Rg1 (C₄₂H₇₂O₁₄) was obtained from Shanghai YaJi Biotechnology Co., Ltd. (Shanghai, China; cat. no. MUST-11041201); CCl₄ was purchased from Shanghai ShenXiang Chemical Reagent Co., Ltd. (Shanghai, China; cat. no. 20090510); minimum essential medium with Earle's balanced salts (MEM-EBSS) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA); fetal bovine serum (FBS) was from Hangzhou Evergreen Biological Material Co., Ltd. (Hangzhou, China); penicillin and streptomycin were from North China Pharmaceutical Co., Ltd. (Shijiazhuang, China); dimethyl sulfoxide (DMSO) was from Beijing YiLi Fine Chemicals Co., Ltd. (Beijing, China); NF-κB p65 antibody (rabbit anti-mouse) was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany; cat. no. SAB4502609); bicinchoninic acid protein concentration assay kit was from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China); NF-κB electrophoretic mobility shift assay (EMSA) kit (cat. no. 20148) was from Thermo Fisher Scientific, Inc.; biotin-labeled NF-κB probes (oligonucleotide sequences: 3′-TCAACTCCCCTGAAAGGGTCCG-5′ and 5′-AGTTGAGGGGACTTTCCCAGGC-3′) were purchased from Shanghai Sangon Biological Engineering Co., Ltd. (Shanghai, China); TNF-α (cat. no. MTA000B) and IL-6 (cat. no. M6000B) ELISA kits were from R&D Systems, Inc. (Minneapolis, MN, USA); and ALT (cat. no. C009-3), AST (cat. no. C010-3), SOD (cat. no. A001-2), and MDA (cat. no. A003-1) kits were from Nanjing JianCheng Bioengineering Institute (Nanjing, China).

A CCl₄-induced acute liver injury model of mice was established as described by Li et al (22). Male Kunming mice (20±2 g; n=60) were purchased from the Experimental Animal Center of Anhui Medical University [animal license no. SCXK (Anhui) 2011–002; Hefei, China]. The facilities and the protocol for these experiments were consistent with the regulations of animal use for biomedical experiments as issued by the Ministry of Science and Technology of China and were also approved by the animal ethics committee of Bengbu Medical College (Bengbu, China). The mice were kept in the dark and had free access to standard chow and water. Following 3 days of adaptive feedings, the 60 mice were randomly divided into five groups: i) Control group [olive oil (Hong Rui Men; Jiangxi Hongrui Oil Food Co. Ltd., Nanchang, China), 0.1 ml/10 g body weight]; ii) model group (CCl₄, 0.1 ml/10 g body weight), iii) low Rg1 group (Rg1, 10 mg/kg body weight; CCl₄ 0.1 ml/10 g body weight), iv) middle Rg1 group (Rg1, 20 mg/kg; CCl₄ 0.1 ml/10 g body weight), v) high Rg1 group (Rg1, 40 mg/kg; CCl₄ 0.1 ml/10 g body weight). Oral doses of Rg1 were administered once a day for 7 days and all groups, except the control group, were intraperitoneally injected with 0.1% CCl₄ in olive oil (v/v) 2 h subsequent to the last dose of Rg1 in order to induce acute liver injury, while the mice in control group were injected with an equal volume of olive oil. All mice were fasted and had free access to water overnight. The blood was obtained from the orbital sinus, centrifuged at 268.3 × g for 5 min at 37°C and prepared for ALT and AST content detection. The liver was rapidly removed subsequent to the mice being sacrificed, and was rinsed three times with saline to remove blood. The left lobe of the liver was prepared for hematoxylin and eosin (H&E) staining and the right lobe of the liver for SOD, MDA, TNF-α and IL-6 content detection.

