ABSTRACT: Nutraceuticals are gaining recognition as promising alternatives for the prevention and treatment of atherosclerosis, particularly given the limitations of current pharmacotherapies, which are often associated with a significant residual risk and various adverse side effects. Among these, pomegranate-derived polyphenols—especially punicalagin (PC) have demonstrated notable health benefits. However, PC exhibits poor absorption and bioavailability, with its beneficial effects largely attributed to gut microbiota-derived metabolites, particularly urolithins. Unfortunately, in-depth studies on PC and its metabolites in atherosclerosis and cellular processes associated with disease are lacking. This study investigates the anti-atherogenic effects and underlying mechanisms of PC and its key metabolites—ellagic acid and urolithins A, B, C, and D (UA–UD). In vitro analyses revealed that these compounds predominantly attenuate chemokine-induced monocyte migration, macropinocytosis, oxidised LDL uptake in macrophages, and the production of reactive oxygen species (ROS) and pro-inflammatory gene expression in both macrophages and endothelial cells. Notably, some metabolite-specific effects were observed, such as increased ROS production by urolithin B in various cell types. Given its broad inhibitory effects on pro-atherogenic processes, urolithin A (UA) was further evaluated in vivo using LDL receptor-deficient mice fed a high-fat diet. UA supplementation led to reduced circulating levels of natural killer cells, granulocytes, and macrophages, alongside elevated levels of gut microbiota-derived short-chain fatty acids in plasma and faeces. Histological and immunohistological analysis showed that UA significantly reduced plaque content and inflammation, as evidenced by decreased macrophage and T-cell infiltration, and increased markers of plaque stability, including smooth muscle cell and collagen content. RNA sequencing of the thoracic aorta, followed by bioinformatic analysis, identified key genes and pathways involved in the anti-inflammatory and anti-atherogenic actions of UA. These findings provide novel insights into the therapeutic potential of PC and its metabolites particularly UA and support further investigation in human clinical trials.
