Studies On Mechanisms Of Apoptosis Of PC12Induced By H/R And Activation Of MTOR/P70S6K Pathway

ObjectiveTo study the relationship between apoptosis of PC12cell induced by hypoxia/reoxygenation (H/R) and mTOR/p70S6K pathway.MethodsPC12was cultured in vitro for6days and induced by NGF(50ng/ml). If the nervous process of PC12was twice in length than the diameter of cell body, it was considered as differentiated neuron-like cells, and cound be used in the next experiments. Differentiated PC12cells randomly divided into6groups:normal control group(treated with0.9%physiological saline), hypoxia/reoxygenation (H/R) group,NAC(a ROS scavenger) disposaled+H/R group,Rap(mTOR inhibitor) disposaled+H/R group,NAC group,Rap group.NAC disposaled+H/R group differentiated PC12cells were incubated in5mM NAC for1hour first and then were disposaled by hypoxia/reoxygenation. Rap disposaled+H/R group differentiated PC12cells were incubated in3μM rapamycin for1hour,and then treated by hypoxia/reoxygenation. NAC group and Rap group were not treated via hypoxia/reoxygenation.The activity of cells was measured by the MTT assay. The apoptosis of PC12was detected by Hoechst33342staining. The activity of reactive oxygen species (ROS) were determined by fluorescent ROS detection kit. Simultaneously,Western Blotting was used to investigate protein(p-P70S6K) expression which is relate to the mTOR/p70S6K pathway. All statistical analyses were performed using the SPSS13.0software package.Differences were considered significant if p values less than0.05were obtained.Results1. NGF could induce PC12cell to defferentiate into neuron like cells.2. Hypoxia/reoxygenation(H/R) could decrease the cell activity of differentiated PC12. Either NAC or Rap can inhibit significantly the degression of the activity of Differentiated PC12cells.3. Differentiated PC12cells were induced apoptosis. Either NAC or Rap can inhibit significantly the apoptosis of Differentiated PC12cells.4. Hypoxia/reoxygenation(H/R) increased the lever of ROS in Differentiated PC12cells. Either NAC or Rap can inhibit significantly the augmentation of the lever of ROS in Differentiated PC12cells.5. Hypoxia/reoxygenation(H/R) increased the expression of p-p70S-6K (Thr389) in Differentiated PC12cells. Either NAC or Rap can inhibit significantly the augmentation of the expression of p-p70S6K (Thr389) in Differentiated PC12cells.Conclusions1. Hypoxia/reoxygenation(H/R) induced apoptosis in PC12(pheo-chromocytoma) increased ROS via activing mTOR/p70S6K pathway signaling pathway.2. NAC and Rap protected against H/R-induced apoptosis and decreased protein(p-p70S6K) expression via inhibiting mTOR/p70S6K apoptotic signaling pathway.