Objective: To investigate the effects of SINC, a secreted protein of Chlamydia psittaci, on autophagy of host cells and the role of MAPK/ERK signaling pathway in mediating SINC-induced autophagy.
Methods: RAW 264.7 cells treated with recombinant SINC were examined for changes in expression levels of LC3-II, Beclin-1, phosphorylated and total ERK1/2 using Western blotting. The expression level of LC3 in the treated cells was detected using immunofluorescence analysis, and the formation of autophagosomes and autolysosomes was observed with transmission electron microscopy (TEM). The effect of pretreatment with U0126 (a specific ERK inhibitor) on the expression levels of LC3-II and Beclin-1 in RAW 264.7 cells exposed to different concentrations of SINC was examined using Western blotting, and LC3 puncta in the cells was detected with immunofluorescence analysis.
Results: The expression levels of LC3-II and Beclin-1 were the highest in RAW 264.7 cells treated with 2 μg/mL SINC for 12h. Immunofluorescence analysis showed exposure to SINC significantly increased the number of cells containing LC3 puncta, where the presence of autophagosomes and autolysosomes was detected. Exposure to 2 μg/mL SINC for 15 min resulted in the most significant increase of the ratios of p-ERK1/2/ERK1/2 in RAW 264.7 cells. Pretreatment of the cells with U0126 prior to SINC exposure significantly decreased the ratio of p-ERK1/2/ERK1/2, lowered the expression levels of LC3-II and Beclin-1, and decreased LC3 aggregation in the cells.
Conclusions: SINC exposure can induce autophagy in RAW 264.7 cells by activating the MAPK/ERK signaling pathway.
目的: 探讨鹦鹉热衣原体（Cps）SINC蛋白对宿主细胞自噬的影响，及MAPK/ERK信号通路在其中的调控作用。
方法: 用重组的SINC蛋白刺激小鼠单核巨噬细胞（RAW 264.7），Western blot检测LC3-II和Beclin-1的水平及ERK1/2的磷酸化程度，并用间接免疫荧光法检测SINC蛋白刺激后细胞LC3的水平，透射电镜检测自噬特殊结构。用50 μmol MEK1/2抑制剂U0126预处理RAW 264.7细胞后1 h，再用不同浓度SINC蛋白刺激不同时间，用间接免疫荧光检测LC3的水平，并用Western blot检测LC3-II和Beclin-1的表达水平。
结果: Western blot检测结果显示，以2 μg/mL SINC蛋白刺激RAW 264.7细胞12 h时，LC3-II和Beclin-1的表达水平显著上调，并达到峰值；间接免疫荧光检测发现SINC刺激后可使细胞内LC3荧光斑点数明显增多，透射电镜下可见更多的自噬小体和自噬溶酶体。2 μg/mL SINC蛋白刺激RAW 264.7细胞15 min时p-ERK1/2/ERK1/2比值明显上调；加入抑制剂U0126后，LC3-II和Beclin-1表达下调，LC3-II荧光斑点聚集减少。
结论: SINC蛋白通过激活MAPK/ERK信号通路促进RAW 264.7细胞发生自噬。
Keywords: Chlamydophila psittaci; MAPK/ERK signaling Pathway; SINC; autophagy.