千金藤素抗病毒作用的免疫学机制研究  

Immunological mechanism of antiviral effect of cepharanthine

作　　者：王靳勇 何昱廷 谢芳 成翠芹 张美琦 初英杰 罗政 李俊良 徐安龙 王遥 WANG Jinyong;HE Yuting;XIE Fang;CHENG Cuiqin;ZHANG Meiqi;CHU Yingjie;LUO Zheng;LI Junliang;XU Anlong;WANG Yao(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;School of Life Science,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]北京中医药大学生命科学学院,北京102488

出　　处：《药物评价研究》2024年第6期1232-1239,共8页Drug Evaluation Research

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A类资助项目(2023-QNRC2-A02)。

摘　　要：目的探索千金藤素体内外的抗病毒作用,并基于抗病毒天然免疫通路探究其抗病毒作用的分子机制。方法CCK-8法检测千金藤素(0.0625~64.0000μmol·L^(-1))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(VSV-GFP)感染A549细胞模型,结合流式细胞术检测千金藤素对病毒复制的影响并探究预处理、吸附过程及吸附后加药对VSV-GFP病毒复制的影响;实时荧光定量PCR(qRT-PCR)检测千金藤素对甲型流感病毒(H1N1)、脑心肌炎病毒(EMCV)和单纯疱疹病毒I型(HSV-1)复制的影响;构建VSV感染小鼠模型探究千金藤素的体内抗病毒作用;A549细胞中利用生物信息学方法探究其抗病毒机制;qRT-PCR检测药物处理A549和原代胚胎成纤维细胞(MEF)后IFNB1及干扰素刺激基因(ISGs)表达变化;免疫印迹法(Immunoblotting)检测人源单核细胞白血病细胞(THP-1)中TBK1和STAT1的磷酸化水平。结果与模型组相比,千金藤素在A549细胞中显著抑制VSV、H1N1、EMCV和HSV-1复制;千金藤素不影响VSV的吸附过程,而预处理或吸附后给药可以显著抑制病毒复制;千金藤素提高VSV感染小鼠的存活率;千金藤素激活基于IFN-I通路的抗病毒天然免疫应答。结论千金藤素通过激活基于IFN-I通路的抗病毒天然免疫发挥体内外抗病毒作用。Objective To investigate the antiviral effects of cepharanthine(Cep)in vivo and in vitro and explore its immunological mechanism based on the innate antiviral immune pathway.Methods The viability of A549 cells was assessed using the CCK-8 assay to determine the impact of Cep(0.0625—64.00000μmol·L^(-1)).Using a vesicular stomatitis virus expressing green fluorescent protein(VSV-GFP)infected cell models,combined with flow cytometry to investigate the impact of Cep on virus replication.We also examined how pretreatment,the adsorption process,and post-adsorption treatment affect virus replication..Real-time fluorescence quantitative PCR(qRT-PCR)was employed to detect the replication of influenza A virus(HINI),encephalomyocarditis virus(EMCV),and herpes simplex virus type 1(HSV-1)under the influence of Cep.The in vivo antiviral effects of Cep were investigated using the VSV infection mouse model.Bioinformatics analysis was conducted in A549 cells to explore the mechanism of the antiviral function of Cep.qPCR was used to detect changes in mRNA expression of IFNBI and the interferon-stimulated genes(ISGs)in A549 and MEF cells with Cep treatment.Immunoblotting was performed to measure changes in the phosphorylation level of TBKI and STAT1 in THP-1 cells.Results In vitro experiments showed that Cep significantly inhibited the replication of VSV,H1N1,EMCV,and HSV-1 viruses compared to the model group in A549 cells.Cep did not affect the adsorption process of VsV,but pre-treatment or post-adsorption administration effectively inhibited virus replication.Cep increased the survival rate of VSV-infected mice.Cep activates the innate antiviral immune response through the IFN-I pathway.Conclusion This study demonstrates that Cep exhibits antiviral effects both in vivo and in vitro by activating the innate antiviral immune response through the IFN-I pathway.

关 键 词：千金藤素 抗病毒天然免疫 I型干扰素通路 干扰素刺激基因 抗病毒 

分 类 号：R285.5[医药卫生—中药学]