天青B对淀粉样前体蛋白代谢和Aβ生成的影响

Effects of azure B on metabolism of amyloid precursor protein and generation of amyloid-β protein

摘要:

目的： 探究天青B对淀粉样前体蛋白（amyloid precursor protein，APP）代谢及β淀粉样蛋白（amyloid-β，Aβ）生成的影响。方法： 用天青B处理能稳定表达APP695基因的Neuron-2a细胞（N2a-APP695）。利用Western blot检测APP、可溶性APPα（soluble APPα，sAPPα）和β-分泌酶1（β-site APP cleaving enzyme 1，BACE1）的水平，利用ELISA检测Aβ的水平。采用BACE1酶活性检测试剂盒检测BACE1酶活性的抑制。采用t检验分析各组数据。结果： ①天青B在5、10、15、20 μmol/L的终浓度下不会影响N2a-APP695细胞的活力。②ELISA检测结果显示天青B能够降低Aβ_1-40（P=0.002）和Aβ_1-42（P=0.036）的水平。③Western blot检测结果显示天青B能够降低全长APP（P=0.018）和sAPPα（P=0.006）蛋白的水平，但不影响BACE1蛋白的水平（P>0.05）。④BACE1酶活性检测试剂盒检测结果显示天青B能降低BACE1的活性，其活性抑制率为66.0%（P=0.000）。结论： 天青B通过抑制APP的表达和BACE1的活性从而抑制Aβ的生成。

Abstract:

Objective: To explore the effects of azure B on metabolism of amyloid precursor protein (APP) and generation of amyloid-beta (Aβ) protein. Methods: Neuron-2a cells that stably expressed APP695 gene (N2a-APP695) was treated with azure B.Levels of APP, soluble APPα (sAPPα) and β-site APP cleaving enzyme 1 (BACE1) were measured by Western bolt. Aβ levels were measured by ELISA. The BACE1 enzyme inhibition was determined by using BACE1 activity assay kit. Data were analyzed by t test. Results: ①Azure B was not able to influence the cell viability of N2a-APP695 cells with final concentration of 5, 10, 15 and 20 μmol/L.②According to ELISA results, azure B was able to decrease levels of Aβ_1-40 (P=0.002) and Aβ_1-42 (P=0.036). ③Western blot demonstrated that azure B was able to reduce levels of full length APP (P=0.018) and sAPPα (P=0.006), but not influence the protein level of BACE1 (P>0.05).④BACE1 activity assaying results showed that the activity of the BACE1 enzyme was significantly inhibited by azure B, with inhibition rate of 66.0% (P=0.000). Conclusion: Azure B inhibits the generation of Aβ by inhibiting the expression of APP and the activity of BACE1.