Following intraperitoneal injection with CCl₄ olive oil solution into mice for 1 h, Kupffer cells from each group of animals were collected (n=4). Hepatic perfusion and digestion was performed according to the method described by Fukada et al (23). Following intraperitoneal injection with CCl₄ olive oil solution into the mouse for 1 h, the mouse was anesthetized with chloral hydrate (5% chloral hydrate, 0.1 ml/10 g body weight) and the abdomen opened to confirm the location of the portal vein. The portal vein was cannulated with a 22 G catheter, ~1 cm away from the hepatic portal with 0.8-cm insertion and an arterial clamp. The liver was perfused with the first infusion, composed of NaCl (137 mmol/l), KCl (2.68 mmol/l), Na₂HPO₄·12H₂O (0.7 mmol/l), HEPES (10 mmol/l), glucose (10 mmol/l), EDTA-Na₂ (0.5 mmol/l) and heparin (2,000 U/l), pH 7.4, until the liver volume had expanded to ~2X the original volume and a slight white color was observed. The inferior vena cava was subsequently cut. Finger pressure was applied to the segments, and they were intermittently opened to enlarge and retract the liver alternately following liver retraction. When the liver had turned white, with 15 min reperfusion, the liver was removed to a sterile petri dish (reserving the portal vein catheterization). Perfusion of the liver was continued with the second infusion, composed of NaCl (137 mmol/l), KCl (2.68 mmol/l), Na₂HPO₄·12H₂O (0.7 mmol/l), HEPES (10 mmol/l), glucose (10 mmol/l), CaCl₂ (5 mmol/l) and collagenase IV (0.05%), pH 7.4, for ~15 min, until the liver became soft and structural collapse was observed. Liver cells were scattered to form a cell suspension using stirring and percussion with a dropper.

For the isolation of Kupffer cells, the method described by Fukada et al (23) and Smedsrød and Pertoft (24) in 1985 was employed and improved. The cell suspension was subjected to low-speed centrifugation (16.7 × g for 3 min at 4°C) and the supernatant was removed prior to the precipitate being resuspended in 10 ml PBS. Subsequently, 15 ml 50% Percoll liquid (Shanghai Xinyu Biotechnology Co., Ltd., Shanghai, China) was added into a new tube and 20 ml 25% Percoll liquid was carefully added along the tube wall, followed by 10 ml cell suspension. Following centrifugation at 268.3 × g for 15 min at 4°C, four apparent partitions were observed: From top to bottom these were cell debris, non-parenchymal cells enriched in endothelial cells, Kupffer cells and red cells, which precipitated at the bottom of the tube. The Kupffer cells were collected, ~15 ml, and were diluted with 15 ml PBS. Following centrifugation at 268.3 × g for 10 min at 4°C, the precipitate was resuspended in culture medium.

Subsequently, purification of Kupffer cells was performed; the survival rate of the cells was >90% as observed with Trypan blue staining (3 min at 4°C). The cells were counted and the concentration adjusted to 2×10⁶/ml, and 1 ml cell suspension/well was seeded on 12-well culture plates. Following incubation for 2 h at 37°C in an atmosphere of 5% CO₂ in air, the cells were gently washed with fresh culture medium, and the adherent cells were considered to be Kupffer cells. Cell culture was continued, with the medium being changed every 2–3 days.

The left lobe of the removed liver was fixed in neutral formalin, paraffin-embedded and sliced. A 2.0×2.0×0.3 cm block of tissue was selected from the same site of each mouse, washed with saline and placed in 10% neutral formalin buffer at 37°C. Following dehydration, the blocks were wax embedded, sliced into 5-µm sections, dewaxed, H&E stained for 5 min at 37°C, dehydrated, and mounted in neutral balsam. The tissues were observed using an optical microscope in the Department of Pathology at the First Affiliated Hospital of Bengbu Medical College and images of morphological changes were captured using a light microscope (magnification, ×100).

The blood obtained from the orbital sinus was centrifuged at 1,341.6 × g at 4°C for 10 min and the serum content of ALT and AST was measured in accordance with the manufacturer's protocol of the ALT and AST activity kits.

The right liver lobes of the sacrificed mice were cut and weighed and 400 mg liver tissue was combined with saline (1:9 v/v) and crushed on ice using a glass homogenizer. The homogenized solutions were centrifuged at 1,341.6 × g at 4°C for 15 min, and the resulting 10% liver homogenate supernatants were measured for SOD and MDA content in accordance with the manufacturer's protocol for the kits.

