目的 探究紫苏叶醇提物（PFEE）的体内外抗病毒作用，阐明其激活抗病毒天然免疫进而发挥抗病毒作用的分子机制。方法 采用CCK-8法检测PFEE（100～800 μg·mL^-1）对A549细胞活力的影响；通过流式细胞术、实时荧光定量PCR（qRT-PCR）、Western blotting探究PFEE（200、400、600 μg·mL^-1）对水疱性口炎病毒（VSV）感染A549细胞的抑制作用，通过qRT-PCR、Western blotting探究PFEE对甲型流感病毒（H1N1）的抑制作用，通过qRT-PCR探究PFEE对脑心肌炎病毒（EMCV）的抑制作用。构建VSV感染小鼠模型，qRT-PCR法检测肝、肺、脾组织中VSV-G的mRNA表达水平，检测肺组织白细胞介素-6（Il6）、肿瘤坏死因子α（Tnfα）、趋化因子配体10（Cxcl10） mRNA表达水平，Western blotting检测肺组织VSV-G蛋白表达。PFEE处理成纤维细胞（MEF）细胞12 h，转录组测序分析基因通路富集变化，qRT-PCR检测干扰素刺激基因（ISGs）（Ifnb1、Ifit1、Ifit2、Isg15、Ddx58）的mRNA表达，免疫荧光检测干扰素调节因子3（IRF3）核转位情况，Western blotting检测Ⅰ型干扰素（IFN-Ⅰ）通路上游转录因子IRF3、p-核因子-κB活化激酶（TBK1）蛋白磷酸化情况； PFEE处理VSV感染的干扰素受体敲除（IFNAR1^-/-） A549细胞和野生型A549细胞，利用流式细胞术检测PFEE对VSV的抑制作用差异。结果 PFEE对A549细胞800 μg·mL^-1以下无明显细胞毒性。与模型组比较，PFEE具有体外抑制VSV、H1N1、EMCV病毒作用（P＜0.01、0.001）； PFEE在VSV感染小鼠体内能抑制病毒和炎症因子的mRNA表达水平、显著降低VSVG蛋白表达（P＜0.01、0.001）。转录组测序分析结果显示，PFEE上调干扰素相关基因及信号通路，结合qRT-PCR、Westernblotting和免疫荧光发现，与对照组比较，PFEE能诱导ISGs mRNA的表达、促进TBK1、IRF3的磷酸化以及核转位（P＜0.05、0.01、0.001）。与A549细胞比较，在IFNAR1^-/- A549细胞中，PFEE的抗病毒作用被显著抑制（P＜0.05、0.01、0.001）。结论 PFEE通过激活基于IFN-Ⅰ通路的抗病毒天然免疫应答在体内外发挥坑病毒作用。

Objective To research the antiviral effects of ethanol extracts from Perillae Folium (PFEE) in vitro and in vivo, and elucidate the underlying mechanism based on innate immunity response. Methods The effect of PFEE (100—800 μg·mL^-1) on the viability of A549 cells was detected by CCK-8 assay; the inhibitory effect of PFEE (200, 400, 600 μg·mL^-1) on vesicular stomatitis virus (VSV) infection of A549 cells was investigated by flow cytometry, real-time fluorescence quantitative PCR (qRT-PCR), and Western blotting; the inhibitory effect of PFEE on influenza A virus (H1N1) was investigated by qRT-PCR and Western blotting; the inhibitory effect of PFEE on encephalomyocarditis virus (EMCV) was investigated by qRT-PCR. A VSV-infected mouse model was constructed, and the mRNA expression levels of VSV-G in liver, lung, and spleen tissues were detected by qRT-PCR, and the mRNA expression levels of interleukin-6 (Il6), tumor necrosis factor α (Tnfα), and chemokine ligand 10 (Cxcl10) in lung tissue were detected by qRT-PCR, and the expression of VSV-G protein in lung tissue was detected by Western blotting. MEF cells were treated with PFEE for 12 h, and transcriptome sequencing was used to analyze the enrichment changes of gene pathways. The mRNA expression of interferon-stimulated genes (ISGs) (Ifnb1, Ifit1, Ifit2, Isg15, Ddx58) was detected by qRT-PCR, and the nuclear translocation of interferon regulatory factor 3 (IRF3) was detected by immunofluorescence. Results PFEE showed no significant cytotoxicity to A549 cells at concentrations below 800 μg·mL^-1. Compared with the model group, PFEE had an inhibitory effect on VSV, H1N1, and EMCV in vitro (P < 0.01, 0.001); PFEE could inhibit the mRNA expression levels of virus and inflammatory factors and significantly reduce the expression of VSV-G protein in VSV-infected mice (P < 0.01, 0.001). Transcriptome sequencing analysis showed that PFEE upregulated interferon-related genes and signaling pathways. Combined with qRT-PCR, Western blotting, and immunofluorescence, it was found that compared with the control group, PFEE could induce the expression of ISGs mRNA, promote the phosphorylation and nuclear translocation of TBK1 and IRF3 (P < 0.05, 0.01, 0.001). Compared with A549 cells, the antiviral effect of PFEE was significantly inhibited in IFNAR1^-/- A549 cells (P < 0.05, 0.01, 0.001). Conclusion PFEE resists viral infection in vitro and in vivo by activating IFN-Ⅰ signaling.

R285.5

国家自然科学基金面上项目（82474153）;2023年度北京中医药大学-优莎纳联合研究中心（ BURC）基金重点项目（ BUCM-2023-JS-KF-032）;北京市科技新星计划课题（20230484342）;北京市自然科学基金资助项目（7242239）;中华中医药学会青年人才托举工程项目（ CACM-2023-QNRC2-A02）