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

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),2μmol/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,Beclin-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。

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 protein-tagged pneumolysin(RFP-PLY) or/and GFP-LC3. The expression levels of phosphoinositide 3-kinase-Ⅰ/Ⅲ(PI3 K-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 PI3 K-I,Akt,and mTOR(tPI3 K-I=16.544,PPI3 K-I=0.004;tAkt=22.679,PAkt=0.002;tm TOR=19.503,Pm TOR=0.003),but there were no significant differences between the above groups in the expression level of PI3 K-Ⅲ or Beclin-1(tPI3 K-Ⅲ=3.572,PPI3 K-Ⅲ=0.070;tBeclin-1=0.799,PBeclin-1=0.508). Conclusion:Wild-type Sp may induce autophagy in A549 cells through the PI3 K-I/Akt/mTOR signaling pathway.