Role Of MTOR Pathway In The Optic Nerve Head Astrocyte Activation

Objective:To investigate m TOR signal pathway related proteins expression in EGF induced optic nerve head astrocyte activation,and study the mechanism of RAPA regulating m TOR signal pathway downstream proteins.Methods:Cultivate primary astrocytes from the optic nerve head of adult male SD rats,then the cells were cultured in selective culture.Purified cells were identified by immunofluorescence targeting GFAP.Astrocytes treated with EGF were divided into three groups:control,20ng/ml,40ng/ml.The cell viability was detected through western blot after 1h stimulation.Western blot analysis detected the expression of phosphorylated-Akt,phosphorylated-m TOR and phosphorylated-S6 K.Then determine the optimal EGF concentration of our experiment.Using 10?M rapamycin pretreat of astrocytes,divided into four groups: normal control group,EGF group,RAPA group and EGF + RAPA group,then western blot analysis phosphorylated-Akt,phosphorylated-m TOR and phosphorylated-S6 K protein expression.Results:Immunofluorescence analysis showed that more then 95% of the cells stain positive of GFAP.The concentration of EGF that significantly increased the expression of GFAP is 40 ng/ml western blotting.In addition,p-Akt expression are raised after two different concentration of EGF stimulation,and both are statistically significant,p-m TOR and p-S6 K protein expression also increased,but only in the concentration of EGF 40ng/ml had statistical significance.After RAPA pretreated for 30 min,EGF+RAPA group of p-m TOR and p-S6 K expression quantity decreased obviously compared with the EGF group and had significant statistical difference(p<0.01).Expression of p-m TOR and p-S6 K decreased obviously when RAPA group compared with control group,and there were statistically significant(p < 0.01),but p-Akt protein expression has no obvious change.Conclusions : Primary optic nerve head astrocyte become activated by EGF induction,m TOR signaling pathways related protein p-Akt,p-m TOR and p-S6 K expression was uprugulated in the process of astrocyte activation.Rapamycin(m TOR inhibitors)inhibited p-m TOR and p-S6 K expression of astrocytes induced by EGF,and rapamycin might inhibit astrocyte activation through m TOR signaling pathway and participated in glaucoma optic nerve protection.