氧糖剥夺再灌注对神经元Wnt5a/FZD2/Ca^(2+)通路的影响

The impact of oxygen-glucose deprivation/reperfusion on the Wnt5a/FZD2/Ca^(2+)pathway in neurons

目的:探讨氧糖剥夺再灌注(OGD/Rep)后神经元细胞内Wnt5a/FZD2/Ca^(2+)通路的变化。方法:采用OGD/Rep模型构建神经元细胞局部缺血/再灌注损伤,分正常组、再灌注(0、3、6、9、12、24 h)组。免疫荧光鉴定皮层神经元细胞,CCK-8法确定神经元细胞OGD时间,Western blotting检测非经典Wnt/Ca^(2+)信号通路中Wnt5a、FZD2、IP3-R和p-CaMKⅡ/CaMKⅡ蛋白的变化,免疫荧光和酶标仪检测细胞内Ca^(2+)和活性氧(ROS)的含量。结果:MAP-2免疫荧光鉴定原代皮层神经元细胞纯度较高。与正常组比较,神经元细胞OGD 3 h后细胞存活率为55.46%;与正常组比较,OGD/Rep后非经典Wnt/Ca^(2+)信号通路相关蛋白Wnt5a(3、6、9、12、24 h)、FZD2(3、6、9 h)、IP3-R(6、9、12、24 h)和p-CaMKⅡ/CaMKⅡ(6 h)的相对表达量上调(P<0.05~P<0.01);与正常组比较,OGD/Rep后神经细胞内Ca^(2+)和ROS的表达量均升高(P<0.01)。结论:神经元细胞OGD/Rep能激活Wnt5a/FZD2/Ca^(2+)信号通路。

Objective:To investigate the changes in the Wnt5a/FZD2/Ca^(2+)pathway in neuronal cells after oxygen glucose deprivation reperfusion(OGD/Rep).Methods:The OGD/Rep model was used to construct neuronal cell local ischemia/reperfusion injury,and cells were divided into normal group and reperfusion(0,3,6,9,12,24 h)group.Cortical neuron cells was identified by immunofluorescence.The OGD time was determined by CCK-8 method.Western blotting was used to detect the changes of Wnt5a,FZD2,IP3-R,and p-CaMKⅡ/CaMKⅡproteins in non-classical Wnt/Ca^(2+)signaling pathways.Immunofluorescence and enzyme-linked immunosorbent assay(ELISA)were used to detect the levels of intracellular Ca^(2+)and ROS.Results:MAP-2 immunofluorescence identification showed that the purity of primary cortical neurons was relatively high.Compared with the normal group,the survival rate of neuronal cells after 3 hours of OGD was 55.46%.Compared with the normal group,the relative expression levels of non classical Wnt/Ca^(2+)signaling pathway related proteins Wnt5a(3,6,9,12,24 h),FZD2(3,6,9 h),IP3-R(6,9,12,24 h),and p-CaMKⅡ/CaMKⅡ(6 h)were upregulated after OGD/Rep(P<0.05 to P<0.01).Compared with the normal group,the expression levels of Ca^(2+)and ROS in nerve cells increased after OGD/Rep(P<0.01).Conclusions:OGD/Rep in neuronal cells can activate the Wnt5a/FZD2/Ca^(2+)signaling pathway.