Zhen Qingjie, Cao Xiuqin, Lu Jingjing, Yang Zhiwei
Department of Pathogenic Biology and Immunology, School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, China.
Ministry-of-Education Key Laboratory for Fertility Preservation and Maintenance, Department of Pathogenic Biology and Immunology, School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Jun;34(6):488-494.
Objective To investigate the effect of the main outer membrane protein (MOMP) of Legionella on the phagocytosis and chemotaxis of RAW264.7 macrophages and explore its mechanism. Methods MOMP and RAW264.7 macrophages were cultured in vitro. The toxicity of MOMP to RAW264.7 macrophages was detected by CCK-8 assay and 50% inhibitory concentration (IC) was determined. The RAW264.7 macrophages were treated by MOMP (1.14, 0.57, 0.28) μg/mL and the control group was established. The cells and cultivate supernatants were collected 24, 48 and 72 hours after the RAW264.7 macrophages were treated by MOMP. The phagocytic function of macrophages was detected by the neutral red phagocytosis experiment; the chemotaxis function of macrophage was examined by Transwell assay, and the levels of monocyte chemoattractant protein 1 (MCP-1) and interleukin 10 (IL-10) in cell culture supernatant monocytes were detected by ELISA. Real-time quantitative PCR was used to check the mRNA level of macrophage nucleotide-binding oligomerization domain 1 (NOD1), NOD2 and receptor-interacting protein 2 (RIP2). The protein levels of NOD1, NOD2 and RIP2 were detected by Western blot analysis. Results The IC of MOMP on RAW264.7 macrophages was 5.69 μg/mL. Compared with the control cells, MOMP treatment caused a decrease of RAW264.7 macrophage phagocytosis in a dose- and time-dependent manner. With the increase of MOMP dosage, the chemotaxis of macrophages and the secretory levels of MCP-1 and IL-10 in the cell culture supernatant increased, and peaked in 36 hours. The mRNA and protein expression levels of NOD2, RIP2 also increased, mRNA levels of NOD2 and RIP2 peaked in 12 hours, and protein levels peaked in 24 hours. Conclusion MOMP can inhibit the phagocytosis of RAW264.7 macrophages and enhance its chemotaxis function, which is related to the activation of NOD2/RIP2 signaling pathway.
目的 探讨嗜肺军团菌主要外膜蛋白（MOMP）对RAW264.7巨噬细胞吞噬作用和趋化功能的影响并探究其机制。方法 体外培养MOMP和RAW264.7巨噬细胞。采用CCK-8法检测MOMP对RAW264.7巨噬细胞的毒性并测定50%抑制浓度（IC）。用（1.14、0.57、0.28）μg/mL的MOMP处理RAW264.7巨噬细胞并设立对照组。在RAW264.7巨噬细胞经MOMP处理后24、48和72小时收集细胞及培养上清。通过中性红吞噬实验检测巨噬细胞的吞噬功能；采用Transwell实验检测巨噬细胞的趋化功能，并用ELISA法检测细胞培养上清单核细胞中单核细胞趋化蛋白1（MCP-1）和白细胞介素10（IL-10）的水平。运用实时定量PCR检测巨噬细胞核苷酸结合寡聚化结构域1（NOD1）、NOD2和受体相互作用蛋白2（RIP2）的mRNA水平。通过蛋白质免疫印迹分析检测NOD1、NOD2和RIP2的蛋白水平。结果 MOMP对RAW264.7巨噬细胞的IC为5.69μg/mL。与对照细胞相比，MOMP处理使RAW264.7巨噬细胞的吞噬作用呈剂量和时间依赖性降低。随着MOMP剂量增加，巨噬细胞的趋化作用以及细胞培养上清中MCP-1和IL-10的分泌水平升高，并在36小时达到峰值。NOD2、RIP2的mRNA和蛋白表达水平也升高，NOD2和RIP2的mRNA水平在12小时达到峰值，蛋白水平在24小时达到峰值。结论 MOMP可抑制RAW264.7巨噬细胞的吞噬作用并增强其趋化功能，这与NOD2/RIP2信号通路的激活有关。
