2.2. Animal Treatments

Seven-week-old male ICR mice were obtained from the National Laboratory Animal Center (Mahidol University, Thailand). All mice were housed on wood chip bedding in polysulfone cages with water and commercial regular diet (RD, SmartHeart® from Perfect Companion Pet Care Company, Thailand; 24% crude protein, 4.5% crude fat, 5% fiber, 10% moisture, 10% ash, 1% calcium, 0.7% phosphorous, and vitamins A, D3, and E) supplied ad libitum in the Northeast Laboratory Animal Center, Khon Kaen University, Thailand, and acclimated for a week before dosing under the supervision of the Animal Ethics Committee for Use and Care of Khon Kaen University (approval number AEKKU 92/2555). The mice quarters were air conditioned (23 ± 2°C) and controlled for humidity 45 ± 2% RH and had a 12 h light/dark cycle.

The high-fat, high-fructose diet (HFFD) mice were additionally given hydrogenated soybean oil intragastrically (1 mL/day), which consisted of 44.1% (w/w) saturated fat and 0.2% (w/w) trans fatty acid (certified by Institute of Nutrition, Mahidol University, Thailand), with the addition of 20% (w/v) fructose in their drinking water supplied ad libitum for 2, 4, and 8 weeks (n = 5 for each group). The control mice (n = 5) were fed with commercial regular diet (RD) and water ad libitum for 4 weeks. The ages of all of the mice at the end of treatment were between 10 and 16 weeks, consistent with adult-aged mice (2–4 months). Levels of antioxidant enzymes and lipid peroxidation, including the GSH/GSSG ratio, have been proposed to be equivalent in the same age-range, for example, young (4–6 weeks), adult (3 months), middle-aged (9 months), and aged (18–24 months) [14]. All mice were sacrificed 24 h after the last treatment, and their organs (livers and brains) were immediately excised and either used for the isolation of total RNA or immediately stored at −80°C for further analysis.