CCl4-Induced Liver Injury Model in Mice

Eight-week-old male C57BL/6NCrSlc mice (Japan SLC, Inc., Hamamatsu, Japan) were caged with free access to food and water and kept at 23 ± 3 °C with 50 ± 20% humidity and a 12 h light/dark cycle. Overall, 50 mice were randomly divided into 5 experimental groups (n = 10) and underwent treatment for 8 weeks as follows (Figure 1A): (i) intraperitoneal injection of corn oil and oral gavage of saline as a vehicle (C/O group); (ii) intraperitoneal injection of CCl₄ (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) twice a week (1 mL/kg) and vehicle (CCl₄ + Veh group); (iii) CCl₄ and oral gavage of sacubitril (30 mg/kg) (Tokyo Chemical Industry, Tokyo, Japan) (CCl₄ + SAC group); (iv) CCl₄ and oral gavage of valsartan (30 mg/kg) (Tokyo Chemical Industry) (CCl₄ + VAL group); and (v) CCl₄ and oral gavage of sacubitril and valsartan (30 mg/kg each) (CCl₄ + SAC/VAL group) [24 (link),25 (link),26 (link)]. After 8 weeks, all of the mice were intravenously injected with pentobarbital sodium (150 mg/kg), subjected to anesthesia, and then their blood and livers were collected immediately after euthanasia. Serum levels of biochemical markers were assessed at SRL, Inc. (Tokyo, Japan).