The Regulatory Effects Of Two-pore-domain Potassium Channels TREK-1on Astrocytes And Neurons After Oxygen And Glucose Deprivation In Vitro

Objective:Under the hypoxic culture conditions, to investigate the influence of two-pore-domain potassium channel TREK-1on proliferation rate and glutamate uptake in primary cultured rat cortical astrocytes. To observe the functional status of astrocytes and the interaction between neurons and astrocytes after oxygen-glucose deprivation injury in order to determine the involved role of TREK-1channel that whether it is a new potential therapeutic target for cerebral ischemic diseases.Methods:Oxygen and glucose deprivation (OGD) model was established as in vitro model of cerebral ischemia to detect the survival and glutamate uptake of astrocytes. TREK-1channel agonist arachidonic acid and antagonist methionine were used to investigate the changes of proliferation rate and glutamate uptake before and after OGD. Astrocytes and neurons co-cultured were adopted to investigate the expression of TREK-1, NR2B, mGluR, GLT-1, caspase-3and ERK on primary cultured astrocytes and neurons, giving the agonist arachidonic acid and the antagonist methionine to observe the changes in the above indicators.Results:TREK-1was expressed in primary cultured astrocytes and neurons under normal and hypoxia conditions, the arachidonic acid (5μmol/L,10μmol/L,20μmol/L,40μmol/L) and methionine (1mmol/L,3mmol/L,10mmol/L,30mmol/L) had no effect on the survival of astrocytes under normal conditions, when astrocytes subjected to oxygen and glucose deprivation, arachidonic acid and methionine could increase the survival of astrocytes. Under oxygen and glucose deprivation conditions, astrocytes appeared compensatory increase in the uptake of glutamate, arachidonic acid could increase the glutamate uptake and methionine could inhibit the ability of glutamate uptake. Compared with neurons cultured alone, the co-cultured neurons had lower LDH release, giving methionine, the LDH release decreased from47.84U/L to23.95U/L. Under the conditions of OGD in co-cultured neurons, the mRNA content of TREK-1channel and GLT-1were reduced, NR2B mRNA and mGluR mRNA expression levels were increased. In co-cultured astrocytes, the mRNA content of TREK-1channel and mGluR were reduced, GLT-1mRNA and ERK mRNA expression levels were increased. Giving arachidonic acid could inhibit the expression of TREK-1, NR2B and ERK, giving methionine, the mRNA content of TREK-1channel and NR2B were reduced.Conclusions:1. Arachidonic acid and methionine improve the survival rate of astrocytes after OGD.2. Arachidonic acid and methionine increase co-cultured neuronal survival after OGD, suggesting that activity of TREK-1is related with the function of astrocytes and neurons.3. The improving of the survival of astrocytes by methionine has no significant relationship with the receptors, and their proliferation reasons will be clarified for further study.