热带病与寄生虫学

热带病与寄生虫学 ›› 2023, Vol. 21 ›› Issue (4): 204-209.doi: 10.3969/j.issn.1672-2302.2023.04.006

• 论著 • 上一篇    下一篇

乙酰紫草素体外抗2型单纯疱疹病毒机理的研究

廉梅芳1(), 时黎明2, 郑文学1, 李娟1, 李桂霞2, 于广福1()   

  1. 1.山东第一医科大学，山东省医学科学院，山东省高等学校新发传染病重点实验室，山东 泰安 271000
    2.菏泽市立医院
  • 收稿日期:2023-03-10 出版日期:2023-08-20 发布日期:2023-08-23
  • 通信作者: 于广福 E-mail:lian17671175084@163.com;gfyu@sdfmu.edu.cn
  • 作者简介:廉梅芳，女，硕士在读，研究方向：病原生物学。E-mail: lian17671175084@163.com
  • 基金资助:
    国家自然科学基金项目(81902065);山东省自然科学基金项目(ZR2022QH039);山东省中医药科技发展计划项目(2017-247);泰安市科技发展计划项目(2017NS0219);教育部大学生创新创业训练计划项目(201710439014)

Study on antiviral mechanism of acetylshikonin against herpes simplex virus type 2 in vitro

LIAN Meifang1(), SHI Liming2, ZHENG Wenxue1, LI Juan1, LI Guixia2, YU Guangfu1()   

  1. 1. Shandong First Medical University; Shandong Academy of Medical Sciences; Key Laboratory of Emerging Infectious Diseases in Shandong Colleges and Universities, Taian 271000, Shandong Province, China
    2. Heze Municipal Hospital
  • Received:2023-03-10 Online:2023-08-20 Published:2023-08-23
  • Contact: YU Guangfu E-mail:lian17671175084@163.com;gfyu@sdfmu.edu.cn

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摘要：

目的 研究乙酰紫草素（acetylshikonin, AS）体外抗2型单纯疱疹病毒（herpes simplex virus type 2, HSV-2）的活性及作用机理。方法 以阿昔洛韦（acyclovir, ACV）为阳性对照药物，采用CCK-8法检测细胞活力，确定AS对成年非洲绿猴肾细胞（Vero）的最大无毒浓度；致细胞病变法和噬斑形成抑制实验测定AS对HSV-2的抗病毒活性；实时荧光定量PCR及半数组织培养感染剂量（50% tissue culture infective dose, TCID50）法测定病毒载量以评估AS对HSV-2复制周期不同阶段的抑制作用；透射电镜观察AS对HSV-2病毒粒子结构的影响。结果 AS对Vero细胞的最大无毒浓度（maximal atoxic concentration, TC0）为3.785 μmol/L，浓度为1.678~3.785 μmol/L时能有效抑制HSV-2复制，减少噬斑形成，半数有效浓度（50% effective concentration, EC50）为0.334 μmol/L。在12和36 h，病毒释放期加入AS的实验组TCID50低于病毒感染对照组（13.43±10.04 vs. 127.00±0.32；3.47±0.55 vs. 5.80±0.12），差异均有统计学意义（t=8.359、4.161，P均<0.05）；在12、24、36 h，AS直接灭活病毒组病毒载量低于病毒感染对照组（0.24±0.09 vs. 1.35±0.07；4.46±0.06 vs. 6.75±0.04；2.70±0.04 vs. 5.27±0.10），差异均有统计学意义（t=9.920、33.360、24.020，P均<0.05）。此外，AS处理可导致HSV-2病毒粒子形态异常，随着AS浓度升高，作用后的HSV-2超微结构发生渐进改变。结论 AS对HSV-2具有一定抗病毒效应，其机理可能为直接破坏病毒颗粒完整结构发挥抗病毒作用。

关键词: 乙酰紫草素, 2型单纯疱疹病毒, 抗病毒效果, 灭活作用

Abstract:

Objective To investigate the activity and mechanism of acetylshikonin (AS) against herpes simplex virus type 2 (HSV-2). Methods By acyclovir (ACV) being used as positive control agent, we examined the cell viability with CCK-8 in order to determine the maximum safe concentration of AS to Vero cell. Cytopathogenic effect method and plaque-reduction neutralization assay were used to detect the antiviral activity of AS against HSV-2. RT-qPCR incubated with TCID50 was used to determine viral load to evaluate the inhibitory effect of AS on different stages of HSV-2 replication cycle. The effect of AS on virion morphology was visualized under the transmission electron microscope. Results The maximal atoxic concentration of AS to Vero cell was 3.785 μmol/L, and AS significantly inhibited the replication of virus and reduced plaque formation at the safe concentration of 1.678-3.785 μmol/L. Meanwhile, the replication of HSV-2 virus was inhibited by AS with an 50% effective concentration (EC50) value at 0.334 μmol/L. The virus titer of the treatment group was lower than that of the corresponding virus infection control group at 12 h and 36 h of release stage (13.43±10.04 vs. 127.00±0.32, 3.47±0.55 vs. 5.80±0.12, respectively), the difference was statistically significant (t=8.359, 4.161; both P<0.05), and the virus replication load of the treatment group was lower than that of the corresponding virus infection control group at 12 h, 24 h and 36 h of co-incubation with HSV-2 (0.24±0.09 vs. 1.35±0.07, 4.46±0.06 vs. 6.75±0.04, 2.70±0.04 vs. 5.27±0.10, respectively), the difference was statistically significant (t=9.920, 33.360, 24.020; all P<0.05). Besides, treatment with AS resulted in abnormal morphology of certain HSV-2 virions, and the ultrastructure of HSV-2 changed gradually with added AS concentration. Conclusion The results reported here indicated that AS possessed a certain antiviral effect on HSV-2. The potential mechanism is to exert antiviral effect by directly destroying the complete structure of the virus particles.

Key words: Acetylshikonin, Herpes simplex virus type 2, Antiviral effect, Inactivation effect

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