嗜肺军团菌重组免疫原蛋白(IP)对RAW264.7细胞自噬的影响

Effect of Legionella pneumophila recombinant immunogenic protein (IP) on autophagy in RAW264.7 cells

摘要:

【目的】观察嗜肺军团菌重组免疫原蛋白(immunogenic protein，IP)对RAW264.7细胞自噬流及自噬相关因子表达水平的影响并探讨其作用机制。【方法】采用His标签蛋白纯化试剂盒纯化嗜肺军团菌重组免疫原蛋白(IP)，并用CCK-8法检测IP对RAW264.7细胞半数抑制浓度(half maximal inhibitory concentration，IC₅₀)。采用低浓度(0.05×IC₅₀)、中浓度(0.1×IC₅₀)、高浓度(0.2×IC₅₀) IP与RAW264.7细胞进行体外共培养1、3、6、12 h，并设细胞对照组，应用自噬双标质粒pmCherry-C1-EGFP-LC3B检测巨噬细胞自噬流的变化，筛选最佳浓度进行后续实验；最佳浓度IP与RAW264.7细胞进行体外共培养6、12、24 h，并设细胞对照组，RT-qPCR检测各组自噬相关蛋白Beclin1、微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3，LC3)、SQSTM 1 (sequestosome 1，p62)及组蛋白去乙酰化酶6 (histone deacetylase 6，HDAC6)的mRNA表达水平；Western blotting法检测各组自噬相关因子的蛋白表达水平；免疫荧光检测各组自噬相关因子的表达。【结果】根据CCK-8结果计算IC₅₀为0.26μg/μL。自噬双标质粒pmCherry-C1-EGFP-LC3B检测自噬流结果显示，与对照组相比，中浓度(0.026μg/μL) IP与RAW264.7细胞进行体外共培养后自噬流抑制显著。RT-qPCR及Western blotting检测结果显示，与对照组相比，IP与RAW264.7细胞共培养6 h，P62表达水平升高，LC3B、HDAC6、Beclin1表达水平均降低，P<0.05；与6 h组相比，12 h组LC3B表达水平降低，P62、HDAC6及Beclin1表达水平均升高，P<0.05；与12 h组相比，24 h组Beclin1表达水平升高，P<0.05。IP一定程度上以时间依赖的方式降低了LC3-Ⅱ/LC3-Ⅰ比值并升高了P62蛋白的表达水平，P<0.05。免疫荧光结果基本与RT-qPCR及Western blotting检测结果一致。【结论】嗜肺军团菌IP抑制巨噬细胞自噬，可能与通过影响自噬小体-溶酶体融合途径，干扰自噬小体及自噬溶酶体的形成、成熟等过程有关。

Abstract:

[Objective] To investigate the effect of Legionella pneumophila recombinant immunogenic protein (IP) on RAW264.7 cell autophagy and the mechanism.[Methods] We purified the L.pneumophila IP using the His-tag protein purification kit and determined the half maximal inhibitory concentration (IC₅₀) of the IP on RAW264.7 cells with cell counting kit-8(CCK-8) assay.We co-cultured RAW264.7 cells with different concentration (0.05×IC₅₀,0.1×IC₅₀,0.2×IC₅₀) of IP for 1,3,6,and 12 h,respectively,and set up a cell control group.In addition,we used the pmCherry-GFP-LC3B tandem fluorescent protein to monitor changes in autophagy flux in RAW264.7 cells,and chose the optimal concentration for subsequent experiments.We co-cultured RAW264.7 cells with the optimal concentration of IP for 6,12,and 24 h,separately,and established a cell control group.Apart from that,the mRNA and protein expression levels of autophagy-related factors Beclin1,microtubule-associated protein 1 light chain 3B (LC3B),SQSTM1(sequestosome 1,p62),and histone deacetylase 6(HDAC6) were detected by RT-qPCR,Western blotting,and immunofluorescence staining.[Results] The IC₅₀ was calculated to be 0.26 μg/μL.When RAW264.7 cells were co-cultured with medium-concentration (0.026 μg/μL) IP,the autophagy flux was clearly inhibited,as monitored by pmCherry-C1-EGFP-LC3B.As revealed by the results of RT-qPCR and Western blotting analysis,when RAW264.7 cells were co-cultured with IP for 6 h,the expression level of P62 increased,but the expression levels of LC3B,HDAC6,and Beclin1 decreased (P<0.05).The expression of LC3B reduced and the expression of P62,HDAC6,and Beclin1 rose at 12 h compared with those at 6 h (P<0.05).Beclin1 expression was higher at 24 h than that at 12 h (P<0.05).According to the RT-qPCR and Western blotting results,IP decreased the LC3-Ⅱ/LC3-Ⅰ ratio and increased the expression level of P62 in a time-dependent manner (P<0.05).The immunofluorescence results were essentially consistent with the RT-qPCR and Western blotting results.[Conclusion] L.pneumophila recombinant IP can inhibit autophagy flux in macrophages.Moreover,IP may affect the autophagosome-lysosome fusion pathway and interfere with the formation and maturation of autophagosome and autophagolysosome.