野生型肺炎链球菌溶血素通过PI3K-I/Akt/mTOR途径诱导A549细胞自噬

Wild-type Streptococcus pneumoniae pneumolysin induces autophagy in A549 cells through the PI3K-I/Akt/mTOR pathway

摘要:

目的：研究肺炎链球菌（Streptococcus pneumoniae，Sp）诱导人肺上皮细胞自噬发生的现象及其机制。方法：常规培养人肺上皮A549细胞与野生型Sp；将GFP-LC3真核载体转染A549细胞，分为实验组（Sp，MOI=30∶1）、阴性对照组[渥曼青霉素（wortmannin，WT），2 μmol/L 2 h，自噬抑制剂]、阳性对照组[雷帕霉素（rapamycin，Rapa），1 mmol/L 10 h，mTOR抑制剂]和参照组（WT和Sp共同处理），各组处理时间均为1、2、3和4 h；荧光显微镜观察各组自噬点形成，透射电镜观察各组细胞自噬体结构，Western blot检测各组微管相关蛋白轻链-Ⅰ/Ⅱ（microtuble-associated protein light chain 3-Ⅰ/Ⅱ，LC3-Ⅰ/Ⅱ）、磷酸化UNC-51-K激酶1（phosphorylated UNC-51-like kinase1，P-ULK1）和螯体1（sequestosome 1，P62）的表达；将溶血素表达载体RFP-PLY（red fluorescent protein-pneumolysin）、GFP-LC3或（和）RFP-PLY转染/共转染A549细胞，Western blot检测各组磷酸肌醇3-激酶-Ⅰ/Ⅲ（phosphoinositide 3-kinase-Ⅰ/Ⅲ，PI3K-Ⅰ/Ⅲ）、蛋白激酶B（protein kinase B，Akt）、Beclin-1蛋白（Beclin 1 protein，Be－clin-1）和哺乳动物雷帕霉素靶（mammalian target of rapamycin，mTOR）的表达。结果：处理A549细胞3 h后，与阴性对照组（WT）相比，Sp感染后，自噬点明显增多（t=41.313，P=0.001），电镜观察到典型的自噬体结构，LC3-Ⅱ蛋白表达明显上调（t=121.592，P=0.000）；A549细胞转染RFP-PLY处理3 h后，与阴性对照组（WT）相比，Sp感染同样观察到明显的自噬现象，PI3K-I、Akt蛋白和mTOR表达下调（tPI3K-I=16.544，PPI3K-I= 0.004；tAkt=22.679，PAkt=0.002；tmTOR=19.503，PmTOR=0.003），而PI3K-Ⅲ和Beclin-1水平无明显差异（tPI3K-Ⅲ=3.572，PPI3K-Ⅲ=0.070；tBeclin-1=0.799，PBeclin-1=0.508）。结论：野生型Sp诱导A549细胞自噬可能通过PI3K-I/Akt/mTOR信号途径。

Abstract:

Objective：To study the Streptococcus pneumoniae（Sp）-induced autophagy in human pulmonary epithelial cells and its mechanism. Methods：Human pulmonary epithelial A549 cells and wild-type Sp strain were cultured by routine methods. The A549 cells were transfected with the eukaryotic expression vector green fluorescent protein-light chain 3（GFP-LC3） and were divided into experimental group（Sp，MOI=30∶1），negative control group（wortmannin[an autophagy inhibitor]，2 μmol/L，2 h），positive control group（rapamycin[an mTOR inhibitor]，1 mmol/L，10 h），and reference control group（co-treated with wortmannin and Sp）；each group was treated for 1 h，2 h，3 h，or 4 h. Each group was observed for the formation of autophagic spots by fluorescence microscopy，for the structure of autophagosomes by transmission electron microscopy（TEM），and for the expression of microtubule-associated protein light chain 3-Ⅰ/Ⅱ（LC3-Ⅰ/Ⅱ），phosphorylated UNC-51-like kinase1（P-ULK1），and sequestosome 1（P62） by Western blot. The A549 cells were transfected/co-transfected with red fluorescent pro－tein-tagged pneumolysin（RFP-PLY） or/and GFP-LC3. The expression levels of phosphoinositide 3-kinase-Ⅰ/Ⅲ（PI3K-Ⅰ/Ⅲ），protein kinase B（Akt），Beclin 1 protein（Beclin-1），and mammalian target of rapamycin（mTOR） were determined by Western blot. Results：After treatment of the A549 cells for 3 hours，compared with the negative control group，the Sp-infected groups had a significantly increased number of autophagic spots（t=41.313，P=0.001） and a significantly up-regulated expression level of LC3-Ⅱ（t=121.592，P=0.000），with typical structures of autophagosomes observed under the TEM. After transfection of the A549 cells by RFP-PLY for 3 hours，compared with the negative control group，the Sp-infected groups also showed obvious autophagy with significantly down-regulated expression of PI3K-I，Akt，and mTOR（tPI3K-I=16.544，PPI3K-I=0.004；tAkt=22.679，PAkt=0.002；tmTOR=19.503，PmTOR=0.003），but there were no significant differences between the above groups in the expression level of PI3K-Ⅲ or Beclin-1（tPI3K-Ⅲ=3.572，PPI3K-Ⅲ=0.070；tBeclin-1=0.799，PBeclin-1=0.508）. Conclusion：Wild-type Sp may induce autophagy in A549 cells through the PI3K-I/Akt/mTOR signaling pathway.