The remainder of the right lobes of the livers were cut and weighed, and 400 mg was combined with saline (1:9 v/v) and crushed on ice using a glass homogenizer. The homogenates were centrifuged at 1,341.6 × g at 4°C for 15 min, and the optical density of the supernatant was detected at 450 nm and compared to the TNF-α or IL-6 standard curve in accordance with the manufacturer's protocol for the kit.

LO₂ human normal liver cells, donated by the College of Pharmacy, Sun Yat-sen University, were maintained in MEM-EBSS containing 10% FBS and 1% penicillin/streptomycin at 37°C in an atmosphere of 5% CO₂ in air. The cells were trypsinized and diluted to 5×10⁴ cells/ml in the logarithmic phase prior to the experiment.

The viability of LO₂ cells incubated with CCl₄ or Rg1 was measured by MTT assay. A total of 5×10³ cells/well were seeded into 96-well plates for 24 h and then increasing concentrations of CCl₄ in normal saline (2.5, 5, 10, 20 and 40 mM) were added for 3 h. Each group was plated in triplicate and normal saline was used as control group. For Rg1 treatment, 0.1, 1 and 10 µM Rg1 was added to LO₂ cells. After 21 h, 20 mM CCl₄ (the most appropriate concentration from the liver cell injury model) was added and incubated for a further 3 h. An NF-κB inhibitor [caffeic acid phenethyl ester (CAPE); 25 µM for 2 h] was additionally assessed. Subsequently, 20 µl MTT (Shanghai Gefan Biotechnology Co., Ltd., Shanghai, China; 5 mg/ml) was added to each well for a further 4 h, the supernatants were carefully discarded and 150 µl DMSO was added to each well in order to dissolve the crystals. The absorbance at 570 nm was measured and the cell viability was calculated as follows: Survival rate=(absorbance of treated group-absorbance of blank group)/(absorbance of control group-absorbance of blank group).

A total of 5×10⁴ LO₂ cells/well were seeded into 24-well plates and incubated at 37°C for 24 h. Treatment with Rg1 at 0.1, 1 and 10 µM was performed for 21 h, and 20 mM CCl₄ was added for a further 3 h. The supernatant was collected, and AST and ALT expression levels, SOD activity and MDA, content were measured according to the manufacturer's protocols.

The NF-κB activity in LO₂ and Kupffer cells was detected by EMSA as described by Li et al (25). The nuclear proteins were incubated in 1X binding buffer (50 ng/µl poly (deoxyinosinic-deoxycytidylic), 0.05% NP-40, 5 mM MgCl₂, 50 mM KCl, 2.5% glycerol, and ultra-pure H₂O at room temperature for 10 min, and the biotin-labeled NF-κB probe was added for a further 20 min. All reactions were electrophoresed on a 6% polyacrylamide gel, transferred to a positively charged nylon membrane and UV cross-linked to fix the DNA. The bands were visualized by Amersham ECL^™ Plus Detection kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

Cells were harvested using lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM β-glycerophosphate and 1:1,000 protease inhibitors]. Protein concentration was determined using the BCA kit then 25 µg total protein from each sample was analyzed by SDS-PAGE and transferred onto a nitrocellulose membrane followed by immunoblotting. The membranes were probed with antibodies against p65 (1:1,000) and β-actin (A1978; 1:8,000; Sigma-Aldrich; Merck KGaA) and the antibodies were blocked with the primary antibody dilution (Bi Yun Tian Biotechnology Co., Ltd., Shanghai, China), at 4°C overnight. Then the membranes were probed by goat anti-rabbit or anti-mouse IgG antibody HRP conjugate secondary antibodies for p65 (1:2,000) and β-actin (BL003A; 1:10,000; Biosharp, Hefei, China) at room temperature for 1 h. Immunopositive bands were visualized by Amersham ECL^™ Plus Western Blotting Detection kit (RPN2232; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The expression of p65 and β-actin as detected by western blotting were analyzed by Image J software version 1.48 (National Institutes of Health, Bethesda, MD, USA) as was the density of western blotting.

