Helicobacter Pylori VacA Up-regulates Secretion And Apoptosis Of Macrophages By Activating Nuclear Factor Kappa B

Objectives:(1) We constructed a recombinant plasmid containing the N-terminus gene of vacA gene of Helicobacter pylori.(2) We studied the effect of VacA on the secretion and apoptosis of THP-1 macrophages as an individual virulence determinant.(3) The effect of NF-κB on the secretion and apoptosis of THP-1 macrophages was also studied.Methods:Primers were designed by PRIMER5.0 software.VacA gene amplified by PCR from H. pylori was cloned into eukaryotic expression vector pDsRed-Monomer-C1. The recombinant plasmids were verified by restriction endonucleases analysis and nucleotide sequencing. Then the recombinant plasmids pDsRed-Monomer-C1/vacA were transfected into macrophages and their expression in macrophages was examined by Western-blot and fluorescence microscope. Vacuolated phenotype in macrophages was observed by electron microscopy and neutral red uptake. The cytokine content of TNF-αor IL-1βin the culture medium was tested quantitatively with ELISA kit, respectively. NO or ROS was tested with Griess Reagent or DCFH-DA fluorescent probe. The apoptosis rate of macrophages was tested by flow cytometry. The activity of NF-κB was examined in THP-1 cells by EMSA. The effect of PDTC, an inhibitor of NF-κB, on the secretion and apoptosis of macrophages transfected with the recombinant plasmids, was also studied. Results:(1) Restriction endonucleases analysis and nucleotide sequencing showed that the eukaryotic expression recombinant pDsRed-Monomer-C1/vacA was successfully constructed.(2) VacA was expressed in macrophages and located at cytoplasm.The result of Western-blot showed that VacA could be recognized by the polyclonal rabbit anti- H. pylori serum.(3) At 6h after transfection, the level of Neutral Red Uptake in macrophages transfected with the recombinant plasmids was significantly higher than that of the control group (P <0.05). A clear vacuolated phenotype developed in some of macrophages transfected with the recombinant plasmids at 24h after transfection.(4) At 6h after transfection, the level of TNF-αand IL-1βin macrophages transfected with the recombinant plasmids was significantly higher than that of the control group (P <0.05).(5) At 6h or 12h after transfection, the level of NO and ROS in macrophages transfected with the recombinant plasmids was significantly higher than that of the control group (P <0.05).(6) At 16h after transfection, the apoptosis rate of macrophages transfected with the recombinant plasmids was significantly higher than that of the control group (P <0.05).(7) PDTC decreased the production of TNF-α, IL-1β, NO, ROS and apoptosis rate induced by VacA.(8) VacA was found to trigger NF-κB activation, a possible mechanism for the induction of cytokines production and apoptosis of THP-1 macrophages.Conclusions:(1)we have successfully constructed the eukaryotic expression plasmid encoding VacA.(2)The over-expression of VacA fusion protein can up-regulate secretion of macrophages. (3)The over-expression of VacA fusion protein can induce apoptosis of macrophages.(4)Activation of NF-κB is probably involved in the production of TNF-α, IL-1β, NO, ROS and apoptosis induced by VacA.