Apamin was purchased from Sigma (St. Louis, MO, USA). TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA) and dissolved in 4 mM HCl containing 0.1% bovine serum albumin (BSA).

For induction of liver injury, 8-week-old C57BL/6 male mice (20–25 g; Samtako, Osan, Korea) were selected. Male C57BL/6 mice were fed a control diet or a DDC supplemented diet (0.1%) for 4 weeks to induce advanced biliary fibrosis as previously described (15). All animal protocols were approved by the Institutional Animal Care and Use Committee of Catholic University of Daegu (Daegu, Korea). The mice received an intraperitoneal injection of apamin (0.1 mg/kg) dissolved in saline twice a week. Mice were sacrificed after 4 weeks from the first DDC diet administration.

HSC-T6 cells, an immortalized rat hepatic stellate cell line, which has a stable phenotype and biochemical characteristics, was kindly provided by Dr S.L. Friedman (Liver Center Laboratory, San Francisco General Hospital, San Francisco, CA, USA). Cells were cultured at 37°C in a humidified incubator under a 5% CO₂ atmosphere. HSC-T6 cells were seeded in complete medium for 24 h. The cells were changed to fresh serum-free media containing the indicated concentrations of apamin (0.5, 1 and 2 µg/ml). After 24 h, the cells were replaced with fresh serum-free media containing 2 ng/ml of TGF-β1 for 24 h.

Hematoxylin and eosin (H&E), Masson's trichrome and immunohistochemical staining were performed according to a previously described procedure (15). Sections were stained with H&E and Masson's trichrome. For immunohistochemical analysis, sections were incubated with anti-fibroblast specific protein-1 (FSP-1) (ab41532; Abcam, Cambridge, UK) for 1 h at 37°C, processed by an indirect immunoperoxidase technique using a commercial kit (Dako, Carpinteria, CA, USA). The slides were examined with an Eclipse 80i microscope (Nikon, Tokyo, Japan) and analyzed with iSolution DT software (IMT i-Solution, Vancouver, BC, Canada).

Paraffin-embedded mouse liver sections (3-µm thickness) were prepared by a routine procedure. After blocking with 10% donkey serum for 30 min, the slides were immunostained with primary antibodies against cytokeratin (CK)7 (ab9021), CK19 (ab52625) (Abcam), and proliferating cell nuclear antigen (PCNA) (sc-56; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After washing, they were incubated with the secondary antibodies (Alexa Flour 488 and/or Alexa Fluor 594) for 30 min at 37°C. Sections were then counterstained with Hoechst 33342. Stained slides were imaged using a Nikon A1+ confocal microscope (Nikon).

HSC-T6 cells were grown on chamber slides and were fixed with 3.7% paraformaldehyde for 10 min. Cells were permeated with 0.5% Triton X-100 for 10 min and then incubated with primary antibodies against SMA (A5228; Sigma) for 1 h at 37°C. After washing, the cells were incubated with the secondary antibodies (Alexa Fluor 594) for 30 min at 37°C. Cells were counterstained with Hoechst 33342. The cells were imaged using a Nikon A1+ confocal microscope.

Concentrations of interleukin-6 (IL-6) and interferon-γ (IFN-γ) in serum were measured with ELISA kit (R&D Systems). The OD was measured at 450 nm in an ELISA reader (BMG Labtech, Mornington, Germany).

Western blotting was performed as previously described (16). Primary antibodies used in this study were the following; anti-p-Smad2/3 (#8828), anti-Smad2/3 (#8685) (Cell Signaling Technology, Danvers, MA, USA), anti-IL-1β (sc-7884), anti-Smad4 (sc-7966), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778) (Santa Cruz Biotechnology, Inc.), anti-SMA (A5228; Sigma), anti-tumor necrosis factor-α (TNF-α; ab6671), anti-collagen I (ab34710) (Abcam), TGF-β1 (MAB240-100; R&D Systems) and anti-fibronectin (610077; BD Biosciences, San Diego, CA, USA).

The experimental results are expressed as mean ± SE. ANOVA and paired or unpaired t-test were performed for statistical analysis as appropriate. p-value <0.05 was considered to indicate a statistically significant result. All experiments were performed at least three times.

