The Antibacterial Effect Of RIP2 Gene On Escherichia Coli And Its Underlying Mechanism

ObjectiveReceptor-interacting protein (RIP)-2 is a serine□threonine kinase containing a caspase recruitment domain (CARD) that is involved in the NOD-like receptor-signaling pathway, RIP2 is downstream of NLRs and activation of NF-κB signaling pathway, and play an important role in innate immune response and clear the pathogen infection in the inflammatory response. Intestinal infection is one of the most common bacterial infection, NLRs can be identified by the infected host, RIP2 signaling molecules activated by NF-κB nuclear transcripted and expressed inflammatory cytokines, triggering a variety of host defense mechanism. Mitogen-activated protein kinase (MAPK) signaling pathway participated in cell growth and function and other physiological processes simultaneously, including p38MAPK which mainly involved in inflammation and stress response. Our study will transfect RIP2 gene to intestinal cells, using RT-PCR and Western blot to test the level of mRNA and protein; using E. coli infected to transfected RIP2 cells, counting the number of intracellular bacteria; using p38MAPK pathway inhibitor SB203580 in the experimental group, counting the number of intracellular E. coli; detect by Western blot of phosphorylation of p38 in each experimental group, and test the expression level to observe the role of RIP2 in p38MAPK signaling pathway.MethodsRIP2 gene (pEGFP-C2-PIP2) were transfected into cell line SW480 by JetPeiTM. Western blot and RT-PCR analyses were then used to detect RIP2 protein and mRNA expression. These transfected and non-transfected cells were incubated with Escherichia coli(E coli) at 37℃. After 24 hours, viable intracellular bacteria were quantified by plating appropriate dilution on LB agar plates. Then transfected and non-transfected cells were treated with SB203580, the ResultsRT-PCR and Western blot analyses indicated that the expression of RIP2 mRNA and protein was significantly enhanced in SW480 cells transfected with the recombinant plasmid pEGFP-C2-PIP2 when compared with cells transfected with pEGFP-C2 and non-transfected cells. The pEGFP-C2-PIP2 transfected cells had the ability to clear invading E coli, and this antibacterial effect was inhibited by SB203580, the ability has decrease. after stimulation of bacteria, Western blot detected phosphorylation of p38 protein, the results showed that cells transfected with RIP2 of phosphorylated p38 protein was significantly higher than cells transfected with C2 group, and after stimulation with E. coli the p-p38 protein expression highest than the other group, further confirmed that RIP2 clear mechanism of intracellular E. coli may have connection to activation of p38MAPK signal pathway.Conclusion(1) Our results show RIP2 plasmid successfully transfected SW480 cells, mRNA transcription level was significantly higher than the control group (P<0.01) and the protein expression levels higher than control (P<0.05)(2) ATCC25922 in SW480 cells increased as time growing; in PIP2 transfected SW480 cells, the number of intracellular E. coli was significantly reduced compared with the control group, suggesting that RIP2 can clear intracellular E. coli.(3) After using p38MAPK inhibitor SB203580, we found it can also blok the function of RIP2; RIP2 can induce the expression of p38MAPK protein, suggesting that activation of p38MAPK may plays an important role in the role of RIP2 clearence effect.