Diosmin protects against ethanol-induced gastric injury in rats: novel anti-ulcer actions

Abstract

Alcohol consumption has been commonly associated with gastric mucosal lesions including gastric ulcer. Diosmin (DIO) is a natural citrus flavone with remarkable antioxidant and anti-inflammatory features that underlay its protection against cardiac, hepatic and renal injuries. However, its impact on gastric ulcer has not yet been elucidated. Thus, the current study aimed to investigate the potential protective effects of DIO against ethanol-induced gastric injury in rats. Pretreatment with DIO (100 mg/kg p.o.) attenuated the severity of ethanol gastric mucosal damage as evidenced by lowering of ulcer index (UI) scores, area of gastric lesions, histopathologic aberrations and leukocyte invasion. These actions were analogous to those exerted by the reference antiulcer sucralfate. DIO suppressed gastric inflammation by curbing of myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) levels along with nuclear factor kappa B (NF-κB) p65 expression. It also augmented the anti-inflammatory interleukin-10 (IL-10) levels. Meanwhile, DIO halted gastric oxidative stress via inhibition of lipid peroxides with concomitant enhancement of glutathione (GSH), glutathione peroxidase (GPx) and the total antioxidant capacity (TAC). With respect to gastric mucosal apoptosis, DIO suppressed caspase-3 activity and cytochrome C (Cyt C) with enhancement of the anti-apoptotic B cell lymphoma-2 (Bcl-2) in favor of cell survival. These favorable actions were associated with upregulation of the gastric cytoprotective prostaglandin E2 (PGE2) and nitric oxide (NO). Together, these findings accentuate the gastroprotective actions of DIO in ethanol gastric injury which were mediated via concerted multi-pronged actions, including suppression of gastric inflammation, oxidative stress and apoptosis besides boosting of the antioxidant and the cytoprotective defenses.

Fig 1. Diosmin lowers ulcer index (A) and area of gastric lesions (B) in rats with ethanol-induced gastric injury.

Gastric ulcer was induced by a single intragastric administration of absolute ethanol (5ml/kg) whereas the control group received the same volume of physiological saline. Diosmin was orally administered (100 mg/kg/day p.o.), starting 1 week before ulcer induction while sucralfate (100 mg/kg p.o.) was administered 1 hr before ulcer induction. One hour post ethanol instillation, rats were euthanized and their stomachs were immediately excised. Scores of the ulcer index (non-parametric) are expressed as median; n = 6–8. *Significant difference from control gp at p < 0.05, # Significant difference from ethanol gp at p < 0.05. DIO; diosmin, SUCR; sucralfate.

Fig 2. Diosmin alleviates ethanol-induced gastric histopathologic injury in rats.

Representative photomicrographs of sections from gastric wall samples taken 1 h post ethanol administration. (A) Control rats receiving saline vehicle showed normal architecture of mucosa (mu) with intact epithelial surface, submucosa (subm) and muscularis (ml) layers. (B) Control rats receiving DIO (100 mg/kg p.o.) elicited no histologic modifications. (C, D) Ethanol-treated group was characterized by mucosal lesions with marked hemorrhage (H).The mucosa was also infiltrated by inflammatory cells (arrows) that also extended to the submucosa which also displayed extensive edema (o). (E) Ethanol + DIO pretreatment (100 mg/kg p.o.) revealed attenuated morphological modifications, diminished inflammatory cell invasion (arrows) and mucosal preservation. (F) Ethanol + SUCR (100 mg/kg p.o.) pretreatment preserved the architecture of the gastric wall. Hematoxylin and eosin staining, original magnification: × 40. (G) Microscopic damage scores (expressed as median; n = 6–8).

Fig 3. Diosmin attenuates gastric MPO and TNF-α and reinstates IL-10 in rats with ethanol-induced gastric injury.

(A) Activity of myeloperoxidase; MPO. (B) Tumor necrosis factor-α; TNF-α. (C) Interleukin 10; IL-10. Measurements were performed 1 h post ethanol instillation and DIO was administered for 1 week before ulcer induction. Data are expressed as mean ± SEM (n = 6–8) *Significant difference from control gp at p < 0.05, # Significant difference from ethanol gp at p < 0.05. DIO; diosmin, SUCR; sucralfate.

Fig 4. Diosmin downregulates the protein expression of NF-κB p65 in rats with ethanol-induced gastric injury.

Representative images for the immunohistochemical detection of activated NF-κB p65 expression from gastric tissues harvested 1 hr post ethanol instillation (magnification: × 200) (A, B) Control and control + DIO gps: minimal expression; (B) Ethanol gp: extensive expression (brown color); (D, E) Ethanol+ DIO and ethanol + SUCR gps: attenuated expression. DIO; diosmin, SUCR; sucralfate.

Fig 5. Diosmin lowers gastric MDA and enhances GSH, GPx and TAC in rats with ethanol-induced gastric injury.

(A) Lipid peroxides expressed as malondialdehyde; MDA. (B) Reduced glutathione; GSH. (C) Glutathione peroxidase; GPx. (D) Total antioxidant capacity; TAC. Measurements were performed 1 h post ethanol instillation and DIO was administered for 1 week before ulcer induction. Data are expressed as mean ± SEM (n = 6–8) *Significant difference from control gp at p < 0.05, # Significant difference from ethanol gp at p < 0.05. DIO; diosmin, SUCR; sucralfate.

Fig 6. Diosmin suppresses gastric caspase-3 and Cyt C and reinstates Bcl-2 in rats with ethanol-induced gastric injury.

(A) Caspase-3 activity. (B) Cytochrome C protein; Cyt C. (C) B cell lymphoma-2 protein; Bcl-2. Measurements were performed 1 h post ethanol instillation and DIO was administered for 1 week before ulcer induction. Data are expressed as mean ± SEM (n = 6–8) *Significant difference from control gp at p < 0.05, # Significant difference from ethanol gp at p < 0.05. DIO; diosmin, SUCR; sucralfate.

Fig 7. Diosmin boosts the gastroprotective PGE₂ and NO in rats with ethanol-induced gastric injury.

(A) Prostaglandin E₂; PGE₂. (B) Nitric oxide; NO. Measurements were performed 1 h post ethanol instillation and DIO was administered for 1 week before ulcer induction. Data are expressed as mean ± SEM (n = 6–8) *Significant difference from control gp at p < 0.05, # Significant difference from ethanol gp at p < 0.05. DIO; diosmin, SUCR; sucralfate.

Fig 8. Diagram depicting the ameliorative actions of diosmin in ethanol-induced gastric injury.