Wang Fucai, Mao Zhirong, Liu Dongsheng, Yu Jing, Wang Youhua, Ye Wen, Lin Dongjia, Zhou Nanjin, Xie Yong
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Gastroenterology Institute of Jiangxi, Key Laboratory of Digestive Diseases of Jiangxi, Nanchang, Jiangxi 330006, P.R. China.
Department of Immunology, Medical College of Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Mol Med Rep. 2017 May;15(5):3252-3258. doi: 10.3892/mmr.2017.6346. Epub 2017 Mar 21.
The present study aimed to investigate the interaction between T-cell immunoglobulin and mucin-domain-containing molecule-3 (Tim-3) and Toll-like receptor 4 (TLR4)/nuclear factor κB (NF‑κB) signaling in Helicobacter pylori-infected RAW264.7 macrophage cells. RAW264.7 cells were co‑cultured with H. pylori SS1 at different bacteria/cell ratios, and subsequently the mRNA expression of Tim‑3, TLR4, and myeloid differentiation factor 88 (MyD88) was measured by reverse transcription-quantitative polymerase chain reaction (RT‑qPCR). Furthermore, the effect of Tim‑3 overexpression was examined by transfection of RAW264.7 with pLVX-IRES-ZsGreen-Tim-3 and co‑culturing with H. pylori. mRNA and protein expression levels were then analyzed for Tim‑3, TLR4, MyD88, and phosphorylated (p‑) NF‑κB by RT‑qPCR and western blot analysis respectively. The concentrations of pro‑inflammatory cytokines [tumor necrosis factor‑α (TNF‑α), interleukin 6 (IL-6), interferon‑γ (IFN‑γ) and interleukin 10 (IL‑10)] released in the culture supernatants were measured by ELISA. H. pylori stimulation resulted in a significant increase of Tim‑3, TLR4, and MyD88 mRNA expression in RAW264.7 cells. H. pylori stimulation upregulated Tim‑3 expression even in the Tim‑3‑overexpressing RAW264.7 cells compared with unstimulated cells. TLR4, MyD88, and pNF‑κB protein expression and pro‑inflammatory cytokines (TNF‑α, IL‑6, and IFN‑γ) release levels were increased in the control RAW264.7 cells following H. pylori infection, but not in the Tim-3-overexpressing RAW264.7 cells. By contrast, IL‑10 levels were decreased following H. pylori infection in both control and Tim‑3‑overexpressing RAW264.7 cells. Overexpression of Tim-3 reduced H. pylori-associated inflammation in RAW264.7 macrophages, by downregulating expression of proteins in the TLR4 pathway and release of pro‑inflammatory cytokines. These findings suggest that Tim‑3 serves a crucial role in the negative regulation of H. pylori-associated inflammation and may be a novel therapeutic target for H. pylori infection.
本研究旨在探讨T细胞免疫球蛋白黏蛋白结构域分子3（Tim-3）与Toll样受体4（TLR4）/核因子κB（NF-κB）信号通路在幽门螺杆菌感染的RAW264.7巨噬细胞中的相互作用。将RAW264.7细胞与幽门螺杆菌SS1以不同的细菌/细胞比例共培养，随后通过逆转录-定量聚合酶链反应（RT-qPCR）检测Tim-3、TLR4和髓样分化因子88（MyD88）的mRNA表达。此外，通过用pLVX-IRES-ZsGreen-Tim-3转染RAW264.7并与幽门螺杆菌共培养来检测Tim-3过表达的影响。然后分别通过RT-qPCR和蛋白质印迹分析来分析Tim-3、TLR4、MyD88和磷酸化（p-）NF-κB的mRNA和蛋白质表达水平。通过酶联免疫吸附测定（ELISA）测量培养上清液中释放的促炎细胞因子[肿瘤坏死因子-α（TNF-α）、白细胞介素6（IL-6）、干扰素-γ（IFN-γ）和白细胞介素10（IL-10）]的浓度。幽门螺杆菌刺激导致RAW264.7细胞中Tim-3、TLR4和MyD88 mRNA表达显著增加。与未刺激的细胞相比，即使在Tim-3过表达的RAW264.7细胞中，幽门螺杆菌刺激也会上调Tim-3表达。在幽门螺杆菌感染后，对照RAW264.7细胞中TLR4、MyD88和pNF-κB蛋白表达以及促炎细胞因子（TNF-α、IL-6和IFN-γ）释放水平增加，但在Tim-3过表达的RAW264.7细胞中未增加。相比之下，在对照和Tim-3过表达的RAW264.7细胞中，幽门螺杆菌感染后IL-10水平均降低。Tim-3的过表达通过下调TLR4通路中的蛋白质表达和促炎细胞因子的释放，减轻了RAW264.7巨噬细胞中幽门螺杆菌相关的炎症。这些发现表明，Tim-3在幽门螺杆菌相关炎症的负调控中起关键作用，可能是幽门螺杆菌感染的一个新的治疗靶点。
