Histological Assessment of Hepatic Lipid Accumulation

Liver tissue samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2 h, stored overnight in 10% formalin, and were embedded in paraffin. Cross-sections (5 µm) of the tissue were cut and used for staining with hematoxylin and eosin. To evaluate the degree of hepatic lipid accumulation, oil red O staining of the sections was performed using an oil red O staining kit (Nanjing Jiancheng Bioengineering Institute) following the manufacturer's instructions. Images were taken using a light microscope. The NAFLD activity score (NAS) were performed by an experienced pathologist without prior knowledge of the treatments. The scoring includes measurement of steatosis grade (0-no steatosis, 1- < 50% steatosis, 2-> 50% steatosis, 3- > 50% steatosis + microvesicular steatosis), hepatocyte ballooning (0-none, 1- < 66%, 2- > 66% hepatocyte involvement) and inflammation (0-no foci, 1- < 2 foci, 2–2–4 foci, 3-+4 foci). NASH was defined in the cases of NAS of ≥5 (Kleiner et al., 2005 (link); Zhang et al., 2016 (link)).