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.