iTRAQ结合高通量液相色谱-串联质谱技术分析贝沙罗汀抗脑缺血再灌注损伤的作用研究  

Study of Bexarotene against cerebral ischemia reperfusion injury analyzed by iTRAQ combined with high throughput liquid chromatography-tandem mass spectrometry

作　　者：刘洁[1] 刘胜伟[2] 任忠洋 丁玲[1] 夏春勇 LIU Jie;LIU Sheng-wei;REN Zhong-yang;DING Ling;XIA Chun-yong(Department of Pharmacy,Chongqing University Jiangjin Hospital,Chongqing 402260,China;Department of Pharmacy,Yongchuan Hospital of Chongqing Medical University,Chongqing 402160,China;Department of Surgery,Jiangjin Hospital of Traditional Chinese Medicine,Chongqing 402284,China)

机构地区：[1]重庆大学附属江津医院药学部,重庆402260 [2]重庆医科大学附属永川医院药学部,重庆402160 [3]重庆市江津区中医院外科,重庆402284

出　　处：《中国临床药理学杂志》2023年第22期3306-3310,共5页The Chinese Journal of Clinical Pharmacology

基　　金：重庆市自然科学基金资助项目(cstc2020jcyj-msxmX0434);永川区自然科学基金资助项目(2022yc-jckx20055);重庆市临床药学重点专科基金资助项目。

摘　　要：目的联合应用高通量液相色谱-串联质谱的蛋白质定量分析技术和同位素标记的相对及绝对定量技术(iTRAQ)技术,分析贝沙罗汀抗脑缺血/再灌注损伤(CIRI)的作用并对关键通路中的关键靶点进行验证。方法随机将39只小鼠分组:13只假手术组,13只模型组,13只实验组。实验组和模型组小鼠建立CIRI模型。实验组小鼠给予贝沙罗汀5 mg·kg^(-1)·d^(-1),腹腔注射,模型组则腹腔注射等量溶剂,造模后72 h,获取检测标本。各组取3只,联合应用高通量液相色谱-串联质谱的蛋白质定量分析技术和iTRAQ技术,用Proteinpilot软件筛选差异表达的蛋白质,取“模型组vs实验组”与“模型组vs假手术组”的交集差异蛋白,即为贝沙罗汀作用于CIRI的潜在靶点,在STRING 11.5平台上,构建靶点交联互作网络,用Cytoscape 3.8.2软件制图,利用R语言,对贝沙罗汀抗CIRI的潜在靶点进行GO富集分析及KEGG富集通路分析,预测潜在机制。各组取10只,用蛋白质印迹法验证贝沙罗汀抗脑缺血/再灌注损伤的潜在靶点。结果筛选出贝沙罗汀抗CIRI的潜在靶点214个,通过PPI网络分析,获取度值排名前十的关键靶蛋白有Hspa8、Alb、Dlg4、Hsp90aa1、Hsp90ab1等。GO结果显示,细胞成分主要涉及髓磷脂、神经元间的突触等,分子功能主要有肌动蛋白丝绑定、伴侣蛋白绑定等,参与突触的组织、蛋白交联等生物学过程。通过KEGG显示,潜在作用通路有Nod样受体信号通路、坏死性凋亡通路、脂质和动脉粥样硬化通路等。结论贝沙罗汀干预CIRI小鼠后脑组织蛋白质存在明显的差异性表达,通过生物信息学分析,涉及多靶点、多通路,为进一步探索、筛选贝沙罗汀作用于CIRI治疗新靶点提供了重要参考。Objective Using a combination of high throughput liquid chromatography-tandem mass spectrometry and isobaric tags for relative and absolute quantitation(iTRAQ) for protein quantitative analysis, to analyze the effects of Bexarotene on cerebral ischemia/reperfusion injury and verify the key targets in key pathways. Methods The 39 mice were randomly divided into sham operation group(Sham, 13mice), model group(CIRI, 13 mice) and experimental group(Bexarotene, 13 mice). CIRI and Bexarotene groups were used to establish cerebral ischemia/reperfusion injury model. In the Bexarotene group, given Bexarotene 5 mg·kg^(-1)·d^(-1) by intraperitoneally injected, while in CIRI group, the same amount of solvent was given by intraperitoneally injected. At 72 h after modeling,test specimens were obtained.Three rats were selected from each group. Protein Pilot software was used to screen differentially expressed proteins by combining high-throughput liquid chromatography-tandem mass spectrometry and i TRAQ technology. The differential protein of the intersection of " CIRI vs. Bexarotene" and " CIRI vs. Sham" is the potential target of Bexarotene acting on CIRI. On the String 11. 5 platform,the target cross-linking interaction network was built,and the software Cytoscape 3. 8. 2 was used for mapping. Using R language,GO enrichment analysis and KEGG enrichment pathway analysis of the potential targets of Bexarotene anti-CIRI were conducted to predict the potential mechanism. Ten rats were selected from each group. Western Blot was used to verify the potential target of bexarotene against cerebral ischemia/reperfusion injury. Results A total of 214 potential targets of Bexarotene anti-CIRI were screened out.Through the PPI network analysis,the top 10 key target proteins in obtaining degree value included HSPA8,ALB,DLG4,HSP90Aa1,HSP90AB1,etc. GO results showed that cell components were mainly involved in myelin and synapses between neurons,and molecular functions were mainly actin filament binding and chaperone binding,which were in

关 键 词：贝沙罗汀 脑缺血再灌注损伤 蛋白质组学 

分 类 号：R972[医药卫生—药品]