The study was approved by The Ethics Committee of Hainan Medical University (Haikou, China; approval no. HYLL-2021-346). A total of 30 male Wistar rats (age, 6-8 weeks; weight, 200-220 g) were obtained from The Experimental Animal Center of Guangdong Province (Guangzhou, China). Animals were fed in a barrier environment, under a 12-h light-dark cycle, at a constant temperature of 24±2˚C and relative humidity ranging from 50-65%, with free access to chow and water. The animals were acclimatized to the laboratory conditions for 1 week prior to surgery.

The rats were randomly divided into the following three groups (n=10/group): i) Sham group; ii) 1 month post operation (MPO) group; and iii) 2 MPO group. Animals in the 1 MPO and 2 MPO groups underwent p-BDL, whilst animals in the sham group underwent laparotomy but without p-BDL.

The rats fasted for ~12 h prior to surgery. Anesthesia was induced with 2% pentobarbital sodium (40 mg/kg intraperitoneally), and the animals were bound in a supine position on the operating board following the confirmation of complete anesthesia (Fig. 1A). Next, the abdominal hair was moistened with soapy water and removed using a razor, taking care to prevent skin injury. The scope of skin preparation extended from the xiphoid process to the iliac crest, and laterally to the midclavicular lines. Subsequently, the surgical area was disinfected with iodophor (Fig. 1B). An incision was made along the abdominal white line ~1 cm below the xiphoid process, and the abdominal wall muscles were incised along the same line using tissue scissors to open the abdominal cavity. The incision was ~5 cm in length (Fig. 1C). Two sterile cotton swabs were then moistened with saline and used to move the intestines towards the right lower abdomen. The duodenum and common bile duct were identified and gently pulled downward to straighten the common bile duct. A saline-soaked gauze pad was placed beneath the incision to maintain intestinal moisture (Fig. 1D). Next, three ligatures were placed loosely around the common bile duct using 3-0 black silk sutures, with an interval of ~2 mm between adjacent ligatures (Fig. 1E).

The modeling procedure was carried out using a 26G (0.60x15 mm) disposable venous indwelling needle (cat. no. Y-shaped IV-Y26II; Jiangxi Fenglin Medical Technology Co., Ltd.). Prior to insertion, the metal core was withdrawn by ~2 mm, ensuring that the tip of the needle was concealed within the casing to avoid injury to surrounding tissues. The trocar was then positioned above the common bile duct, and the three previously placed surgical sutures were knotted closely around it. The tensile force of the surgical knots was measured to be ~5 kg using a tension meter (Fig. 1F). Following ligation, the metal core was gently extracted, and the excess portion of the cannula was cut off. Approximately 3 mm of the tip and end of the casing was left exposed beyond the ligature points on both sides (Fig. 1G). Finally, a cotton swab was used to move the intestines back into the abdominal cavity and restore them to their original position (Fig. 1H), and the muscle and skin were then sutured sequentially (Fig. 1I). After the surgery, the rats were placed in a cage, kept warm and allowed to wake up. A regular diet was provided, and each rat received a daily intraperitoneal injection of 200,000 units penicillin sodium for three consecutive days to prevent infection.

Animals in the sham and 1 MPO groups were euthanized 1 month post-surgery, while those in the 2 MPO group were euthanized 2 months post-surgery. The 1-2-month postoperative period was selected based on a previous review which indicates that this is a critical time for the progression of hepatic fibrosis and metabolic decompensation (2). Following surgery, the model rats exhibited a noticeable yellow discoloration of the fur, particularly at 2 months post-surgery (Fig. 2A), which was indicative of severe jaundice. Therefore, 1 and 2 months post-surgery were designated as the humane endpoints of the study. All animals were euthanized by cardiac exsanguination following anesthesia with pentobarbital sodium (150 mg/kg intraperitoneally). Death was confirmed by the absence of pain responses, complete cessation of cardiac and respiratory activity, and pupil dilation. Liver tissues were obtained and either fixed with 4% paraformaldehyde at 4˚C for 48 h and embedded in paraffin for histological experiments or snap-frozen in liquid nitrogen and stored at -80˚C for zymography. Serum samples were centrifuged at 2,000 x g for 10 min at 4˚C and then stored at -20˚C.

