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).