Animals and treatment
Male wild-type (WT, C57BL/6) mice (8–12 weeks of age) were purchased from Capital Medical University (Beijing, China) and housed in the Capital Medical University animal facility under specific pathogen-free conditions, and received humane care according to Capital Medical University Animal Care Committee guidelines. To induce ALF, the mice were injected intraperitoneally with d-GalN (700 mg/kg, Sigma, St Luis, MO) and LPS (10 μg/kg, Invivogen, San Diego, CA). Mice were treated with a single dose of freshly prepared N-acetylcysteine (NAC, at 300 mg/kg, by i.p. injection, Sigma, St Luis, MO), a ROS scavenger, immediately after d-GalN/LPS treatment. In some experiments, SB216763 (25 mg/kg, Sigma, St Luis, MO) dissolved in dimethyl sulfoxide (DMSO), a specific inhibitor for GSK3β, was suspended in phosphate-buffered saline (PBS) and administered intraperitoneally 2 h prior to d-GalN/LPS treatment. Mice were sacrificed at various time-points after d-GalN/LPS treatment; liver and serum samples were collected for future analysis.
Serum aminotransferase activities
Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as markers of hepatic damage, were measured by using a multiparameteric analyzer (AU 5400, Olympus, Japan), according to an automated procedure.
Assay of ROS level in liver
To determine the level of ROS in the liver tissue, liver homogenates were made in lysis buffer and analyzed using a chemical fluorescent ROS assay kit (Jiancheng Bio, Nangjing) according to the manufacturer’s instruction, which is based on the oxidation of 2′7′-dichlorodihydrofluorescein diacetate to 2′7′-dichloro-fluorescein.
Assay of MDA,GSH and SOD in liver
A part of liver tissue was prepared for homogenization with a buffer containing 0.15 M KCl to obtain 1:10 (w/v) homogenates. The homogenates were then centrifuged at 12,000×g (4 °C) for 20 min to collect the supernatants. The concentrations of malondialdehyde (MDA), and glutathione (GSH), as well as superoxide dismutase (SOD) activities were measured, as described previously .
Human hepatocyte line HL-7702 (Xiangfu Biological Company, Shanghai) was plated in 48-well or 6-well plates at an appropriate density. After overnight culturing, hydrogen peroxide (H2O2, 1 mmol/L, Habo Company, Shanghai) or antimycin A (2 μg/ml, Sigma, St Luis, MO) was added into the culture wells. To study the effects of GSK3β inhibition on hepatocyte apoptosis induced by oxidative stress, SB216763 (10 mΜ) was added 2 h prior to the H2O2 or antimycin A treatment. Cell apoptosis was evaluated at 12 h by caspase western blots and lactate dehydrogenase (LDH) assay (Biochain Institute, Hayward, CA) of culture supernatants, according to the manufacturer's instructions.
Western blot analysis
Protein was extracted from liver tissue with RIPA buffer with phosphatase inhibitors and protease inhibitors. Proteins in sodium dodecyl sulfate (SDS)-loading buffer were subjected to SDS-12 % polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. Monoclonal rabbit antibodies against p-GSK3β, total GSK3β, p-JNK, total JNK, Cyt c, caspase-3, cleaved caspase 3 and β-actin (Cell Signaling Technology, Santa Cruz, CA) were used at 4 °C overnight. Then the membrane was treated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The relative quantities of proteins were determined by a densitometer and expressed as absorbance units (AU).
Paraffin sections were treated with xylene for three times with 10 min each time. The sections were hydrated through a graded alcohol series and then rinsed three times with distilled water. The slides were incubated for 20 min in 10 % goat serum in PBS and then p-GSK3β rabbit monoclonal antibody (Cell Signaling Technology, Santa Cruz, CA) overnight at 4 °C. The slides were incubated with Alexa Fluor® 568 goat anti-rabbit IgG (1:200, Invitrogen, Grand Island, NY) for 45 min. After three washings with PBS, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml, Shizebio, Shanghai) for 10 min. The images were examined on a Nikon Eclipse E800 fluorescent microscope.
Determination of hepatic GSK3β activity
To determine the activity of GSK3β in the liver tissue, liver homogenates were made in lysis buffer and analyzed using a colorimetric GSK3β assay kit (Jianglaibio Co, Shanghai) according to the manufacturer’s instructions.
Human liver samples
This study meets the ethical guidelines of the 1975 Declaration of Helsinki and the study protocol was permitted by the Medical Ethics Committee of Beijing YouAn Hospital. Informed consent was obtained from all patients. Human liver tissue was obtained from the normal subjects, chronic hepatitis B (CHB) patients and HBV-induced ALF patients. All patients were hospitalized during 2009–2011 at Beijing YouAn Hospital. Clinical characteristics and details of clinical data of the patients in analysis are shown in Table 1.
Results are shown as mean ± SD. The data were analyzed with statistics software SPSS 11.5 using a nonparametric analysis of variance test. Differences were considered significant if the P value was less than 0.05.