活动期克罗恩病患者外周血高表达的hsa_circRNA_103124调控巨噬细胞分化、焦亡和炎症的机制  

The mechanism by which hsa_circRNA_103124 highly expressed in peripheral blood of patients with active Crohn′s disease regulates macrophage differentiation,pyroptosis and inflammation

作　　者：尹娟[1] 胡彤 徐丽娟 张丽平 叶玉兰[1] 庞智[1] Yin Juan;Hu Tong;Xu Lijuan;Zhang Liping;Ye Yulan;Pang Zhi(Department of Gastroenterology,the Affiliated Suzhou Hospital of Nanjing Medical University(Suzhou Municipal Hospital),Gusu School,Nanjing Medical University,Suzhou,215008,China)

机构地区：[1]南京医科大学姑苏学院,南京医科大学附属苏州医院(苏州市立医院)消化内科,苏州215008

出　　处：《中华医学杂志》2023年第43期3478-3486,共9页National Medical Journal of China

基　　金：苏州市科技局项目(SKJY2021123);苏州市临床重点病种诊疗技术专项(LCZX201715)

摘　　要：目的研究活动期克罗恩病(CD)患者外周血单个核细胞(PBMC)中高表达的环状RNA分子103124(hsa_circRNA_103124)在巨噬细胞分化、焦亡和炎症中的作用及相关机制。方法回顾性选取2018年4至9月南京医科大学附属苏州医院收治的活动期CD患者(CD组)和2018年7至10月该院体检中心健康体检人群(对照组),通过实时荧光定量聚合酶链反应(RT-qPCR)检测两组PBMC中hsa_circRNA_103124和Toll样受体4(TLR4)的水平。使用人单核细胞白血病细胞株(THP1)作为hsa_circRNA_103124调控巨噬细胞分化的研究模型。慢病毒感染构建hsa_circRNA_103124过表达或下调表达THP1细胞稳转株,诱导巨噬样分化,根据hsa_circRNA_103124的表达水平,将THP1细胞株分为以下4组:pLC5-ciR为过表达对照组;hsa_circRNA_103124 OE为过表达组;ShRNActrl为下调表达对照组;hsa_circRNA_103124 ShRNA为下调表达组。流式细胞术检测分化簇(CD)68、CD80、白细胞介素(IL)-6和肿瘤坏死因子α(TNF-α)水平及活性氧(ROS)生成水平。RT-qPCR检测IL-6、TNF-α、IL-1β、TLR4和髓样分化因子88(MyD88)水平。免疫荧光、RT-qPCR检测焦孔素D(GSDMD)、IL-18、热蛋白结构域相关蛋白3(NLRP3)水平。采用Pearson相关法分析hsa_circRNA_103124的丰度和TLR4表达水平或克罗恩病活动指数(CDAI)的相关性。结果CD组共纳入50例患者,男36例,女14例,年龄(35±10)岁(19~64岁);对照组共纳入30名,男22名,女8名,年龄(38±9)岁(24~64岁)。CD组PBMC中hsa_circRNA_103124[(0.009±0.016)比(0.003±0.002),P=0.042]和TLR4[(0.005±0.003)比(0.001±0.001),P<0.001]的水平均高于对照组,且hsa_circRNA_103124和TLR4的水平呈正相关(r=0.40,P=0.004)。hsa_circRNA_103124水平与CDAI呈正相关(r=0.32,P=0.024)。CD68(P=0.002)和CD80(P<0.001)的表达均增强。巨噬样M1型分化的THP1细胞中,hsa_circRNA_103124可促进ROS生成及IL-6、TNF-α、IL-1β、TLR4、MyD88、GSDMD、IL-18和NLRP3等表达(均P<0.05)。结论活动期CD患者PBMC中高表达的hObjective To investigate the role and related mechanism of the highly expressed circular RNA molecule 103124(hsa_circRNA_103124)in macrophage differentiation,pyroptosis and inflammation in peripheral blood mononuclear cells(PBMC)of patients with active Crohn′s disease(CD).Methods Patients with active CD(CD group)admitted to the Affiliated Suzhou Hospital of Nanjing Medical University from April to September 2018 and healthy people(control group)from the physical examination center of the hospital from July to October 2018 were retrospectively selected.The levels of hsa_circRNA_103124 and Toll-like receptor 4(TLR4)in PBMC of the two groups were detected by real-time quantitative polymerase chain reaction(RT-qPCR).Tohoku hospital pediatrics-1(THP1)cell line was used as a model for the study of hsa_circRNA_103124 regulating macrophage differentiation.Lentivirus infection was used to construct hsa_circRNA_103124 overexpressed or down-regulated THP1 cells to induce macrophage-like differentiation.According to the expression level of hsa_circRNA_103124,THP1 cell lines were divided into the following four groups:pLC5-ciR was overexpression control group;hsa_circRNA_103124 OE was the overexpression group;ShRNActrl was down-regulated expression control group;hsa_circRNA_103124 ShRNA was the down-regulated expression group.Flow cytometry was used to detect levels cluster of differentiation(CD)68,CD80,interleukin(IL)-6,tumor necrosis factorα(TNF-α)and reactive oxygen species(ROS).The expression levels of IL-6,TNF-α,IL-1β,TLR4 and myeloid differentiation factor 88(MyD88)were detected by RT-qPCR.The levels of gasdermin D(GSDMD),IL-18 and NOD-like receptor thermal protein domain associated protein 3(NLRP3)were determined by immunofluorescence and RT-qPCR.Pearson correlation analysis was used to analyze the correlation between the abundance of hsa_circRNA_103124 and TLR4 expression level or Crohn′s disease activity index(CDAI).Results A total of 50 patients were included in the CD group,including 36 males and 14 females

关 键 词：炎性肠疾病 克罗恩病 环状RNA 巨噬细胞 炎症 焦亡 

分 类 号：R574.62[医药卫生—消化系统]