mTOR/p70S6K通路在缺氧/复氧诱导的分化后PC12神经细胞凋亡中的作用

Role of mTOR/p70S6K signaling pathways in differentiated PC12 cell apoptosis induced by hypoxia/reoxygenation

目的探讨缺氧/复氧(H/R)诱导的分化后肾上腺嗜铬细胞瘤细胞(PC12)凋亡与哺乳动物雷帕霉素靶蛋白(mTOR)/核糖体蛋白S6激酶(p70S6K)通路的关系。方法实验分为正常对照(Con)组、H/R组、H/R+N-乙酰半胱氨酸(NAC)组、H/R+雷帕霉素(Rapa)组、NAC组及Rapa组。应用MTT法检测细胞活性,Hoechst 33342染色检测细胞凋亡率,用活性氧试剂盒检测细胞内活性氧(ROS)生成,Western blot检测Bax、Bcl-2、p-mTOR(Ser2448)及p-p70S6K(Thr389)的表达。结果H/R诱导分化后PC12神经细胞凋亡,Bax表达增加,Bcl-2表达降低,NAC或Rapa显著抑制H/R所致的细胞凋亡,逆转Bax及Bcl-2表达的变化;H/R增加分化后PC12神经细胞内ROS水平,NAC显著抑制H/R所致细胞内ROS水平的增加,而Rapa对细胞内ROS水平无明显影响;H/R增加分化后PC12神经细胞p-mTOR(Ser2448)及p-p70S6K(Thr389)的表达,NAC或Rapa显著抑制H/R所致细胞p-mTOR(Ser2448)及p-p70S6K(Thr389)表达的增加。结论H/R可能通过升高细胞内ROS水平,进而激活mTOR/p70S6K信号通路,诱导分化后PC12神经细胞凋亡。

Objective To determine the relationship between apoptosis of differentiated phaeochromocytoma(PC12)cells induced by hypoxia/reoxygenation(H/R)and mTOR/p70 S6 K signaling pathway.MethodsDifferentiated PC12 cells were randomly divided into 6 groups:a normal control group(Con group),a hypoxia/reoxygenation group(H/R group),a N-acetylcysteine+hypoxia/reoxygenation group(H/R+NAC group),a rapamycin+hypoxia/reoxygenation group(H/R+Rapa group),a N-acetylcysteine group(NAC group),and a rapamycin group(Rapa group).The cells activity was measured by MTT assay.The apoptosis of PC12 cells was detected by Hoechst 33342 staining.The activity of reactive oxygen species(ROS)was determined by fluorescent ROS detection kit.Western blot detected the protein expression related with the expression of Bax,Bcl-2 and mTOR/p70 S6 K pathways.Results H/R induced the apoptosis of differentiated PC12 cells,greatly increased the expression of Bax,and inhibited the expression of Bcl-2.NAC or Rapa significantly inhibited the apoptosis of differentiated PC12 cells induced by H/R and reversed the expression of Bax and Bcl-2.H/R increased the intracellular ROS level,NAC inhibited the ROS level increase induced by H/R,while Rapa had no significant effect on the intracellular ROS level.H/R increased the expression of mTOR/p-p70 S6 K(Thr389)in differentiated PC12 cells,while NAC or Rapa inhibited the expression of mTOR/p-p706 K(Thr389)in differentiated PC12 cells.Conclusion Hypoxia/reoxygenation may activate mTOR/p70 S6 K signaling pathways and induce the apoptosis of differentiated PC12 cells by increasing the ROS level in the cells.