土木香内酯抗病毒作用的体内外研究  

Antiviral effects of alantolactone in vitro and in vivo

作　　者：李艺颖 初英杰 孔令东 谢芳 何昱廷 刘霞 李俊良 罗政 周义翔 王遥 LI Yiying;CHU Yingjie;KONG Lingdong;XIE Fang;HE Yuting;LIU Xia;LI Junliang;LUO Zheng;ZHOU Yixiang;WANG Yao(School of Life Science,Beijing University of Chinese Medicine,Beijing 100029,China;School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学生命科学学院,北京100029 [2]北京中医药大学中药学院,北京100029

出　　处：《中草药》2024年第7期2248-2258,共11页Chinese Traditional and Herbal Drugs

基　　金：北京市科技新星计划课题(20230484342);中华中医药学会青年人才托举工程项目(CACM-2023-QNRC2-A02);北京市自然科学基金资助项目(7242239)。

摘　　要：目的探索土木香内酯体内外的抗病毒作用,并基于Ⅰ型干扰素(typeⅠinterferon,IFN-Ⅰ)通路探究其抗病毒作用机制。方法CCK-8法检测土木香内酯对A549细胞活力的影响;利用表达绿色荧光蛋白的水疱性口炎病毒(vesicular stomatitis virus expressing green fluorescent protein,VSV-GFP)感染细胞模型,结合流式细胞术检测土木香内酯对病毒复制的影响;qRT-PCR检测土木香内酯对甲型流感病毒(influenza A virus,H1N1)、脑心肌炎病毒(encephalomyocarditis virus,EMCV)复制的影响;Western blotting检测土木香内酯对VSV病毒G蛋白和H1N1病毒NP蛋白表达的影响;利用生物信息学初步分析土木香内酯的抗病毒分子机制;qRT-PCR检测药物处理后干扰素α1(interferonα1,Ifna1)、干扰素β1(interferonβ1,Ifnb1)、干扰素诱导蛋白1(interferon-induced protein with tetratricopeptide repeats 1,Ifit1)、干扰素刺激基因15(interferon stimulated gene 15,Isg15)的mRNA表达变化;流式细胞术和qRT-PCR检测Ⅰ型干扰素受体1(typeⅠinterferon receptor 1,IFNAR1)敲除(Ifnar1^(-/-))细胞中,土木香内酯的抗病毒作用与干扰素信号通路的关系;构建小鼠H1N1病毒滴鼻感染模型探究土木香内酯的体内抗病毒作用。结果土木香内酯体外抑制VSV、H1N1和EMCV病毒复制;土木香内酯激活IFN-Ⅰ信号通路;在Ifnar1^(-/-)细胞中土木香内酯抗病毒作用被削弱;土木香内酯提高H1N1感染小鼠的存活率,降低脏器H1N1病毒载量。结论土木香内酯激活基于IFN-Ⅰ信号通路的抗病毒天然免疫反应,发挥抵抗多种病毒复制的作用。Objective To explore the antiviral effects of alantolactone in vitro and in vivo,and investigate its mechanism based on type I interferon(IFN-I)pathway.Methods The CCK-8 assay was used to evaluate the effect of alantolactone on viability of A549 cells.The vesicular stomatitis virus expressing green fluorescent protein(VSV-GFP)was employed to infect cells to establish a model,and flow cytometry was used to assess the influence of alantolactone on virus replication.qRT-PCR was conducted to measure the effects of alantolactone on the replication of influenza A virus(H1N1)and encephalomyocarditis virus(EMCV).Western blotting was performed to determine the effect of alantolactone on the expressions of VSV G protein and H1N1 NP protein.Bioinformatics was used to investigate the antiviral molecular mechanism of alantolactone.qRT-PCR was employed to measure the mRNA expressions of interferonα1(Ifna1),interferonβ1(Ifnb1),interferon-induced protein with tetratricopeptide repeats 1(Ifit1)and interferon stimulated gene 15(Isg15)after alantolactone treatment.Flow cytometry and qRT-PCR were used to investigate the relationship between the antiviral effects of alantolactone and the interferon signaling pathway in type I interferon receptor 1(IFNAR1)knockout cells(Ifnar1^(−/−)).To investigate the antiviral effects of alantolactone in vivo,a mouse model of H1N1 virus intranasal infection was established.Results Alantolactone exhibited inhibition of VSV,H1N1 and EMCV virus replication in vitro.Alantolactone activated IFN-I signaling pathway.The antiviral effects of alantolactone were weakened in Ifnar1^(−/−)cells.Alantolactone increased the survival rate of H1N1-infected mice and reduced the viral load of H1N1 in organs.Conclusion Alantolactone activates the antiviral innate immune response based on IFN-I signaling pathway,exhibiting the ability to resist the replication of various viruses.

关 键 词：土木香内酯 抗病毒先天免疫 干扰素 干扰素刺激基因 病毒 

分 类 号：R285.5[医药卫生—中药学]