远志提取物清除β淀粉样蛋白的途径及其信号通路的研究  

The process and signal pathway of eliminating β-amyloid protein by Polygala Tenuifolia extract

作　　者：赵欢[1] 陈晓宇[1] 姬飞虹 王奎锋 王志成[3] 

机构地区：[1]安徽医科大学组织胚胎学教研室,合肥230032 [2]上海市勤浩生物科技有限公司,上海201203 [3]上海中医药大学实验中心,上海200071

出　　处：《安徽医科大学学报》2014年第7期913-917,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81373421);安徽省自然科学基金(编号:1208085MC53)

摘　　要：目的探讨中药远志提取物(PTE)清除β淀粉样蛋白(Aβ)的途径及其可能的信号通路。方法采用MTT法观察不同浓度(100、40、20、10、5、0μg/ml)的PTE对神经细胞株(SH-SY5Y)的毒性作用。ELISA法检测PTE对转染过表达淀粉样前体蛋白(APP)和β分泌酶(BACE1)的中国仓鼠卵巢(CHO)细胞培养液中Aβ水平的影响。单丹磺酰尸胺(MDC)染色检测PTE处理后的神经细胞内自噬小体的变化。Western blot检测微管关联蛋白轻链3(LC3)的表达水平,以及mTOR、p70s6k、Raptor、Akt、AMPK等蛋白的磷酸化水平。结果 MTT结果显示PTE对神经细胞活力没有影响(P>0.05)。CHO-APP/BACE1经药物处理后,Aβ分泌水平明显降低,并呈浓度依赖性。荧光显微镜下观察药物处理组细胞自噬增强,另外,自噬标志物蛋白LC3Ⅱ/LC3Ⅰ水平升高。结论 PTE通过增强AMPK/Raptor通路,抑制mTOR和p70s6k的磷酸化,诱导自噬发生,使细胞清除Aβ的能力增强,减少Aβ的分泌。Objective To discuss the way of eliminating β-amyloid （ Aβ） protein by traditional Chinese medicine Polygala Tenuifolia extract（ PTE） as well as its possible signal pathway. Methods MTT assay was used to observe the toxicity of different concentration of PTE（100, 40, 20, 10, 5, 0 μg/ml）on SH-SY5Y cells. Aβ peptide level in the supernatant of CHO cells overexpressing β-amyloid pro-protein （APP） and β-secretase（BACE1） after treated with PTE was detected by ELISA method. Autophagic vacuoles in SH-SY5Y were observed by MDC staining, and microtubule-associated protein light chain 3 （LC3） was detected by Western blot. mTOR, p70s6k, Raptor, Akt, AMPK as well as their phosphorylated protein were tested by Western blot to investigate the possible signaling pathway of autophagy which induced by PTE. Results MTT result showed that PTE had no significant toxicity on SH-SY5Y. After treated with drug, CHO cells had significantly decreased the secretion of Aβ, and with dose independent. We observed that autophagic vacuoles boosted in drug treatment group by fluorescence microscopy, in addition, LC3Ⅱ/LC3Ⅰ, the marker protein of autophagy also increased. Conclusion PTE may induce autophagy via enhancing AMPK/Raptor pathway, and inhibit mTOR phosphorylation as well as p70s6k, then strengthen the capacity of cellula to eliminate Aβ, and decrease its secretion.

关 键 词：阿尔茨海默病 Β淀粉样蛋白 自噬 微管关联蛋白轻链3 

分 类 号：R749.1[医药卫生—神经病学与精神病学] R341[医药卫生—临床医学]