异土木香内酯体外抗病毒作用与机制研究  

In vitro antiviral function and mechanism of isoalantolactone

作　　者：孔令东 刘霞 李艺颖 王靳勇 董秋童 李敏 禹艳丽 刘哲 贾鑫 KONG Lingdong;LIU Xia;LI Yiying;WANG Jinyong;DONG Qiutong;LI Min;YU Yanli;LIU Zhe;JIA Xin(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100027,China;School of Life Science,Beijing University of Chinese Medicine,Beijing 100027,China)

机构地区：[1]北京中医药大学中药学院,北京100027 [2]北京中医药大学生命科学学院,北京100027

出　　处：《药物评价研究》2024年第3期521-528,共8页Drug Evaluation Research

基　　金：中国科协青年人才托举工程项目(2020-QNRC1-03)。

摘　　要：目的研究异土木香内酯的体外抗病毒作用及机制。方法CCK-8法检测0.125、0.500、1.000、2.000、4.000、8.000、16.000、32.000、64.000μmol·L^(-1)的异土木香内酯处理24 h对人非小细胞肺癌细胞系(A549)细胞活力的影响;表达绿色荧光蛋白的水疱性口炎病毒(VSV-eGFP)以0.02的感染复数(MOI)感染A549细胞制备模型,流式细胞术检测共同孵育12 h的异土木香内酯5、10、20μmol·L^(-1)对GFP阳性细胞率的影响;VSV感染(MOI=0.1)A549细胞,Western blotting检测异土木香内酯处理16 h对VSV病毒G蛋白表达的影响;分别使用甲型流感病毒(H1N1,MOI=0.1)、脑心肌炎病毒(EMCV,MOI=0.1)感染A549细胞,同时给药共同孵育8 h后,实时荧光定量PCR(qRT-PCR)检测土木香内酯对病毒RNA表达的影响;异土木香内酯分别以预处理12 h、病毒吸附过程中给药2 h、病毒吸附后给药10 h 3种不同方式给药,通过流式细胞术检测其对VSV-eGFP在A549细胞中复制的影响;异土木香内酯20μmol·L^(-1)处理胚胎成纤维(MEF)细胞12 h,进行转录组测序分析;异土木香内酯5、10、20μmol·L^(-1)处理MEF细胞12 h,qRT-PCR检测干扰素α1(Ifna1)、干扰素β1(Ifnb1)、干扰素诱导蛋白44(Ifi44)、干扰素刺激基因15(Isg15)的mRNA表达。结果异土木香内酯对A549细胞的半数抑制浓度(IC_(50))为51.01μmol·L^(-1);与模型组比较,流式结果显示异土木香内酯显著减少VSV-eGFP阳性细胞率(P<0.001),但对病毒的吸附过程没有影响,药物预处理及吸附后给药显著抑制VSV病毒复制(P<0.01、0.001);qRT-PCR结果显示异土木香内酯显著降低H1N1、EMCV病毒的mRNA水平(P<0.001);Western blotting结果显示异土木香内酯显著抑制VSV的G蛋白表达(P<0.001);转录组测序分析和qRT-PCR结果表明,异土木香内酯促进I型干扰素通路的激活,并诱导Ifna1、Ifnb1、Ifi44、Isg15(P<0.001)表达。结论异土木香内酯通过促进基于干扰素通路的抗病毒天然免疫激活,发挥抑制病毒复Objective To examine the antiviral activity in vitro and explore the mechanism of action of isoalantolactone.Methods The CCK-8 assay was used to evaluate the effect of isoalantolactone(0.125,0.500,1.000,2.000,4.000,8.000,16.000,32.000,and 64.000μmol·L^(-1))on the viability of A549 cells.The vesicular stomatitis virus expressing green fluorescent protein(VSV-eGFP)infected A549 cells with 0.02 multiplicity of infection(MOI).Flow cytometry was used to detect the effect of isoalantolactone 5,10,20μmol·L^(-1) co-incubated for 12 h on the proportion of GFP positive cells.VSV infected A549 cells,and the effect of isoalantolactone co-incubated for 12 h on the expression of VSV-G protein was detected by Western blotting.A549 cells were infected with influenza A virus(H1N1,MOI=0.1),myocarditis virus(EMCV,MOI=0.1),respectively.After co-incubation for 8 h,the effect of isoalantolactone on virus RNA expression was detected by real-time fluorescence quantitative PCR(qRT-PCR).Isoalantolactone was administered in three different ways:pre-treatment for 12 h,administration during virus adsorption for 2 h,and administration after virus adsorption for 10 h,and its effect on VSV-eGFP replication in A549 cells was detected by flow cytometry.MEF cells were treated with isophorolide(20μmol·L^(-1))for 12 h and subjected to transcriptome sequencing analysis.Treatment of MEF cells with isophorolide(5,10,and 20μmol·L^(-1))for 12 h,qRT-PCR method was used to detect mRNA expression of interferonα1(Ifna1),interferonβ1(Ifnb1),interferon induced protein 44(Ifi44),and interferon stimulated gene 15(Isg15).Results isoalantolactone exhibited an IC10 of 51.01μmol·L^(-1) in A549 cells.Treatment with isoalantolactone resulted in a significant reduction in VSV-eGFP positive cells(P<0.001),as revealed by flow cytometry.Isoalantolactone had no effect on the adsorption process of virus,while pre-treatment or administration after adsorption can significantly inhibit virus replication(P<0.01,0.001).Isoalantolactone led to a significant decrease i

关 键 词：土木香 异土木香内酯 水疱性口炎病毒(VSV) 甲型流感病毒(H1N1) 脑心肌炎病毒(EMCV) 抗病毒免疫 干扰素 干扰素刺激基因 

分 类 号：R285.5[医药卫生—中药学]