南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (06): 828-836.doi: 10.12122/j.issn.1673-4254.2020.06.09

• • 上一篇    下一篇

肠道病毒71型能诱导THP-1巨噬细胞的自噬和凋亡

罗文英，杨拉维，潘庆军，邱丽红   

  • 出版日期:2020-06-20 发布日期:2020-06-20
  • 基金资助:

Enterovirus 71 can induce autophagy and apoptosis of THP-1 macrophages

  

  • Online:2020-06-20 Published:2020-06-20

HTML

PDF

1155

摘要： 目的 探讨肠道病毒71型（EV71）诱导THP-1巨噬细胞自噬和凋亡及其相关信号通路。方法 设置EV71处理组和DMEM对照组，EV71处理组是用感染复数0.1感染THP-1巨噬细胞2、8和16 h，对照组是用DMEM培养基作为阴性对照即是0 h组。CCK-8检测EV71对THP-1巨噬细胞增殖和毒性的影响；荧光定量PCR法检测EV71在细胞内病毒核酸变化情况；透射电镜观察THP-1巨噬细胞内超微结构变化；Hoechst 33342染色法和AnnexinV/PI 双染法检测EV71诱导THP-1巨噬细胞凋亡的情况； 免疫印迹法分析EV71诱导THP-1巨噬细胞自噬和凋亡相关蛋白水平的变化；用3-MA和Ac-DEVD-CHO抑制剂分别检测EV71对THP-1巨噬细胞自噬和凋亡的影响。结果 EV71作用THP-1巨噬细胞2、8和16h的细胞存活率逐渐下降（P<0.05）；细胞内病毒核酸拷贝数逐渐增多（P<0.01）；可见细胞内自噬小体和病毒颗粒；总细胞凋亡率逐渐增加（P<0.01）；与对照组比较，EV71处理组LC3转化量（LC3-II/LC3-I）逐渐增多，而p62蛋白逐渐减少，cleaved caspase 3蛋白增多，Bcl-2和caspase-3蛋白减少（P<0.01），而cleaved caspase-8无差异（P>0.05）；与PBS对比，在8 h时3-MA明显抑制EV71诱导THP-1巨噬细胞自噬，LC3-II/LC3-I比值减少，而p62蛋白增多（P<0.01）；与DMSO对比，Ac-DEVD-CHO明显抑制EV71诱导THP-1巨噬细胞凋亡，凋亡率分别为15.5%和7.7%（P<0.01）。结论 EV71能感染THP-1巨噬细胞并在其中复制，且能诱导自噬和凋亡，其可能与激活LC3/p62自噬途径和caspase凋亡途径有关。

Abstract: Objective To investigate enterovirus 71 (EV71)-induced of autophagy, apoptosis and the related signaling pathways in THP-1 macrophages. Methods THP-1 macrophages were infected with EV71 at the multiplicity of infection (MOI) of 0.1 for 2, 8 or 16 h, and the cell proliferation and toxicity were analyzed using CCK-8 kit. The intracellular viral nucleic acid in THP-1 macrophages were detected by fluorescence quantitative PCR, and the ultrastructural changes of the cells were observed using transmission electron microscopy. Cell apoptosis induced by EV71 infection was detected using Hoechst 33342 staining and AnnexinV/PI double staining. Western blotting was performed for analysis of changes in autophagy and apoptosis of the cells and in the expressions of the related proteins. The effect of EV71 infection on apoptosis of THP-1 macrophages incubated with 3-MA and Ac-DEVD-CHO inhibitor for 2 h was assessed using Western blotting. Results EV71 infection significantly lowered the cell survival rate of THP-1 macrophages at 2, 8 h and 16 h after the infection (P<0.05). The total copy number of viral nucleic acid in THP-1 macrophages incubated with EV71 increased significantly and progressively over time (P<0.01). Intracellular autophagosomes and virions could be seen in EV71-infected THP-1 macrophages. The total apoptotic rate of the infected cell also increased significantly over time (P<0.01). EV71 infection significantly increased LC3 conversion (LC3-II/ LC3-I) and the expression of cleaved caspase 3 protein and decreased the protein expressions of p62, Bcl-2 and caspase-3 (P< 0.01) without causing obvious changes in cleaved caspase-8 (P>0.05). 3-MA significantly inhibited the EV71-induced autophagy of THP-1 macrophages and reduced LC3 conversion (LC3-II/LC3-I) and p62 protein expression at 8 h after EV71 infection (P<0.01). Compared with DMSO, Ac-DEVD-CHO significantly inhibited EV71-induced apoptosis of THP-1 macrophages (15.5% vs 7.7% , P<0.01). Conclusion EV71 not only can infect and replicate in THP-1 macrophages, but also induces autophagy and cell apoptosis possibly by activating LC3/p62 autophagy pathway and caspase apoptosis pathway.