibition percentage and inhibition of the gastric lesions (%) are indicated in brackets above the columns. Alcian-blue-binding capacity is defined as gastric wall mucus (GWM). Gastric wall mucus groups 1, 2, 3, and 4 represent the negative control, the extract control, the ulcer control, and the reference groups, respectively. The experimental groups are presented as groups (groups 5–8). All values are expressed as mean ± standard error mean. All values are expressed as mean ± standard error mean. Mean difference is significant at the P < 0.05 level (one way between groups ANOVA with post hoc analysis). *Significant when compared with the group 3. ^#Significant when compared with the group 4.

Figure 2

Effects of M. pruriens extract on macroscopic appearance of the gastric mucosa. The negative control group and the extract control group show no injuries to the gastric mucosa ((a) and (b)). Severe injuries are observed in the gastric mucosa of the ulcer control group. Ethanol produced extensive visible hemorrhagic necrosis of the gastric mucosa (c). The reference control group, pretreated with omeprazole (20 mg/kg), shows mild injuries to the gastric mucosa (d). In the experimental groups, rats pretreated with 62.5 mg/kg of the extract shows moderate injuries in the gastric mucosa (e); in the pretreatment with 125 mg/kg of the extract, more moderate injuries are observed in the gastric mucosa (f); rats pretreated with 250 show mild injuries in the mucosa (g); pretreatment with 500 mg/kg of the extract shows no injuries to the gastric mucosa; instead, flattening of the gastric mucosa is observed (h).

Figure 3

Histological effects of M. pruriens on gastric tissue (H&E staining 20x). In the negative control group and the extract control group no injuries to the gastric mucosa are observed ((a) and (b)). The ulcer control group has severe disruption to the surface epithelium (black arrow); necrotic lesions penetrating deeply into the mucosa, extensive edema of the submucosal layer (yellow arrow) and leucocyte infiltration (blue arrow) (c). The reference control group shows mild disruption of the surface epithelium mucosa. There are edema and leucocyte infiltration of the submucosal layer (d). In the experimental groups, rats pretreated with 62.5 mg/kg of the extract show moderate disruption of the surface epithelium with edema and leucocytes infiltration of the submucosal layer (e); in the pretreatment with 125 mg/kg of the extract, rats showed a mild disruption of the surface epithelium with edema and leucocyte infiltration in submucosal layer (f); rats pretreated with 250 shows mild disruption of the surface epithelium with edema and leucocytes infiltration of the submucosal layer (g); pretreatment with 500 mg/kg of the extract showed mild edema and leucocytes infiltration of the submucosal layer but no disruption of the surface epithelium (h).

Figure 4

Effects of M. pruriens on gastric tissue glycoprotein (PAS staining 20x). The negative control group (a); the extract control group (b); the ulcer control group (c); the reference control group (d); the experimental groups pretreated with 62.5, 125, 250, and 500 mg/kg ((e), (f), (g), and (h) resp.). Magenta color in the apical epithelial cells in the pretreated groups with the extract ((e), (f), (g), and (h)) shows gradual increase in mucosal secretion of gastric glands. The intense secretion of mucus in gastric glands is demonstrated in the extract control group and the experimental group pretreated with 500 mg/kg of the extract ((b) and (h)).

Figure 5

Immunohistochemical expression of Hsp70 in the gastric mucosa (20x). The negative control group (a); the extract control group (b); the ulcer control group (c); the reference control group (d); the experimental groups pretreated with 62.5, 125, 250, and 500 mg/kg ((e), (f), (g), and (h) resp.). Upregulation of Hsp70 protein in rats pretreated with omeprazole (d) or M. pruriens extract ((b) and (e)–(h)) compared with the ulcer control (c).

Figure 6

Immunohistochemical expression of Bax in the gastric mucosa (20x). The negative control group (a); the extract control group (b); the ulcer control group (c); the reference control group (d); the experimental groups pretreated with 62.5, 125, 250, and 500 mg/kg ((e), (f), (g), and (h) resp.). Immunohistochemical analysis of Bax protein showed downexpression of Bax protein in rats pretreated with M. pruriens extract ((b) and (e)–(h)).