The Role Of GLT-1 In The Protection Of Resveratrol Against Glutamate Excitotoxicity In Rat Cerebral Cortical Neurons

Glutamate（Glu）is an important excitatory amino acid in the brain and it is essential for maintaining normal brain function.However,excessively high concentration of Glu has a strong excitotoxicity,which is an important mechanism of neuronal damage during cerebral ischemia.Therefore,how to reduce the concentration of Glu in the extracellular fluid during cerebral ischemia is an important strategy for relieving neuronal damage.The clearance of Glu relies mainly on two glutamate transporter located on the membrane of astrocytes:Glutamate transporter-1（GLT-1）and Glutamate-aspartate transporter（GLAST）.The function of GLT-1 and GLAST transporters depends on sodium and potassium pump（Na⁺-K⁺-ATPase,NKA）that can effectively regulate the intracellular and extracellular concentration of Na⁺and K⁺.Recent studies in our department have demonstrated that the upregulation of GLT-1 expression can enhance the uptake of Glu,preventing its excitotoxic effect,and then play a protective role in brain ischemia.Resveratrol（Res）is a kind of natural polyphenol.We have preliminarily clarified that Res has an obvious protective effect against cerebral ischemic injury.However,the concrete mechanism in anti-ischemia diseases is still not completely clear.Therefore,this study was intended to investigate whether GLT-1 participates in the protection of Res against brain ischemia and the role of NKA in this process.Part 1 Glutamate-induced excitotoxicity in rat cerebral cortical neurons in the presence and absence of astrocytesObjevtive:To investigate the excitotoxicity of Glu in rat cerebral cortical neurons in the presence and absence of astrocytes.Methods:Astrocyte cultures,neuron cultures and neuron-astrocyte cocultures were originated from the cerebral cortex within 24 h postnatal Wistar rats.The astrocytes were cultured in DMEM/F12 complete medium for approximately 6 days before being passaged to the next generation.After the cells of the second generation covered with culture plates,the astrocytes were identified by immunofluorescence staining and then the cultures were randomly divided into Control and Glu load groups.Control group was changed into DMEM/F12 basic medium and incubated for 6 h,12 h and 24 h,respectively,and then changed back to normal medium for subsequent culture for 24 h.Glu load groups were changed into DMEM/F12 basic medium and divided into three subgroups of Glu 0.5 mM,1 mM and 2 mM according to the added Glu load.Each group was incubated for 6 h,12 h and 24 h respectively,and then changed back to normal medium for subsequent culture for 24 h.The neuron cultures were conducted in Neurobasal+B27 serum-free medium for 7-9 days.The neurons were identified by immunofluorescence staining and and then the cultures were randomly divided into Control and Glu load groups.Control group was changed into Mg²⁺-free Locke’s buffer and incubated for 15 min,30 min and 60 min,respectively,and then changed back to normal medium for subsequent culture for 24 h.Glu load groups were changed into Mg²⁺-free Locke’s buffer and added glycine 10μM in and then divided into seven subgroups of Glu 10μM,25μM,50μM,100μM,200μM,500μM and 1000μM according to the added Glu load.Each group was incubated for 15 min,30 min and 60 min,respectively,and then changed back to normal medium for subsequent culture for 24 h.In astrocyte-neuron cocultures,the astrocytes of the second generation that were passaged to the glass slides were transferred into culture plates in which neurons were cultured for 9-11 days and then cocultured with neurons in Neurobasal+B27 serum-free medium for 1-2 days.The cocultures were randomly divided into Control and Glu load groups.Control group was changed into Mg²⁺-free Locke’s buffer and incubated for 0.5 h,1 h,and 2 h,respectively,and then changed back to normal medium for subsequent culture for 24 h.Glu load groups were changed into Mg²⁺-free Locke’s buffer and added glycine 10μM in and then divided into eight subgroups of Glu 10μM,25μM,50μM,100μM,200μM,500μM,1000μM and 2000μM according to the added Glu load.Each group was incubated for 0.5 h,1 h,and 2 h,respectively,and then changed back to normal medium for subsequent culture for 24 h.Cell viability was measured using the CCK-8（Cell Counting Kit-8）kit and the excitotoxic effect of Glu was analyzed at the predetermined time point of each group.Results:1.The identification rate of primary neurons was above 85%and that of astrocytes was above 95%.2.In neurons cultures and neuron-astrocyte cocultures,the number of dead cells was increased with the increase in concentration and prolonging in incubation time of Glu（P<0.05）.The IC₅₀（half maximal inhibitory concentration）of Glu after 15 min,30 min,and 60 min incubation of Glu was364.5μM,258.5μM and 138.3μM,respectively in neuron cultures,while in neuron-astrocyte cocultures,the IC₅₀0 of Glu after 0.5 h,1 h and 2 h was 932.8μM,789.3μM and 402.7μM,respectively,which was obviously larger than that in neuron cultures.In astrocyte cultures,no significant cell death was measured even after 24 h of Glu loading.Conclusions:In neurons cultures and neuron-astrocyte cocultures,the excitotoxicity induced by Glu to neurons has concentration and time dependence.In astrocyte-neuron cocultures,neurons are able to tolerate higher concentration of Glu than that in neuron cultures,suggesting astrocytes can protect neurons from glutamate-induced excitotoxicity.Part 2 The role of GLT-1 in the protection of resveratrol against glutamate excitotoxicity in rat cerebral cortical neuronsObjective:To explore the role of GLT-1 in the protection of Res against Glu excitotoxicity in rat cerebral cortical neurons and provide evidence for elucidating mechanism involved in the protective effect of Res on neurons against glutamate excitotoxicity.Methods:According to the method of neuron-astrocyte cocultures in the first part,the neuron-astrocyte cocultures were randomly divided into 6 groups:Control group,Glu group,Res group,Glu+Res group,Ouabain（Oua）group,Glu+Oua group.Each group was incubated in Mg²⁺-free Locke’s buffer containing the corresponding reagents（the concentration of Glu,Res and Oua was 1 mM,100μM,10 nM,respectively）for 1 h,and then changed back to normal medium for subsequent culture for 24 h.Cell viability was measured using the CCK-8 kit and Western Blot was used to analyze GLT-1 and NKA expression.Results:1.Compared with Control group,the number of dead neurons was increased obviously in Glu group（P<0.05）.Compared with Glu group,the number of dead neurons was reduced in Res+Glu group（P<0.05）and Oua+Glu group（P<0.05）.2.Compared with Control group,GLT-1 expression was downregulated significantly in Glu group（P<0.05）.However,in Res+Glu group,GLT-1expression was upregulated obviously compared with Glu group（P<0.05）.3.The total protein expression of the two subtypes of NKAα₁ and NKAα₂ and the membrane protein expression of NKAα₂ were downregulated in Glu group compared with Control group（P<0.05）.There was no significant change in NKAα₁ and NKAα₂ total protein expression in Res+Glu group compared with Glu group.However,NKAα₂ membrane protein expression was upregulated in Res+Glu group compared with Glu group（P<0.05）.Conclusion:In neuron-astrocyte cocultures,Res protects neurons against the excitotoxicity induced by Glu and its mechanism may be related to the upregulation of GLT-1 expression.Moreover,the upregulation of NKA membrane protein expression may be involved in this process.