极长链饱和脂肪酸对人神经母细胞瘤细胞Tau蛋白磷酸化及膜流动性的影响

Effect of very-long-chain saturated fatty acids on Tau protein phosphorylation and membrane fluidity in human neuroblastoma cells

目的观察极长链饱和脂肪酸对人神经母细胞瘤细胞SH-SY5Y Tau蛋白磷酸化和膜流动性的影响,探讨其在阿尔茨海默病(AD)发病中的作用。方法取对数生长期的人神经母细胞瘤细胞SH-SY5Y,随机分为对照组、C220组、C240组,对照组细胞常规培养,C220组、C240组细胞分别加入含10μmol·L^(-1)极长链饱和脂肪酸C220、C240的培养液,培养24 h后收集细胞,采用Western blot法检测各组细胞中总Tau蛋白、丝氨酸396位点磷酸化Tau蛋白(p-Tau-ser396)、糖原合成酶激酶3β(GSK-3β)及GSK-3β丝氨酸9位点磷酸化蛋白(p-GSK-3β-Ser9)的表达水平;采用硫代巴比妥酸法测定各组细胞中丙二醛(MDA)水平;并采用荧光漂白恢复技术测定细胞膜荧光恢复率和扩散系数,评价细胞膜流动性。结果3组SH-SY5Y细胞总Tau蛋白水平比较差异无统计学意义(F=1.807,P>0.05)。3组SH-SY5Y细胞p-Tau-ser396水平比较差异有统计学意义(F=18.397,P<0.05);其中C220组和C240组SH-SY5Y细胞p-Tau-ser396水平显著高于对照组(P<0.05),C240组SH-SY5Y细胞p-Tau-ser396水平显著高于C220组(P<0.05)。3组SH-SY5Y细胞GSK-3β蛋白水平比较差异无统计学意义(F=0.351,P>0.05)。3组SH-SY5Y细胞p-GSK-3β-Ser9水平比较差异有统计学意义(F=13.330,P<0.05);其中C220组、C240组SH-SY5Y细胞p-GSK-3β-ser9水平显著低于对照组(P<0.05),C220组与C240组SH-SY5Y细胞p-GSK-3β-ser9水平比较差异无统计学意义(P>0.05)。C240组SH-SY5Y细胞MDA水平显著高于对照组和C220组(P<0.05);对照组与C220组SH-SY5Y细胞MDA水平比较差异无统计学意义(P>0.05)。对照组、C220组、C240组SH-SY5Y细胞的荧光恢复率和扩散系数有降低趋势,但3组SH-SY5Y细胞的荧光恢复率和扩散系数比较差异无统计学意义(F=3.891、3.649,P>0.05)。结论极长链饱和脂肪酸C220、C240可促进Tau蛋白过度磷酸化,诱导细胞氧化损伤,对细胞膜流动性也有降低趋势,极长链饱和脂肪酸可能是引起AD发病的因素之一。

Objective To investigate the effect of very-long-chain saturated fatty acids on Tau protein phosphorylation and membrane fluidity in human neuroblastoma SH-SY5Y cells,and to explore its role in the pathogenesis of Alzheimer′s disease(AD).Methods Human neuroblastoma SH-SY5Y cells in logarithmic growth phase were randomly divided into control group,C22 GA6FA 0 group,and C24 GA6FA 0 group.Cells in the control group were routinely cultured,while cells in the C22 GA6FA 0 and C24 GA6FA 0 groups were treated with culture medium containing 10μmol·L^(-1)very-long-chain saturated fatty acids C22 GA6FA 0 and C24 GA6FA 0,respectively.After 24 hours of incubation,cells were collected.The expression levels of total Tau protein,phosphorylated Tau protein at serine 396 site(p-Tau-ser396),glycogen synthase kinase 3β(GSK-3β),and phosphorylated GSK-3βprotein at serine 9 site(p-GSK-3β-Ser9)in cells of each group were detected by using Western blot.The malondialdehyde(MDA)level in cells of each group was determined by using the thiobarbituric acid method.The fluorescence recovery rate and diffusion coefficient of cell membranes were measured by using fluorescence recovery after photobleaching technique,and the fluidity of cell membranes was evaluated.Results The total Tau protein level in SH-SY5Y cells showed no statistically significant difference among the three groups(F=1.807,P>0.05).However,there was a statistically significant difference in the level of p-Tau-ser396 in SH-SY5Y cells among the three groups(F=18.397,P<0.05).Specifically,the level of p-Tau-ser396 in SH-SY5Y cells in the C22 GA6FA 0 and C24 GA6FA 0 groups was significantly higher than that in the control group(P<0.05),and the level of p-Tau-ser396 in SH-SY5Y cells in the C24 GA6FA 0 group was significantly higher than that in the C22 GA6FA 0 group(P<0.05).There was no statistically significant difference in the GSK-3βprotein level in SH-SY5Y cells among the three groups(F=0.351,P>0.05).However,there was a statistically significant difference in the level of p-GSK-3β-Ser9 in SH-SY5Y cells among the three groups(F=13.330,P<0.05).Specifically,the level of p-GSK-3β-ser9 in SH-SY5Y cells in the C22 GA6FA 0 and C24 GA6FA 0 groups was significantly lower than that in the control group(P<0.05),and there was no statistically significant difference in the level of p-GSK-3β-ser9 in SH-SY5Y cells between the C22 GA6FA 0 group and C24 GA6FA 0 group(P>0.05).The MDA level in SH-SY5Y cells in the C24 GA6FA 0 group was significantly higher than that in the control group and C22 GA6FA 0 group(P<0.05);there was no statistically significant difference in the MDA level in SH-SY5Y cells between the control group and C22 GA6FA 0 group(P>0.05).The fluorescence recovery rate and diffusion coefficient of SH-SY5Y cells in the C22 GA6FA 0 and C24 GA6FA 0 groups showed a decreasing trend compared to the control group,but there was no statistically significant difference in the fluorescence recovery rate and diffusion coefficient of SH-SY5Y cells among the three groups(F=3.891,3.649,P>0.05).Conclusion Very-long-chain saturated fatty acids C22 GA6FA 0 and C24 GA6FA 0 can promote hyperphosphorylation of Tau protein,induce cellular oxidative damage,and tend to reduce the fluidity cell membranes.Very-long-chain saturated fatty acids may be one of the factors that cause the onset of AD.