BALB/C male mice (n=30, weight, 10–25 g; age, 6–8 weeks) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Mice were housed under a 12-h light/dark cycle at a relative temperature of 23±2°C and 70% humidity. The mice were provided with free access to food and water. The animal experiments were performed strictly in accordance with the Care and Use of Laboratory Animal Regulations (17). This study was approved by the Ethics Review Board for Animal Studies of the Affiliated Yantai Yuhuangding Hospital of Qingdao University (Yantai, China; approval no. SH20186242).

A mouse model of cholestasis was established by bile duct ligation (BDL). Briefly, the mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg; Jiangsu Hengrui Medicine Co., Ltd.,) and xylazine (10 mg/kg; Sigma-Aldrich; Merck KGaA). Next, the common bile duct was separated from its surrounding connective tissue, and the hepatic artery from the portal vein. The common bile duct was then ligated with 5-0 silk thread at two locations close to the beginning of its intrapancreatic portion and was transected between the two ligatures. Mice in the sham group received a laparotomy without subsequent identification and ligation of the bile duct. Mice in the sham and BDL groups were administered 0.9% normal saline (20 ml/kg/day) for 7 days. Mice in the catalpol-high (−H) group were gavaged with 10 mg/kg catalpol (Nanjing Jingzhu Biotechnology Co., Ltd.) for 7 days after undergoing sham surgery, whereas those in the BDL + catalpol groups were gavaged with either 5 mg/kg catalpol [catalpol-low (−L)] or 10 mg/kg catalpol (catalpol-H) for 7 days after the BDL model was established. A total of 1 week after the experiment, all mice were sacrificed with an overdose of pentobarbital sodium (120 mg/kg) followed by collection of blood and liver samples. Blood was collected via the tail vein, and then the blood samples (0.4 ml) were immediately separated by centrifugation at 2,200 × g at 4°C for 10 min to obtain serum samples.

The serum of mice was collected from blood samples by centrifugation at 2,200 × g at 4°C for 10 min. Commercial ELISA kits were used to measure serum levels of alanine aminotransferase (ALT; cat. no. C009-2), aspartate aminotransferase (AST; cat. no. C010-2), alkaline phosphatase (ALP; cat. no. A059-2), total bilirubin (TBIL; cat. no. C019-1) and total bile acid (TBA; cat. no. E003-2) (all Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol.

The blood samples were centrifuged at 2,200 × g for 10 min at 4°C to collect serum, which was then immediately stored at −80°C until use. Tumor necrosis factor-α (TNF-α; cat. no. MTA00B), interleukin (IL)-1β (cat. no. MLB00C) and IL-6 (cat. no. M6000B) were detected in the serum samples using ELISA kits (R&D Systems, Inc.) according to the manufacturer's protocol.

The liver tissues were sliced and fixed in 4% paraformaldehyde overnight at 4°C. Subsequently, tissue sections were dehydrated in a diminishing series of ethanol, embedded in paraffin and sectioned into 4-5-µm slices. The sections were stained with hematoxylin and eosin (H&E) for 10 min at room temperature and observed under a light microscope (magnification, ×100; DM4000B; Leica Microsystems, Inc.).

After resection of the liver, the tissue samples were homogenized in a buffer containing 2 mmol/l HEPES, 0.5 mmol/l EGTA, 70 mmol/l mannitol, 220 mmol/l sucrose and 0.1% BSA (Sigma-Aldrich; Merck KGaA) at a ratio of 10:1 buffer:tissue (v/w), final pH 7.4. The tissue homogenates were centrifuged at 4°C for 10 min at 1,000 × g to pellet the whole cells and nuclei. The supernatant was further centrifuged at 4°C for 10 min at 10,000 × g to precipitate the heavy membrane fractions (mitochondria).

JC-1 staining was performed to detect the mitochondrial membrane potential. The hepatocytes were isolated from the liver tissues. Briefly, the liver tissues were digested in 0.1% trypsin for 10 min at 37°C and filtered (40 µm; Hangzhou Xiaoyou Biotechnology Co., Ltd.) to obtain cell suspension, and the cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. The harvested hepatocytes were subsequently washed with PBS, and then incubated with 5 µmol/l JC-1 (Cell Signaling Technology, Inc.) for 30 min prior to being subjected to flow cytometry (ACEA Bioscience; Agilent Technologies, Inc.).

