基于蛋白质组学的黄芪多糖对TLR4激活的肺癌细胞来源外泌体的作用研究  

Proteomics analysis of Astragalus polysaccharide on TLR4-activated lung cancer cell-derived exosomes

作　　者：胡康蝶 杨凯歌 SOUMIA Cheddah 吴明媛[1] 闫超[1] 李欣燕[1] 王彦[1] HU Kang-die;YANG Kai-ge;SOUMIA Cheddah;WU Ming-yuan;YAN Chao;LI Xin-yan;WANG Yan(School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学药学院,上海200240

出　　处：《中国中药杂志》2022年第21期5908-5915,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81874307,21874088);上海市科委“科技创新行动计划”项目(19142203100,2014220400,19DZ2202200)。

摘　　要：黄芪多糖(Astragalus polysaccharides, APS)是黄芪的主要有效成分之一,可通过调节肿瘤炎症微环境发挥抗肿瘤作用。外泌体是粒径50~200 nm的小细胞外囊泡,携带有来自亲本细胞的核酸和蛋白质成分。当与受体细胞结合,外泌体可以发挥细胞间通讯和调节免疫反应等作用。该研究采用超速离心法,对Toll样受体4(Toll-like receptor 4, TLR4)激活的肺癌细胞H460炎症状态组和APS缓解组H460细胞释放的外泌体进行了提取,并利用蛋白免疫印迹、透射电镜和纳米粒径跟踪分析对外泌体进行表征。进一步通过非标记蛋白质组学方法对3组H460细胞来源的外泌体蛋白进行分析,在3组中分别鉴定到897、800和911个蛋白质,其中88%的蛋白质属于ExoCarta外泌体蛋白质数据库;差异统计分析显示有111个蛋白质表达量在LPS组和APS组均有显著变化(P<0.05)。针对差异蛋白质进行生物学信息分析,与差异蛋白质相关的分子功能、生物过程和信号通路主要涉及病毒过程、蛋白质结合、蛋白酶体和上皮细胞细菌入侵等过程。关键差异蛋白包括纤溶酶原激活物抑制剂-1、层黏连蛋白α5、层黏连蛋白α1和CD44等,说明不同状态下肿瘤细胞会发生系统性变化,并体现在炎症微环境的外泌体中;分析结果也提示APS可能通过TLR4/MyD88/NF-κB信号通路或调节细胞外基质影响炎症微环境。该研究有助于了解炎症状态下肿瘤发展机制,并在外泌体层面上探索APS的抗炎作用。Astragalus polysaccharide(APS), one of the main active components of Astragali Radix, plays an anti-tumor effect by regulating the inflammatory microenvironment of tumors. Exosomes are small extracellular vesicles with a diameter ranging from 50 to 200 nm and carry several biological components from parental cells such as nucleic acids and proteins. When combined with recipient cells, they play an important role in intercellular communication and immune response. In this study, exosomes released from H460 cells at the inflammatory state or with APS addition activated by Toll-like receptor 4(TLR4) were extracted by ultracentrifugation and characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. The exosomal proteins derived from H460 cells in the three groups were further analyzed by label-free proteomics, and 897, 800, and 911 proteins were identified in the three groups(Con, LPS, and APS groups), 88% of which belonged to the ExoCarta exosome protein database. Difference statistical analysis showed that the expression of 111 proteins was changed in the LPS group and the APS group(P<0.05). The biological information analysis of the differential proteins was carried out. The molecular functions, biological processes, and signaling pathways related to the differential proteins mainly involved viral processes, protein binding, and bacterial invasion of proteasome and epithelial cells. Key differential proteins mainly included plasminogen activator inhibitor-1, laminin α5, laminin α1, and CD44, indicating that tumor cells underwent systemic changes in different states and were reflected in exosomes in the inflammatory microenvironment. The analysis results also suggested that APS might affect the inflammatory microenvironment through the TLR4/MyD88/NF-κB signaling pathway or the regulation of the extracellular matrix. This study is conducive to a better understanding of the mechanism of tumor development in the inflammatory state and the exploration of the anti-inflammato

关 键 词：黄芪多糖 TLR4 外泌体 肺癌细胞 蛋白质组学 肿瘤微环境 

分 类 号：R285[医药卫生—中药学]