CAG (2 mg/ml) was produced by the authors in our laboratory [ammonium glycyrrhizin (2.8 g; Shaanxi FUJIE Pharmaceutical Co., Ltd., Xianyang, China), glycine (2 g) and methionine (2 g, Tianjin Tianyao Pharmaceuticals Co., Ltd., Tianjin, China) filled with water to 1,000 g)]. ENR was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). LPS (E. coli L-2880; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM) was purchased from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA). Primary antibodies against apoptosis regulator Bcl-2 (Bcl-2; cat. no. 610538; 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), Bcl-2 associated X-protein (Bax; cat. no. orb334986; 1:1,000; Biorbyt Ltd., Cambridge, UK), caspase-3 (cat. no. ab115183; 1:1,000, Abcam, Cambridge, USA), β-actin (1:5,000; Sigma-Aldrich; Merck KGaA, A5441) and secondary horseradish peroxidase-labeled goat anti-rabbit IgG (cat. no. 4201-100; 1:1,000; Shanghai Pufei Biotechnology Co., Ltd., Shanghai, China) were obtained.

A total of 12 30 day old male Hailan chickens (mean weight, 1.5±0.2 kg) were purchased from Qinglong Mountain (Nanjing, China) and housed at 22˚C with ventilation using 12-h light/dark cycles. Animals had free access to food and water and were fasting 12 h prior to experiments. Hepatocytes (23,24) were isolated from the chickens (using the two-stage, collagenase type IV perfusion method via the portal vein. Briefly, the livers were perfused with solution A [33 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 127.8 mM NaCl, 3.15 mM KCl, 0.7 mM Na₂HPO₄ x 12 H₂O and 0.6 mM EGTA; pH 7.6] and solution B (33 mM HEPES, 127.8 mM NaCl, 3.15 mM KCl, 0.7 mM Na₂HPO₄ x 12 H₂O and 3 mM CaCl₂; pH 7.6) at room temperature at a flow rate of 10-20 ml/min for 15 min. A total of 100 ml 0.5% collagenase type IV buffer (C0016; NanJing HoldBio E-Commerce Co., Ltd., Nanjing, China) was perfused for 20-25 min at 37˚C at a flow rate of 20 ml/min. The hepatocytes were separated from the other cellular components by centrifugation at 167.7 x g for 3 min at 37˚C. Isolated cells were washed twice in solution B supplemented with 0.2% bovine serum albumin (BSA; Sigma Aldrich; Merck KGaA). MTT stock solution (5 µl; 5 mg/ml) was added in each well and cells were incubated in a humidified incubator with an atmosphere of 5% CO₂ for 4 h at 37˚C. Absorbance at 570 nm was measured using a microplate reader. Cell yield varied between 4 and 5x10⁸ cells/liver and the cell viability varied between 90 and 95% as determined by trypan blue exclusion (0.04%; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Adherent cells were digested using trypsin to prepare single cell suspension. Cell suspension was mixed with 0.4% trypan blue solution (9:1). Following 3 min at room temperature, living and dead cells were counted. Dead cells appeared blue and living cells were colorless/transparent. The viable cell rate was defined as: Viable cell rate (%)=total viable cells/total cells.

The present study was approved by the Animal Ethics Committee of Nanjing Agricultural University (Nanjing, China).

Chicken hepatocytes were plated at a density of 5x10⁵ cells in 96-well cell plates and cultured for 48 h at 37˚C in DMEM. LPS (30 µg/ml) and different concentrations of ENR (40-120 µg/ml) were added to determine the optimal concentration. Cytotoxicity was measured using an MTT assay. MTT (0.1%) was added to the cell plates and incubated at 37˚C for 4 h. The medium was added to 150 µl dimethyl sulfoxide to dissolve the purple formazan and the absorbance was measured at a wavelength of 570 nm using a microplate reader.

The cells were divided into four groups: i) Control group, not administered with CAG or exposed to LPS/ENR; ii) model group, administered LPS/ENR (30/80 µg/ml) for 24 h; iii) CAG group, administered CAG 400 µg/ml; and iv) combined group, pretreated with CAG (25, 50, 100, 200 and 400 µg/ml) for 24 h prior to LPS/ENR (30/80 µg/ml).

ALT and AST are liver enzyme markers (1). These two enzymes were measured by spectrophotometric analysis. Cell supernatants of the different groups were obtained by centrifugation (1,006 x g; 10 min; 4˚C) and the activity of ALT (C009-1; Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China) and AST (C010-1; Jiancheng Bioengineering Institute Co., Ltd.) were determined according to the manufacturer's protocol.

