Sex-specific effects of Eugenia punicifolia extract on gastric ulcer healing in rats

Figure 1 Effects of 14-d treatment with hydroalcoholic extract from the leaves of Eugenia punicifolia and lansoprazole on gastric ulcer healing in a rat model. Results were expressed as mean ± standard error of the mean in each group (n = 8-10). Values are given as a percentage indicating healing relative to the corresponding vehicle group. Statistical significance was determined using one-way ANOVA followed by Dunnett's test or Student’s t-test. ^aP < 0.05 and ^bP < 0.01 vs vehicle in males; ^cP < 0.0001 vs vehicle in intact females (cycling); ^dP < 0.01 and ^eP < 0.0001 vs vehicle in ovariectomized females; ^fP < 0.01 for HEEP and lansoprazole in intact females vs males; ^gP < 0.01 and ^hP < 0.001 for HEEP and lansoprazole in intact vs ovariectomized females; Ns: not significant; HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 2 Effects of hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP) on the viability of L929 fibroblasts. L929 cells were treated with vehicle, DMSO, or HEEP of different concentrations and incubated for 24 h. Cell viability was measured using the MTT assay. The results represent mean ± standard error of the mean in triplicate experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett's test. ^aP < 0.001 vs vehicle.

Figure 3 Effects of hydroalcoholic extract from the leaves of Eugenia punicifolia on cell proliferation and migration into the scratch-wound area. A monolayer of L929 fibroblasts was scratched and treated with vehicle or HEEP at different concentrations (3, 10, and 30 μg/mL). A: Images captured at 100 × magnification at 0, 24, and 48 h after incubation; B: Rate of migration, evaluated in terms of the total distance that the cells moved from the edge of the scratch toward the center. The results represent mean ± standard error of the mean in triplicate experiments. Statistical significance was established using analysis of variance (two-way ANOVA) followed by Bonferroni’s test. ^aP < 0.001 vs time-matched vehicle. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 4 Prostaglandin E₂ levels in rats with acetic acid-induced gastric ulcers treated for 14 d with vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia. The results are expressed as mean ± standard error of the mean in each group (n = 6-8). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. ^aP < 0.05 and ^bP < 0.001 vs vehicle in males; Ns: not significant; HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia; PGE₂: Prostaglandin E₂.

Figure 5 Representative zymography results of gastric ulcer tissue from male rats treated with vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. A: Zymography gel showing the typical clear bands of matrix metalloproteinase-2 (MMP-2) in its proenzyme (72 kDa), intermediate (64 kDa), and active form (57 kDa); B: Gelatinolytic activity of MMP-2 in the gastric mucosa after 14 d of treatment. The results represent mean ± standard error of the mean of the integrated optical density (IOD) for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia; MMP-2: Matrix metalloproteinase-2.

Figure 6 Representative zymography results of gastric ulcer tissue from intact female rats treated with vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. A: Zymography gel showing the typical clear bands of matrix metalloproteinase-2 (MMP-2) in its proenzyme (72 kDa), intermediate (64 kDa), and active form (57 kDa); B: Gelatinolytic activity of MMP-2 in the gastric mucosa after 14 d of treatment. The results represent mean ± standard error of the mean of the integrated optical density (IOD) for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia; MMP-2: Matrix metalloproteinase-2.

Figure 7 Representative zymography results of gastric ulcer tissue from ovariectomized rats treated with vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. A: Zymography gel showing the typical clear bands of matrix metalloproteinase-2 (MMP-2) in its proenzyme (72 kDa), intermediate (64 kDa), and active form (57 kDa); B: Gelatinolytic activity of MMP-2 in the gastric mucosa after 14 d of treatment. The results represent mean ± standard error of the mean of the integrated optical density (IOD) for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia; MMP-2: Matrix metalloproteinase-2.

Figure 8 COX-1 expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP) for 14 d. The data represent relative expression of COX-1 normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. No statistically significant differences were found. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 9 COX-2 expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. The data represent relative expression of COX-2 normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. No statistically significant differences were found. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 10 Epidermal growth factor expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. Data represent relative expression of EGF normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test or Student’s t-test. ^aP < 0.01 vs vehicle in males; ^bP < 0.01 vs vehicle in ovariectomized females; ^cP < 0.05 for lansoprazole in males vs intact females; ^dP < 0.05 for lansoprazole in intact vs ovariectomized females. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 11 Vascular endothelial growth factor expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. The data represent relative expression of VEGF normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test or Student’s t-test. ^aP < 0.05 for lansoprazole and HEEP in intact vs ovariectomized females. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 12 Caspase-8 expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. The data represent relative expression of caspase-8 normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test or Student’s t-test. ^aP < 0.05 and ^bP < 0.01 for lansoprazole and HEEP in intact females vs males. ^cP < 0.01 and ^dP < 0.001 for vehicle, lansoprazole, and HEEP in intact vs ovariectomized females. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 13 Caspase-9 expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. Data represent relative expression of caspase-9 normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. No statistically significant differences were found. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 14 Caspase-3 expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia or 14 d. The data represent relative expression of caspase-3 normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. No statistically significant differences were found. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 15 Bcl-2 expression in the gastric mucosa of rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. The data represent relative expression of Bcl-2 normalized to the expression of housekeeping genes (actin). The results represent mean ± standard error of the mean for each group (n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett's test or Student’s t-test. ^aP < 0.05 for lansoprazole in intact vs ovariectomized females. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.

Figure 16 Body weight evolution in rats with acetic acid-induced gastric ulcers treated with oral administration of vehicle, lansoprazole, or hydroalcoholic extract from the leaves of Eugenia punicifolia for 14 d. A: Male rats; B: Intact female rats; C: Ovariectomized female rats. Results are expressed as the means of body weight (g) in each group (n = 8-10). Statistical significance was determined using one-way ANOVA followed by Dunnett's test. No statistically significant differences were found. HEEP: Hydroalcoholic extract from the leaves of Eugenia punicifolia.