目的 探索千金藤素體內(nèi)外的抗病毒作用，并基于抗病毒天然免疫通路探究其抗病毒作用的分子機(jī)制。方法 CCK-8 法檢測(cè)千金藤素（0.062 5～64.000 0 μmol·L^-1）對(duì) A549 細(xì)胞活力的影響；利用表達(dá)綠色熒光蛋白的水皰性口炎病毒（VSV-GFP）感染 A549 細(xì)胞模型，結(jié)合流式細(xì)胞術(shù)檢測(cè)千金藤素對(duì)病毒復(fù)制的影響并探究預(yù)處理、吸附過(guò)程及吸附后加藥對(duì) VSV-GFP 病毒復(fù)制的影響；實(shí)時(shí)熒光定量 PCR（qRT-PCR）檢測(cè)千金藤素對(duì)甲型流感病毒（H1N1）、腦心肌炎病毒（EMCV）和單純皰疹病毒 I 型（HSV-1）復(fù)制的影響；構(gòu)建 VSV 感染小鼠模型探究千金藤素的體內(nèi)抗病毒作用；A549細(xì)胞中利用生物信息學(xué)方法探究其抗病毒機(jī)制；qRT-PCR 檢測(cè)藥物處理 A549 和原代胚胎成纖維細(xì)胞（MEF）后 IFNB1 及干擾素刺激基因 （ISGs） 表達(dá)變化；免疫印跡法 （Immunoblotting） 檢測(cè)人源單核細(xì)胞白血病細(xì)胞（THP-1）中 TBK1 和STAT1 的磷酸化水平。結(jié)果 與模型組相比，千金藤素在 A549 細(xì)胞中顯著抑制 VSV、H1N1、EMCV 和 HSV-1 復(fù)制；千金藤素不影響 VSV 的吸附過(guò)程，而預(yù)處理或吸附后給藥可以顯著抑制病毒復(fù)制；千金藤素提高 VSV 感染小鼠的存活率；千金藤素激活基于IFN-I通路的抗病毒天然免疫應(yīng)答。結(jié)論 千金藤素通過(guò)激活基于IFN-I通路的抗病毒天然免疫發(fā)揮體內(nèi)外抗病毒作用。

Objective To investigate the antiviral effects of cepharanthine (Cep) in vivo and in vitro and explore its immunological mechanism based on the innate antiviral immune pathway. Methods The viability of A549 cells was assessed using the CCK-8 assay to determine the impact of Cep (0.062 5—64.000 0 μmol·L^-1). Using a vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) infected cell models, combined with flow cytometry to investigate the impact of Cep on virus replication. We also examined how pretreatment, the adsorption process, and post-adsorption treatment affect virus replication.. Real-time fluorescence quantitative PCR (qRT-PCR) was employed to detect the replication of influenza A virus (H1N1), encephalomyocarditis virus (EMCV), and herpes simplex virus type 1 (HSV-1) under the influence of Cep. The in vivo antiviral effects of Cep were investigated using the VSV infection mouse model. Bioinformatics analysis was conducted in A549 cells to explore the mechanism of the antiviral function of Cep. qPCR was used to detect changes in mRNA expression of IFNB1 and the interferon-stimulated genes (ISGs) in A549 and MEF cells with Cep treatment. Immunoblotting was performed to measure changes in the phosphorylation level of TBK1 and STAT1 in THP-1 cells. Results In vitro experiments showed that Cep significantly inhibited the replication of VSV, H1N1, EMCV, and HSV-1 viruses compared to the model group in A549 cells. Cep did not affect the adsorption process of VSV, but pre-treatment or post-adsorption administration effectively inhibited virus replication. Cep increased the survival rate of VSV-infected mice. Cep activates the innate antiviral immune response through the IFN-I pathway. Conclusion This study demonstrates that Cep exhibits antiviral effects both in vivo and in vitro by activating the innate antiviral immune response through the IFN-I pathway.

R285.5

北京市科技新星計(jì)劃課題（20230484342）；中華中醫(yī)藥學(xué)會(huì)青年人才托舉工程項(xiàng)目 A 類(lèi)資助項(xiàng)目（2023-QNRC2-A02）#共同