Vitamin D3 eradicates Helicobacter pylori by inducing VDR-CAMP signaling

Abstract

Background: Vitamin D₃ [VitD₃, 1,25 (OH)₂D₃] is known to have immunomodulatory and anti-microbial properties; however, its activity against Helicobacter pylori is unclear. In this study, we established H. pylori infection models in wild-type and VitD₃ receptor (VDR) knockdown mice and analyzed the effects of VitD₃ and their underlying mechanisms.

Methods: VDR^+/+ and VDR^+/- mice were intragastrically infected with the H. pylori SS1 strain. After confirmation of H. pylori infection, mice were treated with different doses of VitD₃. The infection levels in stomach tissues were quantified using the colony-forming assay, and the expression levels of the VDR and cathelicidin antimicrobial peptide (CAMP) in the gastric mucosa were analyzed by immunohistochemistry and western blotting.

Results: The gastric mucosa of VDR^+/- mice was more susceptible to H. pylori colonization and had lower levels of VDR and CAMP expression than that of VDR^+/+ mice. H. pylori infection upregulated VDR and CAMP expression in the stomach of both wild-type and mutant mice, and VitD₃ treatment resulted in further increase of VDR and CAMP levels, while significantly and dose-dependently decreasing the H. pylori colonization rate in both mouse groups, without affecting blood calcium or phosphorus levels.

Conclusion: Our data indicate that oral administration of VitD₃ reduces the H. pylori colonization rate and upregulates VDR and CAMP expression in the gastric mucosa, suggesting a role for VitD₃/VDR/CAMP signaling in the eradication of H. pylori in the stomach. These findings provide important insights into the mechanism underlying the anti-H. pylori activity of VitD₃ and should be useful in the development of measures to eradicate H. pylori.

Keywords: 1α; 25-dihydroxyvitamin D3; H. pylori; cathelicidin antimicrobial peptide; inflammation; vitamin D receptor.

FIGURE 1

Mouse strains. VDR^+/– C57BL/6J mice (upper left and lower right) and VDR^+/+ C57BL/6J mice (lower left and upper right).

FIGURE 2

Cultivation of the Helicobacter pylori Sydney strain 1 (SS1) strain in vitro. (A) Typical white needle tip colonies could be seen on the plates after 72 h of incubation. (B) Bacterial flagella were observed after alkalescent carbolfuchsin staining. Scale bar = 10 μm.

FIGURE 3

Helicobacter pylori successfully colonized mouse stomach tissue. (A) Rapid urease reaction showed positive results in infected mice and negative results in control mice. (B) Several black rod-shaped structures were observed after Warthin-Starry silver staining. Scale bar = 10 μm. (C,D) Hematoxylin-eosin staining of the gastric mucosa of uninfected (C) and infected (D) mice. Chronic gastric mucosal inflammation could be observed in the infected group. Scale bar = 20 μm.

FIGURE 4

Helicobacter pylori eradication efficacy of VitD₃ at different doses in infected mice. (A) Colony-forming assay results showing increased susceptibility of VDR^+/– mice to H. pylori colonization compared to VDR^+/+ mice (P < 0.05). In both mouse strains, H. pylori colonization was significantly reduced after VitD₃ administration and H. pylori numbers decreased in a dose-dependent manner (n = 6; ^#P < 0.05 vs. HP group and ^aP < 0.05 vs. VDR^+/+ group). (B) Serum calcium and phosphorus levels were comparable in VDR^+/+ and VDR^+/– mice and remained in the normal range after VitD₃ administration (n = 6; P > 0.05). HP, H. pylori infection.

FIGURE 5

VitD₃ increased VitD₃ receptor (VDR) protein expression in mouse gastric tissues. (A) Western blotting analysis revealed a significant, dose-dependent increase of VDR expression by VitD₃ in mouse gastric tissues. VDR protein expression was consistently lower in VDR^+/– mice than in VDR^+/+ mice after VitD₃ treatment (n = 6; P < 0.05). (B) Histogram showing quantitative analysis of the results presented in panel (A). (C) Immunohistochemistry analysis of VDR expression in gastric tissues of infected mice treated or not with different doses of VitD₃. *P < 0.05 vs. HP group and ^aP < 0.05 vs. VDR^+/+ group. HP, Helicobacter pylori infection. Upper row, scale bar = 20 μm. Bottom row, scale bar = 50 μm.

FIGURE 6

VitD₃ upregulated cathelicidin antimicrobial peptide (CAMP) expression in the mouse gastric mucosa. (A) CAMP expression in the gastric mucosa was analyzed by western blotting. VitD₃ gavage increased CAMP protein expression in a dose-dependent manner. The CAMP expression level was consistently lower in VDR^+/– than in VDR^+/+ mice (n = 6; P < 0.05). (B) Histogram showing quantitative analysis of the results presented in panel (A). (C) Immunohistochemistry analysis showing VitD₃ dose-dependent increase of CAMP staining intensity in the mouse mucosa. *P < 0.05 vs. HP group and ^aP < 0.05 vs. VDR^+/+ group. HP, Helicobacter pylori infection. Scale bar = 50 μm.