Helicobacter Pylori-secreting Protein Tipα Induces Cytokines TNF-α And IL-8 Production In Macrophages Through Activation Of C23/NF-κB Pathway

Objective To study the effects and molecular mechanisms involving of the tumor necrosis factor-α-inducing protein(Tipα) from Helicobacter pylori on the secretion of TNF-α and IL-8 in macrophages.Methods In vitro cultures of human THP-1 cells were differentiated into macrophage cells with PMA. The m RNA levels of nucleolin(C23) and Toll-like receptor 4(TLR4) were evaluated by real-time quantitative PCR after the macrophage cells has been treated for four hours by the 30~200μg /ml Tipα protein. The effects of Tipα protein on the expressions of TNF-α and IL-6 were studied after the macrophages were transfected with C23 or TLR4 si RNA. The macrophages were pretreated with 5μg/m L of tunicamycin to prevent the synthesis of N-linked oligosaccharide, then we investigated the influence of Tipα protein on the secretions of TNF-α and IL-6 in macrophages which have been treated by Tipα protein for 24 h. The C23 and Tipα protein were analyzed by co-immunoprecipitation. We used small interfering RNA to inhibit the C23 expression in THP-1 cells, then determined the protein expression of IκB and p65 by western blotting. We used the ubiquitin proteasome inhibitor MG-132 pretreated the THP-1 cells, then tested its effect on the expression of TNF-α and IL-8 by ELISA.Results(1) The real-time PCR results indicated that the m RNA levels of C23 was up-regulated in a dose dependent manner when the macrophages were treated by the 30~200μg/ml Tipα protein. The expressions of TLR4 m RNA were not affected by Tipα protein.(2) The protein levels of TNF-α and IL-8 were significantly lower following Tipα treatment when cells were transfected with C23 si RNA, however the TLR4 si RNA had no effect on the productions of TNF-α and IL-8.(3) After the cells were pretreated by 5μg/ml tunicamycin, the ELISA results indicated the productions of TNF-α and IL-8 in Tipα stimulated cells obviously lower than un-stimulated cells.(4) The Tipα protein could be detected when the proteins were precipitated by the C23 antibody. At the same time, the C23 protein could be detected when the protein were precipitated by the Tipα antibody.(5) The protein level of IκB decreased in Tipα stimulated macrophage. The degradation of of IκB was inhibited when the C23 expression was blocked by si RNA. The protein level of p65 in the nuclear were up-regulated by Tipα, while it decreased significantly when the C23 expression was blocked by si RNA.(6) The TNF-α and IL-8 were obvious decreased in Tipα stimulated macrophage when cells were pretreated by 10μmol/L MG-132.Conclusion(1) C23 may be the receptor of Tipα.(2) Tip protein could induce the secretion of TNF-α and IL-8 in macrophages via C23/NF-κB pahway.