1α, 25-Dihydroxyvitamin D3 protects gastric mucosa epithelial cells against Helicobacter pylori-infected apoptosis through a vitamin D receptor-dependent c-Raf/MEK/ERK pathway

Abstract

Context: Due to the resistance of Helicobacter pylori to antibiotics, it is difficult to eradicate this pathogenic bacterium from the host. The role of 1α, 25-dihydroxyvitamin D3 (1,25-D3) in H. pylori-infected gastric mucosa epithelial cells remains unknown.

Objective: This study investigates the protective property of 1,25-D3 against H. pylori-infected apoptosis in gastric mucosa epithelial cells and its potential molecular mechanisms.

Materials and methods: GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with 1,25-D3 at 100, 200, and 300 nM for 24 h. Mice were orally gavaged with 10⁸ CFUs of H. pylori and 25 µg/kg 1,25-D3 every other day for 1 month. CCK-8, LDH assay, TUNEL assay and western blot were used to determine the effect of 1,25-D3 on H. pylori-induced apoptosis.

Results: H. pylori infection decreased cell viability to 59.2%, while 100-300 nM 1,25-D3 increased cell viability to 62.2%, 78.4% and 87.1%, respectively. Compared with positive control (4.53-fold), 1,25-D3 reduced caspase-3 activity to 4.49-, 2.88- and 1.49-fold, reduced caspase-6 activity to 2.36-, 1.88- and 1.50-fold, reduced caspase-9 activity to 4.55-, 2.91- and 2.01-fold. 1,25-D3 alters Bcl-2 family, caspase protein expression and c-Raf/MEK/ERK phosphorylation levels in vivo and in vitro. Suppression of 1,25-D3 in apoptosis was reliant on binding to vitamin D receptor. The pharmacological inhibition of c-Raf/MEK/ERK phosphorylation blocked the anti-apoptotic effect of 1,25-D3.

Discussion and conclusion: 1,25-D3 protected gastric mucosa epithelial cells against H. pylori-infected apoptosis through a VDR-dependent c-Raf/MEK/ERK pathway.

Keywords: Bcl-2 family; Cell proliferation; caspase.

Figure 1.

1,25-D3 promotes cell proliferation in H. pylori-infected GES-1 cells. (A) GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with differernt concetrations of 1,25-D3 for 24 h, the cell viability was determined by CCK-8 assay. (B) GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, LDH releasing was determined by a commercially available LDH assay kit. Bars represent means ± S.E.M of three independent experiments. ***p < 0.001 vs. H. pylori alone treatment.

Figure 2.

1,25-D3 inhibits H. pylori-induced cell apoptosis in GES-1 cells. (A) GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, the levels of apoptosis were analysed using an TUNEL detection kit. (B) GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, caspase-3, caspase-6 and caspase-9 activities were determined by commercially available kits. (C) GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, caspase-3, caspase-6 and caspase-9 expression were determined by western blot. Bars represent means ± S.E.M of three independent experiments. *p < 0.01 vs. H. pylori treatment.

Figure 3.

Bcl-2 families are involved in the anti-apoptotic effect of 1,25-D3 in H. pylori-treated GES-1cells. (A) GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, Bcl-2, Bcl-xL, Bax and Bak levels were determined by western blot. (B) GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, Cytochrome C (Cyto C) and apoptosis inducing factor (AIF) levels were determined by western blot. Bars represent means ± S.E.M of three independent experiments. *p < 0.01, **p < 0.05, ***p < 0.001 vs. H. pylori alone treatment.

Figure 4.

1,25-D3 promotes c-Raf/MEK/ERK phosphorylation in H. pylori-treated GES-1 cells. GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, (A) c-Raf, MEK and ERK phosphorylation levels and (B) RXRα and VDR levels were determined by western blot. Bars represent means ± S.E.M of three independent experiments. **p < 0.01, ***p < 0.001 vs. H. pylori alone treatment.

Figure 5.

1,25-D3 exerts an anti-apoptotic effect in H. pylori-treated GES-1 cells through binding to VDR. GES-1 cells were transfected with siRNA-VDR for 24 h, and then infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, (A) the levels of VDR expression was determined by western blot, (B) the cell viability was determined by CCK-8 assay, (C) LDH release was determined by a commercial kit and (D) the levels of apoptosis were analysed using an TUNEL detection kit. Bars represent means ± S.E.M of three independent experiments. ***p < 0.001 vs. H. pylori treatment + siRNA-NC.

Figure 6.

The inhibition of c-Raf/MEK/ERK phosphorylation blocks the anti-apoptotic effect of 1,25-D3 in H. pylori-treated GES-1 cells. GES-1 cells were treated with MEK inhibitor (U0126) for 24 h, and then infected with H. pylori SS1 strain (MOI: 100) and treated with different concentrations of 1,25-D3 for 24 h, (A) the cell viability was determined by CCK-8 assay, (B) LDH release was determined by a commercial kit and (C) the levels of apoptosis were analysed using an TUNEL detection kit. Bars represent means ± S.E.M of three independent experiments. ***p < 0.001 vs. H. pylori alone treatment, ###p < 0.001 vs. H. pylori + 1,25-D3 treatment.

Figure 7.

1,25-D3 protects against H. pylori-infected apoptosis through a vitamin D receptor-dependent c-Raf/MEK/ERK pathway in mice. Mice were orally gavaged with 10⁸ CFUs of H. pylori and 25 µg/kg 1,25-D3 every other day for 1 month. Bcl-xL, Bak, c-Raf, MEK and ERK phosphorylation levels in the stomach of mice were determined by western blot. Bars represent means ± S.E.M of three random mice. **p < 0.01 vs. H. pylori alone treatment.