敲低MUC5B表达对分泌性中耳炎大鼠炎症反应的影响  

Effect of MUC5B knockdown on inflammatory responses in rats with secretory otitis media

作　　者：王丹 杨启梅[2] 刘晓娜 陈菁华 王玉霞 WANG Dan;YANG Qimei;LIU Xiaona;CHEN Jinghua;WANG Yuxia(Department of Otolaryngology,North-West Women and Children's Hospital,Xi'an 710061,China;Department of Otorhinolaryngology-Head and Neck Surgery,Shaanxi Provincial People's Hospital)

机构地区：[1]西北妇女儿童医院耳鼻喉科,西安710061 [2]陕西省人民医院耳鼻咽喉头颈外科

出　　处：《山西医科大学学报》2025年第1期57-65,共9页Journal of Shanxi Medical University

基　　金：陕西省重点研发计划项目(2023-YBSF-081)。

摘　　要：目的 探究黏蛋白5B(MUC5B)对分泌性中耳炎(SOM)炎症反应的调控作用,以及与中性粒细胞胞外诱捕网(NETs)形成的关系。方法 采用腹腔注射卵清蛋白构建SOM大鼠模型,随机分为模型组、shRNA-NC组和shRNA-MUC5B组,每组10只,另取10只健康SD大鼠作为对照组。末次注射完成后24 h,进行大鼠炎症评分,采用HE染色检测大鼠中耳黏膜组织病变,ELISA法检测中耳灌洗液中炎性因子的表达,流式细胞术检测Th1和Th2细胞分布比例,免疫荧光染色检测中耳黏膜组织中NETs标志物髓过氧化物酶(MPO)和瓜氨酸化组蛋白H3(CitH3)的表达,DCFH-DA探针检测中耳黏膜组织中ROS水平。RTqPCR检测MUC5B基因的表达水平,Western blot检测干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)、IL-4、IL-13、MUC5B、MPO、CitH3和中性粒细胞弹性蛋白酶(NE)蛋白的表达水平。结果 与对照组比较,模型组和shRNA-NC组大鼠表现出中耳黏膜增厚,炎症程度评分升高(P<0.05);中耳黏膜组织中MUC5B mRNA水平,CitH3、MPO、MUC5B、IL-4和IL-13蛋白水平,Th2细胞比例以及ROS水平均升高(P<0.05),IFN-γ和IL-2蛋白水平以及Th1细胞比例和Th1/Th2比值均降低(P<0.05);中耳灌洗液中MPO、NE和CitH3蛋白水平以及IL-4和IL-13含量均升高(P<0.05),IFN-γ和IL-2含量以及IFN-γ/IL-4比值均降低(均P<0.05)。与shRNA-NC组比较,shRNA-MUC5B组大鼠中耳黏膜厚度和炎症程度评分均降低(P<0.05);中耳黏膜组织中MUC5B mRNA水平,CitH3、MPO、MUC5B、IL-4和IL-13蛋白水平,Th2细胞比例以及ROS水平均降低(P<0.05),IFN-γ和IL-2蛋白水平以及Th1细胞比例和Th1/Th2比值均升高(P<0.05);中耳灌洗液中MPO、NE和CitH3蛋白水平以及IL-4和IL-13含量均降低(P<0.05),IFN-γ和IL-2含量以及IFN-γ/IL-4比值均升高(P<0.05)。结论 MUC5B敲低表达可以缓解SOM大鼠中耳黏膜组织炎症反应,该作用可能与抑制NETs的形成有关。Objective To investigate the effect of Mucins 5B(MUC5B)on inflammatory response in secretory otitis media(SOM)and its relationship with the formation of neutrophil extracellular traps(NETs).Methods SOM rat model was constructed by intraperito-neal injection of ovalbumin.SOM rats were divided into model group,shRNA-NC group and shRNA-MUC5B group,with 10 rats in each group,and ten healthy SD rats were selected as control group.At 24 h after the last injection,the inflammation was scored,the lesion of middle ear mucosa tissue was detected by HE staining,the expressions of inflammatory factors in middle ear irrigation solu-tion were detected by ELISA,the distribution of Th1 and Th2 cells was detected by flow cytometry,and the expressions of NET mark-ers myeloperoxidase(MPO)and citrullinated histone H3(CitH3)in middle ear mucosa were detected by immunofluorescence staining.The ROS level in middle ear mucosa tissue was detected by DCFH-DA probe.The expression level of MUC5B gene was detected by RT-qPCR,and the expressions of interferon-γ(IFN-γ),interleukin-2(IL-2),IL-4,IL-13,MUC5B,MPO,CitH3 and neutrophil elastase(NE)proteins were detected by Western blot.Results Compared with control group,the middle ear mucosa was thickened,the in-flammation degree score was increased in model group and shRNA-NC group(P<0.05),the mRNA level of MUC5B,the protein levels of CitH3,MPO,MUC5B,IL-4 and IL-13,Th2 cell proportion and ROS levels were all increased in middle ear mucosa tissues(P<0.05),and the protein levels of IFN-γand IL-2,Th1 cell proportion and Th1/Th2 ratio were decreased(P<0.05);the protein levels of MPO,NE and CitH3,and the contents of IL-4 and IL-13 were increased in middle ear lavage fluid(P<0.05),while the contents of IFN-γand IL-2,and IFN-γ/IL-4 ratio were decreased(P<0.05).Compared with shRNA-NC group,the mucosal thickness and the in-flammatory degree score were decreased in shRNA-MUC5B group(P<0.05),the mRNA level of MUC5B,the protein levels of CitH3,MPO,MUC5B,IL-4 and IL-13,Th2 cell proportion and ROS level we

关 键 词：黏蛋白5B(MUC5B) 分泌性中耳炎 炎症反应 中性粒细胞胞外诱捕网(NETs) 中耳黏膜 TH1/TH2 

分 类 号：R764.2[医药卫生—耳鼻咽喉科]