A total of 60 normal Balb/c mice were purchased from Shanghai Jiesijie Experimental Animals Co., Ltd. (Shanghai, China) and 40 HBV transgenic mice were obtained from the Guangzhou 458th Hospital of Chinese People's Liberation Army (Guangzhou, China). Concanavalin A was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The primary antibodies used in the western blot analysis were as follows: Anti-TLR4 (cat. no. NB100-56566), anti-TLR3 (cat. no. NB100-56571) and anti-TLR9 (cat. no. nbp2-24729), purchased from Novus Biologicals LLC (Littleton, CO, USA); anti-TNF-associated factor (TRAF)6 (cat. no. ab33915), anti-IFN regulatory factor (IRF)3 (cat. no. ab25950), anti-TBK1 (cat. no. ab109735), anti-TRAF-interacting protein (TIRP) (cat. no. ab205100) purchased from Abcam (Cambridge, UK); anti-nuclear factor (NF)-κB (cat. no. 8242), anti-MyD88 (cat. no. 4283) purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The secondary antibodies were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). ELISA kits to detect interleukin (IL)-6, IL-1, TNF-α and IFN-γ were purchased from Wuhan Huamei Biotech Co., Ltd. (Wuhan, China). Alanine aminotransferase Assay Kit (cat. no. C009-2) and Cholinesterase assay kit (cat. no. A023-2) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The experimental mice were fed in Specific Pathogen Free Animal Rooms of Animal Experimental Center of Shanghai University of Traditional Chinese Medicine (Shanghai, China) with free access to clean drinking water and food. All animal experiments were carried out according to the regulations of and approved by Laboratory Animal Administration of Shanghai University of Traditional Chinese Medicine. All the mice were divided into two groups: Group 1 and group 2. The group 1 were all normal and the group 2 were all HBV transgenic mice: A replicative HBV transgenic mouse produced by the Department of Genetics Engineering, Infectious Disease Center, Guangzhou 458th Hospital of Chinese People's Liberation Army (Guangzhou, China). The HBV genome was transferred into the genotype of 1.3 times D genotype by microinjection of fertilized eggs, serum HBs Ag >2,000 U/ml, HBV DNA levels of 10⁴−10⁶ copies/ml, HBV replication intermediates exist in the liver. All the experimental mice were germ-free grade, 6–8 weeks old, weighing 20–22 g, and purchased following testing for HBs Ag. Animals of both sexes (males/females 1:1) were included. Mice in both groups were divided into seven sub-groups: A, Control; B, Concanavalin A; C, Bushen recipe (BS); D, Bushen-yang (BSY); E, Bushen-yin (BSC); F, QingHua (QH); G, GanYanLing (GYL). Each sub-group in group 1 had 8 mice, while 5 mice in each sub-group in group 2; the remaining mice were not employed. Acute liver injury was induced using tail vein injection of Concanavalin A in both normal and HBV transgenic mice from B to G sub-groups and the mice in each sub-group were treated with different traditional Chinese medicines (diluted in saline), respectively: A, (Control): Normal Saline; B, (Concanavalin A): with Normal Saline, 15 mg/kg; C, (BS): the Whole Bushen recipe; D, (BSY): the disassembled prescriptions of Bushen recipe; E, (BSC): the disassembled prescriptions of Bushen recipe; F, (QH): The disassembled prescriptions of Bushen recipe; G, (GYL).

These traditional Chinese medicines were prepared in our laboratory or were purchased from different companies and their effective constituents and manufacturer were enumerated as follows:

The Whole Bushen recipe (BS) was prepared according to the formula of ‘Bushen Granule’ (Shanghai Pharmacy) which was produced in our laboratory and is comprised of 15 g herba epimedii, 15 g cuscuta chinensis lam, 15 g cistanche deserticola ma, 15 g fallopia multiflora, 15 g radix rehmanniae recen, 15 g lyceum barbaruml, 15 g giant knotweed rhizome, 15 g radix scutellariae, 15 g radix sophorae flavescentis and 10 g pericarpium citri reticulatae viride.

The Bushen-yang group (BSY) of Bushen recipe was boil-free particles of Chinese medicine and comprised of 15 g herba epimedii, 15 g cuscuta chinensis lam, 15 g cistanche deserticola ma which was produced by Shenzhen Resources Sanjiu Modern Chinese Medicine Co., Ltd. (Shenzhen, Guangdong, China).

The Bushen-yin group (BSC) of Bushen recipe was also boil-free particles of Chinese medicine and comprised of 15 g fallopia multiflora, 15 g radix rehmanniae, 15 g lyceum barbaruml that was produced by Shenzhen Resources Sanjiu Modern Chinese Medicine Co., Ltd.

The QingHua group (QH) of Bushen Recipe has 15 g giant knotweed rhizome, 15 g radix scutellariae, 15 g radix sophorae flavescentis and was produced by Shenzhen Resources Sanjiu Modern Chinese Medicine Co., Ltd. These were all produced as boil-free particles of Chinese medicine.