Paraffin-embedded liver sections (3 µm) were subjected to staining with hematoxylin and eosin (H&E), Sirius red (cat. no. PH1098; Fuzhou Phygene Biotechnology, Co., Ltd.) and Masson's trichrome stain (cat. no. DC0032; Leagene; Beijing Regen Biotechnology Co., Ltd.). The staining procedure was performed as previously described (12). Stained sections were viewed under a microscope (Olympus BX53; Olympus Corporation). For each section, five fields of view were randomly selected and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.). The degree of hepatic fibrosis was assessed using Ishak's scoring system (13), which ranges from 0-6, with higher scores denoting more severe fibrosis.

Cytokeratin 19 (CK-19) protein is a recognized marker of biliary epithelial cells and is associated with the progression of liver fibrosis (3). In the present study, immunohistochemical staining of CK-19 was performed to investigate intrahepatic biliary cell proliferation. Liver fibrosis was further evaluated by the immunohistochemical staining of collagen I, collagen III and α-smooth muscle actin (α-SMA). In addition, the expression levels of matrix metalloproteinase (MMP)-2 and -9 are significantly upregulated in liver fibrosis and commonly used as biomarkers to assessing the extent of fibrotic progression (14). Therefore, the expression levels of MMP-2 and -9 were also evaluated by immunohistochemistry (IHC).

After dewaxing and hydration using xylene and a descending alcohol series, 3 µm thick liver sections were incubated with 3% H₂O₂ at room temperature for 10 min to quench endogenous peroxidase activity. The sections were then blocked with 10% goat serum (cat. no. PV-9001; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 15 min. Subsequently, the sections were incubated overnight at 4˚C with the following diluted primary antibodies: CK-19 (1:800; cat. no. 10712-1-AP), collagen I (1:800; cat. no. 14695-1-AP), collagen III (1:3,200; cat. no. 22734-1-AP), α-SMA (1:1,600; cat. no. 14395-1-AP), MMP-2 (1:400; cat. no. 10373-2-AP) and MMP-9 (1:800; cat. no. 10375-2-AP), all from Proteintech Group, Inc. The samples were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (ready-to-use; cat. no. PV-9001) at 37˚C for 30 min. Finally, antigen detection was performed using a Diaminobenzidine Substrate Kit (cat. no. ZLI-9018; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). Positive staining was visualized as brown staining on the cytomembrane. Under a light microscope (Olympus BX53; Olympus Corporation), five fields of view were randomly selected from each section for analysis, and analyzed using the software of Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

The gelatinolytic activities of intrahepatic MMPs were investigated using gelatin zymography as previously described (15). This assay detects the proenzyme, active enzyme, active fragments and complex forms of gelatinases based on their distinct molecular weights. Equal amounts of non-denatured protein (20 µg) from each liver sample were mixed with 2X SDS loading buffer (without reducing agent) and subjected to SDS-PAGE on 8% gels containing 1 mg/ml gelatin (MilliporeSigma). After electrophoresis, the gels were washed four times (15 min/wash) in 2.5% Triton X-100 at room temperature to remove SDS. The gels were then incubated at 37˚C in incubation buffer containing 50 mM Tris-HCl, 5 mM CaCl₂, 1 µM ZnCl₂ and 0.02% Brij-35 at pH 7.5 for ~24 h. Subsequently, the gels were stained with Coomassie Brilliant Blue R250 (0.25%) in 30% methanol and 10% acetic acid for ~1 h at room temperature, then destained overnight with 30% methanol and 10% acetic acid at room temperature to clearly visualize the digested bands. Proteolytic activities of MMPs were visualized as clear bands against the blue background of stained gelatin. Proteolytic activities were normalized to those of total proteins, as assessed by Coomassie Brilliant Blue R250 staining of the SDS-PAGE gels, and were analyzed using Quantity One (Version 4.6.2; Bio-Rad Laboratories, Inc.).

To assess the impact of p-BDL on liver function, the following parameters were analyzed using the specified kits: Total bilirubin (TBIL; cat. no. G1224W), aspartate aminotransferase (AST; cat. no. G0424W) and alanine aminotransferase (ALT; cat. no. G0423W). All kits were from Suzhou Grace Biotechnology Co., Ltd. The results were obtained using a microplate reader (Epoch 2; Agilent Technologies, Inc.).