The mitochondria suspension and 1X Reaction buffer solution were prepared and subjected to a luciferase luciferin kit (cat. no. FF2021; Enliten^®; Promega Corporation) to determine mitochondrial ATP content, according to the manufacturer's protocol. Subsequently, luminous intensity of the sample was detected at λ=560 nm using the FLUOstar Omega^® multi-function microplate reader (BMG Labtech GmbH).

Production of intracellular ROS was examined using the fluorescent probe DCFH-DA (cat. no. HY-D0940; MedChemExpress). Liver mitochondria were isolated as aforementioned and incubated with a final concentration of 10 µmol/l DCFH-DA for 30 min at 37°C and the fluorescence intensity was measured by flow cytometry (ACEA Bioscience; Agilent Technologies, Inc.) (λ excitation, 488 nm; λ emission, 525 nm).

The level of GSH in mitochondria and serum was determined by spectrophotometry. The chemical 5,50-dithio-2-nitrobenzoic acid (DTNB; cat. no. JD499-5G; Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was used as the indicator of GSH. To extract mitochondrial GSH, the mitochondrial suspension was treated with trichloroacetic acid to a final concentration of 10% (w/v) at room temperature for 5 min. The denatured proteins were removed by centrifugation at 15,000 × g for 1 min at 4°C and 100 µl DTNB (0.04% phosphate buffer) was then added at room temperature for 15 min. The yellow intensity was recorded at 412 nm using an ultraviolet spectrophotometer (ultrospec 2000^® ultraviolet spectrophotometer; Pharmacia Biotechnology; GE Healthcare).

After isolation of liver mitochondria, MDA in mitochondria and serum was detected using a thiobarbituric acid reactive substance (TBARS) assay kit (cat. no. KA4409; AmyJet Scientific, Inc.) according to the manufacturer's protocol. Isolated mitochondria were washed in an ice-cold 3-(N-norpholino)propanesulfonic acid (MOPS)-KCl buffer (50 mM MOPS, 100 mM KCl) to remove sucrose, and re-suspended in fresh MOPS-KCl buffer. Then, the mitochondrial suspension was mixed with 15% trichloroacetic acid, 0.375% thiobarbituric acid, 0.24 M HCl, and 0.5 mM Trolox. The mixture was heated for 15 min at 100°C. Following centrifugation (15,000 × g; 5 min; 4°C), the absorbance of the supernatant was assessed, MDA in reaction with thiobarbituric acid produces the MDA-TBA adduct, which could be read at 532 nm using an Epoch plate reader (BioTek Instruments, Inc.).

Liver tissues (0.1 g) were homogenized by incubation with 1 ml protein lysis buffer (Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g for 10 min at 4°C to collect the supernatant. The concentration of the protein was evaluated using a BCA protein assay kit (Beyotime Institute of Biotechnology). Proteins (30 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Sigma-Aldrich; Merck KGaA). The membranes were then blocked with 5% skimmed milk powder for 3 h at room temperature and incubated with the relevant primary antibodies [cytochrome c (1:1,000; cat. no. ab90529; 15 kD), Bcl-2 (1:1,000; cat. no. ab59348; 26 kD), Bax (1:1,000; cat. no. ab32503; 21 kD), cleaved caspase-3 (1:1,000; cat. no. ab2302; 17 kD) and caspase-3 (1:5,000; cat. no. ab32351; 32 kD) (all from Abcam)] overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (1:2,000; cat. no. ab205718; Abcam) secondary antibodies for 1 h. GAPDH (1:1,000; cat. no. ab181602; 36 kD; Abcam) served as the internal control. Chemiluminescence detection was conducted using an ECL Western Blotting Substrate kit (cat. no. ab65623; Abcam). Protein expression levels were semi-quantified using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

The experiments were performed in triplicate. Data are presented as the mean ± standard deviation. The data were analyzed using one-way ANOVA followed by Bonferroni's post-hoc test using SPSS 20.0 software (IBM Corporation). P<0.05 was considered to indicate a statistically significant difference.

