目的 研究異土木香內(nèi)酯的體外抗病毒作用及機(jī)制。方法 CCK-8法檢測0.125、0.500、1.000、2.000、4.000、8.000、16.000、32.000、64.000 μmol·L^-1的異土木香內(nèi)酯處理24 h對人非小細(xì)胞肺癌細(xì)胞系（A549）細(xì)胞活力的影響；表達(dá)綠色熒光蛋白的水皰性口炎病毒（VSV-eGFP）以0.02的感染復(fù)數(shù)（MOI）感染A549細(xì)胞制備模型，流式細(xì)胞術(shù)檢測共同孵育12 h的異土木香內(nèi)酯5、10、20 μmol·L^-1對GFP陽性細(xì)胞率的影響；VSV感染（MOI=0.1） A549細(xì)胞，Western blotting檢測異土木香內(nèi)酯處理16 h對VSV病毒G蛋白表達(dá)的影響；分別使用甲型流感病毒（H1N1，MOI=0.1）、腦心肌炎病毒（EMCV，MOI=0.1）感染A549細(xì)胞，同時給藥共同孵育8 h后，實(shí)時熒光定量PCR （qRT-PCR）檢測土木香內(nèi)酯對病毒RNA表達(dá)的影響；異土木香內(nèi)酯分別以預(yù)處理12 h、病毒吸附過程中給藥2 h、病毒吸附后給藥10 h 3種不同方式給藥，通過流式細(xì)胞術(shù)檢測其對VSV-eGFP在A549細(xì)胞中復(fù)制的影響；異土木香內(nèi)酯20 μmol·L^-1處理胚胎成纖維（MEF）細(xì)胞12 h，進(jìn)行轉(zhuǎn)錄組測序分析；異土木香內(nèi)酯5、10、20 μmol·L^-1處理MEF細(xì)胞12 h，qRT-PCR檢測干擾素α1（Ifna1）、干擾素β1（Ifnb1）、干擾素誘導(dǎo)蛋白44（Ifi44）、干擾素刺激基因15（Isg15）的mRNA表達(dá)。結(jié)果 異土木香內(nèi)酯對A549細(xì)胞的半數(shù)抑制濃度（IC₅₀）為51.01 μmol·L^-1；與模型組比較，流式結(jié)果顯示異土木香內(nèi)酯顯著減少VSV-eGFP陽性細(xì)胞率（P<0.001），但對病毒的吸附過程沒有影響，藥物預(yù)處理及吸附后給藥顯著抑制VSV病毒復(fù)制（P<0.01、0.001）；qRT-PCR結(jié)果顯示異土木香內(nèi)酯顯著降低H1N1、EMCV病毒的mRNA水平（P<0.001）；Western blotting結(jié)果顯示異土木香內(nèi)酯顯著抑制VSV的G蛋白表達(dá)（P<0.001）；轉(zhuǎn)錄組測序分析和qRT-PCR結(jié)果表明，異土木香內(nèi)酯促進(jìn)I型干擾素通路的激活，并誘導(dǎo)Ifna1、Ifnb1、Ifi44、Isg15（P<0.001）表達(dá)。結(jié)論 異土木香內(nèi)酯通過促進(jìn)基于干擾素通路的抗病毒天然免疫激活，發(fā)揮抑制病毒復(fù)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

中國科協(xié)青年人才托舉工程項目（2020-QNRC1-03）