The hexane extract was concentrated under AZD6244 solubility dmso reduced pressure, yielding an oil (26.9 g). The oil was then purified via silica gel column chromatography (Merck 7734) and eluted with 20% acetone/hexane. It was further purified using the same method (Merck 9385), followed by octadecyl silica gel column chromatography (YMC GEL ODS-A) with a gradient of methanol in water to yield urushiols. The final concentration of extracted urushiol was 10 mg/mL. Age-matched 6-week-old male C57BL/6 mice (Dooyeol Biotech, Inc., Seoul, Korea) were used in all experiments. Only male mice were used, given the hormonal changes of female mice. The mean body weights of the mice in each group are listed in Table 1. A total of 60 male C57BL/6 mice were housed individually

in steel microisolator cages maintained at 22°C with a 12-hour/12-hour light/dark cycle. The mice were randomly assigned to six dietary groups (n = 10). Each group of mice received one of the following six diets for 10 weeks:

(1) standard chow diet (normal feed); (2) alcohol diet (a Lieber–DeCarli liquid diet with 10% alcohol); (3) control diet (a Lieber–DeCarli liquid diet with PI3K inhibitor 10% alcohol and normal feed); (4) KRG diet (200 mg/kg/day of KRG with normal feed for 4 weeks after a Lieber–DeCarli liquid diet with 10% alcohol for 6 weeks); (5) urushiol diet (0.128 mg/mL/day of urushiol with normal feed for 4 weeks after a Lieber–DeCarli liquid diet with 10% alcohol for 6 weeks); and (6) probiotics diet (1 mg/mL/day Anacetrapib of L. rhamnosus R0011 and L. acidophilus R0052 with normal feed for 4 weeks after a Lieber–DeCarli liquid diet with 10% alcohol for 6 weeks; Fig. 1). The liquid diets were based on the Lieber–DeCarli ethanol formulation and purchased from Dooyeol Biotech, Inc. Protein, fat, and carbohydrates constitute, respectively, 18.9%,

16.5%, and 64.5% of the calories of the Lieber–DeCarli liquid diet. Lacidofil, KRG, and urushiol suspended in distilled water were orally administered using a gastric tube five times a week, for 4 weeks. At the end of the treatment period, the animals were sacrificed via isoflurane inhalation. A midline abdominal incision was performed, and blood was collected through the orbital canal. Whole-blood (600 μL) samples were centrifuged at 1,500 × g for 15 minutes to collect the serum. The liver was rapidly excised and stored at −80°C. The animals received humane care, and all procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of the Hallym University College of Medicine, Gangwon Do, Korea approved this study. Levels of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase were analyzed with a biochemical blood analyzer (KoneLab 20, Thermo Fisher Scientific, Waltham, Finland). After rinsing the tissue samples with a cell wash buffer once, they were cut into 3 mm × 3 mm pieces and transferred to a 2 mL tissue grinder.