钙蛋白酶与谷氨酸的兴奋性毒性

Calpain: an executor of glutamate inducing neurotoxicity

目的研究钙蛋白酶在谷氨酸兴奋性毒性中的表达及钙蛋白酶抑制剂MDL-28170对神经元的保护作用。方法乳大鼠大脑皮质神经元体外培养7 d后用于实验,建立谷氨酸损伤模型,MTT法测定细胞活力,Hoechst33258核染色观察细胞凋亡的形态学改变,Western蛋白印迹法检测钙蛋白酶蛋白水平,免疫荧光染色观察钙蛋白酶在细胞内的表达及定位。结果谷氨酸引起80 kD与76 kD钙蛋白酶蛋白水平明显增加,且分布于凋亡的细胞核内。钙蛋白酶抑制剂MDL-28170明显提高细胞生存率及降低神经元凋亡百分比。结论谷氨酸兴奋性毒性引起钙蛋白酶大量激活,钙蛋白酶抑制剂MDL-28170对谷氨酸引起的神经元损伤具有保护作用。

Objective To investigate the expression of Calpain in cultured cortical neurons injured by glutamate and the protective effect of MDL-28170, an calpain inhibitor. Methods The cortical neurons were taken from new born rats and used for experiments 7 days after culture. The neurons were randomly divided into control, glutamate, MDL-28170 ＋ glutamate groups. MTF was used to measure cell viability. Neurons were dyed with Hoechst 33258 and observed under a fluorescence microscope to explore morphology changes. Western blotting was performed to investigate the protein level of ealpain. Immunostaining was used to investigate the localization of p^-calpain. Re- suits The protein level of 80 kD and 76 kD Calpain were upregulated after glutamate insult. MDL-28170 increased the cell viability and decreased the apoptotic rate. Conclusions Calpain is severely activated due to the glutamate neurotoxicity. MDL-28170 provides protective effect for the neurons injured by glutamate.