Effect Of Helicobacter Pylori CagA Protein On NLRP3 Inflammasome Activation

Objective: To investigate the effect of Helicobacter pylori(H.pylori)cytotoxin associated gene A protein(CagA)protein on activating of NLRP3 inflammasome in gastric mucosal epithelial cells and undelying mechanisms.Methods:(1)Bioinformatics softwares DNAWorks and Jcat were used to optimize the humanized codon of H.pylori cagA gene.The human-codon-optimized cagA eukaryotic expression vector was constructed and then transfected in human gastric epithelial cells GES-1 and gastric cancer cells AGS respectively.The expression of the CagA protein in the cell was verified by Western Blot.(2)Western Blot was used to analyze the protein expression of NLRP3 inflammasome related proteins NLRP3,caspase-1,ASC,and NF-?B pathway-associated proteins p65 and phosphorylated p65 as well.The secretion of IL-1? and IL-18 in the supernatant of transfected cells was detected by Enzyme-Linked ImmunoSorbent Assay(ELISA).The levels of reactive oxygen species(ROS)and cell pyroptosis rate were measured by flow cytometry.(3)After pretreated with ROS inhibitor N-acetylcysteine(NAC)for 2 h,GES-1 and AGS cells were transfected with cagA expression vector,and cells and supernatant were collected after 48 h.The expression of NLRP3,caspase-1 and ASC were detected by Western Blot.The changes of IL-1? and IL-18 in cell supernatant were detected by ELISA.The levels of reactive oxygen species(ROS)and cell pyroptosis rate were measured by flow cytometry.Results:(1)CagA protein can be expressed intracellularly in codon-optimized cagA eukaryotic expression vector transfected cells.(2)The human gastric epithelial cells GES-1 and gastric cancer cells AGS were transfected by cagA vector for 48 h,after which the expression of intracellular NLRP3,caspase-1,ASC protein expression increased,as well as intracellular phosphorylated p65 protein,the expression of p65 and phosphorylated p65 protein in the nucleus.Compared with the control vector group,the IL-1? and IL-18 levels in the supernatant of the cagA transfection group were increased,the intracellular ROS production was increased,and the cell pyroptosis rate was increased(P < 0.05).(3)Compared with the direct transfection of cagA-plasmid group,NAC pretreatment reduced intracellular ROS production,prevented CagA-induced up-regulation of intracellular protein expression of NLRP3,caspase-1,and ASC,decrease IL-1?,IL-18 release,and decreased cell atrophy rate as well(P < 0.05).Conclusions:(1)CagA protein activated NF-?B signaling pathway and NLRP3 inflammatory corpuscles,thus,promoted the release of IL-1? and IL-18 inflammatory cytokines,and promoted cell pyroptosis.(2)CagA activation of NLRP3 inflammasome may be mediated through the ROS pathway.