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

LIU Zhengyun, WEN Juan, CHEN Guoli, YU Wan, LUO Guo, GONG Qihai, WANG Huan

Abstract

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.

 

Keywords: Hepatitis B virus, Epimedium herb, Mitochondria

 

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