地高辛和欧夹竹桃苷抑制RNA病毒复制的功能研究

Function of inhibitioii of RNA virus replication by digoxin and oleandrin

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摘要目的研究强心苷类药物地高辛和欧夹竹桃苷的抗病毒能力。方法采用人肺泡腺癌基底上皮细胞(A549细胞)和非洲绿猴肾细胞(vero细胞),利用细胞增殖-毒性检测试剂盒(CCK-8法)确定强心苷类药物地高辛和欧夹竹桃苷的细胞毒性,利用逆转录-实时荧光定量PCR(RT-qPCR)、蛋白免疫印迹法检测地高辛、欧夹竹桃苷对水疱性口炎病毒(VSV)和脑心肌炎病毒(EMCV)以及仙台病毒(SeV)和甲型流感病毒(H1N1)复制的影响。利用天然的IFN信号通路缺陷的vero细胞结合荧光显微技术、RT-qPCR、蛋白免疫印迹法分析地高辛、欧夹竹桃苷抗病毒的作用机制是否依赖IFN信号通路。在敲低钠钾腺苷三磷酸(ATP)酶α1亚基(ATP1A1-shRNA)的vero细胞中,通过RT-qPCR、蛋白免疫印迹法等检测细胞内病毒复制及细胞的抗病毒IFN反应。结果地高辛和欧夹竹桃苷可抑制VSV、H1N1、SeV和EMCV基因表达;结合使用IFN缺陷的vero细胞证明地高辛和欧夹竹桃苷发挥抗病毒作用不依赖于IFN信号通路;在敲低ATP1A1的vero细胞中,地高辛和欧夹竹桃苷不再有明显的抗病毒作用。结论地高辛、欧夹竹桃苷等强心苷类药物具有较广谱的抗病毒能力,主要通过抑制细胞膜内外钠/钾离子转运蛋白发挥抗病毒作用。 Objective To evaluate the antiviral ability of cardiac glycosides of digoxin and oleandrin.Methods Human adenocarcinoma alveolar basal epithelial cell line(A549)and African green monkey kidney cell line(vero)were used for subsequent experiment.The cytotoxicity of cardiac glycosides of digoxin and oleandrin was assessed by CCK-8 assay.The effects of digoxin and oleandrin on the replication of vesicular stomatitis virus(VSV),encephalomyocarditis virus(EMCV),Sendai virus(SeV)and influenza A virus(H1N1)were determined by reverse transcription-real-time quantitative PCR(RT-qPCR)and Western blot.Vero cells with natural interferon(IFN)signaling pathway defects combined with fluorescence microscopy,RT-qPCR and Western blot were employed to analyze whether the antiviral mechanism of digoxin and oleandrin depended on IFN signaling pathway.In vero cells with ATP1A1(sodium potassium ATPase protein 1)(ATPlAl-shRNA)knockdown,intracellular virus replication and antiviral IFN response were detected by RT-qPCR and Western blot.Results Digoxin and oleandrin could inhibit the expression levels of VSV,H1N1,SeV and EMCV mRNA.The combination of IFN-deficient vero cells demonstrated that digoxin and oleandrin played an antiviral role independent of IFN signaling pathway.Meantime,digoxin and oleandrin exerted no antiviral effect in vero cells with ATP1A1 knockdown.Conclusion Digoxin,oleandrin and other potent cardiac glycosides have a broad spectrum of antiviral capability mainly through the inhibition of Na7K*ion transporters intra-and extra-cell membrane.

作者廖夏琳梁巍陈嘉雯刘坤鹏 Liao Xialin;Liang Wei;Chen Jiawen;Liu Kunpeng(Biotherapy Center,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区中山大学附属第三医院生物治疗中心

出处《新医学》 CAS 2023年第5期326-332,共7页 Journal of New Medicine

基金国家自然科学基金(81901613)。

关键词地高辛欧夹竹桃苷抗病毒先天免疫钠钾腺苷三磷酸酶水疱性口炎病毒甲型流感病毒呼吸道病毒脑心肌炎病毒 Digoxin Oleandrin Antivirus Innate immunity Na^(+)/K^(+)-ATPase Vesicular stomatitis virus Influenza A virus Respiratory virus Encephalomyocarditis virus