GYL was purchased from Changshu Lei Yun Shang Pharmaceutical Co., Ltd (Changshu, China) and had 35 mg matrine in 2 ml solution of GYL.

The administration dose of these drugs was calculated according to the human and mice dose conversion method in the second edition of ‘Experimental Zoology’. The conversion coefficient between human (~70 kg) and mice (~20 g) is 0.0026. Assuming the dose for human (70 kg) is n, the dose for mice may be calculated as: n mg/kg × 70 kg ×0.0026/20 g=9.1n mg/kg. Therefore, the dosage which was used in this study was 9 times of the adult daily dose. The mice in sub-groups A to F underwent intragastric administration, while intraperitoneal injection occurred for sub-group G. The drug was administered once daily for 7 days at a dose of 18 mg/kg.

The mice were sacrificed by excessive ether inhalation at 18 h after injection with Concanavalin A and administration with Bushen recipe and its disassembled prescriptions. Peripheral blood samples (0.5 ml) were collected from hearts of mice and centrifuged at 375 × g for 30 min at 4°C to obtain serum for the following experiments. Subsequently, serum was seeded into 96-well plates and detection reagents were added according to the manufacturer's protocol of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Finally, these plates were analyzed at a wavelength of 505 nm by a Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, Inc., Waltham, MA, USA) to detect the content of alanine aminotransferase (ALT) and cholinesterase (ChE).

Mice were sacrificed at 18 h following injection with Concanavalin A and administration with Bushen recipe and its disassembled prescriptions, liver tissues of all mice were obtained to inspect liver injury and antiviral effect of Bushen recipe and its disassembled prescriptions, and they were fixed with 10% paraformaldehyde. Subsequently, the paraffin-embedded sections were prepared for histological analysis. The slices were cut in 5 µm sections and stained in dye liquor of hematoxylin (3 min) and eosin (15 sec) at room temperature for histological evaluation. Finally, these slices were observed under an inverted microscope (ix73, Olympus Corporation, Tokyo, Japan) and evaluated by a pathologist blindly. A total of five random fields of hepatic lobule were imaged at ×200 magnification.

Liver tissues of all mice were homogenized and cells were suspended in PBS. The cell suspension was centrifuged with PBS at 600 × g at 4°C and were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) for 30 min at 4°C. Subsequently, cell lysates were centrifuged at 9,000 × g at 4°C for 15 min and a Takara BCA Protein Assay Kit (cat. no. T9300A; Takara Bio, Inc., Otsu, China) was used to detect the total protein concentrations. Subsequently, total protein was mixed with an equal volume of loading buffer and boiled for 5 min. Equal amounts of proteins (20 µg) in each lane were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to Immobilon-polyvinylidene fluoride Transfer Membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in TBS with Tween 20 (TBST) for 1 h at room temperature and incubated with the indicated primary antibodies overnight at 4°C in a dilution of 1:1,000. Following washing 3 times with TBST, these membranes were incubated with secondary antibodies, including IgG(H+L) horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (cat. no. A0216) and IgG(H+L) HRP-labeled goat anti-rabbit IgG (cat. no. A0208), purchased from Beyotime Institute of Biotechnology, for 1 h at room temperature at a dilution of 1:10,000. GAPDH served as the loading control. Finally, membranes were visualized using the enhanced chemiluminescence system (ECL; PerkinElmer Inc., Waltham, MA, USA).

Mouse IL-6 ELISA kit (cat. no. EK0411) and mouse TNF-α ELISA kit (cat. no. EK0527) were purchased from Boster (Hubei, China). Mouse IFN-γ ELISA kit was purchased from R&D Systems, Inc. (cat. no. MIF00). Mouse IL-1 ELISA kit was purchased from Mlbio (cat. no. ml037875, Shanghai, China). ELISA analysis in the present study was conducted according to the manufacturer's protocol of Wuhan Sanying Bioengineering (Wuhan, China). In brief, blank wells without any solution were set in a 96-well plate and 100 µl negative control (PBS with 0.05% Tween-20), positive control or serum from rats of the aforementioned experimental groups per well were added into the test wells. The plate was covered with the adhesive strip and incubated in a humidified atmosphere of 5% CO₂ at 37°C for 1 h. Subsequently, 50 µl of HRP-conjugated antibodies were added into each well (apart from the blank well). Subsequently, plates were covered with another adhesive strip and incubated for 30 min at 37°C again. Following this, wells were washed with wash buffer (included in kit; 200 µl per well) three times and were left to dry for 20 sec completely removing the liquid. Following washing, any remaining wash buffer was removed through inverting the plate on clean paper towels. In addition, 50 µl substrate A and 50 µl substrate B were added to each well and plates were incubated for 30 min at 37°C in the dark. Finally, 50 µl of stop solution was added into each well, and plates were gently tapped to mix the solution. Finally, optical density (OD) of each well was determined within 10 min (25°C) using a Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, Inc.) at a wavelength of 450 nm.

