2.1. A Two-Factor Design for an Experimental Mouse in Vivo Colitis Model

One hundred and twelve six-week-old female C57BL/6J mice were purchased from Daehan Bio Link Co., Ltd., South Korea, which adopted the strain in 2010 from Taconic Farms, Inc. Mice were maintained in a controlled animal facility under specific pathogen-free conditions. This study was carried out in accordance with the guidelines established by the Korean Association for Laboratory Animals. The protocol was approved by the Committee on the Ethics of Animal Experiments of Handong Global University (approval No. 20190424-008). The in vivo experiment was carried out in a 4 × 7 design, which had two factors (DSS concentration and duration), each with four and seven levels, respectively. After stabilization for two weeks, mice were divided into four groups (n = 28 per group), and colitis was induced by administration of DSS (molecular weight 36 to 50 kDa; MP Biomedicals, Santa Ana, CA, USA) at 1%, 2% and 3% concentrations in drinking water ad libitum. The 0% DSS group was provided with only the vehicle, which was autoclaved distilled water (DW). Each treatment group was kept in the same cage with enough space to accommodate all of the mice to avoid ‘cage effects’ on the composition of the gut microbiota [35,36]. The experiment lasted 12 days, and four mice from each DSS dose group were randomly selected, weighed, and sacrificed at two-day intervals. After mice were euthanized, total blood per mouse was collected in a serum separator tube (BD Microtainer; Becton Dickinson, Sparks, MD, USA), and the serum was isolated with centrifugation at 15,000× g for 2 min. Cecum samples were immediately placed at –80 °C for extracting DNA, and the distal end of the colon was fixed in 10% buffered formalin phosphate (Sigma-Aldrich, St. Louis, MO, USA) solution for histological observations. Mouse fecal samples were collected on alternate days and maintained in a deep freezer until subsequent assessment.