CAJ | 학술논문

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。
목적：탐토중약원지제취물( PTE)청제β정분양단백( Aβ)적도경급기가능적신호통로。방법채용MTT법관찰불동농도(100、40、20、10、5、0μg/ml)적PTE대신경세포주(SH-SY5Y)적독성작용。 ELISA법검측PTE대전염과표체정분양전체단백(APP)화β분비매(BACE1)적중국창서란소( CHO)세포배양액중Aβ수평적영향。단단광선시알( MDC)염색검측PTE처리후적신경세포내자서소체적변화。 Western blot검측미관관련단백경련3( LC3)적표체수평,이급mTOR、p70s6k、Raptor、Akt、AMPK등단백적린산화수평。결과 MTT결과현시PTE대신경세포활력몰유영향(P>0．05)。 CHO-APP/BACE1경약물처리후,Aβ분비수평명현강저,병정농도의뢰성。형광현미경하관찰약물처리조세포자서증강,령외,자서표지물단백LC3Ⅱ/LC3Ⅰ수평승고。결론 PTE 통과증강 AMPK/Raptor 통로,억제 mTOR화p70s6k적린산화,유도자서발생,사세포청제Aβ적능력증강,감소Aβ적분비。
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 trea-ted 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 inde-pendent. We observed that autophagic vacuoles boosted in drug treatment group by fluorescence microscopy, in ad-dition, 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.