氯化钴诱导的N2a细胞缺氧损伤模型的机制研究

Mechanism of cobalt chloride induced hypoxia on N2a cells

目的研究氯化钴(CoCl 2)诱导的N2a细胞缺氧损伤模型的作用机理,为永久性缺血性脑卒中提供治疗思路。方法用0~1200μmol/L的CoCl 2处理24 h建立N2a细胞缺氧损伤模型。根据细胞活力与细胞凋亡率确定缺氧模型的最适宜浓度,免疫荧光法测定细胞线粒体膜电位及胞内活性氧(ROS)的表达,蛋白印迹法测相关蛋白含量。结果300μmol/L的CoCl 2是建立缺氧损伤的最适宜浓度。细胞经过处理后,线粒体膜电位降低,ROS含量明显升高,IKK蛋白被激活,磷酸化水平增加,促进了NF-κB的入核,同时Bcl-2蛋白含量降低、Bax、C-Caspase-3含量上升(P<0.05)。结论CoCl 2对N2a细胞的缺氧损伤主要通过其促凋亡作用产生,胞内线粒体膜电位的下调,ROS含量的增加以及NF-κB的入核和促凋亡蛋白含量的增加共同作用诱导CoCl 2对N2a细胞的损伤。

Objective To evaluate the mechanism of cobalt chloride(CoCl 2)-induced hypoxic injury in N2a cells and provide treatment ideas for permanent ischemic stroke.Methods Different concentration of CoCl 2 was used to induce hypoxia in N2a cells as an in vitro hypoxia model.Cell viability rate and apoptosis rate were used to identify the appropriate concentration of CoCl 2.Immunofluorescence was conducted to determine the mitochondrial membrane potential(MMP)and ROS.Western blot was carried out to confirm the relative protein expression.Results 300μmol/L CoCl 2 was suitable for evaluation of neurological damage effect of cells in vitro.The content of ROS,the protein levels of IKK,p-IKK,NF-κB,p-NF-κB and downstream inflammation and apoptosis-related proteins Bax,Cleaved caspase-3 were elevated after CoCl 2 treatment for 24 h,but the levels of MMP and Bcl-2 were decreased(P<0.05).Conclusion The hypoxic injury of N2a cells induced by CoCl 2 is mainly due to its apoptotic effects,including the down-regulation of MMP,increase of ROS content,the enhancing of NF-κB into the nucleus and the increase the expression of apoptosis proteins.