吉林大学学报(医学版) ›› 2021, Vol. 47 ›› Issue (6): 1446-1454.doi: 10.13481/j.1671-587X.20210614

• 基础研究 • 上一篇    下一篇

瑞德西韦对感染肠道病毒71型的人横纹肌瘤细胞和 ICR乳鼠的抗病毒活性

任晓风1,闫赟政2,李微2,李月香2,肖军海2,曹瑞源2(),李永刚1()   

  1. 1.锦州医科大学基础医学院病原生物学教研室,辽宁 锦州 121000
    2.军事科学院军事医学研究院毒物药物研究所,北京 100850
  • 收稿日期:2021-04-07 出版日期:2021-11-28 发布日期:2021-12-14
  • 通讯作者: 曹瑞源,李永刚 E-mail:21cc@163.com;lygjo@hotmail.com
  • 作者简介:任晓风(1993-),女,河北省邯郸市人,在读硕士研究生,主要从事抗病毒药物方面的研究。
  • 基金资助:
    辽宁省教育厅基础研究项目(JYTJCZR201909)

Antiviral activity of remdesivir against human rhabdomyosarcoma cells and ICR suckling mice infected with enterovirus 71

Xiaofeng REN1,Yunzheng YAN2,Wei LI2,Yuexiang LI2,Junhai XIAO2,Ruiyuan CAO2(),Yonggang LI1()   

  1. 1.Department of Pathogenic Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China
    2.Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China
  • Received:2021-04-07 Online:2021-11-28 Published:2021-12-14
  • Contact: Ruiyuan CAO,Yonggang LI E-mail:21cc@163.com;lygjo@hotmail.com

摘要: 目的

探讨瑞德西韦(RDV)在细胞与动物水平对肠道病毒71型(EV71)的抗病毒活性,并阐明其抗病毒作用机制。

方法

基于人横纹肌瘤(RD)细胞进行RDV抗肠道病毒活性评价,检测RDV对EV71、柯萨奇病毒A6型(CA6)、肠道病毒D68型(EVD68)和柯萨奇病毒16型(CA16)的半数毒性浓度(CC50)和半数有效浓度(EC50),计算选择指数(SI)。将RD细胞分为细胞对照组(不处理)、病毒对照组和给药组。病毒对照组RD细胞给予EV71病毒液,给药组RD细胞再给予不同浓度(0.005、0.015、0.046、0.137、0.410、1.230、3.700、11.110、33.330和100.000 μmol·L-1)RDV。72 h后,采用CellTiter-Glo? Luminescent检测试剂盒测定各组RD细胞活性。在抗EV71细胞活性评价实验中,将RD细胞分为给药组和病毒对照组,给药组RD细胞给予不同浓度(0.03、0.10、0.30、0.80和2.50 μmol·L-1)RDV。30 h后,采用实时荧光定量PCR(RT-qPCR)法检测各组RD细胞中EV71 RNA表达水平,采用Western blotting法和免疫荧光法检测各组RD细胞中EV71结构蛋白VP1表达量。采用加药时序实验验证RDV的抗病毒作用阶段。在抗EV71动物药效学实验中,将35只ICR乳鼠随机分为安慰剂组(给予2% Tween 80,n=12)、3.0 mg·kg-1RDV组(给予3.0 mg·kg-1 RDV,n=12)和1.5 mg·kg-1组RDV组(给予1.5 mg·kg-1 RDV, n=11),每只乳鼠经腹腔攻毒5×103 PFU。4 h后进行第1次给药,连续给药2周,观察并记录乳鼠生存率。在攻毒后第3 天采用RT-qPCR法检测各组乳鼠各种组织中病毒载量(即EV71 RNA拷贝数)。

结果

RDV对EV71、CA6、EVD68和CA16的EC50分别为(0.05±0.01)、(0.14±0.06)、(0.02±0.01)和(0.10±0.03) μmol·L-1。与病毒对照组比较,0.10、0.30、0.80和2.50 μmol·L-1RDV组RD细胞中EV71 RNA表达水平降低(P<0.05或P<0.01),0.80和2.50 μmol·L-1RDV组RD细胞中EV71结构蛋白VP1表达量降低。与病毒对照组比较,0.80 μmol·L-1RDV组RD细胞中Ⅲ和Ⅳ阶段(病毒复制阶段)时EV71病毒基因组RNA拷贝数明显降低(P<0.05)。与安慰剂组比较,各给药组乳鼠生存率差异无统计学意义(P>0.05),但有一定的保护趋势。与安慰剂组比较,3.0 mg·kg-1RDV组乳鼠肺和肌肉组织中病毒载量明显降低(P<0.05或P<0.01)。

