目的 觀察小檗胺的體外抗病毒作用，并基于Ⅰ型干擾素（IFN-Ⅰ）通路的抗病毒天然免疫反應(yīng)探討其作用機制。方法 CCK-8法檢測0.001、0.010、0.100、1.000、10.000、100.000 μmol·L^-1的小檗胺對A549細胞活力的影響；表達綠色熒光蛋白的水皰性口炎病毒（VSV-GFP）以0.05的感染復(fù)數(shù)（MOI）感染A549細胞制備模型，流式細胞術(shù)檢測共同孵育12 h的小檗胺2.5、5.0、10.0 μmol·L^-1對GFP陽性細胞比例的影響；VSV感染A549細胞，Western blotting檢測小檗胺對VSV病毒G蛋白表達的影響；小檗胺使用預(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，通過流式細胞術(shù)檢測其對VSV-GFP在A549細胞中復(fù)制的影響；分別使用甲型流感病毒（H1N1）（MOI＝0.05），腦心肌炎病毒（EMCV）（MOI＝3）和單純皰疹病毒1型（HSV-1）（MOI＝1）感染A549細胞，同時給藥共同孵育12 h后，實時熒光定量PCR（qRT-PCR）檢測小檗胺對病毒RNA表達的影響；小檗胺10.0 μmol·L^-1處理A549細胞24 h，進行轉(zhuǎn)錄組測序分析；10 μmol·L^-1小檗胺處理MEF細胞12 h后，qRT-PCR法檢測Ifnb1、Ifit1、Ifit2、Ifi44基因的mRNA表達；利用IFN刺激性DNA （ISD）轉(zhuǎn)染THP-1細胞，預(yù)先激活I(lǐng)FN-I信號通路，4～6 h加入小檗胺5、10 μmol·L-1處理12 h，qRT-PCR法檢測IFNB1、IFIT1、IFIT2、IFI44基因的mRNA表達。結(jié)果 濃度為10 μmol·L^-1及以下時，小檗胺對A549細胞均未顯示出明顯的細胞毒性；與模型組相比，小檗胺劑量相關(guān)性地減少了VSV-GFP陽性細胞的比例（P＜0.05、0.001），明顯減少了病毒的VSV-G蛋白表達；小檗胺對VSV的吸附過程沒有影響，而預(yù)處理或吸附后給藥可以顯著抑制病毒復(fù)制；與模型組比較，小檗胺劑量相關(guān)性地抑制了H1N1、EMCV和HSV-1的病毒基因表達（P＜0.001）；轉(zhuǎn)錄組測序和qRT-PCR結(jié)果表明，小檗胺促進細胞基于IFN-I信號通路的抗病毒免疫激活；在ISD刺激后，小檗胺誘導(dǎo)更高水平的IFNB1、IFIT1、IFIT2、IFI44 mRNA表達（P＜0.05、0.01、0.001）。結(jié)論 小檗胺可能通過促進基于IFN-I通路的抗病毒天然免疫反應(yīng)抑制多種病毒復(fù)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

中國科協(xié)青年人才托舉工程項目（2020-QNRC1-03）；國家自然科學(xué)基金資助項目（82001663）