Data were processed using SPSS software (version 16; SPSS, Inc., Chicago, IL, USA). The results are expressed as the mean ± standard error of the mean. Two-group comparisons were analyzed using an unpaired Student's t test. P<0.05 was considered to indicate a statistically significant difference. All analyses were plotted using SigmaPlot version 10.0 (Jandel Scientific, San Rafael, CA, USA).

In order to characterize the function of Rg1 in acute CCl₄-induced liver injury in mice, histopathology of liver tissues was performed using H&E staining (Fig. 1A). The control group exhibited normal liver morphology and intact lobular architecture, neat hepatic cords, clear boundaries of liver cells and round and well-defined nuclei in the absence of inflammatory cell infiltration. In the liver of the CCl₄-treated mouse presented in the second panel, normal liver tissue structure disappeared and lobular structure was disrupted, inflammatory cell infiltration was observed at lobular and portal areas, including disordered hepatic cords, and significant swelling of liver cells suggested high levels of necrosis. However, with the addition of Rg1, particularly for the higher dose group, liver cell degeneration and necrosis was markedly reduced, a clearer lobular structure and neatly arranged liver cells were observed, and the inflammatory cell infiltration was also reduced.

The effect of Rg1 on acute CCl₄-induced liver injury in LO₂ cells was also investigated (Fig. 1B and C). Increasing concentrations of CCl₄ (2.5–40 mM) were added to LO₂ cells for 3 h, and the effect of CCl₄ cytotoxicity on LO₂ cells was detected by MTT assay. As presented in Fig. 1B, CCl4 led to an apparent concentration-dependent decrease in cell viability. The cell survival rates with 2.5 and 20 mM CCl₄ were 91.23 and 53.39%, respectively. When the 40 mM CCl₄ cells were observed under the microscope, the survival rate was 20.12%. A concentration of 20 mM CCl₄ was used for the following CCl₄-induced LO₂ cell injury model.

The addition of Rg1 at a range of concentrations (0.1–10 µM) to LO₂ cells for 21 h prior to exposure to 20 mM CCl₄, and detection of cell viability by MTT assay, demonstrated that incubation with CCl₄ alone for 3 h decreased cell viability to 53.08%; the addition of Rg1 markedly improved cell viability in a concentration-dependent manner. The cell survival rate of the 0.1 µM Rg1 group was 66.67% and the 10 µM group reached 84.69% (Fig. 1C).

The serum contents of ALT and AST presented in Table I demonstrated that, compared with levels of ALT and AST in the control group (34.54±5.63 and 75.76±7.64 U/l, respectively), the expression levels of ALT and AST in CCl₄-treated mice significantly increased (89.90±8.18 and 182.20±8.33 U/l, respectively; P<0.05). However, oral administration of Rg1 gradually reduced the serum levels of ALT and AST; particularly in the high-dose group (40 mg/kg), the serum ALT expression level reduced to 50.88±10.85 U/l and the AST expression level reduced to 100.64±8.53 U/l.

ALT and AST levels in the cell culture supernatant of CCl₄-treated LO₂ cells were assessed following pretreatment with increasing concentrations of Rg1 (0.1–10 µM). Consistent with the results of ALT and AST expression levels in vivo, 20 mM CCl₄ caused a significant increase in the levels of ALT and AST, while pretreatment with Rg1 decreased ALT and AST expression levels in a concentration-dependent manner (Table II).

In order to investigate the effect of Rg1 on SOD activity and MDA content in acute CCl₄-induced liver injury in mice, 10% liver homogenate supernatants of the different treatment groups were prepared, and the results presented in Table III demonstrated that SOD activity in mice that were intraperitoneally injected with CCl₄ was decreased (113.21±11.31 U/mg) compared with control group (212.31±5.65 U/mg); however, pretreatment with Rg1 caused a dose-dependent enhancement of SOD activity. By contrast, the MDA expression level was increased in CCl₄-treated mice (8.78±0.62 nmol/mg) compared with the control group (3.42±0.88 nmol/mg), while pretreatment with Rg1 caused a dose-dependent reduction in the MDA expression level.

