In vitro antiviral function and mechanism of isoalantolactone
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 IC1o 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.
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