Animals and treatments
Male C57BL/6 mice, 6–8 weeks old, weighting 20–22 g, were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China. Experiments and procedures were approved by the Animal Ethics Committee of the Second Military Medical University. All animal handling and study procedures complied with the current Chinese regulation, GB14925-2010: Laboratory animal requirements of environment and housing facilities (Chinese version). For acute ethanol-induced liver injury model, ethanol binge was conducted as previously described  that mice were administrated by 50% (v/v) ethanol gavage at a dose of 5 g/kg BW after 6 h of fasting. BBD mixed powder, donated by Shanghai Pharmaceutical Group Inc., was dissolved in 0.9% saline at normal temperature and pressure to obtain BBD suspension with different concentrations for next experiments. To determine the preventive effect of BBD on acute ethanol liver damage, some mice were given BBD with increasing concentrations respectively by gavage prior to ethanol at 1 h apart. Control mice were given the same volume of normal 0.9% saline. They were sacrificed 16 h after ethanol administration. In the meanwhile, sleep latency (disappear of righting reflex) and sleep time on drunkenness (polysomnography) of mice was observed. Blood samples and liver tissues were collected at the end of experiments. The livers were dissected quickly, and portions of liver tissue were fixed in neutral buffered formalin, while others were frozen in liquid nitrogen. Plasma samples for further analysis were obtained by centrifugation (1750g, 15 min, 4 °C) and stored at − 80 °C.
Cell culture and treatments
AML-12 cell line was obtained from the Chinese Academy of Sciences Cell Bank and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C in a 5% CO2 humidified atmosphere. We collected portal vein serum from normal male C57BL/6 mice (normal-serum) and only BBD gavage treated male C57BL/6 mice (BBD-serum) at a dose of 0.5 g/kg BW for 1 h. Cells were treated with BBD-serum or other agents when they were 50–60% confluence. BBD-serum was added 1 h prior to the treatment of ethanol. Cells were harvested with lysis buffer after washing by PBS for three times, then were used for subsequent experiments.
Reagents and biochemical assay
Chloroquine (CQ), 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St Louis, MO). The serum biochemical markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and triglyceride (TG) levels were measured with a biochemical autoanalyzer (Fuji Medical System, Tokyo, Japan). The activity of MDA, TG, ROS, GSH, GPx, SOD and CAT in liver tissues or cells were determined by kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturers’ instructions.
Liver tissues were sectioned and mounted on glass slides then stained with H&E. Each sample was observed at a 200× magnification of microscopic field in 10 randomly selected areas. The hepatic steatosis was evaluated by hepatic steatosis score, assigning from 0 to 3 where 0 = no steatosis, 1 = slight steatosis, 2 = moderate steatosis and 3 = severe steatosis. The liver cryostat section (8 mm) was stained with Oil Red O (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) staining.
The cells were transfected with Nrf2 siRNA (Santa Cruz Biotechnology, sc-37049) or non-targeting siRNA (Santa Cruz Biotechnology, sc-37007) using Lipofectamine 2000 according to the manufacturer’s instructions. Transfection efficiency in Cells were confirmed by western blot measurement.
Fugene HD transfection reagent (Calbiochem, La Jolla, CA) was used to transfect cells with GFP-LC3 expressing plasmids according to the manufacturer’s instructions. After initial treatment, autophagy was detected by counting the number of GFP-LC3-positive dots per cell under fluorescence microscope (Olympus IX71).
Electron microscopic analysis
AML-12 cells were fixed in 2.5% glutaraldehyde in PBS (pH = 7.4) for 2 h at room temperature, then postfixed in 1% osmium tetroxide in water for 1 h, dehydrated in an ascending series of ethanol, and at last embedded in araldite (Basel, Switzerland). After solidified, 50 nm sections were cut on a LKB-I ultramicrotome and picked up on copper grids, post-stained with uranyl acetate and lead citrate, and observed in a Philips CM-120 TEM.
Total RNA was isolated by using Trizol reagent (Invitrogen) according to the manufacturer’s specifications. cDNA was reverse-transcribed using the Revert Aid RT-PCR system (Fermentas, Pittsburgh, PA, USA). Real-time PCR was performed by mixing cDNA with primers and Maxima SYBR Green qPCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). The sequences of the primers were as follows: CYP2E1 (forward: 5ʹ-CGTTGCCTTGCTTGTCTGGA-3ʹ; reverse: 5ʹ-AAGAAAGGAATTGGGAAAGGTCC-3ʹ), Nrf-2 (forward: 5ʹ-ACCAAGGGGCACCATATAAAA G-3ʹ; reverse: 5ʹ-CTTCGCCGAGTTGCACTCA-3ʹ), HO-1 (forward: 5ʹ-AGGTACAC ATCCAAGC CGAGA-30; reverse: 5ʹ-CATCACCAGCTTAAAGCCTTCT-3ʹ), GCLC (forward: 5ʹ-CTACCACG CAGTCAAGGACC-3ʹ; reverse: 5ʹ-CCTCCATTCAGTAACAACTGGAC-3ʹ) and β-actin (forward: 5ʹ-GGCTGTATTCCCCTCCATCG-3ʹ; reverse: 5ʹ-CCAGTTGGTAACAATGCCATGT-3ʹ). Real-time PCR was performed using a Stratagene Mx3000P Real-time PCR System with supplied software (Applied Biosystems), according to the manufacturer’s instructions. β-actin was used as internal control for RNA integrity and loading normalization.
Western blot analysis
Cells and tissues were lysed in RIPA lysis buffer (Beyotime) with 1 mM PMSF. Equal amount of protein was separated by SDS-PAGE and transferred to NC membrane. The membranes were washed, blocked and incubated with specific primary anti-human antibodies against CYP2E1, p62/SQSTM1 (Cell Signaling Technology, Inc., Danvers, MA, USA), LC3 (Novus Biologicals, Littleton, CO), Nrf2, HO-1, GCLC and β-actin (Abcam, Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Hangzhou HuaAn Biotech). Signals were visualized by chemiluminescent detection (Beyotime).
All of the experiments were repeated at least three times. Final data were expressed as mean ± standard deviation (SD). Statistical analysis of the data was done by using GraphPad Prism 5. Student’s t-test was used to compare between mean values of two groups. Value of at least P < 0.05 was considered statistically significant.