免疫相关鸟苷三磷酸酶通过NF-κB通路调节流感病毒感染后的细胞炎性因子表达  

Immunity-related GTPase family M regulates the expression of inflammatory cytokines in influenza virus-infected cells through the NF-κB signaling pathway

作　　者：刘世博 黄逸伦 杨新燕 朱明利 Liu Shibo;Huang Yilun;Yang Xinyan;Zhu Mingli(Affiliated Hangzhou Xixi Hospital Open Laboratory,Hangzhou 310000,China;Alberta Institute,Wenzhou Medical University,Wenzhou 3252035,China;School of Laboratory Medicine and Bioengineering,Hangzhou Medical College,Hangzhou 311399,China)

机构地区：[1]杭州市西溪医院开放实验室,杭州310000 [2]温州医科大学阿尔伯塔学院,温州325035 [3]杭州医学院检验医学院生物工程学院,杭州311399

出　　处：《国际流行病学传染病学杂志》2025年第1期17-23,共7页International Journal of Epidemiology and Infectious Disease

基　　金：浙江省医药卫生科技计划(2022KY1024、2021KY636、2020KY098);浙江省基金-青山湖科技城联合基金(LQQ20H190001);杭州市卫生科技计划(oo20190555)。

摘　　要：目的:探讨免疫相关鸟苷三磷酸酶(IRGM)在流感病毒感染后引发的细胞炎性因子表达中的作用。方法:通过Western印迹法和RT-PCR检测PR8流感病毒株感染A549后IRGM基因和蛋白水平的表达,其次构建IRGM敲除的A549细胞并进行转录组测序,对所得到的差异基因进行KEGG富集分析。IRGM敲除的A549细胞感染PR8流感病毒株,PCR检测炎性因子IL-1b、IL-6,以及趋化因子CCL2、CCL5的基因表达,免疫印迹检测NF-κB、p-NF-κB的蛋白水平表达。STRING在线构建蛋白互作网络,Cytoscape软件解析核心子网络,drugbank数据库寻找预测小分子化合物。结果:相比未感染状态下,流感病毒感染12、18、24和48 h后,A549细胞中IRGM基因的mRNA表达分别上调了2.0、3.5、4.1和4.2倍,差异均具有统计学意义(t=3.13、7.73、9.57和9.94;P值分别为0.034、<0.001、<0.001和<0.001),蛋白表达水平也均明显升高。与未敲除IRGM并感染PR8的细胞相比,IRGM敲除后炎性因子IL-1b、IL-6、CCL2和CCL5表达分别上调了2.6、2.8、3.3和2.8倍,差异均具有统计学意义(t=3.75、5.07、6.60和6.75;P值分别为0.020、0.007、0.003和0.002)。IRGM敲除后的差异基因进行KEGG富集分析结果显示其主要富集在细胞周期、胞质DNA传感、甲型流感、细胞因子与趋化因子信号通路、Toll样受体信号通路。与未敲除IRGM并感染PR8的细胞相比,IRGM敲除后NF-κB信号通路活性升高。通过构建PPI蛋白互作网络并得到核心子网络,根据核心子网络基因使用drugbank数据库预测了10种小分子化合物,分别为N-甲基-L-亮氨酸、埃迪鲁单抗、肝素二糖Ⅰ-S、肝素二糖Ⅲ-S、二硫赤藓醇、4-(2,4-二甲基-5-噻唑)-2-氨基嘧啶、O6-环甲基己基鸟嘌呤、[4-(2-氨基-4-甲基-5-噻唑乙酸乙酯)-乙酰基嘧啶]-(3-硝基苯硼酸)-胺、4-(2,4-二甲基-1,3-噻唑)-N-[4-三氟甲基苯乙胺]-2-氨基嘧啶、4-[(7-氧-7氢-噻唑并[5,4-E]吲哚-8-甲基)-磺胺嘧啶])作为潜在的治疗Objective:To explore the role of immunity-related GTPase family M(IRGM)in the expression of cellular inflammatory factors induced by influenza virus infection.Methods:Western blotting and RT-PCR were used to detect the expression of IRGM gene and protein levels in A549 cells infected with PR8 influenza virus.Then,IRGM-knockout A549 cells were constructed and transcriptome sequencing was performed.The differential genes obtained were subjected to KEGG enrichment analysis.IRGM-knockout A549 cells were infected with PR8 influenza virus.PCR was used to detect the gene expression of inflammatory factors IL-1b,IL-6,and chemokines CCL2 and CCL5.Western blotting was used to detect the protein expression levels of NF-κB,p-NF-κB.A protein-protein interaction network was constructed using STRING,and the core subnetwork was analyzed with Cytoscape software.Potential small molecule compounds were predicted using the drugbank database.Results:Compared to the uninfected cells,the mRNA expression of the IRGM gene in A549 cells was upregulated by 2.0,3.5,4.1,and 4.2 times after influenza virus infection for 12,18,24,and 48 hours,respectively,with statistically significant differences(t=3.13,7.73,9.57 and 9.94;P=0.034,<0.001,<0.001 and<0.001).Additionally,the protein expression levels also significantly elevated.Compared to IRGM-intact cells infected with PR8,IRGM-knockout cells showed increased levels of inflammatory factors IL-1b,IL-6,CCL2,and CCL5 by 2.6,2.8,3.3,and 2.8 times,respectively,with statistically significant differences(t=3.75,5.07,6.60 and 6.75;P=0.020,0.007,0.003 and 0.002).The KEGG enrichment analysis of differentially expressed genes after IRGM knockout showed that they mainly enriched in cell cycle,cytoplasmic DNA sensing,influenza A,cytokine and chemokine signaling pathways,and Toll like receptor signaling pathways.Compared to IRGM-intact cells infected with PR8,the activity of NF-κB signaling pathway increased in IRGM-knockout cells.By constructing a PPI protein interaction network and obtaining a core subn

关 键 词：流感病毒 免疫相关鸟苷三磷酸酶 细胞炎性因子 NF-ΚB 信号通路 

分 类 号：R373.1[医药卫生—病原生物学]