All data are presented as the mean ± standard deviation and were analyzed using SPSS 21.0 software (IBM Corporation). One-way ANOVA followed by Tukey's post hoc tests were used to analyze normally distributed continuous data and Kruskal-Wallis followed by Dunn's post hoc tests were used to analyze ordinal data. P<0.05 was considered to indicate a statistically significant difference.

One rat was excluded from the study following mortality caused by accidental hepatic portal vein puncture during the surgical procedure. No post-operative mortality occurred prior to scheduled sacrifice, indicating that the surgical safety of the p-BDL technique was markedly improved compared with that of conventional BDL.

Prior to sacrifice, the hair color of the animals became yellowish, indicating that biliary obstruction was effectively established by the p-BDL procedure (Fig. 2A), particularly in the 2 MPO group. When the abdominal cavity was opened for sampling, adhesion between the inferior margin of the liver and the bowel was clearly observed in the surgical groups (Fig. 2B), which was indicative of an immune reaction caused by the non-absorbable surgical sutures and the plastic casing. Additionally, enlargement of the common bile duct was observed in the surgical groups, indicating the successful establishment of biliary obstruction (Fig. 2C).

Serum test results revealed that the liver function indices AST, ALT and TBIL were significantly elevated in the two surgical groups (Fig. 2D-F), particularly in the 2 MPO group, compared with those in the sham group. This upregulation signified the impairment of the liver following the p-BDL procedure.

H&E, Sirius red and Masson's trichrome staining of the livers were conducted to visualize the histopathological alterations and quantify the degree of liver fibrosis induced by the p-BDL procedure (Fig. 3).

The livers in the sham group exhibited a normal histological structure, with only a minor amount of collagen visible in the portal area (Fig. 3A). The portal areas of the livers in the 1 MPO and 2 MPO groups appeared expanded compared with those in the sham group due to pseudo bile duct proliferation, particularly in the 2 MPO group. In addition, no evidence of hepatic necrosis was observed in the 1 MPO and 2 MPO groups. Both Sirius red and Masson's trichrome staining revealed an abundant deposition of collagen in the 1 MPO and 2 MPO groups, particularly in the 2 MPO group (Fig. 3B and C). Notably, the areas of fibrotic lesions and percentages of collagen in the 1 MPO group and particularly in the 2 MPO group, were significantly higher than those in the sham group.

The biliary epithelial cells marker CK-19 was mainly expressed in normal bile duct endothelial cells in the sham group (Fig. 3D). By contrast, high expression of CK-19 was observed not only in normal bile duct cells but also in pseudo bile ducts in the 1 MPO and 2 MPO groups, indicating an abundance of reactive bile ductules in the portal regions.

Based on these staining results, Ishak's score was utilized to assess the level of liver fibrosis. The scores ranged from 0 for a normal liver to 6 for a cirrhotic liver. The sham group exhibited no fibrosis and received a score of 0. The 1 MPO group presented fibrous expansion in most of the portal area, along with occasional portal-to-portal bridging, and had an average score of 2.8±0.42. Finally, the 2 MPO group displayed nodule formation and probable cirrhosis, and had an average score of 5.00±0.67. These results indicate that obstructive jaundice was effectively induced by p-BDL in a manner similar to that induced by BDL.

IHC assays for collagen I, collagen III and α-SMA were conducted to further assess the extent of liver fibrosis. The results demonstrated that collagen I and α-SMA were only weakly expressed in the portal area in the sham group but exhibited significant increases in the 1 MPO and 2 MPO groups, with the 2 MPO group showing markedly and significantly higher expression than the 1 MPO group (Fig. 4A-C). In addition, collagen III was uniformly distributed throughout the hepatic lobules in the sham group, whereas it was abundantly expressed and significantly upregulated in the portal area and interlobular septum in the 1 MPO and 2 MPO groups.

The expression levels of MMPs were evaluated to investigate the molecular mechanisms underlying liver fibrosis following p-BDL. The IHC results demonstrated that MMP-2 and MMP-9 exhibited only minimal positivity in the sham group, whereas the number of positive cells was markedly elevated in the 1 MPO and 2 MPO groups (Fig. 4D and E). Furthermore, gelatin zymography revealed significantly increased activity of the MMP complex and MMP-9 in the liver tissues of both the 1 MPO and 2 MPO groups compared to the sham group. Additionally, active-MMP-2 activity was significantly elevated in the 2 MPO group vs. the sham group. These findings indicate severe damage to the hepatic extracellular matrix following p-BDL.