Targeting Host Sulphonyl Urea Receptor 2 Can Reduce Severity of Helicobacter pylori Associated Gastritis

Abstract

Background and aims: While most Helicobacter pylori-infected individuals remain asymptomatic throughout their lifetime, in a significant proportion, the resulting severe chronic gastritis drives the development of gastric cancer. In this study, we examine a new therapeutic target, a host potassium channel regulatory subunit, SUR2 (encoded by ABCC9), with potential to protect against H pylori-associated diseases.

Methods: SUR2 gene (ABCC9) expression in human gastric biopsies was analyzed by quantitative polymerase chain reactions. Helicobacter-infected mice were administered the SUR2-channel agonists, pinacidil and nicorandil, then gastric tissues analyzed by histology, immunohistochemistry and quantitative polymerase chain reaction, and splenic tissues by enzyme-linked immunosorbent assays. In vitro studies were performed on human and mouse macrophages, human gastric epithelial cells and mouse splenocytes.

Results: ABCC9 expression in human and mouse stomachs is downregulated with H pylori infection. Treatment of Helicobacter-infected mice with SUR2 channel modulators, pinacidil or nicorandil, significantly reduced gastritis severity. In gastric epithelial cells, nicorandil-induced opening of the SUR2 channel increased intracellular K⁺ and prevented H pylori-mediated Ca²⁺ influx and downstream pro-inflammatory signaling.

Conclusion: SUR2 is a novel host factor that regulates Helicobacter pathogenesis. Pharmacological targeting of SUR2 provides a potential approach for reducing the severity of H pylori-associated gastritis, without eradicating infection.

Keywords: Gastritis; Helicobacter pylori; Potassium channel; SUR2; Therapeutic.

Figure 1

Reduced ABCC9 expression in stomachs infected with H pylori SS1. H pylori infection significantly reduced overall ABCC9 gene expression (all transcripts) in (A) human gastric mucosa (normal = 16, H pylori = 15) and (B) mouse stomachs at 2-, 4-, and 12-month postinfection (mpi) as determined by quantitative polymerase chain reaction (Mann-Whitney). SUR2B splice variant expression was significantly higher than SUR2A in both (C) humans and (D) mice at 4 mpi (Wilcoxon matched-pairs test).

Figure 2

Nicorandil inhibits the inflammatory effect of H pylori on epithelial cells. A) K_ATP channel subunit transcript expression in human AGS gastric epithelial and murine RAW 264.7 macrophage cell lines. B) Immunofluorescence images showing expression of SUR2 and Kir6.1 proteins in AGS cells. Blue, 4′,6-diamidino-2-phenylindole; red, actin; green, SUR2/Kir6.1 as labeled. C, D) Nicorandil (100 μM) had no effect on (C) IL-6 and (D) MIP-2 secretion by RAW264.7 macrophages, primary mouse intraperitoneal (IP) macrophages or splenocytes (as annotated) stimulated for 24 hours with H pylori SS1 lysate. E–G) Nicorandil treatment (100 μM) significantly reduced the IL-8 response of AGS cells to 24 hours stimulation with (E) H pylori SS1 lysate, (F) live H pylori SS1 and (G) live Cag-positive H pylori 251(MOI = 10). Graphs show group medians (horizontal bar), interquartile range (box), 10th and 90th percentiles (bars). P values were calculated using Mann-Whitney tests (ns, P > .05).

Figure 3

Nicorandil treatment prevents H pylori-induced Ca²⁺ influx in AGS cells. Intracellular potassium K⁺ (A) and Ca²⁺ (B) in untreated (clear box) or nicorandil-treated (100 μM; shaded box) AGS cells, expressed as a percentage of initial baseline reads from each well. Intracellular K⁺ spiked immediately after nicorandil addition and stayed elevated at 24 hours post-treatment. Ca²⁺ increased slightly but this effect was not significant. C) Intracellular Ca²⁺ levels increased upon H pylori 251 stimulation (MOI = 10) at 1 hour post infection (hpi) and D) 24 hpi. Nicorandil treatment significantly reduced H pylori induced Ca²⁺ influx, comparable to uninfected cells. Intracellular K⁺ at 1 hpi (E) and 24 hpi (F). P values were calculated by Mann-Whitney tests. G) Intracellular K⁺ in uninfected and H pylori-stimulated cells (without nicorandil treatment). All differences among infected cells and uninfected controls were nonsignificant (ns, P > .05) as calculated with two-way ANOVA followed by Sidak’s multiple comparisons test.

Figure 4

SUR2-K_ATP channel agonists do not impact H felis CS1 colonization but counteract infection-related SUR2 downregulation in a mouse gastritis model. A) Mice were infected with H felis CS1 for 4 weeks, then treated for another 8 weeks with either pinacidil or nicorandil (via drinking water). Age-matched uninfected controls (with or without drug treatment) were analyzed in parallel. Drug treatment had no effect on (B) mouse body weights, or (C) daily water intake as measured by ‘area under curve’ analysis. D) Gastric colonization with H felis, measured by qPCR, was not affected by drug treatment. E) Sur2B expression in stomach halves, measured by qPCR. F) SUR2 expression in the stomach mucosa as detected by immunofluorescence staining in a 5μm cross-section through the corpus epithelium; SUR2 (green), cytokeratin 19 (CK19, an epithelial cell marker; red) and cell nuclei (4′,6-diamidino-2-phenylindole, blue). G) SUR2 expression was significantly reduced upon H felis infection (3 mpi) and restored upon pinacidil and nicorandil treatment. Infection groups as labeled. Scale bars, 100μm. qPCR data analyzed by Mann-Whitney tests: ns, not-significant P > .05; other P values as annotated.

Figure 5

Anti-inflammatory effects of pinacidil and nicorandil are associated with a reduced local gastric cytokine response. (A–D) mRNA levels of cytokines in stomach halves from H felis CS1 infected, drug-treated mice (as described in Figure 4A) were quantified by qPCR. (E–H) Cytokines in splenic homogenates were quantified by enzyme-linked immunosorbent assay. Graphs show each mouse as an individual marker and group medians are depicted by horizontal lines. P values were calculated using Mann-Whitney tests.

Figure 6

Histopathological analysis of mouse stomachs. A) Representative gastric histopathology (H&E-stained sections) from H felis CS1 infected mice and uninfected control at 12 weeks postinfection (as annotated). B–D) Analysis of mouse gastric sections at 12 weeks postinfection showed that both pinacidil and nicorandil significantly reduced immune cell infiltration (B) and atrophy (C) compared to untreated/infected controls. D) Loss of Atp4b parietal cell marker expression in H felis infected gastric tissues was restored by nicorandil treatment. Data points on each graph show individual mice with group medians depicted by horizontal lines. P values were calculated using Mann-Whitney tests.