Aβ_(1-42)诱导的阿尔茨海默病细胞模型中神经损伤及凋亡的机制

Mechanism of neurologic injury and apoptosis in Aβ_(1-42)-induced cell model of Alzheimer′s disease

目的 使用Aβ_(1-42)诱导HT22细胞建立阿尔茨海默病(Alzheimer′s disease, AD)细胞模型,并探讨该模型中Aβ导致的神经损伤及细胞凋亡的靶向信号通路。方法 分别使用不同浓度的Aβ_(1-42)(0,0.625,1.25,2.5,5,10,20,40μmol/L)干预HT22细胞,确定建立AD细胞模型的最适浓度;随后在最适浓度干预后6,12,24,48 h观察细胞形态,检测细胞存活率,确定建立AD细胞模型的最适时间。将细胞分为AD组(20μmol/L Aβ_(1-42)+PBS)和NC组(PBS),生长对数期的HT22细胞分别干预24 h,进行后续实验。Western blot检测细胞凋亡指标(cleaved-Caspase-3)、神经营养因子(NRN1)、神经突触指标(SYP、MAP-2)的表达情况;免疫荧光检测MAP-2的表达及分布情况。生物信息学的方法预测AD相关信号通路,并通过Western blot验证ERK信号通路的磷酸化水平。结果 Aβ_(1-42)诱导HT22细胞建立AD细胞模型的最适浓度为20μmol/L,最适时间为24 h。与NC组相比,AD组cleaved-Caspase-3表达升高(P<0.01),NRN1表达降低(P<0.000 1),SYP、MAP-2表达降低(P<0.01)。生物信息学的分析显示,ERK1(MAPK3)与APP和CASP9基因表达呈正相关(r=0.634,P<0.01;r=0.513,P<0.05)。Western blot显示,与NC组相比,AD组ERK磷酸化水平较高(P<0.01)。结论 Aβ_(1-42)诱导HT22细胞建立的AD细胞模型中,引起神经损伤及细胞凋亡的机制可能通过激活ERK信号通路来实现。

Objective To establish a cell model of Alzheimer′s disease(AD)in HT22 cells induced by Aβ_(1-42),and explore the targeted signaling pathway of Aβ-induced neuronal injury and cell apoptosis in this model.Methods HT22 cells were treated with different concentrations of Aβ_(1-42)(0,0.625,1.25,2.5,5,10,20,40μmol/L)to screen the optimal concentration for AD cell model.Subsequently,the cell morphology was observed and the cell viability was detected at 6,12,24,48 h after the intervention of the optimal concentration to screen the optimal time for AD cell model.HT22 cells in the logarithmic phase of growth were intervened with 20μmol/L Aβ_(1-42)+PBS and PBS for 24 h for subsequent experiments in AD group and NC group,respectively.The expressions of apoptosis indicator(cleaved-Caspase-3),neurotrophic factor(NRN1),and synaptic indicators(SYP,MAP-2)were detected by Western blot.The expression and distribution of MAP-2 was detected by immunofluorescence.The AD-related signaling pathway was predicted by bioinformatics,and the phosphorylation level of ERK signaling pathway was verified by Western blot.Results The optimal concentration of Aβ_(1-42) to establish an AD HT22 cell model was 20μmol/L,and the optimal time was 24 h.Compared with NC group,the expression of cleaved-Caspase-3 was elevated(P<0.01),NRN1 was decreased(P<0.0001),and the expressions of SYP,MAP-2 were decreased in AD group(P<0.01).Bioinformatics analysis showed that ERK1(MAPK3)was positively correlated with APP expression(r=0.634,P<0.01)and CASP9 expression(r=0.513,P<0.05).Western blot showed that compared with NC group,the level of ERK phosphorylation was increased in AD group(P<0.01).Conclusion In the AD model of HT22 cells,neuronal injury and cell apoptosis may be achieved by activating ERK signaling pathway.