To investigate the effects of apamin treatment on liver fibrosis, a mouse model induced by DDC diet feeding was used. When challenged with the DDC diet for 4 weeks, the structure of the hepatic lobule was clear in the NC group (Fig. 1A) and there was a large amount of bile duct proliferation, accompanied by inflammatory cell infiltration in the DDC-fed group as shown by H&E staining (Fig. 1C). In addition, the above pathological changes were reduced in the apamin-treated group (Fig. 1D) compared with these changes noted in the DDC-fed group. Masson's trichrome staining indicated collagen deposition surrounding the proliferated bile duct in the DDC-fed group (Fig. 2C). In contrast, apamin treatment resulted in diminished fibrosis and collagen deposition (Fig. 2D).

In cholangiopathies, inflammation and reactive proliferation of bile ducts are closely related with the development of biliary fibrosis (17). ELISA and western blot analyses indicated that expression levels of IL-6, IFN-γ, TNF-α and IL-1β were significantly higher in the DDC-fed group compared with these levels in the NC group (Fig. 3). However, apamin treatment attenuated inflammatory cytokine expression, including IL-6, IFN-γ, TNF-α and IL-1β compared with expression levels in the DDC-fed group. Taken together, these data confirm the anti-inflammatory and moderate anti-fibrotic effects of apamin on the DDC-fed mice.

We next determined the effect of apamin on ductular reaction in the DDC-fed mice by immunofluorescence of CK19 expression. Chronic DDC feeding in mice was previously demonstrated to result in cholangitis and immune responses against BECs with the destruction of bile ducts and ductules (18). CK19 is regarded as a hallmark of bile epithelial cells (19). Immunofluorescence staining showed that CK19 was highly expressed in the BECs in bile ductules in enlarged portal tracts (Fig. 4). The DDC-fed group had significantly increased expression of CK19 compared with the NC group. In contrast, apamin treatment significantly reduced biliary activation and proliferation as evidenced by CK19 staining, indicating a defect in the ductular reaction. In addition, immunofluorescence staining of PCNA showed that apamin treatment suppressed the proliferation of BECs compared with the DDC-fed group. These results indicate that apamin may inhibit cholestatic liver fibrosis by suppressing BEC proliferation and ductular reaction induced by the DDC diet.

To investigate the anti-fibrotic effect of apamin on ECM deposition in the DDC-fed mice, we used western blot analysis, immunohistochemistry and immunofluorescence assay to determine the effects of this compound on ECM molecules. Liver fibrosis induced by DDC was confirmed by induction of fibrogenic genes, FSP-1, α-smooth muscle actin (α-SMA) and collagen I expression. Expression of α-SMA was strongly expressed in the myofibroblasts and HSCs around the proliferated bile duct in the DDC-fed group and clearly with the apamin treatment (Fig. 5). Moreover, expression of collagen I in the DDC-fed group was significantly increased, especially in the portal tracts. Compared to the DDC group, apamin treatment inhibited collagen I expression. During tissue remodeling in liver fibrosis, FSP-1 is considered as a marker of fibroblasts in the fibrotic liver. DDC feeding increased the number of cells positive for FSP-1 expression (Fig. 6C). In contrast, apamin treatment resulted in a reduction in FSP-1-positive cells (Fig. 6D). Furthermore, western blot results showed that the expression levels of TGF-β1, collagen I, and fibronectin were significantly higher in the DDC-fed group, whereas apamin treatment markedly decreased the protein level of TGF-β1, collagen I, and fibronectin compared with the DDC-fed group (Fig. 6F). Taken together, the data suggest that apamin may protect liver fibrosis during DDC feeding by suppressing fibrotic gene expression.

TGF-β1 is a Smad family member, and it is known to stimulate the activation of HSCs (20). We next investigated whether the inhibitory effect of apamin on the activation of HSCs by TGF-β1 is through the Smad signaling pathway. α-SMA and collagen I expression was increased by TGF-β1 and was decreased by apamin treatment in the HSC-T6 cells (Fig. 7A). Also, immunofluorescence showed that TGF-β1 induced activation of HSC-T6 cells through increased α-SMA expression (Fig. 7C). However, apamin treatment markedly reduced the expression of α-SMA in the TGF-β1-induced HSC-T6 cells. Western blot analysis showed that phosphorylation of Smad2/3 and Smad4 were stimulated by 2 ng/ml TGF-β1 (Fig. 7B). Apamin treatment abrogated the activation of p-Smad2/3 and Smad4 induced by TGF-β1. These results indicated that apamin may attenuate TGF-β1-activated HSCs by inhibiting the Smad signaling pathway.