All experiments in the present study were repeated at least three times. All data are presented as the mean ± standard deviation. Data were analyzed by SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). Two-way analysis of variance and Student-Neuman-Keuls method were used to determine statistical significance among experimental groups. P<0.05 was considered to indicate a statistically significant difference.

ALT and ChE are both synthesized in liver and may reflect the degree of liver injury. Following injection of Concanavalin A, liver tissues of all mice presented liver injury and the Bushen recipe and its disassembled prescriptions have previously been demonstrated to decrease liver injury. Therefore, the expression levels of ALT and ChE were investigated in peripheral blood of normal and HBV transgenic mice following treatment with Concanavalin A, Bushen Recipe and its disassembled prescriptions. As presented in Fig. 1, the results demonstrated that the content of ALT was increased immediately following administration of Concanavalin A, whereas the ChE declined in the liver tissues of both normal and HBV transgenic mice compared with the control. However, this increase of ALT was suppressed following administration of Bushen Recipe and its disassembled prescriptions (Fig. 1A), while the expression of ChE was upregulated (Fig. 1B). These results indicated that Bushen Recipe and its disassembled prescriptions alleviated the liver injury induced by Concanavalin A as observed within the Bushen Recipe and disassembled prescription groups compared with in the Con A group.

Livers of mice demonstrated acute immune hepatitis following injection of Concanavalin A. The results obtained in this experiment are presented in Fig. 2. Liver cells of normal mice were well-formed and the structure of hepatic cord was clear and intact. The hepatic lobule had no necrosis (which is a form of cell injury resulting in the premature death of cells in living tissue by autolysis), bleeding, and infiltration of inflammatory cells. However, the opposite phenomenon was observed in the sections of mice injected with Concanavalin A, whereas Bushen recipe and its disassembled prescriptions relieved the pathological features in the HBV transgenic mice, for example, the hepatic necrotic area was significantly decreased in the Bushen recipe and its disassembled prescriptions treated groups. The results indicated that Bushen recipe and its disassembled prescriptions relieved the liver injury induced by Concanavalin A.

Protein expression levels of MyD88, TBK1, TRAF6, IRF3, TLR3, TLR9, TLR4, NF-κB and TIRP that are members of TLR3/9 signaling pathway in all mice were detected by western blot analysis. In Figs. 3–5, the protein expression levels of MyD88, TBK1, IRF3, TLR4, TLR3, TRAF6 and NF-κB were increased in normal mice following administration of Concanavalin A compared with the mice in control group without any treatment. However, these expression levels did not increase in HBV transgenic mice following administration of Concanavalin A (Figs. 6–8). The increased expression of MyD88, IRF3, TBK1 and NF-κB due to immunological hepatitis has been reported previously, which is in agreement with the present findings (17). Furthermore, following administration with Bushen recipe and its disassembled prescriptions, these proteins had no upregulated trend in either normal or HBV groups. NF-κB further decreased in HBV groups following treatment with Bushen recipe and its disassembled prescriptions (Figs. 6–8). The expression of TRAF6 in QH group and GYL group decreased more notably upon interaction of corresponding treatment. Additionally, compared with the control group, the expression levels of MyD88, TBK1, TRAF6, IRF3, TLR3, TLR9 and TIRP were decreased to varying extents, indicating the pharmaceutical effect of Bushen recipe and all its disassembled prescriptions. In conclusion, these results indicated that the TLR3/9 signaling pathway may be inhibited by Bushen Recipe and its disassembled prescriptions to repair liver injury induced by Concanavalin A.

IL-6, TNF-α and IL-1 are primary inflammatory factors that affect the downstream pathway of NF-κB, and IFN-γ affects the downstream pathway of IRF3 (18,19). These inflammatory factors were detected by ELISA as demonstrated in Fig. 9. The expression levels of IL-6, IL-1, TNF-α and IFN-γ were significantly increased in normal and HBV mice following injection with Concanavalin A, compared with in the control group. However, following treatment with Bushen recipe and its disassembled prescriptions, the Concanavalin A-induced upregulated expression levels of IL-6, IL-1 and TNF-α decreased significantly in the BS group, while only IL-1 in BSY group decreased significantly and only IL-6 in the BSC and QH group decreased significantly. These results are consistent with activation state of NF-κB in western blot analysis, and the downstream pathway of NF-κB requires further investigation and validation. Furthermore, the increase of IFN-γ expression was evident following Concanavalin A injection in normal and HBV mice, while the content of IFN-γ in the mice treated with the Bushen recipe and its disassembled prescriptions decreased only slightly in BSC and BSY groups. Therefore, the results of the present study suggested that the Bushen recipe and its disassembled prescriptions (BSC and BSY in particular) may repair liver injury induced by Concanavalin A by inhibiting the expression of inflammatory factors. Variations in the effects exhibited by Bushen recipe and its disassembled prescription may be due to the differing compunds present; however further research is required.