小檗胺的体外抗病毒作用及基于Ⅰ型干扰素通路的机制研究

Antiviral effect in vitro and mechanism study based on type I interferon pathway of berbamine

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、100.000μmol·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、10.0μmol·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10.0μmol·L^(-1)处理A549细胞24 h,进行转录组测序分析;10μmol·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、10μmol·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10μmol·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 0.001,0.010,0.100,1.000,10.000,100.000μmol·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 2.5,5.0,10.0μmol·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 10.0μmol·L^(-1) for 24 h,and transcriptome sequencing was performed.After treating MEF cells with 10μmol·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 10μmol·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 10μmol·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.