神经节苷脂对染砷人脑皮层神经元的保护作用

 目的研究神经节苷脂对As2O3体外干预的皮质神经元的保护作用及可能机制。方法将人原代脑皮质神经细胞分为 5组,空白对照组、5 μmol·L^-1 As₂O₃干预组和5μmol·L^-1 As₂O₃加神经节苷脂50,100,200 μg·L^-1组,Fluo-3/AM荧光探针标记皮质神经元胞浆钙([Ca²⁺]i),激光共聚焦显微镜实时测定[Ca²⁺]_i的变化。磷基转移法测定细胞膜和胞浆的蛋白激酶C(PKC) 的活性。流式细胞仪检测细胞凋亡百分比。结果 5 μol·L^-1的As₂O₃,作用3 min,[Ca²⁺]_i开始升高,并随时间延长逐渐明显,As₂O₃加神经节苷脂200 μg·L^-1,作用9 min,[Ca²⁺]i才开始升高,且程度远没有As2O3组明显。5 μmol·L^-1的As₂O₃组的 PKC活化程度明显高于As₂O₃加神经节苷脂组,以细胞膜相明显。24 h凋亡率分别为:As₂O₃组78.5%,As₂O₃加神经节苷脂 200μg·L^-1组13.5%。结论神经节苷脂抑制As₂O₃引起的皮质神经元的[Ca²⁺]i升高和PKC活化,抑制细胞凋亡。并通过对细胞膜靶点的保护来实现对神经细胞的保护作用,这进一步证明砷剂最初的作用靶点是细胞膜。

Protective Effect of Ganglioside on Human Cortical Neuron Treated by Arsenic Trioxide

OBJECTIVE To investigate the protective effect and the possible mechanism of ganglioside on cortex neuron lesions induced by arsenic trioxide. METHODS Five groups of human cortex neuron isolated from human cortex brain tissue and incubated in vitro were enrolled, including the control,5 μmol·L^-1 As₂O₃ group, and 5 μmol·L^-1 As₂O₃+ ganglioside 50,100,200 μg·L^-1 groups. The cytosolic calcium [Ca²⁺]i of cortex neurons were loaded by fluorescent probe Fluo-3/AM, the changes of [Ca²⁺]i were monitored by laser confocal microscopy in real time, the activation of protein kinase C on these changes was assayed by phosphorus radioisotope assay, the percentage of apoptosis was analyzed by flow cytometry. RESULTS The [Ca²⁺]i of cortex neurons began to increase at the 3th minute in 5 μmol·L^-1 As₂O₃ group,and at the 9th min in As₂O₃+ ganglioside group. Moreover, the degree of [Ca²⁺]i increasing was lower in As₂O₃+ ganglioside group than that in As₂O₃ group. The PKC activation was higher in As₂O₃ group than that in As₂O₃+ gangliosied group, especially the PKC in cell membrane. The apoptosis rates of cortex neurons was 78.5% in As₂O₃ group, and 13.5% in As₂O₃+ gangliosied 200 μg·L^-1 group after being incubated in vitro for 24h.CONCLUSION Ganglioside inhibites the increasing of [Ca²⁺]i, the activation of PKC ,and the apoptosis of cortex neurons is induced by arsenic trioxide concentration-dependently, and the preventive target is the neuron membrane. It shows that the neuron membrane is the initial target of As₂O₃ interference.