NAT10 promotes pyroptosis and pancreatic injury of severe acute pancreatitis through ac4C modification of NLRP3

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Abstract Severe acute pancreatitis (SAP) is a highly morbid acute digestive disorder linked to pyroptosis. N-acetyltransferase 10 (NAT10) facilitates the production of N4-acetylcytidine (ac4C) modifications in mRNA, thereby contributing to the progression of various diseases. However, the specific role of NAT10 in SAP remains to be elucidated. This study aimed to elucidate the mechanism through which NAT10 mediates pyroptosis in SAP. Sprague-Dawley rats and AR42J rat pancreatic exocrine cells were used to establish in vivo and in vitro models of SAP. The levels of ac4C and NAT10 expression were quantified using dot blot analysis and quantitative real-time PCR (qPCR). Assessment of cell viability, apoptosis, amylase content, and concentrations of lactate dehydrogenase (LDH), interleukin (IL)-1β, and IL-18 was conducted to evaluate the severity of SAP both in vivo and in vitro. Pyroptosis was assessed by measuring caspase-1 and gasdermin D (GSDMD)-N-terminal (GSDMD-N) expression. Further mechanistic insights were gained using methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), and dual-luciferase reporter assays. Our findings indicate that the levels of ac4C modification and NAT10 were elevated in both in vivo and in vitro SAP models. Knockdown of NAT10 inhibited cell death and reduced the levels of amylase, LDH, IL-1β, and IL-18 as well as the protein expression of caspase-1 and GSDMD-N, suggesting that NAT10 knockdown suppresses pyroptosis in SAP cell models. Mechanistically, NAT10 knockdown decreased the expression and stability of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) mRNA by inhibiting ac4C modification of NLRP3. Moreover, NAT10 knockdown alleviated pancreatic tissue pathology, mitigated SAP severity, and suppressed pyroptosis in an SAP rat model. Collectively, these results demonstrate that NAT10 exacerbates pancreatic injury in SAP by promoting pyroptosis through ac4C modification of NLRP3, thereby enhancing its expression. These findings suggest a potential novel therapeutic target for SAP.