基于磷酸化蛋白质组学技术研究益糖康防治糖尿病肾脏疾病的机制  

Study on the mechanism of Yitangkang in preventing and treating diabetic nephropathy based on phosphorylated protein genomics technology

作　　者：王玮鼐 于嘉祥 石岩[2] 曲超 张文顺 WANG Weinai;YU Jiaxiang;SHI Yan;QU Chao;ZHANG Wenshun(Graduate School,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;Liaoning Province Traditional Chinese Medicine Research Institute,Shenyang 110034,China)

机构地区：[1]辽宁中医药大学研究生院,辽宁沈阳110847 [2]辽宁中医药大学,辽宁沈阳110847 [3]辽宁省中医药研究院,辽宁沈阳110034

出　　处：《沈阳药科大学学报》2023年第11期1486-1497,共12页Journal of Shenyang Pharmaceutical University

基　　金：国家重点基础研究发展计划(973计划)项目(2013CB532004);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(2023-BS-141);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：目的观察中药复方益糖康干预后模型大鼠肾组织磷酸化修饰位点的变化,探讨中药复方益糖康对糖尿病肾脏疾病的影响机制中的关键磷酸化修饰蛋白和通路。方法80只8周龄雄性SD大鼠(体重范围为160~220 g),分别设置模型组、益糖康组、空白组、阳性对照药(氯沙坦)组,按照要求进行干预后取各组大鼠的肾组织,所有样本经SDS-PAGE凝胶电泳、蛋白质酶解及肽段脱盐、磷酸化肽段富集、LC-MS/MS分析后,用数据库检索并进行生信分析。结果组织学评价结果显示:HE染色结果表明相较于空白组,模型组大鼠呈现严重肾损现象。与模型组相比,益糖康组大鼠肾损程度发生了显著减轻,肾脏损伤情况与阳性对照药(氯沙坦)组相似。益糖康组vs模型组差异比较显示磷酸化蛋白质组学分析共检测到显著差异表达磷酸化肽段389个,其中上调166个;下调223个。GO富集分析结果显示这些差异磷酸化修饰蛋白质主要行使结合,催化活性等分子功能,主要参与的生物过程是细胞过程;KEGG富集通路分析显示:差异磷酸化修饰蛋白质主要富集在糖酵解/糖异生KEGG通路、肌动蛋白细胞骨架调控以及MAPK信号通路等信号通路。在蛋白质相互作用层面,参与度最高的前三位蛋白为ALDOA、ALDOB及DLD。结论中药复方益糖康可能通过靶向ALDOA调控糖酵解途径;还可以通过调控ALDOB基因及蛋白的表达,改善由于ALDOB的突变导致的糖代谢异常;亦可以作用于DLD分子,影响TCA循环和糖酵解过程,通过这些途径调节糖尿病肾脏疾病能量代谢的过程。Objective To observe the changes of phosphorylation modification sites in kidney tissue of model rats after the intervention of traditional Chinese medicine compound Yitangkang,and to explore the key phosphorylation modification proteins and pathways in the influence mechanism of traditional Chinese medicine compound Yitangkang on diabetic kidney disease.Methods Eighty 8-week-old male SD rats(weight range 160-220 g)were divided into model group,Yitangkang group,blank group and positive control drug(losartan)group.After intervention,the kidney tissue of each group was collected.All samples were searched by database after SDS-PAGE gel electrophoresis,protein enzymolysis,peptide desalination,phosphorylated peptide enrichment and LC-MS/MS analysis.Results Histological evaluation showed that compared with the blank group,the rats in the model group showed severe renal damage.Compared with the model group,the degree of renal damage in Yitangkang group was significantly reduced,and the renal damage was similar to that in the positive control drug(losartan)group.A total of 389 differentially expressed phosphorylated peptide fragments were detected by phosphorylated protein proteomics analysis,of which 166 were up-regulated and 223 were down-regulated.The results of GO enrichment analysis showed that these differentially phosphorylated proteins mainly performed molecular functions such as binding and catalytic activity,and the biological process they mainly participated in was cell process.KEGG enrichment pathway analysis showed that differentially phosphorylated protein was mainly enriched in glycolysis/gluconeogenesis KEGG pathway,actin cytoskeleton regulation and MAPK signaling pathway.At the level of protein interaction,the top three proteins with the highest participation were ALDOA,ALDOB and DLD.Conclusion Yitangkang may regulate glycolytic pathway by targeting ALDOA.

关 键 词：糖尿病肾脏疾病 磷酸化蛋白组学 益糖康 

分 类 号：R28[医药卫生—中药学] R96[医药卫生—中医学]