Adiponectin alleviates inflammatory response in metabolic dysfunction-associated steatohepatitis by inhibiting NLRP3 inflammasome-mediated hepatocyte pyroptosis

Abstract

Background: Activation of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasomes induced by pyroptosis is crucial in metabolic dysfunction-associated steatohepatitis (MASH) progression. Adiponectin possesses an anti-inflammatory role in various liver diseases. This study aimed to evaluate the effects of adiponectin on MASH.

Methods: Adiponectin-mediated anti-inflammatory mechanisms, effects on pyroptosis-related proteins, and activation of NLRP3 inflammasomes were investigated using methionine-choline-deficient (MCD)-induced MASH murine model and in vitro models. The degree of MASH inflammation in liver tissue of C57BL/6J mice was assessed using histopathology. Enzyme-linked immunosorbent assay was performed to measure levels of inflammatory factors [interleukin-18 (IL-18), IL-1β, and tumor necrosis factor-α (TNF-α)] in mice serum and culture medium. Western blot and quantitative polymerase chain reaction were performed to analyze the expression of pyroptosis-related genes and proteins in liver tissues of mouse model and in vitro models. Macrophage recruitment in vitro was evaluated using co-culture of upper and lower chambers.

Results: MASH developed in MCD diet mice [metabolic dysfunction-associated steatotic liver disease (MASLD) activity score = 6] but not in methionine-choline-sufficient (MCS) diet mice (MASLD activity score = 3). Compared to MCS-fed mice, MCD-fed mice showed increased serum levels of aspartate aminotransferase, IL-18, IL-1β, and TNF-α and higher MASLD activity score (P < 0.001). Adiponectin inhibited these increases (P < 0.05) and suppressed mRNA and protein levels of NLRP3, gasdermin-D (GSDMD), and GSDMD-N in liver tissues (P < 0.05). In vitro, lipopolysaccharide (LPS)/palmitic acid (PA) increased the levels of IL-18, IL-1β, and TNF-α, mRNA expressions of CASP1 and GSDMD, and production of CASP1, NLRP3, GSDMD, and GSDMD-N (P < 0.01). Adiponectin reduced the levels of these inflammatory factors and downregulated the mRNA expression and protein generation of pyroptosis-related markers (P < 0.05). HepG2 cells pretreated with LPS/PA recruited more J774A.1 cells (P < 0.001) and increased inflammatory factor secretion by J774A.1 cells (P < 0.001). Adiponectin inhibited this recruitment and reduced inflammatory factor secretion (P < 0.001).

Conclusions: Adiponectin inhibits hepatocyte pyroptosis by reducing the production and activation of NLRP3 inflammasomes, CASP1, and GSDMD, thus improving the inflammatory response in MASH and possibly delaying or reversing MASLD progression.

Keywords: Adiponectin; Gasdermin-D; NLRP3; Nonalcoholic steatohepatitis; Pyroptosis.