In Vivo Evidence for the Preventive Role of Vaccinium macrocarpon Aiton in Indomethacin-Induced Gastric Ulcer: Focusing on Antioxidant, Anti-Inflammatory and Anti-Apoptotic Mechanisms

Abstract

The present study aimed to unveil the gastroprotective potential of Vaccinium macrocarpon (VM) extract and its mechanism of action against indomethacin (INDO)-induced gastric ulcers in rats. To achieve this goal, rats were pretreated with either omeprazole (20 mg/kg) or VM (100 mg/kg) orally for 14 consecutive days. Gastric tissue samples were collected and various parameters were evaluated to understand the mechanism of VM's action, including the levels of superoxide dismutase, malondialdehyde, glutathione, CAT and transforming growth factor beta (TGF-β), as well as the mRNA expression levels of tumour necrosis factor alpha, interleukin 1 beta, nuclear factor kappa B (NF-κB) and inhibitor kappa B (IκB). Additionally, the immunopositivity of cyclooxygenase (COX)-1, COX-2, PGE2, proliferating cell nuclear antigen (PCNA) and caspase-3 was assessed. The total amount of phenolic compounds present in the VM extract was high (58.08 µg/mL gallic acid equivalent/mg extract). The healing effect of VM was demonstrated by an increase in the expression of PCNA. Furthermore, the level of TGF-β was found to increase upon treatment with VM. Analyses of COX-1, COX-2 and PGE2 expression in gastric tissue confirmed the gastroprotective effect of VM. Notably, the expression of NF-κB was markedly reduced, whereas that of IκB was substantially increased. Overall, the findings of this study demonstrate that VM extract has gastroprotective and curative effects against INDO-induced ulcers through its antioxidant, anti-inflammatory, mucosal regenerative and anti-apoptotic activities. Therefore, VM may serve as a useful adjuvant treatment for nonsteroidal anti-inflammatory drugs-induced gastric ulcer disease.

Keywords: cyclooxygenase (COX); nuclear factor kappa B (NF‐κB)/inhibitor kappa B (IκB); oxidative stress; ulcer; vaccinium macrocarpon.

FIGURE 1

Effects of VM on lipid peroxidation and endogenous antioxidant enzymes in gastric tissues. Data are expressed as the means ± SDs ^* p < 0.001 versus control group, ^# p < 0.001 versus INDO group. GSH, glutathione; INDO, indomethacin; MDA, malondialdehyde; OME, omeprazole; SDs, standard deviations; SOD, superoxide dismutase; VM, Vaccinium macrocarpon.

FIGURE 2

Effects of VM on TGF‐β levels in each group detected with ELISA. Relative mRNA expression levels of IL‐1β and TNF‐α in the gastric tissues of the control and experimental groups. Data are expressed as the means ± SDs ^* p < 0.001 versus control group, ^# p < 0.001 versus INDO group. IL‐1β, interleukin 1 beta; INDO, indomethacin; OME, omeprazole; SDs, standard deviations; TGF‐β, transforming growth factor beta; TNF‐𝛼, tumour necrosis factor alpha; VM, Vaccinium macrocarpon.

FIGURE 3

Effects of VM on the relative mRNA expression levels of NF‐κB and IκB in the gastric tissues of the control and experimental groups. Data are expressed as the means ± SDs ^* p < 0.001 versus control group, ^# p < 0.001 versus INDO group. INDO, indomethacin; IκB, inhibitor kappa B; NF‐κB, nuclear factor kappa B; OME, omeprazole; SDs, standard deviations; VM, Vaccinium macrocarpon.

FIGURE 4

Histopathological analysis of gastric sections from the control and experimental groups (haematoxylin and eosin [H&E], ×10): (A) control; (B) INDO; (C) OME; (D) VM. Arrowheads: leukocyte infiltration. Arrows: necrosis. Big arrow: haemorrhage. Asterix: oedema. Blue arrow: ulceration. (E–G): Histopathological scores. Data are expressed as the means ± SDs; ^a–c: Differences between the groups (p < 0.05). Scale bar = 50 µm. INDO, indomethacin; OME, omeprazole; SDs, standard deviations; VM, Vaccinium macrocarpon.

FIGURE 5

Immunohistochemical analysis of COX‐1 in the control and experimental groups (×40): (A) control group; (B) INDO group; (C) OME group; (D) VM group; (E) immunohistochemistry scoring for COX‐1. Data are expressed as the means ± SDs; ^a–c: **Differences between the groups (p < 0.05). Scale bar = 50 µm. COX‐1, cyclooxygenase‐1; INDO, indomethacin; OME, omeprazole; SDs, standard deviations; VM, Vaccinium macrocarpon.

FIGURE 6

Immunohistochemical analysis of COX‐2 in the control and experimental groups (×40): (A) control group; (B) INDO group; (C) OME group; (D) VM group; (E) immunohistochemistry scoring for COX‐2. Data are expressed as the means ± SDs; ^a–c: Differences between the groups (p < 0.05). Scale bar = 50 µm. COX‐2, cyclooxygenase‐2; INDO, indomethacin; OME, omeprazole; VM, Vaccinium macrocarpon.

FIGURE 7

Immunohistochemical analysis of PGE₂ in the control and experimental groups (×40): (A) control group; (B) INDO group; (C) OME group; (D) VM group; (E) immunohistochemistry scoring for PGE₂. Data are expressed as the means ± SDs; ^a–c: Differences between the groups (p < 0.05). Scale bar = 50 µm. INDO, indomethacin; OME, omeprazole; SDs, standard deviations; VM, Vaccinium macrocarpon.

FIGURE 8

Immunohistochemical analysis of caspase‐3 in the control and experimental groups (×40): (A) control group; (B) INDO group; (C) OME group; (D) VM group; (E) immunohistochemistry scoring for caspase‐3. Data are expressed as the means ± SDs; ^a–c: Differences between the groups (p < 0.05). Scale bar = 50 µm. INDO, indomethacin; OME, omeprazole; SDs, standard deviations; VM, Vaccinium macrocarpon.

FIGURE 9

Immunohistochemical analysis of PCNA in the control and experimental groups (×40): (A) control group; (B) INDO group; (C) OME group; (D) VM group; (E) immunohistochemistry scoring for PCNA. Data are expressed as the means ± SDs; ^a–d: Differences between the groups (p < 0.05). Scale bar = 50 µm. INDO, indomethacin; OME, omeprazole; PCNA, proliferating cell nuclear antigen; SDs, standard deviations; VM, Vaccinium macrocarpon.