人参二醇对APP-SH-SY5Y细胞中Tau蛋白磷酸化及Fyn/GluN2B信号通路的影响  

Effects of Panaxadiol on Tau Protein Phosphorylation and Fyn/GluN2B Signaling Pathway in APP-SHSY5Y Cells

作　　者：梁喜才 蔺莹[2] 肖洪贺 孔亮[2] 杨静娴[2] LIANG Xicai;LIN Ying;XIAO Honghe;KONG Liang;YANG Jingxian(Laboratory Animal Center,Liaoning University of TCM,Shenyang 110847,China;College of Pharmacy,Liaoning University of TCM,Liaoning Dalian 116600)

机构地区：[1]辽宁中医药大学实验动物中心,沈阳110847 [2]辽宁中医药大学药学院,辽宁大连116600

出　　处：《中国药房》2021年第12期1485-1491,共7页China Pharmacy

基　　金：辽宁省教育厅科学技术研究项目(No.L202036)。

摘　　要：目的:研究人参二醇(PD)对转染APP基因的人神经母细胞瘤细胞SH-SY5Y(APP-SH-SY5Y)中Tau蛋白磷酸化的影响及其作用机制。方法:采用分子对接技术验证PD与非受体酪氨酸激酶Fyn的靶向性。在体外培养SH-SY5Y细胞,构建APP-SH-SY5Y细胞模型和绿色荧光蛋白(GFP)-SH-SY5Y细胞模型(对照细胞),通过检测细胞中β淀粉样蛋白(Aβ1-42)的表达来验证APP-SH-SY5Y细胞模型是否构建成功。以GFP-SH-SY5Y细胞为对照,采用CCK-8法检测5、10、20、30、40μmol/L的PD和125、250、500、1000、2000 nmol/L的PP2(Fyn抑制剂,阳性对照)作用24 h对APP-SH-SY5Y细胞存活率的影响,以确定最佳给药浓度。以APP-SH-SY5Y细胞为对象,分别检测最佳浓度PD、PP2作用24后细胞中Ca2+浓度及磷酸化Tau蛋白(p-Tau)/Tau、磷酸化非受体酪氨酸激酶Src(p-Src)/Fyn、磷酸化谷氨酸受体2B(p-GluN2B)/GluN2B比值。结果:分子模拟对接结果显示,PD可靶向结合Fyn蛋白。与GFP-SH-SY5Y细胞比较,APP-SH-SY5Y细胞中Aβ1-42蛋白表达水平显著升高(P<0.01)。PD和PP2的最佳作用浓度分别为20μmol/L和500 nmol/L。20μmol/L PD、500 nmol/L PP2均可显著升高细胞的存活率,显著降低细胞中Ca2+浓度以及p-Tau/Tau、p-Src/Fyn、p-GluN2B/GluN2B比值。结论:PD可通过抑制Fyn/GluN2B信号通路来降低APP-SH-SY5Y细胞中Ca2+浓度及Tau蛋白的磷酸化水平。OBJECTIVE:To study the effects and mechanism of panaxadiol(PD)on Tau protein phosphorylation in the SH-SY5Y cells transfected with APP gene(APP-SH-SY5Y).METHODS:The target of PD and non-receptor tyrosine kinases Fyn was verified by molecular docking.SH-SY5Y cells were cultured in vitro,and the APP-SH-SY5Y cell models and green fluorescent(GFP)-SH-SY5Y cell model(control cell)was constructed.The expression of Aβ1-42 was detected so as to verify the success of APP-SH-SY5Y cell model.Taking GFP-SH-SY5Y cells as control,the effects of 5,10,20,30,40μmol/L PD and 125,250,500,1000,2000 nmol/L PP2(Fyn inhibitor,positive control)on the survival rate of APP-SH-SY5Y cells were detected by CCK-8 assay after treated for 24 h,so as to confirm the optimal concentration.The concentration of Ca2+,the ratio fophosphorylated Tau protein(p-Tau)/Tau,phosphorylatedn Src(p-Src)/Fyn and phosphorylated glutamate receptor2B(p-GluN2B)/GluN2B were detected in APP-SH-SY5Y cells after trated with the optimal concentration of PD and PP2 for 24 h.RESULTS:The results of molecular simulation docking showed that PD could target Fyn protein.Compared with GFP-SH-SY5Y cells,the protein expression of Aβ1-42 in APP-SH-SY5Y cell were increased significantly(P<0.01).The optimal concentration of PD and PP2 were 20μmol/L and 500 nmol/L.The 20μmol/L PD and 500 nmol/L PP2 could increase the survival rate of the cells and reduced the concentration of Ca2+,the ratio of p-Tau/Tau,p-Src/Fyn,and p-GluN2B/GluN2B.CONCLUSIONS:PD can reduce the the phosphorylation of Tau protein through inhibiting Fyn/GluN2B signaling pathway.

关 键 词：人参二醇 转染APP基因的人神经母细胞瘤细胞SH-SY5Y TAU蛋白 磷酸化 非受体酪氨酸激酶Fyn/谷氨酸受体2B信号通路 

分 类 号：R966[医药卫生—药理学]