Helicobacter pylori (H. pylori) infection is a major cause of chronic gastritis. This study aims to determine whether H. pylori infection induces the expression of G protein-coupled receptor class C group 5 member C (GPRC5C) in gastric epithelial cells, to verify the regulatory role of the upstream NF-κB signaling pathway, and to elucidate the biological function of GPRC5C in H. pylori-associated gastritis, thereby providing a potential new therapeutic target for the prevention and treatment of this disease.

A total of 190 SPF-grade female C57BL/6J mice (6 to 8 weeks old, weighing 18 to 22 g) were randomly divided into an uninfected control group (gavaged with PBS) and an H. pylori-infected group (gavaged with H. pylori), with 95 mice per group. The mice were gavaged 5 times consecutively, twice a day. To establish a chronic in vivo infection model, 5 mice from each group were randomly euthanized weekly from week 1 to week 19 post-infection, for 19 weeks. The expression of GPRC5C in mouse gastric mucosal tissues was examined by quantitative real-time PCR, Western blotting, and immunohistochemical staining. Human gastric tissue specimens were obtained from normal gastric tissues at least 5 cm from the tumor margin in gastric cancer patients hospitalized in Department of General Surgery of First Affiliated Hospital of Army Medical University. Immunohistochemical staining was employed to evaluate the protein expression of GPRC5C in human gastric tissues. Mouse primary gastric epithelial cells were isolated from gastric tissues of uninfected mice by enzymatic digestion and mechanical dissociation, followed by filtration to prepare single-cell suspensions, which were then purified by CD326 based magnetic-activated cell sorting (MACS) system. An H. pylori infection model was established in both the GES-1 cell line and mouse primary gastric epithelial cells. The expression of GPRC5C in GES-1 cells and mouse primary gastric epithelial cells was examined by qRT-PCR and Western blotting. Signaling pathway blocking experiments were performed to screen and verify the intracellular signaling pathway activated during H. pylori-induced GPRC5C expression in gastric epithelial cells. Dual-luciferase reporter gene assay was used to analyze the GPRC5C promoter activity of the full-length and truncated sequences of GPRC5C promoter region after H. pylori infection, and to determine the effect of signaling pathway inhibitors on GPRC5C promoter activity. Furthermore, another female C57BL/6J WT mice and Gprc5c^-/- mice (n=5, 6 to 8 weeks old, weighing 18 to 22 g) were used to establish the H. pylori infection model. Gastric tissue inflammation was observed after HE staining to preliminarily investigate the effector function of GPRC5C in H. pylori infection-associated gastritis.

Compared with the uninfected group, the mRNA levels of GPRC5C in mouse gastric mucosal tissues were significantly upregulated at week 14 post-H. pylori infection (P<0.0001), and the protein levels were also elevated in both mouse and human gastric mucosal tissues. In vitro experiments showed that, compared with the corresponding uninfected group, H. pylori infection upregulated the expression of GPRC5C at both mRNA and protein levels in GES-1 cells and mouse primary gastric epithelial cells, with mRNA expression levels increased 4.1-fold and 3.1-fold, respectively (P<0.01). This upregulation was dependent on the bacterial dose and infection duration (P<0.05). In terms of the underlying mechanism, pretreatment with an NF-κB signaling pathway inhibitor significantly suppressed H. pylori-induced GPRC5C upregulation in gastric epithelial cells, with an inhibitory rate of 64.0% (P<0.01). Dual-luciferase reporter assay revealed that H. pylori infection significantly increased GPRC5C promoter activity to 4.1 times of that in the uninfected group (P<0.001). When the NF-κB signaling pathway was blocked, this promoter activity was significantly suppressed, with an inhibitory rate of 66.1% (P=0.0002); In functional validation, compared with H. pylori-infected WT mice, Gprc5c^-/- mice exhibited significantly reduced gastric inflammation after infection (P<0.001).

H. pylori infection induces GPRC5C expression in gastric epithelial cells through activation of the NF-κB signaling pathway, thereby promoting the progression of H. pylori-associated gastritis.

To explore the regulatory and functional mechanism of angiopoietin-like 4 (ANGPTL4) expression in gastric epithelial cells infected with Helicobacter pylori (H.pylori).

H.pylori-positive gastric specimens from Department of Gastroenterology of second Affiliated Hospital of Army Medical University in November 2021 and the gastric tissues of H.pylori-infected mice were collected, and the expression locations of ANGPTL4 in these gastric tissues were detected by immunofluorescence assay. After gastric epithelial cells and gastric organoids were infected with H.pylori, the expression of ANGPTL4 was detected by real-time PCR and Western blotting. The regulatory mechanism of H.pylori-induced ANGPTL4 expression was investigated by cellular models above. Recombinant ANGPTL4 was used to stimulate gastric epithelial cells to investigate the effect of ANGPTL4 on the claudin 1 (CLDN1) expression.

H.pylori infection significantly increased the expression of ANGPTL4 in gastric epithelial cells in a CagA-dependent, bacterial concentration-dependent and infection time-dependent manner (P<0.05). Compared to uninfected cells and CagA knockout (△cagA) H.pylori-induced ones, the ANGPTL4 expression was significantly increased in WT H.pylori-infected human and mouse gastric organoid (P<0.05). Blocking NF-κB signaling pathway significantly inhibited the enhanced ANGPTL4 expression in H.pylori-infected gastric epithelial cells (P<0.05). Recombinant ANGPTL4 activated ERK signaling pathway to down-regulate the CLDN1 expression in gastric epithelial cells (P<0.05).

H.pylori activates NF-κB signaling pathway to up-regulate ANGPTL4 expression in gastric epithelial cells in a CagA-dependent manner, and ANGPTL4 activates ERK signaling pathway to down-regulate the CLDN1 expression in gastric epithelial cells, which may contributes to the development of gastric diseases induced by H.pylori infection.

Regulated in development and DNA damage responses-1 (REDD1) is a conserved and ubiquitous protein, and is induced in response to multiple stimuli. However, its regulation, function and clinical relevance in Helicobacter pylori (H. pylori)-associated gastritis are presently unknown. The study aimed to investigate its expression and regulation mechanism in H. pylori infection.

C57 mice and gastric epithelial cells (AGS cells) were respectively infected with H. pylori to establish infected models. Then the expression of REDD1 at mRNA and protein levels was detected by quantitative PCR, immunohistochemical staining and Western blotting. The mechanism of REDD1 up-regulation induced by H. pylori infection was investigated by transfection with signaling pathway inhibitor, plasmid or dual-luciferase reporting analysis in cell models.

The expression of REDD1 was increased in gastric mucosa of the H. pylori-infected mice than the un-infected ones. Compared with the wild-type strain of H. pylori, the strain with cagA knockout (△cagA H. pylori 11637 cells) showed decreased effect on the upregulation of REDD1 (P<0.05). H. pylori infection induced up-regulation of REDD1 in AGS cells in a time-, cagA- and pathogen dose-dependent manner (P<0.05). Blocking of P38/MAPKp38 signaling pathway could significantly inhibit the upregulation of REDD1 induced by the infection (P<0.05).

H. pylori induces the up-regulation of REDD1 through activation of phosphorylated cagA in MAPKp38 pathway in the gastric mucosa of mouse with H. pylori infection.