中国中药杂志

2021, (14) 3650-3659

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基于蛋白质组学研究葛根素逆转Aβ_(1-42)损伤SH-SY5Y细胞的机制
Mechanism of puerarin reversing SH-SY5Y cell injury induced by Aβ_(1-42) based on proteomics

张林;方德宇;柳春;赵丹玉;王艳杰;陈文娜;崔勇;郭隽馥;丛培玮;冯晓帆;张云亭;
ZHANG Lin;FANG De-yu;LIU Chun;ZHAO Dan-yu;WANG Yan-jie;CHEN Wen-na;CUI Yong;GUO Jun-fu;CONG Pei-wei;FENG Xiao-fan;ZHANG Yun-ting;Liaoning University of Traditional Chinese Medicine;

摘要(Abstract):

葛根素具有逆转β-淀粉样蛋白(amyloidβpeptide,Aβ)诱导的神经损伤、抑制神经元凋亡的抗阿尔茨海默病(Alzheimer's disease,AD)活性,然而,其潜在药效机制依然有待进一步的研究。AD的发生发展是由于体内多个代谢环节发生变化而导致平衡的破坏,而葛根素可能作用于多靶点、多代谢过程来达到治疗目的,如何尽可能全面了解其作用机制,定量蛋白质组学分析提供了新的选择。该研究采用Aβ_(1-42)诱导SH-SY5Y细胞建立AD细胞模型,并通过Aβ免疫荧光检测发现葛根素干预后Aβ显著减少。利用Label-free非标记定量技术,并基于生物信息学分析结果采用Western blot检测,来进一步探究葛根素逆转Aβ_(1-42)损伤SH-SY5Y细胞的机制。结果表明,大部分差异蛋白与cellular component organization or biogenesis、cellular component organization、cellular component biogenesis等生物过程相关,并主要参与pathogenic Escherichia coli infection、m TOR signaling pathway、regulation of autophagy、regulation of actin cytoskeleton、spliceosome、hepatocellular carcinoma、tight junction、non-small cell lung cancer、apoptosis、gap junction等P排在前10的通路中。Annexin V/PI流式细胞仪以及TUNEL检测结果显示,葛根素干预后Aβ显著减少,凋亡率显著下降;Western blot检测发现自噬相关蛋白LC3Ⅱ蛋白表达水平在Aβ诱导后上调,其上调程度在葛根素干预组出现了进一步的增强;LC3Ⅱ/LC3Ⅰ的比值在各组间趋势与LC3Ⅱ蛋白表达水平相同;p62蛋白的表达水平在对照组、AD模型组及葛根素干预组依次降低。蛋白互作网络分析表明,CAP1与TUBA1B、HSP90AB2P、DNM1L、TUBA1A、ERK1/2之间的功能存在相关性,其中CAP1与ERK1/2的相关性最高。Western blot检测发现,葛根素干预后p-ERK1/2、Bax、CAP1的表达显著下调,而Bcl-2蛋白的表达水平显著上调。因此,葛根素可能在细胞内多个位置,通过多个生物学过程及通路的共同作用来改善Aβ_(1-42)对SH-SY5Y细胞的损伤,其中CAP1或许扮演着重要角色。
Puerarin has the anti-Alzheimer's disease (AD) activity,which can reverse nerve injury induced by Aβand inhibit neuronal apoptosis.However,its potential pharmacodynamic mechanism still needs to be further researched.The occurrence and development of AD is due to the change of multiple metabolic links in the body,which leads to the destruction of balance.Puerarin may act on multiple targets and multiple metabolic processes to achieve therapeutic purposes.Quantitative proteomic analysis provides a new choice to understand the mechanism as completely as possible.This research adopted SH-SY5Y cells induced by Aβ_(1-42)to establish AD cell model,and Aβimmunofluorescence detection showed that Aβdecreased significantly after puerarin intervention.The mechanism of puerarin reversing SH-SY5Y cell injured by Aβ_(1-42)was further explored by using label-free non-labeled quantitative technology and Western blot detection based on bioinformatics analysis result.The results showed that most of the differential proteins were related to biological processes such as cellular component organization or biogenesis,cellular component organization and cellular component biogenesis,and they mainly participated in the top ten pathways of P value such as pathogenic Escherichia coli infection,m TOR signaling pathway,regulation of autophagy,regulation of actin cytoskeleton,spliceosome,hepatocellular carcinoma,tight junction,non-small cell lung cancer,apoptosis and gap junction.Annexin V/PI flow cytometry and TUNEL were used to detect apoptosis,and the results showed that Aβdecreased significantly and the rate of apoptosis decreased significantly after puerarin intervention.Western blot analysis found that the protein expression level of autophagy related protein LC3Ⅱwas up-regulated after Aβinduction,and the degree of this up-regulation was further enhanced in puerarin intervention group.The trend of the ratio of LC3Ⅱ/LC3Ⅰamong groups was the same as the protein expression level of LC3Ⅱ,the protein expression level of p62 in the control group,AD model group and puerarin intervention group decreased successively.Protein interaction network analysis showed that CAP1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.Western blot showed that the expressions of p-ERK1/2,Bax and CAP1 were significantly down-regulated and the protein expression level of Bcl-2 was significantly up-regulated after puerarin intervention.Therefore,puerarin might improve the SH-SY5Y cells injured by Aβ_(1-42)through the interaction of multiple biological processes and pathways in cells multiple locations,and CAP1 might play an important role among them.

关键词(KeyWords): 葛根素;蛋白质组学;SH-SY5Y细胞;β-淀粉样蛋白
puerarin;proteomics;SH-SY5Y cell;amyloid β peptide

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81803854);; 辽宁省自然科学基金项目(20180551277);; 辽宁省教育厅科学技术研究项目(L201720,L202026)

作者(Authors): 张林;方德宇;柳春;赵丹玉;王艳杰;陈文娜;崔勇;郭隽馥;丛培玮;冯晓帆;张云亭;
ZHANG Lin;FANG De-yu;LIU Chun;ZHAO Dan-yu;WANG Yan-jie;CHEN Wen-na;CUI Yong;GUO Jun-fu;CONG Pei-wei;FENG Xiao-fan;ZHANG Yun-ting;Liaoning University of Traditional Chinese Medicine;

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