SOD activity and MDA content in the cell culture supernatants of the CCl₄-induced LO₂ cell injury and Rg1 treatment groups were also examined (Table IV). CCl₄ inhibited SOD activity and increased the MDA expression levels of LO₂ cells; however, Rg1 alleviated the inhibited SOD activity and reduced the increased MDA level, which was consistent with the results of SOD activity and MDA content experiments in vivo.

Liver Kupffer cells serve a role as a barrier to liver injury. In order to investigate whether NF-κB is involved in the protective effect of Rg1 in acute CCl₄-induced liver injury in mice, liver Kupffer cells were isolated from the different treatment groups. NF-κB p65 protein expression was determined using western blotting and NF-kB binding activity using EMSA analysis. The results of the western blotting demonstrated that NF-κB p65 protein expression was significantly increased in the CCl₄ model group, although pretreatment with Rg1 (10–40 mg/kg) reduced the protein expression of p65 in a dose-dependent manner (Fig. 2A). Additionally, the results of the EMSA analysis demonstrated that CCl₄ (0.2 mg/10 g) used alone enhanced NF-kB binding activity, however, pretreatment with Rg1 alleviated the enhanced NF-kB binding in a dose-dependent manner (Fig. 2B).

NF-κB function in the protective effect of Rg1 on acute CCl₄-induced liver injury was further confirmed in vitro. CAPE, an inhibitor of NF-κB, was used and its role observed on CCl₄-induced LO₂ cell injury. The results of the MTT assay presented in Fig. 2C demonstrated that exposure of LO₂ cells to 20 mM CCl₄ for 3 h inhibited cell growth, and CAPE (25 µM, 2 h) attenuated this cell growth inhibition. P65 expression in LO₂ cells treated with CCl₄ alone or in combination with Rg1 was observed by western blotting. When treated with 20 mM CCl₄ for 15 min, there was no significant alteration in p65 expression; however, when the incubation time was prolonged to 30 or 60 min, the expression level increased, and 10 µM Rg1 alleviated the increased p65 expression (Fig. 2D and E). The present results were also confirmed by EMSA assay. Rg1 (0.1–10 µM) used alone did not affect NF-κB binding activity; however, it significantly alleviated the enhanced binding of NF-κB when used in combination with CCl₄; the NF-κB inhibitor CAPE served a comparable role and inhibited CCl₄-induced NF-κB binding (Fig. 2F).

The results presented in Table IV demonstrated that pretreatment with Rg1 alleviated the inhibited SOD activity and reduced MDA expression level in CCl₄-induced LO₂ cell injury. Inhibition of NF-κB activity by CAPE demonstrated comparable results: CCl₄ inhibited SOD activity and increased the MDA expression level compared with the control group; however, using 25 µM CAPE for 2 h enhanced the inhibited SOD activity and reduced the MDA expression level (Table V). The expression levels of TNF-α and IL-6 in 10% liver homogenate supernatants of the different treatment groups of mice were measured by ELISA, and it was observed that CCl₄ injection led to an increase in the serum levels of TNF-α and IL-6; however, pretreatment with Rg1 reduced this increase in a dose-dependent manner (Table VI).

It is well known that NF-κB expression and activity in liver Kupffer cells may be modulated by TNF-α, IL-6 and other cytokines, and NF-κB may, in turn, activate these inflammatory regulators (26,27). The results of the present study, displayed in Fig. 2A and B, demonstrated that CCl₄ increased NF-κB p65 protein expression and enhanced NF-kB binding activity, while pretreatment with Rg1 reduced p65 protein expression and alleviated the enhanced NF-κB binding. Therefore, Rg1 protected CCl₄-induced liver injury, by directly alleviating the inhibition of SOD activity and reducing MDA, and possibly by inhibiting NF-κB activity, thereby further alleviating the inhibited SOD activity and reducing MDA, TNF-α and IL-6 expression levels.