The hepatocytes were lysed in a radioimmunoprecipitation assay buffer (RIPA; Jiancheng Bioengineering Institute Co., Ltd.) on ice and centrifuged at 7,280 x g for 15 min at 4˚C. The cell lysate supernatant was aspirated and stored at -20˚C prior to glutathione (GSH) assays (reduced glutathione assay kit; A006-1; Jiancheng Bioengineering Institute Co., Ltd.), superoxide dismutatse (SOD) assays (SOD assay kit; A001-3; Jiancheng Bioengineering Institute Co., Ltd.), catalase (CAT) assays (CAT assay kit; A007-1-1; Jiancheng Bioengineering Institute Co., Ltd.), glutathione peroxidase (GPx) assays (GPx assay kit; A005; Jiancheng Bioengineering Institute Co., Ltd.) and malondialdehyde (MDA) assays (MDA assay kit; A003-1; Jiancheng Bioengineering Institute Co., Ltd.) according to the manufacturer's protocols. The protein concentration of each sample was determined using the bicinchoninic acid protein quantitation cassette.

In normal viable cells propidium iodide (PI) and Annexin V are negative, while in early apoptotic cells Annexin V is positive and PI is negative. In late apoptotic cells Annexin V and PI are positive, while in dead cells Annexin V is negative and PI is positive. Cells following 24 h LPS/ENR treatment were collected (560 x g; 5 min; 4˚C) and stained using an Annexin V-fluorescein (FITC)/PI Apoptosis Detection kit (BD Biosciences) according to manufacturer's protocol. The cells collected were then analyzed by flow cytometry using a BD FACS Calibur Cell Sorting system (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo v10 (FlowJo LLC, Ashland, OR, USA).

Chicken hepatocytes following 24 h treatment with LPS/ENR were collected (7,280 x g; 15 min, 4˚C) and total RNA was extracted using an RNAiso Plus kit (Takara Biotechnology Co., Ltd., Dalian, China). RT was performed using the PrimeScript^™ RT reagent Kit with gDNA Eraser (RR037A; Takara Biotechnology Co., Ltd.) at 37˚C for 15 min, followed by 85˚C for 5 sec. RNA concentrations were detected using a Qubit RNA Assay kit and a Qubit 2.0 fluorometer (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primer sequences of the genes used for PCR are listed in Table I. SYBR green premix (RR820A; Takara Biotechnology Co., Ltd.) was used for qPCR analysis. The PCR thermocycling conditions were as follows: 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec and 60˚C for 30 sec. Quantification was based on the 2^-ΔΔCq method (25) and β-actin was used as the housekeeping gene.

Total protein was extracted from chicken hepatocytes using a RIPA buffer. Protein concentrations were determined using bicinchoninic acid assays. Equal amounts of protein (100 µg) were separated on 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA at room temperature for 2 h followed by incubation with primary antibodies overnight at 4˚C. Membranes were incubated with secondary antibodies for 1 h at room temperature. Following, membranes were washed with TBST and immunoreactive bands were detected by using enhanced chemiluminescence (Vazyme, Piscataway, NJ, USA). The western blot bands were analyzed using ImageJ software (1.46r; National Institutes of Health, Bethesda, MD, USA).

Caspase-3 activity was measured in all groups following 24 h of LPS/ENR treatment using a commercial Caspase-3 Colorimetric Assay kit (C1115; Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol.

Cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4˚C. Fixed cells were subsequently cut into sections for TEM (10-100 nm). The sections were stained with 2% uranyl acetate at 37˚C for 30 min and embedded in osmium tetroxide at 37˚C for 30 min and observed under a Hitachi TEM (Hitachi, Ltd., Tokyo, Japan).

Hepatocyte MMP was measured using the membrane-sensitive JC-1 dye and analyzed with a fluorescence microscope. JC-1 staining solution (1 ml; Mitochondrial Membrane Potential kit; Beyotime Institute of Biotechnology) was added to each well and mixed. Cells were incubated for 20 min at 37˚C in an incubator with the JC-1 stain. Following incubation the supernatant was aspirated, cells were washed twice with JC-1 buffer solution at room temperature for 20 sec, 2 ml DMEM were added and samples were observed under a fluorescence microscope (magnification, x100).

All data are expressed as the mean ± standard deviation, from ≥3 repeats. Data were analyzed using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance with Dunnett's post hoc test was used for the comparison of multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Assessment of the variations in hepatocyte cell viability and morphological evaluation following LPS/ENR treatment were performed. The results demonstrated that LPS/ENR treatment decreased the viability of cells in a dose-dependent manner (Fig. 1A). When cells were treated with LPS/ENR 30/80-30/120 µg/ml they demonstrated evident reduced cell viability. When the concentration of LPS/ENR was 30/100 µg/ml, the cell viability was 22.57±0.43%. Exposure of cells to 30/120 µg/ml LPS/ENR injury led to a notable reduction in cell number and these cells exhibited morphological alterations, including the disruption of cell membranes and nuclear fragmentation compared with the control cells. At LPS/ENR 30/80 µg/ml the cell viability was 48.27±2.50% and the cell shape was irregular with clear disruption of the cell membrane. Therefore, LPS/ENR 30/80 µg/ml was determined to be the most appropriate concentration and was used in the model groups for all further experimentation.

