Liver histology

H&E staining was conducted to analyze the pathologic alterations. Briefly, liver tissues were fixed with 4% paraformaldehyde for 24 h at room temperature and then embedded in paraffin. Subsequently, tissues were sliced into 4 µm sections followed by deparaffinization. Deparaffinization was performed by incubation with xylene for 40 min, anhydrous ethanol for 10 min and 75% alcohol for 5 min. Following washing with running water, tissues were stained with hematoxylin for 2–3 min and eosin for 1 min at room temperature. Stained tissues were observed using an OLFV-34CM/XE light microscope (Olympus Corporation).

The frozen fresh tissue was used for oil red O staining to analyze the accumulation of lipids in the liver. Liver tissues were frozen and cut into 8 µm sections. The sections placed on slides and air-dried for 30 min. Subsequently, tissues were fixed with paraformaldehyde for 15–20 min at room temperature. Following washing with running water, the sections were stained with oil red O for 8–10 min and counterstained with hematoxylin for 3–5 min at room temperature. Images were captured using an OLFV-34CM/XE light microscope. TG levels in liver tissues were determined according to the instructions of the TG quantitative kit (Applygen Technologies, Inc.).