鞘氨醇激酶1在丙烯酰胺致神经细胞损伤的保护作用

The study of the protection function of the sphingosine kinase 1 in the nerve cell damage caused by acrylamide

目的研究鞘氨醇激酶1(SphK1)过表达在丙烯酰胺(ACR)致神经细胞损伤中的保护作用以及影响。方法将纯度为99%的ACR用生长液制备成浓度为1.25、2.5 mmol/L溶液;将人神经母细胞瘤(SH-SY5Y)细胞分为对照组(NC组)、实验组和SphK1激活剂组。NC组加入(12-)十四酸佛波酯(-13-)乙酸盐(PMA)溶液[SphK1特异性激活剂,二甲基亚砜(DMSO)配制,终浓度为100 nmol/L]。实验组给予终浓度分别为1.25和2.5 mmol/L的ACR溶液,染毒24 h。SphK1激活剂组在实验组染毒浓度的基础上,每个染毒浓度分别加入PMA溶液,其他处理与实验组一致。各组采用免疫印迹(Western blot)法检测SphK1蛋白的表达含量;CCK-8检测SH-SY5Y细胞的增殖活性;Hoechst33342法观察神经细胞形态学改变;流式细胞术分析细胞的凋亡。结果与NC组比较,实验组和SphK1激活剂组细胞SphK1蛋白表达均降低,差异有统计学意义(P<0.05)。与实验组比较,SphK1激活剂组细胞在1.25和2.5 mmol/L浓度的SphK1蛋白表达均增高,差异均有统计学意义(P<0.05)。与NC组比较,实验组和2.5 mmol/L浓度SphK1激活剂组的细胞相对生长存活率均更低,差异有统计学意义(P<0.05)。与实验组比较,SphK1激活剂组细胞相对生长存活率均更高,差异有统计学意义(P<0.05)。随着染毒剂量的增加,实验组细胞在ACR 1.25 mmol/L浓度时呈现出早期凋亡、在ACR 2.5 mmol/L浓度时呈现出晚期凋亡的形态学特征。与NC组比较,实验组和SphK1激活剂组在ACR 2.5 mmol/L浓度时凋亡率差异均有统计学意义(P<0.05);与实验组比较,SphK1激活剂组在ACR 2.5 mmol/L浓度时细胞凋亡率更低,差异有统计学意义(P<0.05)。结论SphK1过表达可以对丙烯酰胺引起的神经细胞损伤起到保护作用。

Objective To study the protective effect and effect of SphK1 overexpression on the injury of nerve cells induced by acrylamide.Methods ACR with 99%purity was prepared into 1.25 mmol/L and 2.5 mmol/L solutions.SH-SY5Y cells were divided into control group(NC group),experimental group and SphK1 activator group.The experimental group was given ACR solution with final concentration of 1.25 mmol/L and 2.5 mmol/L respectively for 24 h.In the SphK1 activator group,on the basis of the exposure concentration of the experimental group,the SphK1 specific activator(12-)phorbol tetradecanoate(-13-)acetate(PMA)solution[prepared by dimethyl sulfoxide(DMSO),the final concentration was 100 nmol/l],and other treatments were the same as the experimental group.Control group(NC group)added PMA solution into normal cells.Western blot was used to detect the expression of SphK1 protein;CCK-8 was used to detect the proliferation of SH-SY5Y cells;hoechst33342 method was used to observe the morphological changes of nerve cells;flow cytometry was used to analyze the apoptosis of cells.Results Compared with NC group,the expression of SphK1 protein in the experimental group and the SphK1 activator group was significantly lower(P<0.05).Compared with the experimental group,the expression of SphK1 protein in each concentration of SphK1 activator group was increased,and the difference was statistically significant(P<0.05).In addition to 1.25 mmol/L SphK1 activator group,compared with NC group,the relative growth survival rate of experimental group and 2.5 mmol/L SphK1 activator group were lower,the difference was statistically significant(P<0.05).Compared with the experimental group,the relative survival rate of cells in the SphK1 activator group was higher,and the difference was statistically significant(P<0.05).With the increase of exposure concentration,the cells in the experimental group showed the morphological characteristics of early apoptosis at ACR 1.25 mmol/L and late apoptosis at ACR 2.5 mmol/L.Compared with NC group,the apoptosis rate of experimental group and SphK1 activator group at ACR 2.5 mmol/L was significantly different(P<0.05);compared with experimental group,the apoptosis rate of SphK1 activator group at ACR 2.5 mmol/L was lower,the difference was statistically significant(P<0.05).Conclusion The SphK1 excessive expression plays the protective function to the nerve cell damage caused by acrylamide.