An in vitro study to evaluate the role of helicobacter pylori secretory proteins in oxidative stress and altered immune responses

Abstract

Helicobacter pylori is associated with various gastrointestinal diseases, including gastritis, peptic ulcers, and gastric cancer. Oxidative stress and immune response modulation are hallmarks of H. pylori-induced infection. The role of H. pylori virulence factors in the regulation of oxidative stress is not clearly understood. The involvement of H. pylori-induced oxidative stress in the modulation of host immune responses is also not understood clearly. Reactive oxygen species (ROS) regulate cytosolic sensor proteins complex NLRP3 inflammasome, which is responsible for pro-inflammatory immune responses and pyroptosis. THP-1 (human monocyte) and AGS (human gastric epithelium) cell lines were used to demonstrate the role of H. pylori-secreted concentrated proteins (HPSCP) induced oxidative stress in the regulation of NLRP3 inflammasome and immune cell modulation. HPSCP was prepared from H. pylori broth culture and its effect on cell growth (THP-1 and AGS) and vacuolation (HeLa) was evaluated using different concentrations. THP-1 and AGS were treated with 25 µg of HPSCP in the presence of oxidative stress inhibitors, NLRP3 inflammasome activators, and inhibitors. 2and#8242;,7and#8242;-Dichlorodihydrofluorescein diacetate (H2DCFDA), nitro blue tetrazolium (NBT), Griess, thiobarbituric acid reactive substances (TBARS), myeloperoxidase (MPO), 2,4-dinitrophenylhydrazine (DNPH) assays, and ELISA for pro and anti-inflammatory cytokines were performed to evaluate the HPSCP-induced oxidative stress, inflammation, and NLRP3 involvement. The expression of ROS- iv producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunit gp91phox and nitric oxide (NO) producing nitric oxide synthase 2 (NOS2) enzyme was analyzed with confocal microscopy. Similarly, M1 and M2 macrophage differentiation markers CD86 and CD163 presence were analyzed with confocal microscopy. Nucleotide-binding domain leucine-rich repeat-containing (NLR) family pyrin domain containing 3 (NLRP3) inflammasome expression was analyzed through confocal microsc