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淫羊藿次苷Ⅱ体外抗乙型肝炎病毒的作用及对线粒体分裂的影响

Icariside Ⅱ Inhibits Hepatitis B Virus and Modulates Mitochondrial Fission in vitro

    摘要
  • 摘要:
    目的 探讨淫羊藿次苷Ⅱ（icariside, ICS Ⅱ）体外抗乙型肝炎病毒（hepatitis B virus, HBV）的作用及对线粒体分裂的影响。
    方法 采用HBV阳性的肝癌细胞HepAD38作为细胞模型，CCK8法检测ICS Ⅱ的细胞毒性，ELISA和荧光定量PCR法检测ICS Ⅱ以及ICS Ⅱ联合恩替卡韦（entecavir, ENT）作用细胞后乙型肝炎病毒表面抗原（HBsAg）、乙型肝炎病毒E抗原（HBeAg）的分泌水平和HBV DNA拷贝量；使用激光共聚焦显微镜和透射电子显微镜观察ICS Ⅱ对细胞线粒体形态和运动的影响；采用Western blot分析ICS Ⅱ作用后细胞线粒体动力学相关关键蛋白的表达水平；采用荧光染色法评估ICS Ⅱ对细胞内活性氧（reactive oxygen species, ROS）生成的影响。
    结果 CCK8结果显示ICSⅡ 25 μmol/L作用72 h对细胞增殖无明显影响；ICS Ⅱ显著抑制细胞HBsAg（抑制率54.90%）和HBeAg（抑制率39.65%）的分泌水平（P<0.05），ICS Ⅱ对细胞HBV DNA拷贝量的抑制率为15.19%，ENT对细胞HBV DNA拷贝量的抑制率为34.11%，ICSⅡ联合ENT后对HBV DNA拷贝量的抑制率为55.81%（P<0.05）；ICS Ⅱ诱导HepAD38细胞线粒体变短，增强线粒体运动（P<0.05）；促进线粒体运动相关蛋白Mfn1、Fis1及p-Drp1(ser 616)表达升高（P<0.05），Mfn2、Drp1及Drp1(ser 637)表达无明显变化（P>0.05）；抑制HepAD38细胞中ROS的产生（P<0.05）。
    结论 ICS Ⅱ抑制HepAD38细胞中HBV的复制，其机制可能与促进细胞线粒体分裂和抑制ROS的产生有关。

     

    Abstract:
    Objective To investigate the in vitro anti-hepatitis B virus (HBV) effects of icariside Ⅱ (ICS Ⅱ) and its impact on mitochondrial fission.
    Methods HBV-positive hepatocellular carcinoma HepAD38 cells were used as the cellular model. The cytotoxicity of ICS Ⅱ was assessed via CCK8 assay. The secretion levels of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), as well as HBV DNA copy numbers, were measured by ELISA and qPCR after treatment with ICS Ⅱ alone or ICS Ⅱ in combination with entecavir (ENT). The effects of ICS Ⅱ on mitochondrial morphology and motility were observed using confocal laser scanning microscopy and transmission electron microscopy (TEM). After ICS Ⅱ treatment, Western blot was performed to analyze the expression levels of key proteins involved in mitochondrial dynamics. Additionally, intracellular reactive oxygen species (ROS) production was evaluated via fluorescence staining.
    Results The CCK8 assay results showed that ICS Ⅱ treatment at 25 μmol/L had no significant effect on cell proliferation after 72 h. ICS Ⅱ significantly inhibited the secretion levels of HBsAg and HBeAg, with the respective inhibition rates reaching 54.90% and 39.65% (P < 0.05). Additionally, ICS Ⅱ alone reduced HBV DNA copy numbers by 15.19%, while ENT alone achieved a 34.11% inhibition rate. Notably, ICS Ⅱ in combination with ENT reduced HBV DNA copy numbers by 55.81% (P < 0.05). Furthermore, ICS Ⅱ induced mitochondrial shortening and enhanced mitochondrial motility in HepAD38 cells (P < 0.05). ICS Ⅱ significantly increased the expression levels of mitochondrial motility-related proteins, including Mfn1, Fis1, and phosphorylated Drp1 (ser 616) (P < 0.05), while no significant changes were observed in the expression levels of Mfn2, total Drp1, or Drp1 (ser 637) (P > 0.05). Additionally, ICS Ⅱ significantly suppressed the production of intracellular ROS in HepAD38 cells (P < 0.05).
    Conclusion ICS Ⅱ inhibits HBV replication in HepAD38 cells, and the underlying mechanism may be associated with the promotion of mitochondrial fission and suppression of ROS production.

     

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