The mice were given free access to control diet or alcohol Lieber–DeCarli liquid Dinaciclib nmr diet for 4 weeks with or without RGE (250 mg/kg or 500 mg/kg, per os, n = 8) The mice were randomly assigned to the groups specified. The second was a mouse model of chronic–binge EtOH intake. The mice were fed with the control diet for 5 days, and then divided into four groups. The EtOH groups were fed with the Lieber–DeCarli liquid diet containing 5% EtOH for 10 days with or without RGE (250 mg/kg or 500 mg/kg, per os, n = 8). The control groups were pair-fed the

control diet for 10 days. At Day 11, mice in EtOH groups were gavaged a single dose of EtOH (5 g/kg body weight, 20% EtOH), whereas mice in control groups were gavaged isocaloric dextrin maltose. The mice were sacrificed 9 hours after gavage. AML12 cell lines were purchased from ATCC (Manassas, VA, USA). Cells were plated at a density of 3 × 105/well in 60 mm dishes and grown to 70–80% confluency. Cells were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 containing 10% fetal bovine serum (Hyclone, Logan, UT, USA), 50 units/mL penicillin, 50 μg/mL streptomycin, check details 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone at 37°C in a humidified atmosphere with 5% CO2. RGE or ginsenosides were dissolved in phosphate-buffered saline (PBS) and added to the cells. The cells were then incubated at

37°C for the indicated time period, and washed twice with ice-cold PBS prior to sample preparation. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase Prostatic acid phosphatase (AST) were analyzed using Spectrum, an automatic blood chemistry analyzer (Abbott Laboratories, Abbott Park, IL, USA). Samples from the liver

were separated and fixed in 10% neutral buffered formalin. The samples were then embedded in paraffin, sectioned (3–4 μm), and stained with hematoxylin and eosin (H&E) for general histopathological analysis. In addition, the effect of RGE treatment on the 4-HNE and nitrotyrosine immunoreactivity was also observed by immunohistochemical methods. For the analysis of fat accumulation in the liver, 10-μm sections were cut from frozen samples and stained with Oil Red O for 10 min. The slides were rinsed in water and counterstained with Mayer’s hematoxylin, followed by analysis using light microscopy. Lipid droplet formation in hepatocytes was determined by Oil Red O staining. Cells were grown on a six-well plate. After treatment, the cells were fixed 4% formaldehyde in PBS for 1 h and rinsed with 60% isopropanol. Cells were then stained with Oil Red O solution. Hepatic lipid content was measured as described previously [25]. Briefly, lipids from the total liver homogenate were extracted using chloroform/methanol (2:1), evaporated, and dissolved in 5% triton X-100. Triglyceride content was determined using Sigma Diagnostic Triglyceride Reagents (Sigma).