Rapamycin Mediates Astrocyte Activity By Regulating MTOR Signaling And Reduces Retinal Ganglion Cell Apoptosis

Objective: The purpose of this study was to investigate the role of rapamycin in the regulation of astrocyte activity by regulating the m TOR pathway in astrocytes and further study the protective effect of rapamycin on retinal ganglion cells(RGC).mechanism.Methods: Cells induced by epidermal growth factor(EGF)were used as a model of cell activation.Cells were randomly divided into control group(CON),EGF group,drug group(RAPA group/LY group/PD group)and EGF+ drug group.In the four groups,the cells stimulated by EGF were treated with m TOR pathway inhibitor rapamycin(RAPA,10μM)and their two upstream inhibitors LY294002(50μM)and PD98059(25μM),and m TOR pathway was detected by protein immunoblotting.Two pathway-related proteins were detected: expression levels of p-Erk,p-Akt,p-S6 K,and p-m TOR.In vivo experiments,rats were randomly assigned to normal control(CON)group,ischemia-reperfusion(6h,3d,or 7d I/R)group,rapamycin(RAPA)group,and ischemia-reperfusion after treatment with rapamycin(6h,3d or 7d I/R+RAPA)group,and the expression of m TOR-associated proteins were detected:p-Akt,p-S6 K and p-m TOR.And the apoptosis-related protein in the retina of each group were detected by Western blotting: Caspase-3 expression levels;Immunofluorescence staining was used to detect: the expression levels of p-S6 and GFAP in the retina or optic nerve head;Astrocyte activation was assessed by immunostaining retinal flat mounts or radial sections with antibodies against GFAP and Vimentin,and we also used Western blots to detect the expression of GFAP.RGCs were retrogradely labeled with fluorescent gold and the cell viability was calculated.Results:1.Changes of p-m TOR and p-S6 K protein after EGF and RAPA treatment: compared with CON group,the EGF group was significantly increased(p<0.05),while RAPA group had no significant change(p>0.05);The EGF+RAPA group was significantly lower than the EGF group(p<0.01 or p<0.05).2.Changes of p-Erk and p-Akt in EGF-dependent astrocytes after treating with two inhibitors: EGF group was significantly higher than the CON group(p<0.01 or p<0.05);and compared with the EGF group,EGF+LY group and EGF+PD group were significantly lower(p<0.01 or p<0.001).Compared with EGF group,the expression of p-S6 K in EGF+LY group was significantly lower(p<0.01),and the expression of p-S6 K in EGF+PD group was not significantly changed(p>0.05).3.On the 3rd day of the model protein immunoblot: Compared with the I/R group,the expression of p-Akt(Thr308)in the RAPA+I/R group was significantly increased(p<0.01)and the expression of p-m TOR and p-S6 K was significantly increased.Significantly lower(p<0.01 or p<0.001).4.Immunofluorescence results of the 6h and 3d models showed that p-S6 expression levels were significantly higher in the I/R group than in the CON group,and decreased in the I/R+RAPA group compared with the I/R group.The results of GFAP on 6h and 3d models showed that the expression of GFAP in the I/R group increased(p<0.05)and the cell morphology changed compared with the CON group.Compared with the I/R group,the expression of GFAP in the I/R+RAPA group was decreased(p<0.05).The cellular morphology showed a tendency of retraction.And the morphology was closer to the normal group,but the effect of rapamycin on the expression of Vimentin was not significant.5.The results of fluorescence gold retrograde labeling showed that compared with the CON group,the survival of RGCs in the I/R group was significantly reduced(p<0.01).However,the RGC survival in the I/R+RAPA group was decreased but there was no significant statistical difference compared with CON group(p>0.05);The survival of RGCs in I/R+RAPA group was significantly higher than that in I/R group(P<0.05).Conclusion: The epidermal growth factor-induced activation of m TOR signaling pathway in astrocytes may be mainly activated by PI3 K / Akt pathway,but not by Erk / MAPK pathway.Rapamycin mediates the regulation of astrocyte activity through inhibition of the m TOR signaling pathway.Rapamycin effectively reduces the occurrence of retinal ganglion cell apoptosis and rapamycin may have the potential to treat diseases associated with retinal ganglion cell loss.