Porcine serum

Male Wistar rats (120–150 g) were purchased from the Vital River Laboratories (Beijing, China). All rats were raised in an environment with a 12-h light/dark cycle at a temperature of 22 ± 1°C with free access to food and water. Animal care and all experiments were performed according to the guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee of Peking Union Medical College & Chinese Academy of Medical Sciences (PUMC&CAMS). Porcine serum was purchased from Solarbio Science & Technology (Beijing, China). Lipopolysaccharide (LPS), D-galactosamine (D-gal), and N-(4-hydroxyphenyl) retinamide (4-HPR) were purchased from Sigma-Aldrich (St Louis, MO, USA). Cytochrome C primary antibody was purchased from Santa Cruz (Dallas, Texas, USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were purchased from Biosino (Beijing, China). Prothrombin times (PTs) determination kit and plasma ammonia kit were purchased from the Nanjing Institute of Jiancheng Bioengineering (Nanjing, China).

Male Wistar rats were randomly divided into 2 groups (control: n=10; ACLF model: n=60). Rats in the ACLF group were administered porcine serum (Beijing Solarbio Science &Technology Co., Ltd., Beijing, China) intraperitoneally at a dose of 0.5 mL twice per week for 11 weeks to generate an immune liver fibrosis model. After 11 weeks, rats with immune liver fibrosis were injected intravenously with LPS (Sigma-Aldrich Co., USA) at a dose of 50 μg/kg. Thirty minutes later, d-gal (Sigma-Aldrich Co., USA) was injected intraperitoneally at a dose of 600 mg/kg to induce acute liver failure via chronic liver cirrhosis. The control group was administered physiological saline at the same time points. Animals were killed at 0, 4, 8 and 12 h post-treatment for blood and hepatic tissue collection. The remaining rats were observed for survival time every hour for 48 h.