The relationship between non-steroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori (H. pylori) in causing gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells (Ricci et al. 2000). VacA may act as a channel-forming toxin: endocytosed VacA channels could stimulate the turnover of endosomal V-ATPase by increasing the permeability of the endosomal membrane to anions. This would lead to the accumulation of osmotically active species causing an osmotic imbalance of late endosomes with subsequent vacuole formation. This study was designed to investigate whether and how NSAIDs interfere with VacA-induced vacuolation of human gastric epithelial cells in culture.

MKN 28 cells were incubated for 16 h at 37 °C with different concentrations of NH₄Cl in the absence or presence of VacA and of different NSAIDs (0.1Ð1 mM indomethacin; 0.1Ð1 mM aspirin; 0.01Ð1 mM NS-398, a selective cyclooxygenase-2 inhibitor), prostaglandin E₂ (PgE₂; 10^-7Ð10^-5 M) or arachidonic acid (AA; 0.01Ð0.05 mM). To study NSAID action on already developed cell vacuolation, cells were incubated for 16 h with 4 mM NH₄Cl or 4 mM NH₄Cl plus VacA and then for an additional 8 h in the absence or presence of different NSAIDs. Cell vacuolation was quantified by neutral red uptake assay (Ricci et al. 2000). We also investigated NSAID action on cell binding and internalization of VacA, using a protease-protection assay and SDS-PAGE followed by Western blotting. Moreover, we compared NSAID action with that of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), an NSAID itself known as a specific inhibitor of VacA action via its chloride channel blocking activity and exhibiting structural similarities with NSAIDs we used. Results were expressed as the mean ± S.E.M. of three independent experiments. The statistical significance of the differences was evaluated by ANOVA and Newman-Keuls Q-test.

We found that (1) NSAIDs dose-dependently and significantly (P < 0.05) both prevented and reverted VacA-induced vacuolation of MKN 28 cells, causing a 55Ð70 % reduction in neutral red uptake with the highest dose used (e.g. VacA: 2.05 ± 0.10; VacA + 1 mM indomethacin: 0.61 ± 0.04 µg/µg cell protein; (2) neither PgE₂ nor AA affected NSAID-dependent inhibition of VacA vacuolating action; (3) none of NSAIDs used impaired cell binding or internalization of VacA; (4) NSAIDs and NPPB, in a very similar manner, significantly (P < 0.05) inhibited ammonia-dependent cell vacuolation and reduced the VacA potentiating effect on ammonia action.

Our findings may suggest that NSAIDs counteract VacA cytotoxicity through inhibition of VacA channel activity as well as of endogenous anionic channels required for vacuole genesis.