4.2. Experimental Model of Liver Injury

Chronic liver injury was induced by intraperitoneally injecting increasing doses of carbon tetrachloride (CCl₄, cat. N. 289116, Sigma-Aldrich, Saint Louis, MO, USA) in corn oil (from 0.17 to 0.72 mL/kg × body weight) twice a week for a maximum of 12 weeks. Male and female mice were randomly divided into two groups, which included the CCl₄-treated fibrotic mice (n = 5 per time point per gender) and control corn-oil-treated mice (n = 5 per time point per gender). The selected time points for suppression were baseline (healthy mice), 6 weeks, 12 weeks (end of CCl₄ treatment), and 20 weeks (recovery groups, i.e., 12 weeks of CCl₄ treatment was administered, followed by 8 weeks of recovery to allow for fibrosis regression). Then, the mice were sacrificed, and their livers were excised, weighed, and stored in 10% formalin (cat. N. HT501128-4L, Sigma-Aldrich, Saint Louis, MO, USA) or snap-frozen with liquid nitrogen and stored at −80 °C until further analysis.