FoxO3a影响过敏性鼻炎中巨噬细胞M2型极化及铁死亡水平  

Effects of FoxO3a on M2 polarization and ferroptosis levels of macrophages in allergic rhinitis

作　　者：黎庆辉 曾永亮 陈雪梅[1] 肖斌[1] 易述军 LI Qinghui;ZENG Yongliang;CHEN Xuemei;XIAO Bin;YI Shujun(Department of Otolaryngology-Head and Neck Surgery,Chongqing University Jiangjin Hospital,Chongqing 402260,China)

机构地区：[1]重庆大学附属江津医院耳鼻咽喉头颈外科,402260

出　　处：《免疫学杂志》2024年第11期831-838,共8页Immunological Journal

基　　金：重庆市科卫联合医学科研项目(2020FYYX097);重庆市江津区科技局基金(Y2021055)。

摘　　要：目的探讨叉头盒蛋白O3a(FoxO3a)对过敏性鼻炎(AR)中巨噬细胞M2型极化及铁死亡的影响。方法采集30例AR患者以及30位健康志愿者鼻腔灌洗液,收集3例AR患者以及3位健康志愿者的鼻黏膜。酶联免疫吸附试验(ELISA)检测鼻腔灌洗液中FoxO3a水平。M0型巨噬细胞分别转染siRNA阴性对照质粒(si-Con)、siRNA FoxO3a质粒(si-FoxO3a)、pcDNA3.1-vector或pcDNA3.1-FoxO3a质粒后,用白介素-4(IL-4)诱导向M2型极化。M0型巨噬细胞设为对照组、IL-4模型组、IL-4+si-Con组、IL-4+si-FoxO3a组、IL-4+si-FoxO3a+核转录因子E2相关因子2(Nrf2)抑制剂ML385组、IL-4+pcDNA3.1-vector组和IL-4+pcDNA3.1-FoxO3a组。建立M0型巨噬细胞与人鼻黏膜上皮细胞(NECs)共培养体系,分为NECs+M0型巨噬细胞(对照组)、NECs+M2型巨噬细胞(IL-4模型组)、NECs+si-FoxO3a转染巨噬细胞(IL-4+si-FoxO3a组)、NECs+si-FoxO3a转染巨噬细胞+ML385(IL-4+si-FoxO3a+ML385组),流式细胞仪检测NECs凋亡。C57BL/6小鼠分为正常组、AR模型组和shRNA-FoxO3a腺相关病毒(AAV-sh-FoxO3a)组,HE染色考察鼻黏膜组织病理变化,流式细胞仪检测小鼠鼻腔灌洗液中巨噬细胞M2型比例。ELISA检测小鼠鼻腔灌洗液IL-5、IL-13及NECs粘蛋白5AC(MUC5AC)水平。Western blot检测FoxO3a、IL-10、精氨酸酶1(Arg-1)、Nrf2、血红素氧合酶-1(HO-1)和谷胱甘肽过氧化物酶4(GPX4)蛋白水平。结果与正常组对比,FoxO3a在AR患者鼻腔灌洗液和鼻黏膜组织中表达升高。与对照组相比,IL-4模型组M2型巨噬细胞比例、FoxO3a表达水平升高,Nrf-2、HO-1和GPX4蛋白表达水平降低,Fe2+和丙二醛(MDA)水平升高(P<0.05)。与IL-4模型组相比,IL-4+si-FoxO3a组上述指标被逆转。与IL-4模型组相比,IL-4+pcDNA3.1-FoxO3a组M2型巨噬细胞比例增加。在共培养体系中,与IL-4模型组相比,IL-4+si-FoxO3a组NECs凋亡及MUC5AC分泌减少(P<0.05)。ML385逆转si-FoxO3a对NECs的保护效应。与模型组相比,AAV-sh-FoxO3a组小鼠鼻黏膜组织损伤�Objective To investigate the effects of Forkhead box protein O3a(FoxO3a)on M2 polarization and ferroptosis of macrophages in allergic rhinitis(AR).Methods Nasal lavage fluids were collected from 30 patients with AR and 30 healthy volunteers,while nasal mucosa samples were obtained from 3 AR patients and 3 healthy volunteers.The levels of FoxO3a in nasal lavage fluids were detected by ELISA.M0 macrophages were transfected with siRNA negative control plasmid(si-Con),siRNA FoxO3a plasmid(si-FoxO3a),pcDNA3.1-vector,or pcDNA3.1-FoxO3a plasmid,and then induced to polarize towards M2 type by interleukin-4(IL-4).M0 macrophages were set as the control group,IL-4 model group,IL-4+si-Con group,IL-4+si-FoxO3a group,IL-4+si-FoxO3a+nuclear factor erythroid 2-related factor 2(Nrf2)inhibitor ML385 group,IL-4+pcDNA3.1-vector group,and IL-4+pcDNA3.1-FoxO3a group.A co-culture system of M0 macrophages and human nasal epithelial cells(NECs)was established,and then divided into the NECs+M0 macrophages(control group),NECs+M2 macrophages(IL-4 model group),NECs+si-FoxO3a-transfected macrophages(IL-4+si-FoxO3a group)and NECs+si-FoxO3a-transfected macrophages+ML385(IL-4+si-FoxO3a+ML385 group).Apoptosis of NECs was detected by flow cytometry.C57BL/6 mice were divided into normal group,AR model group and shRNA-FoxO3a adeno-associated virus(AAV-sh-FoxO3a)group.HE staining was used to observe histopathological changes in nasal mucosa tissue,and flow cytometry was used to detect the proportion of M2 macrophages in mouse nasal lavage fluids.The levels of IL-5,IL-13 and NECs mucin 5AC(MUC5AC)in mouse nasal lavage fluids were detected by ELISA;Western blot was used to detect the levels of FoxO3a,IL-10,arginase 1(Arg-1),Nrf2,heme oxygenase-1(HO-1),and glutathione peroxidase 4(GPX4).Results Compared with the normal,the expression of FoxO3a was increased in nasal lavage fluids and nasal mucosa tissues of AR patients.Compared with the control group,the proportion of M2 macrophages and the levels of FoxO3a were increased in the IL-4 model group,while t

关 键 词：过敏性鼻炎 M2巨噬细胞 FOXO3A 铁死亡 凋亡 

分 类 号：R765.21[医药卫生—耳鼻咽喉科] R364.5[医药卫生—临床医学]