Key Word(s): 1. Helicobacter pylori; 2. outer-membrane protein; 3. HomB Presenting Author: YONG XIE Additional Authors: ZHI RONG click here MAO, NAN JIN ZHOU, DONG SHENG LIU, FU CAI WANG Corresponding Author: YONG XIE Affiliations: The First Affiliated Hospital of Nanchang University, Institute of Medical Sciences of Jiangxi Province, The First Affiliated Hospital of Nanchang University, The First Affiliated Hospital of Nanchang

University Objective: Macrophages play an important role in H. pylori infection. Toll-like receptor 4 (TLR4) activated macrophages to secrete plenty of cytokines which regulated inflammation and immunity reaction; T-cell immunoglobulin and mucin-domain-containing molecule-3 (Tim-3) , an important member of TIM family, was also expressed on macrophages and could impact macrophages function through interacting

with TLR4 pathways. Until now, It is unclear that how H. pylori impacts Tim-3 and TLR4 pathways in macrophages. Methods: ①RAW264.7 cells were co-cultured with H.pylori EPZ-6438 price SS1 at different bacteria/cell ratio (MOI) at 3h, 6h, 12h, 24h and 48h were detected by MTT assay, respectively. At 12h, the mRNA expressions of Tim-3\TLR4\MyD88 were measured by RT-PCR; ②Tim-3-overexpressing RAW264.7 cells were constructed by transfer pLVX-IRES-ZsGreen-Tim-3 and co-cultured with H.pylori. The mRNA and protein expressions 上海皓元 of Tim-3\TLR4\MyD88 were determined

by RT-PCR and Western Blot. The concentrations of cytokines (TNF-α, IL-6, IFN-γ and IL-10) in supernatants were measured by ELISA. Results: ①RAW264.7 cells were co-cultured with H.pylori SS1 at different bacteria/cell ratio (MOI) at 3h, 6h, 12h, 24h and 48h were detected by MTT assay, respectively. At 12h, the mRNA expressions of Tim-3\TLR4\MyD88 were measured by RT-PCR; ②Tim-3-overexpressing RAW264.7 cells were constructed by transfer pLVX-IRES-ZsGreen-Tim-3 and co-cultured with H.pylori. The mRNA and protein expressions of Tim-3\TLR4\MyD88 were determined by RT-PCR and Western Blot. The concentrations of cytokines (TNF-α, IL-6, IFN-γ and IL-10) in supernatants were measured by ELISA. Conclusion: Over-expression of Tim-3 reduces H. pylori-induced inflammation through down-regulating TLR4 pathways expressions and pro-inflammatory cytokines release from RAW264.7 infected with H. pylori. Key Word(s): 1. Helicobacter pylori; 2. RAW264.7; 3. Tim-3; 4.