结论

RDV对以EV71为代表的肠道病毒具有良好的细胞水平抗病毒活性,并主要作用于病毒感染后的复制阶段。RDV能明显降低小鼠肺和肌肉组织中病毒载量,提示其在动物水平有一定的治疗潜力。

关键词: 瑞德西韦, 肠道病毒71型, 人横纹肌瘤细胞, ICR乳鼠

Abstract: Objective

To explore the antiviral activity of remdesivir(RDV) against enterovirus 71 (EV71) in the cellular and animal levels,and to clarify its antiviral mechanism.

Methods

The anti-enterovirus activity of RDV was evaluated based on the human rhabdomyosarcoma(RD) cells. The half toxic concentration (CC50) and half effective concentration (EC50) of RDV for EV71, Coxackie virus 6(CA6), enterovirus 68(EVD68),and Coxackie virus 16(CA16) were detected, and the selection index (SI) was calculated. The RD cells were divided into cell control group,virus control group(without treatment) and administration groups. The RD cells in virus control group were given EV71, and the RD cells in administration groups were given different concentrations (0.005, 0.015, 0.046, 0.137,0.410, 1.230,3.700,11.110,33.330,and 100.000 μmol·L-1) of RDV. After 72 h, CellTiter-Glo? Luminesent assay kit was used to determine the activities of RD cells in various groups. In the cell activity evaluation experiment of anti-EV71, the RD cells were divided into administration groups and virus control group.The RD cells in administration groups were given different concentrations(0.30, 0.10, 0.30, 0.80 and 2.50 μmol·L-1) of RDV. After 30 h, the expression levels of EV71 RNA in the RD cells in various groups were detected by Real-time fluorescence quantitative PCR (RT-qPCR) method, and the expression levels of EV71 structural protein VP1 in various groups were detected by Western blotting and immunofluorescence methods. Time of addition assay was used to conform the antiviral stage of RDV.In anti-EV71 animal pharmacodynamics, 35 ICR suckling mice were randomly divided into placebo group (given 2% Tween 80, n=12),3.0 mg·kg-1 RDV group (given 3.0 mg·kg-1 RDV,n=12) and 1.5 mg·kg-1 RDV group (given 1.5 mg·kg-1 RDV, n=11);each suckling mouse was challenged intraperitoneally with 5×103 PFU.The first dose was given after 4 h,and the treatment lasted for 2 weeks. The survival rates of the sucking mice in various groups were observed and recorded. On the 3rd day after challenge, the viral loads(copy number of EV71 RNA) in different tissues of the suckling mice in various groups were detected by RT-qPCR method.

Results

The EC50 of RDV for EV71, CA6, EVD68,and CA16 were (0.05±0.01),(0.14±0.06),(0.02±0.01),and(0.10±0.03) μmol·L-1, respectively. Compared with virus control group, the expression levels of EV71 RNA in the RD cells in 0.10, 0.30, 0.80,and 2.50 μmol·L-1 RDV groups were decreased (P<0.05 or P<0.01), and the expression levels of EV71 structural protein VP1 in the RD cells in 0.80 and 2.50 μmol·L-1 RDV groups were decreased. Compared with virus control group, the copy numbers of EV71 RNA in the RD cells at stages Ⅲ and Ⅳ (virus replication stage) were decreased significantly (P<0.05). There were no significant differences in the survival rates of the suckling mice between administration groups and placebo group (P>0.05),but there was a certain protective trend. Compared with placebo group, the viral loads in lung and muscle tissues of the suckling mice in 3.0 mg·kg-1 RDV group were decreased significantly (P<0.05 or P<0.01).

Conclusion

RDV has an excellent antiviral activity against enteroviruses (such as EV71)at the cellular level, and mainly acts on the replication stage of viral infection. RDV can significantly reduce the viral loads in lung and muscle tissues, suggesting its therapeutic potential at the animal level.

Key words: remdesivir, enterovirus 71, human rhabdomyosarcoma cells, ICR suckling mouse

中图分类号: 

  • R512.5