Reagents
GBDP (batch number: A01J180506) were provided by Wanbangde Pharmaceutical Group Co., Ltd (Wenling, China). Dimethylsulfoxide (DMSO) and carboxymethyl cellulose sodium (CMC-Na) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Primary antibodies of Bax (2772S), caspase-3 (9662S), and β-actin (4970S) were purchased from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China).
Animals
Eight week-old male C57BL/6 mice weighing 22–25 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and maintained in a controlled environment (20–26 °C, 12-hour light/dark cycle) with ad libitum access to food and water. The animal experiments were approved by the Animal Care and Use Committee of Zhejiang University School of Medicine.
Isolation of primary hepatocytes and cell culture
Primary hepatocytes were isolated from C57BL/6 mice according to the method reported previously [4]. Briefly, primary hepatocytes were obtained by perfusion with 0.05% collagenase type IV (Sigma-Aldrich, St. Louis, MO). Then hepatocytes were filtrated through a 70 µm cell strainer and were resuspended in Media mixed with 42% percoll (Sigma-Aldrich, St. Louis, MO) followed by centrifugation for 5 minutes at 1300 rpm. Hepatocytes were plated in six-well culture dish at a density of 1.5 × 106 cells/well and were cultured in Medium 199 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 23 mM N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid (HEPES) (Sigma-Aldrich, St. Louis, MO), and 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO). The mouse hepatocyte cell line alpha mouse liver 12 (AML-12) cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco, USA) containing 10% FBS, 1% penicillin-streptomycin, 1% Insulin-Transferrin-Selenium-G Supplement (ITS) (Sigma-Aldrich, St. Louis, MO), and 40 ng/mL dexamethasone. Both AML-12 cells and primary hepatocytes in flasks were cultured in the cell incubator (Thermo Fisher, USA) at 37 °C with 5% CO2.
Model of hypoxia/reoxygenation (H/R) injury
AML-12 cells or primary hepatocytes were incubated at 37 °C in a closed hypoxia chamber filled with N2 in a tri-gas incubator (94% N2, 5% CO2, 1% O2), followed by reoxygenation in normal culture conditions to establish the H/R model. Drug intervention with GBDP of two concentrations (60 or 120 µg/mL) was performed 2 hours before the onset of hypoxia. The blank solution DMSO was served as control.
Model of liver I/R injury and drug treatment
The mice were divided into four groups at random: sham, I/R, I/R + GBDP (100 mg/kg), I/R + GBDP (200 mg/kg). Mice in I/R and I/R + GBDP groups went through 70% warm hepatic I/R injury as described previously [31]. Briefly, the mice were placed supine for midline laparotomy after anesthetization to expose the liver. Murine hepatic artery and portal vein were isolated and clipped with microvascular clamp. After 45 minutes of ischemia, the clamp was removed. Blood or liver tissue samples were collected at 6 or 24 hours after reperfusion for subsequent experiments. Blood samples were centrifuged for 10 minutes at 4000 rpm, and the plasma was obtained for liver damage assessment. Mice in I/R + GBDP group were treated with GBDP in 1% CMC-Na by gavage once per day for two weeks, and 2 hours before surgery on the 15th day. The 1% CMC-Na solution was given to the other two groups served as control.
Cell viability assay
Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay as described [32]. AML-12 cells or primary hepatocytes were seeded into a 96-well plate at a density of 1 × 104 or 8 × 104 cells/well respectively. Cells were cultured in H/R conditions after 2-hour pretreatment of GBDP in different concentrations for 12 or 6 hours. Then, medium containing 0.5% MTT reagent was added to the each well. After incubation for 4 hours, the supernatants were removed and 100 µL DMSO was added to dissolve the formed formazan crystals at room temperature for 10 minutes. The absorbance of the solution was measured at 580 nm using the Infinite M1000 Pro (TECAN, Germany).
Annexin V staining
AML-12 cells were cultured in hypoxia chamber for 12 hours, and then reoxygenated for 22 hours. Cells were stained with an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit according to the manufacturer’s instructions (BD Biosciences, San Jose, CA). Briefly, AML-12 cells seeded in 6-well plates were collected and resuspended in 1 × binding buffer to the final concentration of 1 × 106 cells/mL. Then 100 µL cell resuspension was incubated with 5 µL Annexin V-FITC and 5 µL PI for 15 minutes at room temperature in dark. Next, 400 µL 1 × binding buffer was added to end the incubation. Apoptotic rate was assayed by Accuri™ C6 flow cytometer (BD Bioscience, San Jose, CA) in 1 hour.
Cell lysis and Western blot analysis
Preparation of whole cell or tissue lysates and western blot analysis were performed as described previously [33]. Briefly, the cells or tissues were lysed in the lysis buffer, which was made from Tris-HCL, NaCl, EDTA, glycerol, Triton X-100, Nonidet P-40, dithiothreitol, and phenylmethylsulfonyl fluoride, supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Germany). The lysates were centrifuged for 15 minutes at 10000 g, and the protein concentrations were quantified by Bradford method using Quick Start™ Bradford 1 × Dye Reagent (Bio-Rad, Hercules, CA). Lysates with equal amount of six mice from the same group were mixed together to be one sample. Protein samples were electrophoresed by 9–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (10–20 µg of protein/lane) and separated protein was transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany). The membranes were blocked in Tris-Buffered Saline with Tween (TBST) containing 5% skim milk for 1 hour at room temperature, which was followed by incubation with primary antibodies overnight at 4 °C. After washing three times in TBST, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies for 1 hour at room temperature. All primary antibodies were used at a 1:1000 dilution, and secondary antibodies were used at the dilution of 1:2000. Protein bands were visualized by using a ChemiDocTM XRS + system (Bio-Rad, Hercules, CA) with chemiluminescence substrate reagents (Bio-Rad, Hercules, CA) and quantified by using ImageJ software.
Liver damage assessment
Plasma concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicators of hepatocellular injury, were assayed by an automated biochemical analyzer (Cobas C8000, Roche, USA) following the manufacturer’s instructions.
Histological analysis
After fixation in 10% formaldehyde, liver sections were embedded in paraffin for hematoxylin and eosin (H&E), MPO immunohistochemical, and Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling (TUNEL) staining. The liver tissues were sectioned at 5 µm thickness and stained with H&E to visualize the pattern in necrotic areas of the liver. The infiltration of neutrophils was detected by MPO staining. Liver sections were incubated with MPO primary antibody (1:1000, Servicebio) at 37℃ for 1 hour, followed by incubating with HRP-conjugated goat anti-mouse secondary antibody (1:200, Servicebio). TUNEL staining was performed to determine DNA fragmentation using an In Situ Cell Death Detection Kit (Roche Diagnostics, USA) according to the manufacturer’s instructions as described previously [34]. Images were captured using a fluorescence microscope (Nikon Eclipse Ti-SR, Japan).
Statistical analysis
All statistical analyses were performed with GraphPad Prism 5.0, and the data was expressed as the mean ± standard deviation (SD). Statistical differences between two groups were calculated by two-tailed Student's t-test, and one-way ANOVA was used for comparisons among multiple groups followed by Dunnett's multiple comparison test. Statistical differences with p values less than 0.05 were considered significant.