Resveratrol Protects against Restraint Stress Effects on Stomach and Spleen in Adult Male Mice

Abstract

The objectives were to investigate whether restraint stress (which is known as a mixture of psychologic and physical stress) exerts negative effects on the stomach and spleen, and whether the phenolic compound resveratrol (RES) exerts any protective roles. Fifty adult male mice were divided into five groups, with 10 mice per group as follows: control (C), restraint stress (RS), RS with vehicle (RS + V), RS with 2 mg/kg of resveratrol (RS + 2 mg RES), and RS with 20 mg/kg of resveratrol (RS + 20 mg RES). Mice were restrained in conical centrifuge tubes for 4 h daily to establish the RS model. RS + 2 mg RES, RS + 20 mg RES, and RS + V groups were given an oral dose of resveratrol or vehicle for 15 consecutive days, while the control group was not exposed to restraint stress. Herein, we showed that restraint stress decreased body weight and food and water consumption in stressed groups RS and RS + V compared to controls, while the groups treated with resveratrol showed improvements. Moreover, restraint stress caused acute damage to the morphology of gastric cells and reduced the quantitative distribution of parietal cells along with their decreased size and diameter, pointing to gastritis or ulcer. Furthermore, the antibody against the apoptosis-inducing factor (AIF) was highly attached in the RS groups. Splenic size, weight, and length were also greatly augmented in the stressed groups compared to the controls, while these phenomena were not observed in the RS + 2 mg RES group. Our findings proved significant ameliorating effects of resveratrol against restraint stress in adult male mice.

Keywords: DAB intensity; apoptosis-inducing factor; parietal cells; restraint stress; resveratrol; splenomegaly.

Figure 1

Effects of resveratrol on the body weights of non-stressed mice (C), restraint-stressed mice (RS), restraint-stressed mice treated with vehicle (RS + V), restraint-stressed mice treated with 2 mg/kg body weight of resveratrol (RS + 2 mg RES), and restraint-stressed mice treated with 20 mg/kg body weight of resveratrol (RS + 20 mg RES). Statistical differences were determined by 1-way ANOVA followed by Tukey’s multiple-comparison test. Different superscript letters represent significant differences among groups (p < 0.05).

Figure 2

Effects of resveratrol on food consumption index of non-stressed mice (C), restraint-stressed mice (RS), restraint-stressed mice treated with vehicle (RS + V), restraint-stressed mice treated with 2 mg/kg body weight of resveratrol (RS + 2 mg RES), and restraint-stressed mice treated with 20 mg/kg body weight of resveratrol (RS + 20 mg RES). The columns represent the average food consumption index during the 15-day treatment. Food consumption index (g/g) = total food consumed per day/total body weight.

Figure 3

Effects of resveratrol on water consumption index of non-stressed mice (C), restraint-stressed mice (RS), restraint-stressed mice with vehicle (RS + V), restraint-stressed mice treated with 2 mg/kg body weight of resveratrol (RS + 2 mg RES), and restraint-stressed mice treated with 20 mg/kg body weight of resveratrol (RS + 20 mg RES). The columns represent the average water consumption index during the 15-day treatment. Water consumption index (mL/g) = total water consumed per day/total body weight.

Figure 4

Histopathologic analysis of parietal cells of non-stressed mice (Control), restraint-stressed mice (RS), restraint-stressed mice with vehicle (RS + V), restraint-stressed mice treated with 2 mg/kg body weight of resveratrol (RS + 2 mg RES), and restraint-stressed mice treated with 20 mg/kg body weight of resveratrol (RS + 20 mg RES) using H&E staining. Red arrowheads point to an acute loss of parietal cells. Black arrowheads point to severe damage to the general structure of the gastric mucosa. Scale bar is 100 µm.

Figure 5

Effects of resveratrol on the parietal cell diameter of non-stressed mice (Control), restraint-stressed mice (RS), restraint-stressed mice with vehicle (RS + V), restraint-stressed mice treated with 2 mg/kg body weight of resveratrol (RS + 2 mg RES), and restraint-stressed mice treated with 20 mg/kg body weight of resveratrol (RS + 20 mg RES). Statistical differences were determined by 1-way ANOVA followed by Tukey’s multiple-comparison test. Different superscript letters represent significant differences among groups (p < 0.05).

Figure 6

Immunohistochemical observations of apoptosis-inducing factor (AIF) in parietal cells of non-stressed mice (Control), restraint-stressed mice (RS), restraint-stressed mice with vehicle (RS + V), restraint-stressed mice treated with 2 mg/kg body weight of resveratrol (RS + 2 mg RE), restraint-stressed mice treated with 20 mg/kg body weight of resveratrol (RS + 20 mg RES), and the negative control (NC). Red arrows point to the nucleus and black arrows point to the cytoplasm. Scale bar is 100 µm (A–F) and 50 µm (a–f).

Figure 7

Representative digital images of the histogram profile showing 3, 3′-diaminobenzidine (DAB) brown staining and color pixel intensity for AIF in the parietal cells of the mouse stomach. The histogram profile corresponds to the pixel intensity value vs. the corresponding number of counts of pixel intensity. From top to bottom rows, panels show the digital image masks stained with hematoxylin, DAB, and threshold, respectively. The threshold function of ImageJ was used to place red spots on the DAB stains by setting different threshold levels, with the lower threshold at 0 and upper threshold at 110. (A) AIF is expressed in a limited quantity at the tight junction in parietal cells. Each bar in the given graph (B) represents the mean ± SEM (n = 12). Statistical differences were determined by 1-way ANOVA followed by Tukey’s multiple-comparison test. Different superscript letters represent significant differences among groups (p < 0.05).

Figure 8

Resveratrol and restraint stress effects on spleen size (A), length (B), and weight (C) of non-stressed mice (Control), restraint-stressed mice (RS), restraint-stressed mice with vehicle (RS + V), and restraint-stressed mice treated with 2 mg/kg body weight resveratrol (RS + 2 mg RES), and restraint-stressed mice treated with 20 mg/kg body weight of resveratrol (RS + 20 mg RES). Each bar in the given graphs represents the mean ± SEM (n = 10). Statistical differences were determined by 1-way ANOVA followed by Tukey’s multiple comparison test. Different superscript letters represent significant differences among groups (p < 0.05).

Figure 9

Histopathologic analysis of H&E staining of the splenic structures of non-stressed control mice (C), restraint-stressed mice (RS), and restraint-stressed mice treated with 2 mg/kg body weight of resveratrol (RS + 2 mg RES). Scale bar is 100 µm (A,D,G), 50 µm (B,E,H), and 20 µm (C,F,I). (A,D,G) Black arrows point to the white pulp. (A,D,G) Red arrows point to the red pulp. There is disruption of the normal splenic architecture by a diffuse infiltrate predominantly expanding the white pulp (D, green arrows), loss of typical structure of the germinal center (G,C) (D, orange arrows), proliferation of large atypical lymphoid cells with bizarre nuclei (E and F, brown arrows), and prominent eosinophilic nucleoli surrounded by abundant cytoplasm (E and F, green arrows). The red pulp was also enlarged and increased (D, red arrows), containing extramedullary hematopoiesis and prominent megakaryocytes (D, yellow arrows) in the RS group relative to the controls (A–C) and RS + 2 mg RES (G–I) groups, which show normal histology in the white and red pulp of the spleen.