蟾酥注射液抗SARS-CoV-2病毒感染的体外活性研究

In vitro Activity Study of Chansu Injection against SARS-CoV-2 Virus Infection

    摘要
  • 摘要:
    目的 探究蟾酥注射液抗SARS-CoV-2病毒感染的体外活性，研究蟾毒灵是否为其发挥活性的关键成分。
    方法 采用CellTiter-Glo细胞活力检测法测定蟾酥注射液及蟾毒灵抗SARS-CoV-2病毒感染的半数有效浓度（EC50）；通过qPCR检测蟾酥注射液和蟾毒灵对SARS-CoV-2病毒感染的细胞中病毒NP蛋白、干扰素、炎症因子等的mRNA水平的影响；构建细胞-细胞膜融合模型，采用荧光素酶报告基因方法检测蟾酥注射液和蟾毒灵对HEK-293T / HEK-293T-ACE2细胞膜融合的影响。
    结果 蟾酥注射液具有抗SARS-CoV-2病毒感染活性，EC50为85.56 ng·mL-1。蟾酥注射液显著降低SARS-CoV-2感染的Calu-3细胞中病毒NP蛋白的mRNA水平（P < 0.05），下调干扰素IFN-λ2/3（P < 0.05）以及ISG-15、RIG-I（P < 0.000 1）的mRNA水平，并能显著降低炎症因子IL-6和TNF-α的mRNA水平（P < 0.000 1）。蟾毒灵抑制SARS-CoV-2病毒感染细胞的EC50为13.3 nmol·L-1，可能为蟾酥注射液抗SARS-CoV-2病毒感染的主要活性成分。蟾酥注射液能显著抑制SARS-CoV-2病毒S蛋白介导的细胞膜融合，从而阻断病毒入侵。
    结论 蟾酥注射液具有体外抗SARS-CoV-2病毒感染的活性，能抑制NP蛋白表达、降低炎症因子水平并阻断膜融合入侵宿主细胞，蟾毒灵可能是其发挥抗SARS-CoV-2作用的关键活性物质。

     

    Abstract:
    OBJECTIVE To investigate the in vitro activity of Chansu injection against SARS-CoV-2 infection and determine whether bufalin is the primary active component responsible for its efficacy.
    METHODS The half-maximal effective concentration (EC50) of Chansu injection and bufalin against SARS-CoV-2 infection was determined using the CellTiter-Glo cell viability assay. qPCR was performed to assess the effects of Chansu injection and bufalin on mRNA levels of SARS-CoV-2 NP protein, inflammatory factors, and interferons in virus-infected cells. A cell-cell membrane fusion model was established, and the impact of the drugs on membrane fusion between HEK-293T and HEK-293T-ACE2 cells was evaluated using a luciferase reporter gene assay.
    RESULTS Chansu injection exhibited anti-SARS-CoV-2 virus infection activity, with an EC50 of 85.56 ng·mL-1. Chansu injection significantly reduced the mRNA levels of viral NP protein (P < 0.05), IFN-λ2/3 (P < 0.05), ISG-15 and RIG-I (P < 0.000 1) in SARS-CoV-2 infected Calu-3 cells, and the mRNA levels of inflammatory factors IL-6 and TNF-α were significantly reduced (P < 0.000 1). The EC50 of bufalin in inhibiting SARS-CoV-2 virus-infected cells was 13.3 nmol·L-1, which might be the main active component of Chansu injection in resisting SARS-CoV-2 virus-infected cells. Chansu injection could significantly inhibit the cell membrane fusion mediated by the S protein of SARS-CoV-2 virus, thereby blocking the virus invasion.
    CONCLUSION Chansu injection demonstrates in vitro anti-SARS-CoV-2 activity by suppressing NP protein expression, reducing inflammatory cytokine levels, and blocking viral entry through membrane fusion inhibition. Bufalin is likely the key active component responsible for its anti-SARS-CoV-2 effects.

     

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