基于nano-LC-LTQ/Orbitrap-MS筛选黄芩-黄连药对干预2型糖尿病KKAy小鼠的骨骼肌差异表达蛋白  

Screening of Differentially Expressed Proteins in Skeletal Muscle of Type 2 Diabetes KKAy Mice Treated with Couplet Medicines of Scutellariae Radix and Coptidis Rhizoma Based on nano-LC-LTQ/Orbitrap-MS

作　　者：刘彤彤 肖琴 盛军庆[2] 涂秀英[1] 孙军 魏学鑫[1] 于梅[1] 章常华[1] LIU Tong-tongl;XIAO QinI;SHENG Jun-qing;TU Xiu-ying;SUN Jun;WEI Xue-xinI;YU MeiI;ZHANG Chang-hua(School of Pharmacy,Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China;School of Life Sciences,Nanchang University,Nanchang 330031,China)

机构地区：[1]江西中医药大学药学院,南昌330004 [2]南昌大学生命科学学院,南昌330031

出　　处：《中国实验方剂学杂志》2018年第17期126-131,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81460622,81774194,81260660);江西省自然科学基金项目(20171BAB205095,20151BAB205079,20151BAB205077);江西省卫生计生委科研计划项目(2016A024)

摘　　要：目的采用nano-LC-LTQ／Orbitrap．MS鉴定2型糖尿病KKAy小鼠模型组与给药组的骨骼肌差异表达蛋白，推测黄芩-黄连药对（sc）防治2型糖尿病的作用机制。方法：取20只8-10周龄的雄性KKAy小鼠，给予全价高脂饲料，随机等分为2组，分别为黄芩-黄连药对组（27 g·kg^-1）和模型组（灌胃等量水）。2组均连续灌胃8周，于最后1次给药后1 h处死小鼠，取骨骼肌在液氮中进行研磨，加裂解液及蛋白酶抑制剂提取蛋白，测定蛋白浓度，再进行蛋白变性、还原、烷基化、酶解及脱盐，利用nano-LC-LTQ／Orbitrap．MS检测，利用Xcalibur 2．2软件和Proteome Discoverer 1．4．0．288进行数据处理，采用Sievev2．1（×64）进行无标样定量分析寻找相关差异蛋白，筛选并鉴定差异表达蛋白，并对差异表达蛋白进行生物信息学分析。结果：黄芩．黄连药对组与模型组共鉴定出107个差异表达蛋白质，经生物学分析筛选出与糖尿病相关的差异蛋白共5个，其中上调4个（50磷酸腺苷激活的蛋白激酶、脂联素等），下调1个（脂肪细胞型脂肪酸结合蛋白4）。分析显示差异蛋白质主要涉及炎症反应、信号通路调节等生物过程。结论：通过有效的比较与分析，得到了一些参与胰岛素信号转导等通路的差异表达蛋白质，为研究黄芩一黄连药对防治胰岛素抵抗的分子机制提供了新靶点。Objective： To identify the differentially expressed proteins in skeletal muscle of type 2 diabetes KKAy mice from the model group and the administration group by nano-LC-LTQ/Orbitrap-MS, and speculate mechanism of couplet medicines of Scutellariae Radix and Coptidis Rhizoma against type 2 diabetes. Method： Twenty male KKAy mice aged 8-10 weeks were used. KKAy mice were given full-fledged high-fat diet and randomly divided into 2 groups, included Scutellariae Radix and Coptidis Rhizoma group （27 g-kg-l） and model group （the same amount of distilled water）. Mice in the two groups were continuous intragastricadministrated for 8 weeks. After the last administration for 1 hour, mice were sacrificed, skeletal muscle was taken and placed in -80 ~C refrigerator. Skeletal muscles of the model group and administration group were ground in liquid nitrogen, lysate and protease inhibitors were added to extract the protein, the concentration of protein was determined, and then protein denaturation, reduction, alkylation, enzymolysis and desalting, the samples were identified by nano-LC-LTQ/Orbitrap-MS. Data were processed by Xcalibur 2.2 software and Proteome Discoverer 1.4.0. 288, and Sieve v2.1 （ ~ 64） was adopted to analyze and find relevant differential proteins with quantitative analysis of non-standard samples, differentially expressed proteins were screened and identified, and bioinformatics analysis of the differentially expressed proteins was carried out. Result： A total of 107 differentially expressed proteins were identified from Scutellariae Radix and Coptidis Rhizoma group and model group, and five differential proteins related to diabetes were screened out by biological analysis, including 4 up- regulated [5＇-adenosine-activated protein kinase （AMPK）, adiponectin （APN）, etc] and 1 down-regulated [ adipocyte type fatty acid binding protein4 （FABP4） ]. The differential proteins mainly involved in inflammatory reactions, signal pathway regulation and other biological processes. Conclu

关 键 词：黄芩一黄连 药对 KKAY小鼠 蛋白质组学 差异表达蛋白 

分 类 号：R22[医药卫生—中医基础理论] R289[医药卫生—中医学]