Establishment of in vivo streptozotocin (STZ)-injected, high-fat, high-cholesterol (HFHC)-fed NASH mouse model
Sixteen days pregnant female C57BL/6J mice were purchased from the Jackson Laboratory (Japan). STZ (0.2 mg; Sigma-Aldrich, St. Louis, MO, USA) was injected subcutaneously into 3-day-old male C57BL/6J mice. STZ was dissolved in 0.1 M sodium citrate buffer (pH 4.5) before administration. One month later, the male mice were randomly divided into four groups: normal, STZ-injected normal carbohydrate (Teklad Global 18% Protein Rodent Diet; TD 2018C, Envigo, Indianapolis, IN, USA) diet, STZ-injected HFHC (Western Diet; D12079B, 41 kcal% fat, 43 kcal% carbohydrates; Research Diets, New Brunswick, NJ, USA) diet-fed, and STZ-injected TAF-treated HFHC diet-fed groups (TAF, 5 mg/kg; Gilead Sciences, Foster City, CA, USA). TAF was administered daily for 4 weeks. Blood was collected from the orbital venous plexus every 2 weeks, and random glucose levels were analysed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TRIG) levels were measured using a chemical analyser according to the manufacturer’s protocol (Vettest 8008 Chemistry Analyzer; IDEXX Laboratories, Westbrook, ME, USA). Mice were intraperitoneally anesthetized with Rompun (10 mg/kg) and Zoletil (40 mg/kg), and their livers were removed. Mice livers were dissociated using a dissociation kit (MACS, Miltenyi Biotec) and gentleMACS Dissociator (MACS, Miltenyi Biotec). Fluorescence staining was then performed (CD11b, F4/80, CD45, Ly6G, PD-L1, IAIE, MerTK, and LIVE/DEAD dyes).
The Korea Excellence Animal Laboratory Facility at the Korea Food and Drug Administration was accredited in 2007 by the Institutional Animal Care and Use Committee (IACUC) and the Department of Laboratory Animals (DOLA) at the Catholic University of Korea, Songeui Campus, and acquired full Assessment and Accreditation by the Laboratory Animal Care International accreditation in 2018. All animal care and experimental procedures were conducted according to the Institutional Animal Care and Use Committee and were approved by the Center for Medical Science of the Catholic University of Korea (2021-0329-02).