The Effects Of Rhynchophylline On EAAT2 And NR2B MRNA Expression Of Ischemia-hypoxic Hippocampal Astrocyte In Rats

ObjectiveTo investigate the mRNA expression of hippocampal astrocyte glutamate transporter EAAT2 and NR2 B under the condition of ischemia-hypoxic, and the change of EAAT2 and NR2 B mRNA expression after dealing with Rhynchophylline,to discuss the role of Rhy on EAAT2 and NR2 B gene expression.MethodsCultured the newborn SD rat hippocampal astrocyte, and taken the 4th generational cell for cell growing on the glass slide then identified GFAP by immunofluorescence, the positive rate of astrocyte more than that 90% or 95% of the cells can be used in subsequent experiment. The experiment is divided into four parts:normal control group, ischemia- hypoxia group, the Rhy low-dose group(0.02mg/ml), the Rhy high dose group(0.2 mg/ml). Dealing with sodium dithionite in sugar-free Earle ’s balanced salt solution to establish ischemic-anoxic model, total RNA of each groups is extracted, real-time fluorescent quantitative PCR is used to detect the astrocyte glutamate transporter EAAT2 and NMDA receptor 2B subunit mRNA expression.Results1.Primary cultured astrocyte cell density can be up to 80%-90% after 7-9 days,and astrocyte is in the majority, it’s refraction is good, with irregular shape, soma large and flat, abundant cytoplasm, nucleus biased towards one side of the cell body;2, The expression of astrocyte EAAT2 and NR2 B mRNA in ischemia-hypoxia group and rhy group is increased significantly compared with normal control group; 3.In Rhy low dose group, astrocyte EAAT2 and NR2 B mRNA expression is down-regulated less obviously compared with ischemia-hypoxia group, astrocyte EAAT2 and NR2 B mRNA expression in Rhy high-dose group is significantly down-regulated compared with those of ischemia-hypoxia group and Rhy low-dose group.ConclusionsThe expression of hippocampal astrocyte EAAT2 and NR2 B mRNA are increased Significantly after ischemia-hypoxic,however,Rhy can down-regulated EAAT2 and NR2 B mRNA expression obviously after ischemia-hypoxic, and has the concentration dependence, indicated that Rhy has inhibitory effect on EAAT2 and NR2 B gene expression in ischemia-hypoxic astrocyte.