NAFLD activity score (NAS)

Mouse livers were scored under blinded conditions utilizing the NAFLD activity score (NAS). This score is defined as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3), and ballooning (0–2). Scores of 0–2 are not NASH, 3–4 are considered borderline, and 5–8 are considered NASH⁸.

The pyroptotic cells were detected with FLICA® 660-YVAD-FMK (ImmunoChemistry Technologies, NRM-KF17362) by immunofluorescence, according to the manufacturer’s protocol. The surfaces of the tissues were covered in tissue section staining solution (TSSS) and incubated for 30–60 min while protected from light. The slides were then washed with 1× Cellular Wash Buffer twice for 5 min each. The slides were then set in an incubation dish containing 1× Cellular Wash Buffer. Nuclei were stained with DAPI (ROCHE, 28718-90-3) for 20 min at room temperature and protected from light. The slides were then imaged with a fluorescence microscope (Olympus BX63, 40 × 10).

The pyroptotic cells were detected by flow cytometry (FCM). After 48 h of exposure to As₂O₃, HepG2 cells were harvested in 1.5 mL centrifuge tubes for following experiments: active caspase-1 was measured in HepG2 cells with FLICA 660-YVAD-FMK, and membrane pore formation was measured by staining with propidium iodide (PI, KeyGEN, KGA214) according to the manufacturer’s instructions. After staining, the cells were analyzed using an ACEA NovoCyte flow cytometer (NovoCyte 2040 R), and pyroptosis was defined as double positive for caspase-1 and PI.