葛根素对阿尔茨海默病细胞模型Aβ蛋白的抑制作用

Puerarin Against Alzheimer’s Disease by Inhibition β-amyloid Protein

关键词:  阿尔茨海默病  葛根素  β淀粉样蛋白

Key words:  Alzheimer’s disease  puerarin  amyloid

摘要:

目的 在过表达β淀粉样前体蛋白的SH-SY5Y细胞上(SH-SY5Y/APP695)观察葛根素对β淀粉样蛋白(β-amyloid protein，Aβ)生成的作用，探讨其防治阿尔茨海默病的机制。方法 葛根素2.5，5和10 μmol·L^-1处理SH-SY5Y/APP细胞24 h，MTT法检测细胞活力，ELISA试剂盒测定细胞外Aβ_1-40和Aβ_1-42水平；Western blot蛋白质印迹法检测APP及β-分泌酶的蛋白表达变化；荧光法测β-分泌酶的活性；RT-PCR法检测β-分泌酶转录的变化。结果 葛根素可剂量依赖性的减少SH-SY5Y/APP695细胞外Aβ_1-40、Aβ_1-42的水平；酶活性分析显示2.5，5和10 μmol·L^-1的葛根素分别抑制了15%，30%和40%β-分泌酶的活性。Western blot印迹结果显示，葛根素能剂量依赖性抑制β-分泌酶蛋白表达，2.5，5和10 μmol·L^-1的葛根素使β-分泌酶的蛋白表达分别减少至82%，71%和45%，与空白对照组比较差异均具有统计学意义(P<0.05)。结论 葛根素通过下调β-分泌酶蛋白的表达、抑制β-分泌酶的活性减少Aβ的形成，这可能是葛根素防治阿尔茨海默病作用的重要机制之一。

Abstract:

OBJECTIVE To investigate the effects of puerarin on β-amyloid precursor protein (APP) processing on SH-SY5Y cells transfected with human APP695 cDNAs and to explore the mechanism of puerarin on Alzheimer’s disease(AD). METHODS SH-SY5Y cells transfected with human APP695 cDNAs were cultured with 2.5, 5, 10 μmol·L^-1 puerarin，and cell survival was detected by MTT assay. The levels of Aβ_1-40 and Aβ_1-42 in conditioned were measured medium by using ELISA kit. The expressions of APP and b-secretase were detected by Western blot. The activity of secretase were measured by using a fluorescence spectrophotometer, the mRNA transcription of β-secretase were detected by RT-PCR. RESULTS Puerarin could dose-dependently reduce the levels of extracellular Aβ_1-40 or Aβ_1-42. After treatment with 2.5, 5 and 10 μmol·L^-1 puerarin for 24 h, the activity of β-secretase was decreased 15%, 30% and 40%, respectively(P<0.05). Compared with control group, puerarin 2.5, 5 and 10 μmol·L^-1 decreased β-secretase protein express to 82%, 71% and 45%, respectively(P<0.05). CONCLUSION Puerarin decreases Aβ levels by inhibit b-secretase expression and β-secretase activity, which may account for molecular mechanisms underlying the therapeutic efficacy of puerarin in AD patients.