基于TMT标记定量蛋白质组学技术研究中药复方益糖康改善db/db小鼠骨骼肌胰岛素抵抗的作用机制  

Mechanism of Yitangkang(益糖康)Improving Skeletal Muscle Insulin Resistance in db/db Mice Based on TMT Labeled Quantitative Proteomics

作　　者：于嘉祥 张瀚文[1] 于睿[1] 韩雪莹 张文顺[1] 安继仁 曲超 冀天威[1] 姜楠[1] 吕雪辉 杨宇峰[1] 石岩[1] YU Jiaxiang;ZHANG Hanwen;YU Rui;HAN Xueying;ZHANG Wenshun;AN Jiren;QU Chao;JI Tianwei;JIANG Nan;LYU Xuehui;YANG Yufeng;SHI Yan(Liaoning University of Traditional Chinese Medicine,Shenyang 110847,Liaoning,China)

机构地区：[1]辽宁中医药大学,辽宁沈阳110847

出　　处：《中华中医药学刊》2023年第1期46-51,I0015-I0020,共12页Chinese Archives of Traditional Chinese Medicine

基　　金：国家重点基础研究发展计划(973计划)项目(2013CB532004);中国博士后科学基金面上资助项目(2021M693853,2021M693852);辽宁省“兴辽英才计划”青年拔尖人才项目(XLYC2007121);辽宁省科技厅博士科研启动课题(2021-BS-183);中医脏象理论及应用教育部重点实验室2021年度开放基金项目(zyzx2109)。

摘　　要：目的研究db/db小鼠骨骼肌胰岛素抵抗的发病机制及中药复方益糖康对其的改善作用。方法9周龄健康雄性SPF级db/db小鼠16只、db/m小鼠8只,分别设置模型组、益糖康组、空白组,益糖康组使用30 g·kg^(-1)·d^(-1)的中医复方益糖康煎剂进行灌胃,其余两组予以等体积的生理盐水灌胃,5周后进行取材,分离各组小鼠的骨骼肌组织。所有样本经TMT 10标试剂进行标记定量、蛋白酶解、TMT肽段标记与肽段分级、LC-MS/MS分析后,用数据库检索方法确定差异蛋白,并进行生信分析。结果所选取的样本稳定,有较高的科学价值;与db/m正常小鼠相比,db/db小鼠骨骼肌胰岛素抵抗总共导致Amd1、Serpina1e、Sorbs3、Fabp4、Myl3、Upf2等109个蛋白的表达变化,涉及了糖酵解、脂肪酸氧化、ATP的合成及供能等生物过程及PPAR、支链氨基酸代谢等信号通路;中药复方益糖康可以改善db/db小鼠骨骼肌胰岛素抵抗,总共涉及了Pcolce、Pgk2、Trip11、Tmem41b、Mrps9、Bola2等49个蛋白的表达变化,通过调节三羧酸循环,乙醛酸和二羧酸代谢等通路实现;中药复方益糖康可以产生心血管受益,并能缓解由2型糖尿病及胰岛素抵抗导致的帕金森病、亨廷顿病。结论db/db小鼠是研究2型糖尿病及胰岛素抵抗的理想模型,TMT标记定量蛋白质组学技术可以精准发现相关差异蛋白,结果较权威;脾虚导致的骨骼肌线粒体功能障碍是2型糖尿病及胰岛素抵抗发病的原因之一;中药复方益糖康可以对2型糖尿病及胰岛素抵抗异常的生物过程起到纠正作用,与健脾修复线粒体损伤、促进线粒体自噬有关;整个实验过程筛选的差异蛋白及所涉及的生理病理变化是课题组下一步研究的重点。Objective To research the pathogenesis mechanism of skeletal muscle insulin resistance in db/db mice and the improvement effect of Yitangkang(益糖康).Methods Sixteen healthy male SPF db/db mice and 8 db/m mice at the age of 9 weeks were divided into model group,Yitangkang group and blank group.Yitangkang group was treated with Yitangkang prescription at the dosage of 30 g·kg^(-1)·d^(-1) by gavage and the other two groups were tretaed with the equal volume of normal saline by gavage.After 5 weeks,the samples were taken to separate the skeletal muscle tissue of mice in each group.All samples were labeled with TMT 10 reagent for quantification,proteolysis,TMT peptide labeling and peptide grading and LC-MS/MS analysis.The differential proteins were determined by database retrieval method and analyzed by bioinformatics.Results The selected samples were stable and had high scientific value.Compared with those of db/m mice,the skeletal muscle insulin resistance in db/db mice resulted in the expression changes of 109 proteins such as Amd1,Serpina1e,Sorbs3,Fabp4,Myl3 and Upf2,involving biological processes such as glycolysis,fatty acid oxidation,ATP synthesis and energy supply,PPAR signal pathway and branched chain amino acid metabolism.Yitangkang prescription can improve the insulin resistance of skeletal muscle in db/db mice,involving the expression changes of 49 proteins such as Pcolce,Pgk2,Trip11,Tmem41b,Mrps9 and Bola2,which were realized by regulating the tricarboxylic acid cycle,glyoxylic acid and dicarboxylic acid metabolism.Yitangkang prescription can produce cardiovascular benefits and alleviate Parkinson's disease and Huntington's disease which were caused by type 2 diabetes mellitus and insulin resistance.Conclusion Db/db mice are ideal models for researching type 2 diabetes and insulin resistance.TMT marker quantitative proteomics technology can accurately identify the related differential proteins,and the results are more authoritative.Skeletal muscle mitochondrial dysfunction caused by spleen deficienc

关 键 词：益糖康 胰岛素抵抗 骨骼肌 TMT标记定量蛋白质组学技术 线粒体 

分 类 号：R285.5[医药卫生—中药学]