基于高通量测序研究清润方改善2型糖尿病大鼠肝脏胰岛素抵抗的作用机制  

Mechanism of Qingrun Formula in Improving Hepatic Insulin Resistance of Ratswith Type 2 Diabetes Mellitus Based on High-throughput Sequencing

作　　者：卜祥伟 郝晓晖 张美珍 王泽 王皓朔 史佩玉 张润云[1] 倪青[1] 林兰[1] BU Xiangwei;HAO Xiaohui;ZHANG Meizhen;WANG Ze;WANG Haoshuo;SHI Peiyu;ZHANG Runyun;NI Qing;LIN Lan(Department of Endocrinology,Guang′anmen Hospital of China Academy of Chinese Medicine Sciences,Beijing 100053,China;Department of Endocrinology,the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 300381,China)

机构地区：[1]中国中医科学院广安门医院内分泌科,北京100053 [2]天津中医药大学第一附属医院内分泌科,天津300381

出　　处：《世界中医药》2024年第11期1599-1607,1613,共10页World Chinese Medicine

基　　金：国家自然科学基金面上项目(81973685)。

摘　　要：目的:基于高通量转录组测序(RNA-seq)研究清润方改善2型糖尿病(T2DM)大鼠肝脏胰岛素抵抗的作用机制。方法:采用高脂饲料喂养联合链脲佐菌素腹腔注射构建T2DM大鼠模型,将成模大鼠采用随机数字表随机分为模型组,二甲双胍组(150 mg/kg),清润方大(11.2 g/kg)、中(5.6 g/kg)、小(2.8 g/kg)剂量组,另设正常组,灌胃干预8周。观察空腹血糖(FBG)、胰岛素抵抗指数(IRI)、胰岛素敏感指数(ISI)变化,利用RNA-seq结合生物信息学分析筛选差异表达的长链非编码RNA(lncRNA)、微RNA(miRNA)、信使RNA(mRNA),对差异基因进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析及qPCR验证,构建竞争性内源性RNA(ceRNA)调控网络。结果:与模型组比较,干预第6、8周清润方大剂量组FBG明显降低(P<0.05);第8周清润方各剂量组IRI降低(P<0.01),清润方大、小剂量组ISI升高(P<0.01,P<0.05)。通过差异表达筛选得到85个关键mRNA、12个miRNA、37个lncRNA,通过蛋白质-蛋白质相互作用(PPI)网络得到12个核心基因,并构建lncRNA-miRNA-mRNA网络。KEGG富集分析显示,差异基因主要涉及Janus激酶/信号转导及转录活化因子(JAK-STAT)、过氧化物酶体增殖物激活受体(PPAR)、氨基酸代谢、脂质代谢等通路。结论:清润方可能通过lncRNA-miRNA-mRNA网络调控CYP2、Acer2等基因,并影响Lpin1、Insig1等基因和PPAR、JAK-STAT、氨基酸代谢、脂质代谢等信号通路,改善2型糖尿病大鼠肝脏胰岛素抵抗。Objective:To explore the mechanism of Qingrun Formula in improving hepatic insulin resistance in rats with type 2 diabetes mellitus(T2DM)through high-throughput RNA sequencing(RNA-seq).Methods:A T2DM model of rats was established by feeding rats a high-fat diet combined with intraperitoneal injection of streptozotocin(STZ).The successfully modeled rats were randomly divided into the model group,metformin group(150 mg/kg),and high-dose(11.2 g/kg),medium-dose(5.6 g/kg),and low-dose(2.8 g/kg)groups of Qingrun Formula,and a normal control group was set up.The rats were given intragastric intervention for eight weeks.Changes in fasting blood glucose(FBG),insulin resistance index(IRI),and insulin sensitivity index(ISI)were observed among the groups.RNA-seq technology combined with bioinformatics analysis was used to screen differentially expressed long-chain non-coding RNA(lncRNA),micro RNA(miRNA),and messenger RNA(mRNA).Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses and qPCR verification of differentially expressed genes were performed,and regulatory networks of competitive endogenous RNA(ceRNA)were constructed.Results:Compared with that in the model group,the FBG in the high-dose group of Qingrun Formula decreased significantly in the 6th and 8th week of intervention(P<0.05).In the 8th week of intervention,the IRI in all groups of Qingrun Formula decreased significantly(P<0.01),while that in the high-dose and low-dose groups of Qingrun Formula increased significantly(P<0.01,P<0.05).85 key mRNA,12 miRNA,and 37 lncRNA were obtained through differential expression screening.12 core genes were obtained by protein-protein interaction(PPI)network,and the network of lncRNA-miRNA-mRNA was constructed.KEGG enrichment analysis revealed that differentially expressed genes were mainly involved in pathways such as JAK-STAT,PPAR,amino acid metabolism,and lipid metabolism.Conclusion:Qingrun Formula may improve hepatic insulin resistance of rats with T2DM by regulating CYP2 and Acer2 genes thro

关 键 词：清润方 2型糖尿病 胰岛素抵抗 转录组测序 竞争性内源性RNA网络 差异基因 信号通路 作用机制 

分 类 号：R285.5[医药卫生—中药学] R259[医药卫生—中医学]