GBDP (batch number: A01J180506) were provided by Wanbangde Pharmaceutical Group Co., Ltd (Wenling, Zhejiang, 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, USA), Bcl-2 (ab196495) and PARP-1 (ab191217) were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits of MCP-1 and IL-6 were purchased from Invitrogen (Carlsbad, CA, USA).
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 . Briefly, primary hepatocytes were obtained by perfusion with 0.05% collagenase type IV purchased from Sigma-Aldrich (St. Louis, MO, USA). Then hepatocytes were filtrated through a 70 μm cell strainer and resuspended in media mixed with 42% percoll (Sigma-Aldrich, St. Louis, MO, USA) followed by centrifugation for 5 minutes at 1300 rpm. Hepatocytes were plated in the six-well culture dish at a density of 106 cells/well and cultured in the Medium 199 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Carlsbad, CA, USA), 1% penicillin-streptomycin (Gibco, Invitrogen, Carlsbad, CA, USA), 23 mM N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid (HEPES) (Sigma-Aldrich, St. Louis, MO, USA), and 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). The alpha mouse liver-12 (AML-12) cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in the Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco, Invitrogen, Carlsbad, CA, USA) containing 10% FBS, 1% penicillin-streptomycin, 1% Insulin-Transferrin-Selenium-G Supplement (ITS) (Sigma-Aldrich, St. Louis, MO, USA), and 40 ng/mL dexamethasone. Both AML-12 cells and primary hepatocytes in flasks were cultured in the cell incubator (Thermo Fisher Scientific, Waltham, MA, 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 injury model. Drug intervention with GBDP of two concentrations (60 and 120 μg/mL) was performed 1 hour 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 the I/R and I/R + GBDP groups went through 70% warm hepatic I/R injury as described previously . Briefly, the mice were placed supine for midline laparotomy after anesthetization to expose the liver. The murine hepatic artery and portal vein were isolated and clipped with the microvascular clamp. After 45 minutes of ischemia, the clamp was removed. The samples of blood or liver tissue were collected after 6 or 24 hours of reperfusion for subsequent experiments. The blood samples were centrifuged for 10 minutes at 4000 rpm, and the plasma was obtained for liver damage assessment. Mice in low- and high- dose I/R + GBDP groups 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 . 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 1-hour pretreatment of GBDP in different concentrations for 12 or 6 hours. Then, the 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. The absorbance of the solution was measured at 580 nm using the Infinite M1000 Pro (TECAN, Mannedorf, Zurich, Switzerland).
Annexin V staining
AML-12 cells were cultured in the 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, USA). Briefly, AML-12 cells seeded into 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 the 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, USA) 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 . Briefly, the cells or tissues were lysed in the lysis buffer, which was made from Tris-HCL, NaCl, Ethylene Diamine Tetraacetic Acid (EDTA), glycerol, Triton X-100, Nonidet P-40, dithiothreitol, and phenylmethylsulfonyl fluoride, supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Switzerland). The lysates were centrifuged for 15 minutes at 10000 g, and the protein concentrations were quantified by Bradford method using Quick StartTM Bradford 1× Dye Reagent (Bio-Rad, Hercules, CA, USA). 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, Darmstadt, Germany). The membranes were blocked in Tris-Buffered Saline with Tween 20 (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, USA) with chemiluminescence substrate reagents (Bio-Rad, Hercules, CA, USA) 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 Diagnostics, Switzerland) following the manufacturer’s instructions.
After fixation in 10% formaldehyde, the liver tissues were embedded in paraffin and sectioned at 5 μm thickness for hematoxylin and eosin (H&E), MPO immunohistochemical, and Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling (TUNEL) staining. The liver sections were stained with H&E to visualize the pattern in necrotic areas of the liver. The infiltration of neutrophils was detected by MPO staining. The liver sections were incubated with MPO primary antibody (1:1000, Servicebio, Wuhan, Hubei, China) at 37℃ for 1 hour, followed by incubation with HRP-conjugated goat anti-mouse secondary antibody (1:200, Servicebio, Wuhan, Hubei, China). TUNEL staining was performed to determine DNA fragmentation using an In Situ Cell Death Detection Kit (Roche Diagnostics, Switzerland) according to the manufacturer’s instructions as described previously . Images were captured using a fluorescence microscope (Nikon Eclipse Ti-SR, Tokyo, Japan).
All statistical analyses were performed with GraphPad Prism 5.0, and the data was expressed as the mean ± standard deviation (SD). The statistical significance of the differences between two groups were calculated by two-tailed Student's t-test, and one-way ANOVA with the Turkey’s post hoc test was used for comparisons among multiple groups. The p values less than 0.05 were considered statistically significant.