BIN1基因对阿尔茨海默病细胞模型Tau磷酸化及p38MAPK/NF-κB通路的影响

Influence of Bin1 Gene on Tau Phosphorylation and Signaling Pathway of p38MAPK/NF-κB in Alzheimer’s Disease Cell Model

目的:探讨BIN1基因对AD细胞模型Tau磷酸化及p38MAPK/NF-κB通路的影响。方法:①构建BIN1过表达质粒与BIN1的小干扰RNA(siRNA),qPCR检测BIN1表达效果,选取效果最佳片段转染PC12细胞。②通过Aβ25-35诱导转染的PC12细胞建立AD细胞模型,利用qPCR方法检测Tau和p-Tau的mRNA转录水平,MTT法及Caspase3活性检测判断AD模型是否建立成功。③实验分为4组:空白组,AD模型组,过表达组,siRNA组。④Westernblot法检测各组细胞BIN1表达水平及Tau、磷酸化Tau(p-Tau,Ser396位点)、p38MAPK、核因子κB(NF-κB)蛋白的表达;⑤生化试剂盒检测各组细胞上清中一氧化氮(NO)水平;ELISA法检测各组细胞上清中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β等炎性因子表达。结果:①BIN1过表达质粒显著上调BIN1的转录水平(P<0.01),siRNA明显下调BIN1的转录水平表达(P<0.01)。②MTT实验结果显示,AD模型组细胞存活率较空白组显著下降(P<0.01);Caspase3活性检测结果显示,AD模型组细胞Caspase3活性较空白组显著增加(P<0.01)。③Westernblot检测结果显示,BIN1在AD模型组、过表达组中表达明显高于空白组(P<0.01),在siRNA组中表达明显低于空白组(P<0.01);空白组、siRNA组、AD模型组、过表达组中Tau、p-Tau(Ser396)蛋白表达依次增多,组间两两比较差异均有统计学意义(P<0.05)。④与AD模型组相比较,过表达组中p38MAPK和NF-κB蛋白水平明显上升(P<0.05),siRNA组中p38MAPK和NF-κB蛋白水平明显降低(P<0.05)。⑤空白组、siRNA组、AD模型组、过表达组中IL-1β、TNF-α、NO水平依次增多,单因素方差分析及组间两两比较,差异均有统计学意义(P<0.01)。结论:BIN1可能通过影响p38MAPK/NF-κB信号通路参与了tau蛋白的异常磷酸化过程,导致AD病程持续发展。

Objective:To investigate the effect of Bin1 gene on Tau phosphorylation and the p38MAPK/NF-κB pathway in the Alzheimer’s disease(AD)cell model.Methods:(1)The Bin1 overexpression plasmid and Bin1 siRNA were constructed respectively.The expression of Bin1 was detected by qPCR,and the fragment with the best effect was selected to transfect PC12 cells.(2)The transfected PC12 cells were induced by Aβ25-35 to establish the AD cell model.The mRNA expression of Tau and p-Tau was detected by qPCR to verify the successful construction of the AD model cells.The viability of model cells was detected by MTT and caspase-3.(3)The experiment included 4 groups:blank group,AD model group,overexpression group,and siRNA group.(4)Western blot was used to detect the expression level of Bin1 and the protein levels of Tau,phosphorylated Tau(p-tau,ser396 site),p38MAPK,and NF-κB in each group.(5)The level of NO in the cell supernatant was detected by a biochemical kit.Inflammatory factors such as TNFαand IL-1βin the cell supernatant were detected by ELISA.Re⁃sults:(1)Bin1 overexpression plasmid significantly up-regulated Bin1 protein expression(P<0.01),and siRNA significantly down-regulated Bin1 protein expression(P<0.01).(2)MTT assay showed that cell survival in the AD model group was significantly lower than that in the blank group(P<0.01).Detection of caspase-3 activity showed that it was significantly higher in the AD model group than that in the blank group(P<0.01).(3)Western blot revealed that Bin1 expression was significantly greater in the AD model group and overexpression group than that in the blank group(P<0.01)and significantly lower in the siRNA group than that in the blank group(P<0.01).The protein expression of Tau and p-Tau(Ser396)in the blank group,siRNA group,AD model group,and overexpression group increased in the order listed,and pairwise comparison showed that the difference was statistically significant(P<0.05).(4)Compared with that in the AD model group,the expression of p38MAPK and NF-κB in the overexpression group increased significantly(P<0.05);the expression of p38MAPK and NF-κB in the siRNA group was decreased significantly(P<0.05).(5)The expression levels of IL-1β,TNF-α,and NO in the blank group,siRNA group,AD model group,and overexpression group were increased in that order,and the differences were statistically significant(P<0.01).Conclusion:Bin1 may affect the signaling pathway of p38MAPK/NF-κB and be involved in the abnormal phosphorylation of tau protein,which leads to the sustained progression of AD.