Mice in the catalpol-H treatment group showed no significant alterations in the serum levels of ALT, AST, ALP, TBIL or TBA (Fig. 1A-E) compared with mice in the sham control group. However, addition of catalpol-H and catalpol-L could significantly reduce serum ALT, AST, ALP, TBIL and TBA levels in the BDL model group (Fig. 1A-E), with catalpol-H producing a significantly greater protective effect compared with catalpol-L.

As shown in Fig. 1F-H, it was revealed that mice treated with catalpol-H had no significant change in the serum levels of TNF-α, IL-1β and IL-6 compared with the sham control group. Serum levels of TNF-α, IL-1β and IL-6 all significantly increased following BDL. Furthermore, compared with the BDL model group, catalpol-L and catalpol-H significantly reduced the levels of TNF-α, IL-1β and IL-6, with catalpol-H producing a greater effect compared with catalpol-L.

The histological alterations in liver tissues derived from cholestatic mice were evaluated by H&E staining. As shown in Fig. 2, treatment with catalpol-H alone had no marked effect on the histological morphology of the mouse liver tissues. In the BDL model group, inflammatory infiltration and hepatocyte damage in the liver tissues were observed, and subsequent treatment with catalpol markedly reduced hepatocyte necrosis and neutrophil infiltration, with catalpol-H producing a greater protective effect compared with catalpol-L.

Flow cytometry was performed to detect mitochondrial membrane potential of the extracted hepatocytes. The subsequent results of JC-1 distribution demonstrated that the mitochondrial membrane potential in the BDL model group was reduced compared with that in the sham group. In addition, catalpol-H had no notable effect on mitochondrial membrane potential in normal mouse liver cells; however, treatment with catalpol-L and catalpol-H significantly improved the mitochondrial membrane potential of liver cells in the cholestatic model mice (Fig. 3A). The ATP content of mitochondria was determined using a luciferase luciferin kit; the results revealed that ATP content was significantly lower in mitochondria in the BDL group compared with the sham control group, whereas treatment with catalpol significantly increased the content of ATP in mitochondria within the cholestatic mice compared with the BDL model group (Fig. 3B). The production of ROS in the mitochondria was measured by flow cytometry, and the results indicated that ROS generation was increased in the BDL model group compared with the sham group, whereas catalpol significantly reduced the production of ROS compared with in the BDL model group, with catalpol-H producing a more significant effect compared with catalpol-L (Fig. 3C).

Intracellular GSH contents in the serum were also detected; as shown in Fig. 4A, GSH content was significantly reduced in the BDL model group compared with that in the sham control group, whereas treatment with catalpol significantly increased GSH content compared with the BDL model group. Once again, catalpol-H produced a greater protective effect compared with catalpol-L. Moreover, similar results were found with regards to GSH concentration in the mitochondria (Fig. 4B). The contents of MDA in the serum and mitochondria were also assessed, and the data revealed that catalpol significantly reduced the total MDA content in the BDL cholestatic model mice (Fig. 4C and D), with catalpol-H producing a significantly more protective effect than catalpol-L.

As shown in Fig. 5, expression levels of proteins related to apoptosis and oxidative stress were measured. The results revealed that cytochrome c protein expression was increased in the BDL model group, whereas cytochrome c levels were significantly reduced by treatment with catalpol in the cholestatic model mice, with catalpol-H producing a greater protective effect compared with catalpol-L. With regards to the expression levels of proteins related to cell apoptosis, it was revealed that Bcl-2 expression was significantly reduced, whereas Bax and cleaved caspase-3 were significantly increased in the BDL model group. Conversely, following treatment with catalpol, the protein expression levels were significantly altered and closer to the sham control sample levels. Once again, catalpol-H produced a more protective effect than catalpol-L. There were no notable changes to total caspase-3 expression levels among the different control and treatment groups.