Fig. 1

Expression of ITGBL1 is increased in the HBV expression model. (A) ITGBL1 protein levels were elevated following the induction of HBV replication in HepAD38 cells. Cells were cultured in the presence (1 µg/mL) or absence of tetracycline for 48 h. Western blot analysis revealed increased ITGBL1 expression under HBV-expressing conditions (tetracycline withdrawal). (B) HBV DNA levels in the supernatant of HepAD38 cells were quantified by real-time PCR. HBV DNA levels were significantly elevated in the tetracycline-free group compared to the tetracycline-treated group, indicating effective induction of HBV replication. Data are presented as mean ± SEM from three independent experiments. (C) Huh7 cells were seeded in 6-well plates and transfected with 1 µg of pHBV1.2 plasmid. Cells were harvested 48 h post-transfection, and ITGBL1 expression was analyzed by Western blot. A significant increase in ITGBL1 protein levels was observed in HBV-transfected Huh7 cells compared to control cells. (D–E) HBsAg and HBeAg levels in the supernatant of HBV-transfected Huh7 cells were quantified using the Abbott Architect immunoassay system. Since residual plasmid DNA in the culture medium after transfection could interfere with HBV DNA quantification—even after DNase treatment—viral protein expression was used as an indicator of successful HBV transfection. To investigate the effect of HBV on intrahepatic ITGBL1 expression, two groups of 6-week-old male C57BL/6 mice were intravenously injected with 200 µL saline containing either 5 × 10¹⁰ viral genome equivalents (vge) of rAAV8–1.3×HBV or empty rAAV8, respectively, as described in the Materials and Methods section. A control group of untreated mice was also included. Blood samples (200 µL) were collected at weeks 0, 1, 4, and 8 post-injection via retro-orbital bleeding for the analysis of serum ITGBL1 and HBV DNA levels. Each group consisted of three mice (n = 3). (F) Serum ITGBL1 levels were measured using an ELISA kit, and HBV DNA levels were quantified using a real-time PCR assay (Shanghai Kehua Bio-Engineering Co., Ltd.) with a lower limit of detection of 500 IU/mL. (G) At week 8 post-injection, mice in the MOCK and HBV groups were sacrificed, and liver tissues were collected for immunohistochemistry (IHC) using an anti-ITGBL1 antibody. Representative images demonstrate increased ITGBL1 expression in the liver sections of rAAV8–1.3×HBV-infected mice compared to controls. Relative staining intensity was quantified using ImageJ software. Data are presented as mean ± SEM; p < 0.05 vs. control group. Scale bar: 200 μm (n = 3)