Objective: To investigate the effect of osthole on the expression of amyloid precursor protein (APP) in Alzheimer's disease (AD) cell model and its mechanism.
Methods: The SH-SY5Y cell with over expression of APP was established by transfection by liposome 2000. The cells were treated with different concentrations of osthole, and the cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The differentially expressed miRNAs with and without osthole treatment were detected by miRNA array, and the target genes binding to the differentially expressed miRNAs were identified and verified by databases and Cytoscape. After the inhibitor of the differentially expressed miRNA was transduced into cells, the changes of APP and amyloid β (Aβ) protein were determined by immunofluorescence cytochemistry, and the mRNA expression of APP was determined by RT-PCR.
Results: The AD cell model with over expression of APP was established successfully. The results of MTT and LDH assay showed that osthole had a protective effect on cells and alleviated cell damage. miR-101a-3p was identified as the differentially expressed miRNA, which was binding to the 3'-UTR of APP. Compared with APP group, the expression of APP and Aβ protein and APP mRNA increased in the miR-101a-3p inhibitor group (all P<0.01), while the expression of APP and Aβ protein and APP mRNA decreased in the cells with osthole treatment (all P<0.01).
Conclusions: Osthole inhibits the expression of APP by up-regulating miR-101a-3p in AD cell model.
目的: 研究阿尔茨海默病细胞模型中蛇床子素抑制淀粉样前体蛋白（APP）的作用及机制。
方法: 脂质体2000转染建立APP高表达的SH-SY5Y细胞模型。利用MTT和乳酸脱氢酶（LDH）法检测蛇床子素对APP高表达细胞的存活率和损伤程度的影响。利用基因芯片技术筛选给予蛇床子素治疗后差异表达的微RNA（miRNA），然后通过生物信息学分析预测与差异表达miRNA靶向结合的基因。细胞内转入差异表达miRNA的抑制剂，然后采用免疫荧光细胞化学法观察细胞中APP、β淀粉样蛋白（Aβ）表达情况，RT-PCR法检测细胞中APP mRNA表达。
结果: 实验成功构建了APP高表达的阿尔茨海默病细胞模型。MTT和LDH法检测结果显示，蛇床子素对于APP高表达的细胞具有一定的保护作用，可以减轻细胞的损伤。miRNA-101a-3p是蛇床子素调控较明显的miRNA，其与APP的3'非翻译区靶向结合。与模型对照组比较，miR-101a-3p抑制剂组APP和Aβ蛋白荧光强度增加，APP mRNA表达量增加（均 P < 0.01）；加入蛇床子素后细胞中APP和Aβ蛋白荧光强度降低，APP mRNA表达量减少（均 P < 0.01）。
结论: 在阿尔茨海默病细胞模型中，蛇床子素通过上调miR-101a-3p抑制APP表达。