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 Shenyang 110000,China.
Zhongguo Zhong Yao Za Zhi. 2021 Jul;46(14):3650-3659. doi: 10.19540/j.cnki.cjcmm.20210201.403.
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.
葛根素具有抗阿尔茨海默病（AD）活性，可逆转Aβ诱导的神经损伤并抑制神经元凋亡。然而，其潜在的药效学机制仍需进一步研究。AD的发生发展是由于体内多个代谢环节发生变化，导致平衡破坏。葛根素可能作用于多个靶点和多个代谢过程以达到治疗目的。定量蛋白质组学分析为尽可能全面地了解其机制提供了新的选择。本研究采用Aβ1-42诱导的SH-SY5Y细胞建立AD细胞模型，Aβ免疫荧光检测显示葛根素干预后Aβ显著降低。基于生物信息学分析结果，采用无标记定量技术和蛋白质免疫印迹法进一步探究葛根素逆转Aβ1-42损伤SH-SY5Y细胞的机制。结果显示，大多数差异蛋白与细胞成分组织或生物发生、细胞成分组织和细胞成分生物发生等生物学过程相关，主要参与致病性大肠杆菌感染、mTOR信号通路、自噬调节、肌动蛋白细胞骨架调节、剪接体、肝细胞癌、紧密连接、非小细胞肺癌、凋亡和缝隙连接等P值前十的通路。采用Annexin V/PI流式细胞术和TUNEL法检测细胞凋亡，结果显示葛根素干预后Aβ显著降低，凋亡率显著下降。蛋白质免疫印迹分析发现，Aβ诱导后自噬相关蛋白LC3Ⅱ的蛋白表达水平上调，在葛根素干预组中这种上调程度进一步增强。各组LC3Ⅱ/LC3Ⅰ比值的变化趋势与LC3Ⅱ的蛋白表达水平一致，对照组、AD模型组和葛根素干预组中p62的蛋白表达水平依次降低。蛋白质相互作用网络分析显示，CAP1与TUBA1B、HSP90AB2P、DNM1L、TUBA1A和ERK1/2相关，其中CAP1与ERK1/2的相关性最高。蛋白质免疫印迹显示，葛根素干预后p-ERK1/2、Bax和CAP1的表达显著下调，Bcl-2的蛋白表达水平显著上调。因此，葛根素可能通过细胞多个部位的多个生物学过程和通路的相互作用改善Aβ1-42损伤的SH-SY5Y细胞，其中CAP1可能起重要作用。
