荚膜唾液酸对猪链球菌激活巨噬细胞TLR2-AKT-NF-κB信号通路影响的研究

Research on the role of capsular sialic acid in Streptococcus suis activate macrophage TLR2-AKT-NF-κB signaling pathway

【目的】探索猪链球菌2型感染后单核/巨噬细胞启动信号转导通路机制,探讨荚膜唾液酸对细菌激活巨噬细胞TLR2-AKT-NF-κB信号通路的影响。【方法】以小鼠巨噬细胞系RAW264.7细胞为研究对象,采用RT-PCR、Western blotting、免疫荧光和ELISA法分别检测猪链球菌2型野毒株、唾液酸缺失突变株、唾液酸回复突变株感染后不同时间点巨噬细胞TLR2 mRNA转录水平、AKT磷酸化水平、NF-κB激活程度以及前炎症因子TNF-α分泌水平;再分别用TLR2阻断剂和PI-3K抑制剂预处理巨噬细胞,检测上述分子的表达水平。【结果】唾液酸缺失株可选择性的活化信号转导通路途径。RT-PCR结果表明,缺失株TLR2 mRNA表达水平自1 h开始升高,1.5 h达高峰后有所下降;Westernblotting显示,缺失株TLR2蛋白表达水平7 h达高峰,9 h下降;p-AKT水平1.5 5 h持续稳定在高峰水平,7 h后开始下降;免疫荧光可见15 min NF-κB激活-核转运程度较高;ELISA结果显示,10 h之后TNF-α的水平显著高于野生株和回复株。使用TLR2阻断剂和PI-3K抑制剂,三菌株通路活化程度均明显受抑制。【结论】荚膜唾液酸可抑制宿主免疫细胞TLR2-AKT-NF-κB信号通路的激活,藉此参与细菌逃避宿主的免疫防御作用。

[Objective] To explore the mechanism of signaling pathways Streptococcus suis serotype 2 infected monocytes/macrophages leaded, discuss the role of capsular sialic acid component played in Streptococcus suis activate macrophage TLR2-AKT-NF-κB signaling pathway. [Methods] RAW264.7 as the target cell line, RT-PCR, Western blotting, im- munofluorescence and ELISA were applied to detect different infection time of wild type strain, sialic acid knockout strain and sialic acid complementary strain on macrophages TLR2 mRNA transcription level, AKT phosphorylation level, NF-κB activation level, as well as TNF-α secretion level. Pretreat with TLR2 blocking agent and PI-3K inhibitor on macro- phages, detect the expression level above. [Results] Sialic acid knockout strain activates signal transduction pathways selectively. RT-PCR results show that TLR2 mRNA expression levels began to increase at 1 h, 1.5 h reached its peak then slowly decline. Western blotting showed that TLR2 protein expression level reached its peak at 7 h, 9 h decline. Level ofp-AKT is sta- ble at its peak during 1.5-5 h, 7 h decline. Immuno fluorescence showed high level of NF-κB activation-nuclear translocation at 15 min. ELISA results indicate TNF-α secretion level was significantly higher than the other two strains after 10 h. TLR2 blocking agent and PI-3K in- hibitor significantly suppressed the activation degree of three strains. [Conclusion] Capsular sialic acid could inhibit activation of the TLR2-AKT-NF-κB signaling pathway to some extent, thus participate in bacteria evading the host immune defense.