八肽胆囊收缩素对谷氨酸诱导的海马星形胶质细胞GLT-1表达的影响

Effect of Cholecystokinin Octapeptide on Expression of GLT-1in Glutamate-induced Hippocampal Astrocytes

目的 探讨八肽胆囊收缩素(CCK-8)对谷氨酸(Glu)诱导的海马星形胶质细胞谷氨酸转运体1(GLT-1)表达的影响。方法 分离小鼠海马星形胶质细胞,检测不同浓度CCK-8对小鼠海马星形胶质细胞的毒性。将细胞分为对照组、Glu组、Glu+0.1μmol/L CCK-8组、Glu+0.5μmol/L CCK-8组和Glu+1.0μmol/L CCK-8组。MTT法检测细胞增殖,流式细胞术检测细胞凋亡,生化试剂盒检测细胞培养上清液中Glu含量,qRT-PCR检测GLT-1和谷氨酸/天冬氨酸转运体(GLAST)mRNA的表达,Western blot检测Caspase-3、Bcl-2、GLT-1和GLAST蛋白的表达,ELISA检测细胞培养上清中TNF-α的含量。结果 不同浓度的CCK-8对小鼠海马星形胶质细胞的增殖均无明显影响。与对照组相比,Glu组细胞增殖能力、细胞中Bcl-2蛋白、GLT-1、GLAST mRNA和蛋白表达水平显著降低(均P<0.01),细胞凋亡率、细胞外Glu含量、细胞中Caspase-3蛋白表达水平、细胞上清中TNF-α水平显著升高(均P<0.01);与Glu组相比,Glu+0.5μmol/L CCK-8组和Glu+1.0μmol/L CCK-8组细胞增殖能力、细胞中Bcl-2蛋白、GLT-1、GLAST mRNA和蛋白表达水平显著升高(均P<0.05),细胞凋亡率、细胞外Glu含量、细胞中Caspase-3蛋白表达水平、细胞上清中TNF-α水平显著降低(均P<0.01)。结论 CCK-8能够抑制Glu诱导的星形胶质细胞的炎症反应,促进GLT-1的表达,降低细胞外Glu的浓度,促进细胞增殖并抑制凋亡。

Objective To investigate the effect of cholecystokinin octapeptide(CCK-8)on glutamate transporter 1(GLT-1)expression in hippocampal astrocytes induced by glutamate(Glu).Methods The mouse hippocampal astrocytes were isolated and the toxicity of CCK-8at different concentrations on the mouse hippocampal astrocytes was detected.The cells were divided into control group,Glu group,Glu+0.1μmol/L CCK-8group,Glu+0.5μmol/L CCK-8group and Glu+1.0μmol/L CCK-8 group.MTT assay was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Biochemical kit was used to detect Glu content in the extracellular supernatant,and qRT-PCR was used to detect the mRNA expression of GLT-1 and glutamate/aspartate transporter(GLAST).The protein expressions of Caspase-3,Bcl-2,GLT-1and GLAST were detected by Western blotting,and the expression of TNF-αin the cell supernatant was detected by ELISA.Results CCK-8at different concentrations had no significant effect on the proliferation of mouse hippocampal astrocytes.Compared with the control group,the cell proliferation ability and the expression levels of Bcl-2protein,GLT-1and GLAST mRNA and protein in Glu group were significantly decreased(all P<0.01),the apoptosis rate,extracellular Glu content,Caspase-3protein expression level in cells and TNF-αlevel in cell supernatant were significantly increased(all P<0.01);Compared with the Glu group,the cell proliferation ability and the expression levels of Bcl-2protein,GLT-1and GLAST mRNA and protein in the Glu+0.5μmol/L CCK-8group and Glu+1.0μmol/L CCK-8group were significantly increased(all P<0.05),the apoptosis rate,extracellular Glu content,Caspase-3protein expression level in cells and TNF-αlevel in cell supernatant were significantly decreased(all P<0.01).Conclusion CCK-8can inhibit Glu-induced inflammatory response of astrocytes,promote the expression of GLT-1,reduce the concentration of extracellular Glu,promote cell proliferation and inhibit apoptosis.