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

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、20μmol/L的终浓度下不会影响N2a-APP695细胞的活力。(2)ELISA检测结果显示天青B能够降低Aβ1-40(P=0.002)和Aβ1-42(P=0.036)的水平。(3)Western 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)。(4)BACE1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。

Objective:To explore the effects of azure B on metabolism of amyloid precursor protein(APP) and generation of amyloidbeta(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:(1)Azure B was not able to influence the cell viability of N2a-APP695 cells with final concentration of 5,10,15 and 20 μmol/L.(2)According to ELISA results,azure B was able to decrease levels of Aβ1-40(P=0.002) and Aβ1-42(P=0.036).(3)Western 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).(4)BACE1 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.