目的 探究紫蘇葉醇提物（PFEE）的體內(nèi)外抗病毒作用，闡明其激活抗病毒天然免疫進(jìn)而發(fā)揮抗病毒作用的分子機(jī)制。方法 采用CCK-8法檢測(cè)PFEE（100～800 μg·mL^-1）對(duì)A549細(xì)胞活力的影響；通過(guò)流式細(xì)胞術(shù)、實(shí)時(shí)熒光定量PCR（qRT-PCR）、Western blotting探究PFEE（200、400、600 μg·mL^-1）對(duì)水皰性口炎病毒（VSV）感染A549細(xì)胞的抑制作用，通過(guò)qRT-PCR、Western blotting探究PFEE對(duì)甲型流感病毒（H1N1）的抑制作用，通過(guò)qRT-PCR探究PFEE對(duì)腦心肌炎病毒（EMCV）的抑制作用。構(gòu)建VSV感染小鼠模型，qRT-PCR法檢測(cè)肝、肺、脾組織中VSV-G的mRNA表達(dá)水平，檢測(cè)肺組織白細(xì)胞介素-6（Il6）、腫瘤壞死因子α（Tnfα）、趨化因子配體10（Cxcl10） mRNA表達(dá)水平，Western blotting檢測(cè)肺組織VSV-G蛋白表達(dá)。PFEE處理成纖維細(xì)胞（MEF）細(xì)胞12 h，轉(zhuǎn)錄組測(cè)序分析基因通路富集變化，qRT-PCR檢測(cè)干擾素刺激基因（ISGs）（Ifnb1、Ifit1、Ifit2、Isg15、Ddx58）的mRNA表達(dá)，免疫熒光檢測(cè)干擾素調(diào)節(jié)因子3（IRF3）核轉(zhuǎn)位情況，Western blotting檢測(cè)Ⅰ型干擾素（IFN-Ⅰ）通路上游轉(zhuǎn)錄因子IRF3、p-核因子-κB活化激酶（TBK1）蛋白磷酸化情況； PFEE處理VSV感染的干擾素受體敲除（IFNAR1^-/-） A549細(xì)胞和野生型A549細(xì)胞，利用流式細(xì)胞術(shù)檢測(cè)PFEE對(duì)VSV的抑制作用差異。結(jié)果 PFEE對(duì)A549細(xì)胞800 μg·mL^-1以下無(wú)明顯細(xì)胞毒性。與模型組比較，PFEE具有體外抑制VSV、H1N1、EMCV病毒作用（P＜0.01、0.001）； PFEE在VSV感染小鼠體內(nèi)能抑制病毒和炎癥因子的mRNA表達(dá)水平、顯著降低VSVG蛋白表達(dá)（P＜0.01、0.001）。轉(zhuǎn)錄組測(cè)序分析結(jié)果顯示，PFEE上調(diào)干擾素相關(guān)基因及信號(hào)通路，結(jié)合qRT-PCR、Westernblotting和免疫熒光發(fā)現(xiàn)，與對(duì)照組比較，PFEE能誘導(dǎo)ISGs mRNA的表達(dá)、促進(jìn)TBK1、IRF3的磷酸化以及核轉(zhuǎn)位（P＜0.05、0.01、0.001）。與A549細(xì)胞比較，在IFNAR1^-/- A549細(xì)胞中，PFEE的抗病毒作用被顯著抑制（P＜0.05、0.01、0.001）。結(jié)論 PFEE通過(guò)激活基于IFN-Ⅰ通路的抗病毒天然免疫應(yīng)答在體內(nèi)外發(fā)揮坑病毒作用。

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

國(guó)家自然科學(xué)基金面上項(xiàng)目（82474153）;2023年度北京中醫(yī)藥大學(xué)-優(yōu)莎納聯(lián)合研究中心（ BURC）基金重點(diǎn)項(xiàng)目（ BUCM-2023-JS-KF-032）;北京市科技新星計(jì)劃課題（20230484342）;北京市自然科學(xué)基金資助項(xiàng)目（7242239）;中華中醫(yī)藥學(xué)會(huì)青年人才托舉工程項(xiàng)目（ CACM-2023-QNRC2-A02）