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、64.000 μmol·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、20 μmol·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20 μmol·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、20 μmol·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（IC₅₀）为51.01 μmol·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，发挥抑制病毒复制的作用。

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 64.000 μmol·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, 20 μmol·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 (20 μmol·L^-1) for 12 h and subjected to transcriptome sequencing analysis. Treatment of MEF cells with isophorolide (5, 10, and 20 μmol·L^-1) for 12 h, qRT-PCR method was used to detect mRNA expression of interferon α1 (Ifna1), interferon β1 (Ifnb1), interferon induced protein 44 (Ifi44), and interferon stimulated gene 15 (Isg15). Results isoalantolactone exhibited an IC₁₀ of 51.01 μmol·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 in the mRNA levels of H1N1 and EMCV, as indicated by qRT-PCR results (P< 0.001). Transcriptome sequencing analysis and qRT-PCR results demonstrated that isoalantolactone enhanced the activation of the type I interferon pathway and upregulated the expression of Ifna1, Ifnb1, Ifi44, and Isg15 (P< 0.001). Conclusion Isoalantolactone has the potential to suppress viral replication by enhancing the antiviral innate immunity mediated through the type I interferon pathway.

R285.5

中国科协青年人才托举工程项目（2020-QNRC1-03）