Role of MD-2 in Tool-like receptor 4-dependent signaling in Helicobacter-pylori-associated gastritis

幽门螺杆菌(Hp)脂多糖(LPS)是引起胃炎的主要毒力因子之一。依赖内毒素激活的胃上皮细胞和浸润性巨噬细胞可导致多种促炎细胞因子的分泌。然而，关于幽门螺杆菌内毒素在胃粘膜细胞中的识别系统却知之甚少。最近的研究表明，内毒素通过Toll样受体(TLR)-4启动信号转导，该途径被MD-2激活。本研究的目的是探讨TRL-4/MD-2复合体对幽门螺杆菌内毒素在胃粘膜细胞中的特异性识别作用。胃窦活检标本取自幽门螺杆菌感染和未感染患者。用四种胃癌细胞系(MKN-7、MKN-28、MKN-45和AGS)进行体外研究。用逆转录聚合酶链式反应和免疫印迹法检测TLR-4和MD-2在胃活检组织和胃癌细胞系中的表达。免疫组织化学方法检测TLR-4在组织切片中的定位。用荧光素酶分析法检测幽门螺杆菌脂多糖对胃上皮细胞和瞬时表达TLR-4和/或MD-2的人胃上皮细胞中核因子-kB的激活作用。TLR-4在幽门螺杆菌感染前后的胃组织中呈结构性表达，定位于胃上皮细胞和浸润性单个核细胞。而幽门螺杆菌阳性组MD-2的表达高于阴性组。在体外，所有胃上皮细胞均表达TLR-4和MD-2，幽门螺杆菌脂多糖刺激这些细胞中的核因子-kB活化。同时表达TLR-4和MD-2的转染体的内毒素应答显著高于仅表达TLR-4的转染体。TLR-4/MD-2复合体对幽门螺杆菌脂多糖的识别是必不可少的，MD-2可能在幽门螺杆菌相关性胃炎的发生发展过程中调节TLR-4依赖的信号转导。

Helicobacter pylori (H. pylori) lipopolysccharide (LPS) is one of the major virulence factors in the induction of gastritis. LPS-dependent activation of gastric epithelial cells and infiltrating macrophages leads to secretion of several proinflammatory cytokines. However, little is known about the recognition system of H. pylori LPS in gastric mucosal cells. Recent studies have shown that LPS initiates signal transduction through toll-like receptor (TLR)-4, and this pathway is activated by MD-2. The aim of this study is to investigate TRL-4/MD-2 complex-specific recognition of H. pylori LPS in gastric mucosal cells. Gastric antral biopsy samples were taken from patients with and without H. pylori infection. Four gastric cancer cell lines (MKN-7, MKN-28, MKN-45 and AGS) were used for in vitro study. Expression of TLR-4 and MD-2 in gastric biopsy specimens and gastric cancer cell lines was examined by reverse-transcription polymerase chain reaction and western blot analysis. Localization of TLR-4 in histological sections was also evaluated by immunohistochemistry. Luciferase assay was performed for the assessment of H. pylori LPS-induced NF-kB activation in gastric epithelial cells and transient transfectants expressing TLR-4 and/or MD-2. TLR-4 was constitutively expressed in the stomach with and without H. pylori infection and localized in gastric epithelial cells and infiltrating mononuclear cells. However, the expression of MD-2 in H. pylori-positive subjects was higher than that in H. pylori-negative subjects. In vitro, all gastric epithelial cells expressed both TLR-4 and MD-2 and H. pylori LPS stimulated NF-kB activation in these cells. LPS responses in transfectant expressing both TLR-4 and MD-2 was significantly higher than that expressing only TLR-4. TLR-4/MD-2 complex is essential for the recognition of H. pylori LPS and MD-2 may regulate TLR-4-dependent signaling in the development of H. pylori-associated gastritis.