Ephrin-A3逆向通路对海马星形胶质细胞谷氨酸摄取能力的影响

Ephrin-A3 reverse signaling regulates glutamate clearance capacity of cultured hippocampal astrocytes

目的观察EphA4介导的ephrin-A3逆向通路激活对星形胶质细胞谷氨酸摄取能力的影响。方法采用原代培养的大鼠海马星形胶质细胞,使用免疫荧光双标法定位ephrin-A3在海马星形胶质细胞上的表达,Western blot法观察糖氧剥夺(oxygen-glucose deprivation,OGD)后星形胶质细胞ephrin-A3表达水平的变化,随后实验分为三组:空白对照组(不含星形胶质细胞),药物对照组(加入IgG-Fc)和EphA4组(加入ephrin-A3逆向通路激动剂预聚集化的EphA4-Fc),分别在正常及缺血条件下的特定时间点以谷氨酸浓度测定试剂盒检测不同干预组星形胶质细胞谷氨酸摄取能力的变化。结果 ephrin-A3高表达于海马星形胶质细胞,并在缺血后出现蛋白表达水平一过性上调。与对照组相比,EphA4干预组星形胶质细胞谷氨酸摄取能力较对照组明显下降。结论 Ephrin-A3高表达于海马星形胶质细胞并参与调节星形胶质细胞谷氨酸摄取能力。

Objective To observe the effect of ephrin-A3 reverse signaling mediated by EphA4 on astrocytic glutamate clearance capacity.The ephrin-A3 is highly expressed in hippocampal astrocytes and involved in the regulation of astrocytic glutamate clearance capacity.Methods Primary astrocytes were cultured from rat hippocampus.The expression of ephrin-A3 in cultured astrocytes was analyzed by double immunofluorescence staining.The expression change of ephrin-A3 protein in astrocytes after OGD was semi-quantittatively analyzed by Western blot.Primary astrocytes were divided into three groups:Glu(EBSS medium containing 100μmol/L glutamate acid without astrocytes),Control(EBSS medium containing 100μmol/L glutamate acid and preclusteredIgG-Fc)and EphA4group(EBSS medium containing 100μmol/L glutamate acid and preclustered EphA4-Fc as the agonist of ephrin-A3).Supernatants were harvested to measure glutamate concentrations at different time pointsunder normal and ischemic conditions using the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit.Results Ephrin-A3 was highly expressed in hippocampal astrocytes and increased transitorily after OGD.The astrocytic glutamate clearance capacity was significantly inhibited by activating ephrin-A3 and downstream reverse signaling with preclustered EphA4.Conclusion Ephrin-A3 is highly expressed in hippocampal astrocytes and is involved in the regulation of astrocytic glutamate clearance capacity.