COPD患者m6A甲基化酶水平观察及FTO对PM2.5刺激后MH-S细胞极化和NF-κB信号活化的影响  

Observation of m6A methylases levels in COPD patients and the effect of FTO on the polarization of MH-S cells and activation of NF-κB signal after PM2.5 stimulation

作　　者：陈丽娜 薛岑[2] 杜丽娟 祝劲 CHEN Li-na;XUE Cen;DU Li-juan;ZHU Jin(School of Clinical Medicine,Guizhou Medical University,Guiyang 550004,China;Department of Respiratory Medicine,Guizhou Cancer Hospital,Guiyang 550001,China;Department of Respiratory Medicine,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;The First Affiliated Hospital of Guiyang College of Traditional Chinese Medicine,Guiyang 550001,China)

机构地区：[1]贵州医科大学临床医学院,贵州贵阳550004 [2]贵州省肿瘤医院呼吸科,贵州贵阳550001 [3]贵州医科大学附属医院呼吸科,贵州贵阳550004 [4]贵阳中医学院第一附属医院,贵州贵阳550001

出　　处：《实用药物与临床》2021年第7期577-585,共9页Practical Pharmacy and Clinical Remedies

基　　金：贵州省科技合作计划项目(黔科合LH字[2018]7034号)。

摘　　要：目的观察COPD患者体内N6-甲基腺苷(m6A)甲基化酶水平,并探讨肥胖相关蛋白(FTO)对细颗粒物2.5(PM2.5)刺激后小鼠肺泡巨噬细胞(MH-S)的极化及核因子κB(NF-κB)信号活化的影响。方法收集COPD患者的支气管肺泡灌洗液标本。使用m6A dot blot检测两组mRNA的m6A变化。体外培养MH-S细胞,使用PM2.5刺激细胞。构建FTO的过表达质粒pcDNA3.1-FTO(pc-FTO)及空载体pcDNA3.1-Null(pc-Null),并转染MH-S细胞。将MH-S细胞分为对照组、PM2.5组、PM2.5+pcFTO组、pcFTO组和pcNull组。RT-PCR和ELISA检测人肺泡灌洗液中m6A甲基化调节酶METTL3、METTL14、FTO、ALKBH5、YTHDF2的表达;CCK-8检测MH-S细胞的存活率,蛋白免疫印迹检测MH-S细胞的FTO、一氧化氮合酶2(iNOS2)、肿瘤坏死因子-α(TNF-α)、精氨酸酶1(ARG1)、甘露糖受体C2(MRC2)、NF-κB-p65、磷酸化的(p)-p65、p-IκBα、IκBα的表达。结果FTO在COPD患者体内表达下调(P<0.05),METTL3、METTL14、ALKBH5、YTHDF2无变化(P>0.05);且COPD患者的m6A甲基化增加。不同浓度暴露PM2.5抑制MH-S细胞的存活率和FTO的表达水平(P<0.05),对METTL3、METTL14、ALKBH5、YTHDF2的表达影响不明显(P>0.05)。PM2.5明显促进MH-S细胞M1型极化特征标志物iNOS2和TNF-α、NF-κB信号蛋白NF-κB-p65、p-p65的表达(P<0.05),但是抑制M2型标志物ARG1和MRC2、NF-κB信号蛋白抑制蛋白p-IκBα及IκBα的表达(P<0.05)。PM2.5暴露MH-S细胞过表达FTO后,iNOS2和TNF-α,NF-κB信号蛋白NF-κB-p65、p-p65的表达下调(P<0.05),而M2型标志物ARG1和MRC2,NF-κB信号蛋白抑制蛋白p-IκBα及IκBα的表达上调(P<0.05)。结论COPD患者的去甲基化m6A酶FTO表达降低,过表达FTO可抑制PM2.5诱导MH-S细胞的M1型极化及NF-κB信号通路的激活。Objective To observe the level of N6-methyladenosine(m6 A)methylase in COPD patients,and to discuss the effects of obesity-related protein(FTO)on the polarization of alveolar macrophages(MH-S)and activation of nuclear factor kappa B(NF-κB)signal after fine particle 2.5(PM2.5)stimulation.Methods Bronchoalveolar lavage fluid specimens from patients with COPD characteristics were collected.M6 A dot blot was used to detect the changes of m6 A mRNA in the two groups.MH-S cells were cultured in vitro and stimulated with PM2.5.The FTO overe-expression plasmid pcDNA3.1-FTO(pc-FTO)and the empty vector pcDNA3.1-Null(pc-Null)were constructed and transfected into MH-S cells.MH-S cells were divided into control group,PM2.5 group,PM2.5+pcFTO group,pcFTO group and pcNull group.RT-PCR and ELISA were used to detect the expression of m6 A methylation regulators,including METTL3,METTL14,FTO,ALKBH5,and YTHDF2 in human alveolar lavage fluid;CCK-8 was used to detect the survival rate of MH-S cells.Western blot was used to detect the expression of FTO,nitric oxide synthase 2(iNOS2),tumor necrosis factor-α(TNF-α)and arginase 1(ARG1),mannose receptor C2(MRC2),NF-κB-p65,phosphorylated(p)-p65,p-IκBα,IκBαof MH-S cells.Results FTO expression was down-regulated in patients with COPD(P<0.05),and METTL3,METTL14,ALKBH5,and YTHDF2 had no change(P>0.05);M6 A methylation was also increased in COPD patients.Exposure of PM2.5 at different concentrations inhibited the survival rate and FTO expression level of MH-S cells(P<0.05),but had no significant effect on the expression of METTL3,METTL14,ALKBH5,and YTHDF2(P>0.05).PM2.5 significantly promoted the expression of M1-type polarization markers(iNOS2 and TNF-α),and NF-κB signaling proteins(NF-κB-p65 and p-p65)in MH-S cells(P<0.05);however,it inhibited the expression of M2-type markers(ARG1 and MRC2),and NF-κB signaling protein inhibitor protein(p-IκBαand IκBα)(P<0.05).After PM2.5-exposed MH-S cells overexpressed FTO,the expression of iNOS2 and TNF-α,NF-κB signaling proteins(NF-κB-p65

关 键 词：慢性阻塞性肺疾病 巨噬细胞 极化 m6A去甲基化酶 肥胖相关因子FTO NF-κB信号 

分 类 号：R563.9[医药卫生—呼吸系统]