Chronological activation of the NLRP3 inflammasome/pyroptosis pathway in the progression from metabolic dysfunction-associated fatty liver disease to hepatocellular carcinoma.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a risk factor for hepatocellular carcinoma (HCC). Evidence links the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome/pyroptosis pathway to MAFLD-related fibrosis; however, its role in MAFLD progression to HCC remains poorly understood. This study investigates the temporal activation of the NLRP3 inflammasome/pyroptosis pathway in MAFLD-associated HCC. Rats were assigned to a control group, who received a standard diet with intraperitoneal injections of liquid petrolatum and water, or to experimental groups, subjected to a hepatopathogenic diet, CCl, and diethylnitrosamine. Serum liver enzymes, inflammatory cytokines, NLRP3 inflammasome/pyroptosis pathway, fibrosis, and HCC markers were assessed. Hepatosteatosis and serum liver enzymes increased after 3 weeks. Hepatosteatosis was associated with elevated levels of ALT ( = 0.0051), GGT ( < 0.0001), IL-6 ( = 0.0499), TNF-α ( = 0.0020), and IL-1β ( 0.0001), and the NLRP3 inflammasome/pyroptosis pathway activators (NLRP3,  = 0.0246; ASC,  0.0003, caspase-1,  = 0.0003, and GSDMD  = 0.0111). Levels of fibrosis markers, including TGF-β ( < 0.0001), α-SMA ( = 0.0035), and collagen ( < 0.0001), increased after 7 weeks, while those of HCC markers PTGR1 and KRT19 increased 13 weeks onwards (both  < 0.0001). These findings provide the first evidence of the NLRP3 inflammasome/pyroptosis pathway's involvement in MAFLD-associated HCC development. The data strongly suggest that metabolic dysregulation and NLRP3-driven inflammation lead to pyroptosis, triggering ongoing cycles of cellular damage and regeneration and accelerating the transition from MAFLD to HCC.