目的 观察小檗胺的体外抗病毒作用，并基于Ⅰ型干扰素（IFN-Ⅰ）通路的抗病毒天然免疫反应探讨其作用机制。方法 CCK-8法检测0.001、0.010、0.100、1.000、10.000、100.000 μmol·L^-1的小檗胺对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-GFP）以0.05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的小檗胺2.5、5.0、10.0 μmol·L^-1对GFP阳性细胞比例的影响；VSV感染A549细胞，Western blotting检测小檗胺对VSV病毒G蛋白表达的影响；小檗胺使用预处理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-GFP在A549细胞中复制的影响；分别使用甲型流感病毒（H1N1）（MOI＝0.05），脑心肌炎病毒（EMCV）（MOI＝3）和单纯疱疹病毒1型（HSV-1）（MOI＝1）感染A549细胞，同时给药共同孵育12 h后，实时荧光定量PCR（qRT-PCR）检测小檗胺对病毒RNA表达的影响；小檗胺10.0 μmol·L^-1处理A549细胞24 h，进行转录组测序分析；10 μmol·L^-1小檗胺处理MEF细胞12 h后，qRT-PCR法检测Ifnb1、Ifit1、Ifit2、Ifi44基因的mRNA表达；利用IFN刺激性DNA （ISD）转染THP-1细胞，预先激活IFN-I信号通路，4～6 h加入小檗胺5、10 μmol·L-1处理12 h，qRT-PCR法检测IFNB1、IFIT1、IFIT2、IFI44基因的mRNA表达。结果 浓度为10 μmol·L^-1及以下时，小檗胺对A549细胞均未显示出明显的细胞毒性；与模型组相比，小檗胺剂量相关性地减少了VSV-GFP阳性细胞的比例（P＜0.05、0.001），明显减少了病毒的VSV-G蛋白表达；小檗胺对VSV的吸附过程没有影响，而预处理或吸附后给药可以显著抑制病毒复制；与模型组比较，小檗胺剂量相关性地抑制了H1N1、EMCV和HSV-1的病毒基因表达（P＜0.001）；转录组测序和qRT-PCR结果表明，小檗胺促进细胞基于IFN-I信号通路的抗病毒免疫激活；在ISD刺激后，小檗胺诱导更高水平的IFNB1、IFIT1、IFIT2、IFI44 mRNA表达（P＜0.05、0.01、0.001）。结论 小檗胺可能通过促进基于IFN-I通路的抗病毒天然免疫反应抑制多种病毒复制。

Objective Observe the antiviral effect of berbamine in vitro and explore its mechanism of action based on the antiviral natural immune response of the type I interferon (IFN-I) pathway. Methods CCK-8 method for detecting effect of 0.001, 0.010, 0.100, 1.000, 10.000, 100.000 μmol·L^-1 berbamine on the viability of A549 cells. The vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) infected A549 cells with 0.05 multiplicity of infection (MOI). Flow cytometry was used to detect the effect of berbamine 2.5, 5.0, 10.0 μmol·L-1 co-incubated for 12 h on the proportion of GFP positive cells. VSV infected A549 cells, and the effect of berbamine on the expression of VSV-G protein was detected by Western blotting. Berberine 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-GFP replication in A549 cells was detected by flow cytometry. A549 cells were infected with influenza A virus (H1N1) (MOI = 0.05), myocarditis virus (EMCV) (MOI = 3), and herpes simplex virus type 1 (HSV-1) (MOI = 1), respectively. After co-incubation for 12 h, the effect of berberine on virus RNA expression was detected by real-time fluorescence quantitative PCR (qRT-PCR). A549 cells were treated with berbamine 10.0 μmol·L^-1 for 24 h, and transcriptome sequencing was performed. After treating MEF cells with 10 μmol·L^-1 berbamine for 12 h, qRT-PCR was used to detect the mRNA expression of Ifnb1, Ifit1, Ifit2, and Ifi44 genes. Transfection of THP-1 cells using IFN stimulated DNA (ISD) was performed, and the IFN-I signaling pathway was pre-activated. Berberine 5 and 10 μmol·L^-1 were added after 4—6 h to treat for 12 h. mRNA expression of IFNB1, IFIT1, IFIT2, and IFI44 genes was detected by qRT-PCR. Results At concentrations of 10 μmol·L^-1 and below, berbamine did not exhibit significant cytotoxicity on A549 cells. Compared with the model group, berbamine dose dependently reduced the proportion of VSV-GFP positive cells (P < 0.05, 0.001), and significantly reduced the expression of virus VSV-G protein. Berberine had no effect on the adsorption process of VSV, while pre-treatment or administration after adsorption can significantly inhibit virus replication. Compared with model group, berberine dose-related inhibition of viral gene expression in H1N1, EMCV, and HSV-1 (P < 0.001). The Results of transcriptome sequencing and qRT-PCR showed that berbamine promoted the antiviral immune activation of cells based on IFN-I signaling pathway. After ISD stimulation, berbamine induced higher levels of IFNB1, IFIT1, IFIT2, and IFI44 mRNA expression (P < 0.05, 0.01, 0.001). Conclusions Berbamine may inhibit the replication of multiple virus by promoting antiviral innate immune responses based on the IFN-I signaling.

R965

中国科协青年人才托举工程项目（2020-QNRC1-03）；国家自然科学基金资助项目（82001663）