ALT and AST activities were measured in the supernatant and the cell viability was evaluated using an MTT assay. Following treatment with CAG (400 µg/ml) the cell viability was 97.39±0.75%, which was similar to the negative control group and indicated the non-toxic nature of CAG (Fig. 1B). Treatment with LPS/ENR (30/80 µg/ml) induced a significant elevation in ALT (Fig. 1C) and AST (Fig. 1D) concentrations (P<0.01) compared with the control, while treatment with CAG (50-400 µg/ml) caused a dose-dependent decrease compared with the model group (P<0.05 and P<0.01).

LPS/ENR (30/80 µg/ml) treatment of hepatocytes significantly reduced the cell viability of the hepatocytes compared with the control group (P<0.01; Fig. 1B). Hepatocytes treated with CAG demonstrated a dose dependent improvement in cell growth and all groups treated with CAG had significantly improved cell viability compared with the model group (all P<0.01).

Treating hepatocytes with LPS/ENR (30/80 µg/ml) for 24 h caused a significant reduction in activity of GSH (P<0.01; Fig. 1E), SOD (P<0.01; Fig. 1F), CAT (P<0.01; Fig. 1G) and GPx (P<0.01; Fig. 1H) compared with the control group. Treatment with LPS/ENR caused a significant elevation in MDA levels (P<0.01; Fig. 1I) compared with the control group. CAG treatment provided a protective effect as evidenced by the restoration of these biomarkers. The highest dose of CAG (400 µg/ml) attenuated protein MDA levels and caused an increase in GSH protein levels and the activities of the SOD, CAT and GPx antioxidant enzymes (P<0.01; Fig. 1E-I).

To confirm apoptotic activity, flow cytometry analysis of hepatocytes was performed using dual stain Annexin V-FITC/PI (Fig. 2). In the control group there was a high percentage of surviving cells (91.40±3.74%) and a low percentage of early apoptotic (2.69±0.79%) and late apoptotic (3.05±1.81%) cells. In the LPS/ENR group the percentage of surviving cells (17.26±3.98%) was significantly decreased compared with the control group. The number of early apoptotic (73.03±3.36%) and late apoptotic (10.42±2.15%) cells were significantly increased following LPS/ENR (30/80 µg/ml) treatment in comparison with the control group. The number of early apoptotic cells (66.92±2.73, 41.73±4.07, 31.32±2.74%, 13.82±2.07 and 11.53±1.17%) notably reduced in a dose-dependent manner with increasing concentrations of CAG (25, 50, 100, 200 and 400 µg/ml, respectively). All groups treated with CAG had a significantly reduced number of apoptotic cells compared with the model group (73.03±3.36%; P<0.05).

Treatment with LPS/ENR (30/80 µg/ml) significantly increased hepatocyte caspase-3 (Fig. 3A) and Bax (Fig. 3B) mRNA expression 19.45±1.87- and 11.43±2.35-fold, respectively compared with the control group (P<0.01). Treatment with 25-400 µg/ml CAG significantly inhibited Caspase-3 expression (P<0.01; Fig. 3A). Treatment with ≥50 µg/ml CAG significantly inhibited Bax mRNA expression (P<0.05; Fig. 3B). LPS/ENR (30/80 µg/ml) treatment markedly suppressed Bcl-2 expression, while CAG treatment at doses of 100 and 200 µg/ml significantly increased Bcl-2 mRNA expression compared with the control (P<0.01; Fig. 3C). Interestingly, at 400 µg/ml Bcl-2 mRNA was not significantly decreased (P>0.05).

Western blot analysis was performed to confirm the apoptotic data (Fig. 3D). Bcl-2 and caspase-3 protein expression levels were significantly upregulated in the LPS/ENR group compared with the control group (P<0.01; Fig. 3E and F). The results also revealed that Bax protein expression was notably increased in the LPS/ENR group, while this was significantly reversed by CAG treatment (25, 200 and 400 µg/ml) for 24 h prior to LPS/ENR treatment (P<0.01; Fig. 3G). Caspase-3 activity was further examined using a colorimetric assay; the results demonstrated that 24 h exposure to LPS/ENR caused a significant increase in caspase-3 activity compared with the control (P<0.01). In addition, when the hepatocytes were exposed to CAG (50-400 µg/ml) the caspase-3 activity significantly decreased compared with the LPS/ENR group (all P<0.01; Fig. 3H).

Mitochondria serve an important role in the apoptotic-signaling pathway (26). In the present study, normal ultrastructure features were observed in the control group, including a round nucleus, nuclear membrane integrity and normal mitochondrial cristae (Fig. 4A). In the LPS/ENR treatment group, the mitochondria were swollen with clear vacuolization and loss of the crest (Fig. 4B). Following treatment with CAG (25-400 µm/ml) for 24 h these modifications were alleviated (Fig. 4C-G). The MMP was also investigated using JC-1 dye. In normal mitochondria, JC-1 accumulates in the mitochondrial matrix to form polymers and the polymer emits intense red fluorescence. Unhealthy mitochondria may be present in the cytoplasm in monomeric form due to a decrease or loss of membrane potential and produces green fluorescence (10). In the model group green fluorescence was increased compared with red fluorescence. However, in the CAG group, red fluorescence was increased compared with green fluorescence (Fig. 4H).