泄浊解毒方通过miRNA-155-5p/JAK2/STAT3通路改善溃疡性结肠炎小鼠结肠黏膜炎症反应分析  

Improvement of Colonic Mucosa Inflammatory Response in Mice with Ulcerative Colitis by Xiezhuo Jiedu Recipe Through miRNA-155-5p/JAK2/STAT3 Pathway

作　　者：孙超迪 赵蒙蒙 郎晓猛 任杰[2,3] 康欣 崔建从[2,3] 贾苏杰 马玉景[1,2] 刘悦 揣强 翟文静 刘建平 SUN Chaodi;ZHAO Mengmeng;LANG Xiaomeng;REN Jie;KANG Xin;CUI Jiancong;JIA Sujie;MA Yujing;LIU Yue;CHUAI Qiang;ZHAI Wenjing;LIU Jianping(Hebei University of Chinese Medicine,Shijiazhuang 050091,China;Hebei Key Laboratory of Gastroenterology Research of Integrated Traditional Chinese and Western Medicine,Shijiazhuang 05001l,China;Hebei Provincial Hospital of Traditional Chinese Medicine,Shijiazhuang 05001l,China)

机构地区：[1]河北中医药大学,石家庄050091 [2]河北省中西医结合胃肠病研究重点实验室,石家庄050011 [3]河北省中医院,石家庄050011

出　　处：《中国实验方剂学杂志》2024年第11期174-182,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：第二批国家中医临床研究基地建设项目(国中医药科技函[2018]131号);河北省自然科学基金项目(H2022423326);河北省研究生创新资助项目(XCXZZSS2023023);河北省总工会河北省科学技术厅第八批河北省劳模和工匠人才创新工作室项目(冀工字[2021]42号)。

摘　　要：目的:应用生物信息学方法分析溃疡性结肠炎(UC)活动期与缓解期之间差异表达的微小RNA(miRNAs),筛选差异表达基因(DEGs)构建调控关系,推测泄浊解毒方治疗UC的作用机制,并通过动物实验进行验证。方法:从基因表达数据库(GEO)中获取UC患者结肠黏膜组织miRNAs数据集,运用GEO2R、Excel等工具筛选差异表达最显著的miRNAs作为研究对象,运用TargetScan、miRTarbase、miRDB及STRING、TRRUST、Matescape数据库筛选关键DEGs、预测下游转录因子(TF)、基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析,选取关键信号通路进行动物实验验证。动物实验采用2.5%的葡聚糖硫酸钠(DSS)自由饮方式制备UC小鼠模型,泄浊解毒方及美沙拉嗪灌胃7 d,苏木素-伊红(HE)染色观察结肠黏膜炎性浸润情况,实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠组织中miR-155-5p mRNA表达量;免疫组化法及蛋白免疫印迹法(Western blot)检测结肠组织中细胞因子信号转导抑制因子1(SOCS1)、磷酸化转录信号转导子及激活子3(p-STAT3)、磷酸化JAK激酶2(p-JAK2)、维甲酸受体相关孤儿受体-γt(ROR-γt)蛋白表达量;酶联免疫吸附测定法(ELLSA)检测血清中转换生长因子-β(TGF-β)、白细胞介素-17(IL-17)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)表达量。结果:GEO数据库筛选出GSE48957数据集,从活动期与缓解期样本中筛选到miR-155-5p作为研究对象,筛选出131个DEGs,其GO/KEGG富集分析主要与miRNA转录的正调节、蛋白质磷酸化等生物学过程,干细胞信号通路、IL-17信号通路、辅助性T细胞17(Th17)细胞分化等信号通路密切相关。运用Matescape数据库筛选出关键DEGs 10个,其中SOCS1是miR-155-5p的关键DEGs之一,进一步筛选关键DEGs的TF发现,STAT3是SOCS1的主要TF之一。动物实验结果显示,泄浊解毒方能有效下调UC小鼠结肠组织中miR-155-5p mRNA和p-STAT3、p-JAK2、ROR-γt蛋白表达,上调SOObjective: The differential expression of microRNAs(miRNAs) between the active stage and the remission stage of ulcerative colitis(UC) was analyzed by bioinformatics method, and the regulatory relationship was constructed by screening the differentially expressed genes(DEGs). The mechanism of Xizhuo Jiedu recipe in the treatment of UC was speculated and verified by animal experiments. Method: The miRNAs data set of colonic mucosa tissue of UC patients was obtained from the gene expression database(GEO), and the most differentially expressed miRNAs were screened by GEO2R, Excel, and other tools as research objects.TargetScan, miRTarbase, miRDB, STRING, TRRUST, and Matescape databases were used to screen key DEGs, predict downstream transcription factors(TFs), gene ontology(GO), and conduct Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis. The key signaling pathways were selected for animal experiments. In animal experiments, the UC mouse model was prepared by making the mouse freely drink 2.5%dextran sodium sulfate(DSS). Xiezhu Jiedu recipe and mesalazine were given by gavage for seven days, and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin(HE) staining. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was used to detect the mRNA expression of miR-155-5p in colon tissue. Immunohistochemistry and Western blot were used to detect the protein expression levels of cytokine signal transduction inhibitor(SOCS1), phosphorylated transcriptional signal transductor and activator 3(p-STAT3), phosphorylated Janus kinase 2(p-JAK2), and retinoic acid-associated orphan receptor-γt(ROR-γt). The expression levels of transforming growth factor-β(TGF-β), interleukin-17(IL-17), interleukin-6(IL-6), and interleukin-10(IL-10) in serum were detected by enzyme linked immunosorbent assay(ELISA). Result: The GSE48957 dataset was screened from the GEO database, and miR-155-5p was selected as the research object from the samples in the active and remission s

关 键 词：生物信息学 溃疡性结肠炎 泄浊解毒方 miRNA-155-5p/JAK激酶2(JAK2)/转录信号转导子及激活子3(STAT3)信号通路 

分 类 号：R284[医药卫生—中药学] R285[医药卫生—中医学] R289R287R22R2-031R33R24