Lipid metabolism disorders lead to elevated levels of free fatty acids (FFA) in plasma, which are closely associated with obesity, insulin resistance, and an increased risk of type 2 diabetes (Shulman, 2014). Obesity is the most common chronic metabolic disease caused by excessive accumulation of body fat (Swinburn et al., 2011). Numerous studies have shown that obese individuals are at higher risk of developing cardiovascular diseases (CVD) (Choi et al., 2018; Gao et al., 2020; Khan et al., 2018). Among lipid abnormalities, vascular endothelium is the primary target of FFAs in the blood, and chronically elevated levels of FFA in the bloodstream lead to endothelial dysfunction and damage (Jiang et al., 2024; Wang et al., 2023a). Endothelial dysfunction plays a critical role in the initiation and progression of atherosclerosis (AS) (Batumalaie et al., 2016; Zhang et al., 2024; Wang et al., 2023b). However, some existing drugs, such as statins, can cause liver and kidney damage with long-term use, and some drugs may develop resistance. Therefore, there is an urgent need to find a natural compound to treat endothelial dysfunction.

Pyroptosis, a lytic form of programmed cell death triggered by inflammasomes, is characterized by the release of numerous pro-inflammatory factors (Kovacs and Miao, 2017). Upon activation of NLRP3, the NLRP3 inflammasome triggers the production of active caspase-1 from pro-caspase-1. Active caspase-1 cleaves pro-interleukin (IL)-1β and pro-interleukin (IL)-18 into their mature forms and also cleaves the protein gasdermin D (GSDMD), releasing its active N-terminal domain (GSDMD-N). Subsequently, GSDMD-N binds to lipids in the plasma membrane, forming oligomers that facilitate the formation of pyroptotic pores. These pores disrupt the cell's osmotic balance, leading to the release of inflammatory factors, cell swelling, and eventually pyroptosis (Gaidt and Hornung, 2018; Schroder and Tschopp, 2010; Broz and Dixit, 2016). Studies have demonstrated that elevated palmitic acid (PA) concentrations can significantly induce mitochondrial dysfunction, promote excessive reactive oxygen species (ROS) generation, and trigger oxidative stress(Zeng et al., 2020; Alnahdi et al., 2019). As a crucial upstream signaling molecule for NLRP3 inflammasome activation, abnormal ROS accumulation directly initiates inflammasome assembly and activation, consequently leading to endothelial cell dysfunction(Huang et al., 2021). Notably, ROS-mediated endothelial cell pyroptosis has been identified as a critical pathological mechanism underlying endothelial dysfunction(Zheng et al., 2022). Therefore, targeted inhibition of oxidative stress-induced pyroptosis pathways represents a highly promising therapeutic strategy for intervening in high-fat diet-associated endothelial dysfunction.

SIRT1, an NAD+ dependent deacetylase, plays a critical role in regulating various physiological processes, including cellular senescence (van de Ven et al., 2017), inflammation and oxidative stress (Ubaid et al., 2021), through the deacetylation of target proteins. Studies have indicated that activation of the SIRT1 signaling pathway can attenuate endothelial cell (EC) damage induced by oxidative stress (Chen et al., 2022; Yan et al., 2024). Moreover, numerous studies have shown that SIRT1 can inhibit the activation of the NLRP3 inflammasome by suppressing ROS production (Ping et al., 2024; Dong et al., 2024; Coll et al., 2022).

Pituitary adenylate cyclase activating peptide 38 (PACAP38, or PACAP) is a neuropeptide with pleiotropic functions, widely expressed across vertebrate tissues. Due to the highly conserved nature of the PACAP gene during biological evolution, it plays many critical biological functions in organisms (Vaudry et al., 2000). Evidence indicates that PACAP safeguards blood vessels by exerting both anti-inflammatory and antioxidant effects (Rácz et al., 2007; Bian et al., 2017). However, the precise role and mechanisms by which PACAP ameliorates endothelial cell damage associated with hyperlipidemia remain to be elucidated.

In summary, we hypothesized that PACAP may attenuate PA-induced ROS production through the SIRT1 signaling pathway and thus inhibit endothelial cell death. The aim of this study was to clarify the effect of PACAP on PA-induced endothelial cell death and its mechanism, and to provide a basis for the treatment of obesity-induced endothelial dysfunction with PACAP.