No Correlation of Inflammation With Colonization of Helicobacter pylori in the Stomach of Mice Fed High-salt Diet

Abstract

Background: Previous studies on Helicobacter pylori infection in mice have contributed to better understanding of the pathogenesis of chronic gastritis and gastric carcinoma. The aim of this study was to evaluate H. pylori colonization and subsequent inflammatory responses in the stomachs of C57BL/6 mice depending on inoculation number and the presence of high-salt diet.

Methods: Eighty-four female mice with 4 weeks age were used in this study. The infected mice were gavaged with H. pylori strain Sydney-1 (SS1), and the uninfected mice were dosed with vehicle. In each of these groups, half of the mice were fed ona basal diet (0.25% salt) and the other half were fed on a high-salt diet (7.5% salt). The infected mice were inoculated 4 or 5 times, and infection status and degree of inflammation were checked by culture and histopathology, respectively, after 4 weeks. Gastric mucosal myeloperoxidase and tumor necrosis factor-alpha were measured by ELISA.

Results: The overall infection rate was 95.2%; the infection rate after 5 inoculations (100%) was greater than that after 4 inoculations (91.3%). However, no differences in the degree of inflammation were found between 2 groups. The bacterial density was also significantly increased in mice that were on the high-salt diet and had been inoculated 5 times, respectively. Mean neutrophil infiltration in the infected group was 1.7±0.6 (1, minimal; 2, mild; 3, moderate; 4, marked). However, the high-salt diet was not increase the inflammatory grade in the infected group. Gastric mucosal myeloperoxidase and tumor necrosis factor-alpha levels did not increased by the high-salt diet and increased the number of inoculation.

Conclusions: In spite of well colonization of H. pylori in the stomachs of C57BL/6 mice, the degree of subsequent inflammation was irrelevant to high-salt diet and frequent (5 times) inoculations.

Keywords: Helicobacter pylori; Inflammation; Salt.

Figure 1.

Schematic representation of the experimental design.

Figure 2.

Helicobacter pylori (H. pylori) colonization of the gastric mucosa as determined by quantitative culture in mice on a basal or high-salt diet (A). The histopathological H. pylori density score on a basal or high-salt diet (B). Culture and infection rate of H. pylori according to the number of inoculations (C). BD, basal diet; HSD, high-salt diet. *P<0.05 vs. basal diet.

Figure 3.

Helicobacter pylori colonization on gastric mucosa in C57BL/6 mice (H&E stain); ×200 (A), ×1000 (B). The spiral bacteria were observed in gastric pits at week 4 after inoculation (arrow).

Figure 4.

Gastric histopathology scores according to diet and Helicobacter pylori (H. pylori) infection status regardless of inoculation times. The degree of neutrophil infiltration (A), mononuclear cell infiltration (B) (y-axis) was assessed according to the updated Sydney classification as follows: 0, absent; 1, minimal; 2, mild; 3, moderate; 4, marked. Mice were divided into 4 groups according to the diet and H. pylori infection status (x-axis). *P<0.05 vs. uninfected group.

Figure 5.

Gastric histopathology scores according to the number of inoculations. The degree of neutrophil infiltration (A), mononuclear cell infiltration (B) (y-axis) was assessed according to the updated Sydney classification as follows: 0, absent; 1, minimal; 2, mild; 3, moderate; 4, marked. BD, basal diet; HSD, high-salt diet; 4 times, 4 times inoculation; 5 times, 5 times inoculation.

Figure 6.

Normal gastric mucosa (A) and mucous metaplasia and lymphoid aggregation in the gastric mucosa of Helicobacter pylori (H. pylori) infected C57BL/6 mice on a basal diet (B) and a high-salt diet (C, D) 4 weeks after inoculation (H&E stain); ×100 (A–C), ×200 (D). The glandular epithelium has been replaced by a hyperplastic, hypertrophic mucous epithelium (arrow and black square).

Figure 7.

Gastric mucosal myeloperoxidase (MPO, ng/mL) (A) and tumor necrosis factor alpha (TNFα, pg/mL) (B) levels according to the diet, Helicobacter pylori (H. pylori) infection status and the number of inoculations. *P<0.05 vs. uninfected group.