The MTOR/Akt Pathway Is Involved In Regulating Astrocyte Growth And GLT-1 Upregulation During Oxygen-glucose Deprivation And Reoxygenation

Objective:Astrocyte specific glutamate transporter subtype 2(excitatory amino acid transporter 2,GLT-1)plays an important role in influencing glutamate excitatory toxicity and preventing the death of excitatory toxic neurons.Although it has been confirmed that mammalian target of rapamycin(mTOR)-Akt-nuclear factor-B(NF-κB)signaling cascade is involved in the upregulation of astrocyte GLT-1 in oxygen-glucose deprivation,it is not fully clear whether mTOR-Akt pathway is involved in the astrocyte growth and GLT-1 upregulation in oxygen-glucose deprivation and reoxygenation.Therefore,in this study,we aimed to assess the effect of rapamycin on astrocytes and the regulatory mechanisms of the growth of astrocytes and GLT-1 expression under oxygen-glucose deprivation and reoxygenation(OGD/R)conditions.Methods:(1)Culture of astrocytes and identification of astrocyte purity by immunofluorescence;Neuronal cell culture and immunofluorescence identification,neuronal supernatant collection.(2)The morphology of astrocytes was observed by immunofluorescence technique,cell apoptosis was examined by flow cytometry,and the content of target protein was detected by Western blot.Results:(1)mTOR specific inhibitor rapamycin or Akt specific inhibitor triciribine resulted in shorter and less axial mutation of astrocytes,smaller cell volume and nucleus,and increased apoptosis(P<0.05);Western blot analysis showed that GLT-1,phosphorylated Akt-Ser473(pAkt),cleaved caspase-3 increased significantly and phospho-S6-Ser240/244(pS6)significantly decreased in rapamycin treated group(P<0.05);GLT-1,pAkt,pS6 decreased in triciribine treatment group(P<0.05).(2)After oxygen-glucose deprivation and reoxygenation(OGD/R)treatment,the axial mutation of astrocytes decreased and apoptosis increased significantly(P<0.05).Western blot analysis showed that OGD/R significantly increased the expression of GLT-1,pAkt in astrocytes(P<0.05),and pS6 significantly decreased(P<0.05).After the addition of mTOR inhibitor rapamycin,the axial mutation of astrocytes was shorter and less,apoptosis and cleaved caspase-3 were further increased(P<0.05),while GLT-1 and pAkt changes were not statistically significant(P>0.05).(3)Cultured astrocytes were pretreated with the Akt inhibitor triciribine(10μM)for 24 h to inhibit Akt before OGD/R.Then the morphology of astrocytes was observed and found that the axis mutation of astrocytes was shorter and less.Western blot analysis showed that OGD/R induced GLT-1 and pAkt increased(P<0.05),pS6 significantly decreased(P<0.05).Triciribine treatment significantly reversed OGD/R induced GLT-1 elevation(P<0.05),cleaved caspase-3 was further increased,and pS6 was further decreased(P<0.05).Conclusion:In this study,astrocytes were cultured in vitro and supernatant of neurons was added to induce specific glutamate transporter 2(excitatory amino acid transporter 2,GLT-1)expression.The addition of rapamycin,an mTOR inhibitor,or triciribine,an Akt inhibitor,demonstrated that mTOR/Akt is involved in astrocyte growth and GLT-1 upregulation.At the same time,oxygen-glucose deprivation and reoxygenation(OGD/R)were used to simulate the pathological process of ischemia-reperfusion in vivo.It was found that OGD/R resulted in shorter and fewer axial mutations in astrocytes and increased apoptosis,and increased GLT-1,pAkt,and cleaved caspase-3 expression.The addition of rapamycin,an mTOR inhibitor,or triciribine,an Akt inhibitor,demonstrated that Akt/mTOR pathway is involved in astrocyte growth and GLT-1 expression in oxygen-glucose deprivation and reoxygenation.Our results suggest that astrocytes may adapt to short-term ischemic-reperfusion injury by regulating growth and GLT-1 upregulation.