P2X4 Receptor Antagonists Exert Anti-Inflammatory Effects via NLRP3 Inflammasome in a Murine Model of Colitis

Abstract Pharmacological inhibition of P2X4 receptors (P2X4Rs) has emerged as a potential therapeutic approach for the management of several immune/inflammatory diseases. However, the contribution of P2X4Rs in the pathophysiological mechanisms underlying colitis-associated intestinal inflammation remain partially unclear. Here, we investigated the anti-inflammatory effects of two novel and selective P2X4R antagonists, NC-2600 and NP-1815-PX, in rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis. In addition, the molecular mechanisms of their action were also characterized. Oral treatment with either drug or dexamethasone (DEX) was initiated immediately after DNBS administration and continued for 6 consecutive days. NC-2600 and NP-1815-PX attenuated the body weight loss induced by colitis, whereas DEX had no effect on this parameter. The increase in spleen weight and the macroscopic severity of colonic lesions were reduced by all three treatments. Furthermore, NC-2600, NP-1815-PX, and DEX decreased colonic IL-1β levels and caspase-1 activity. A reduction in tumor necrosis factor levels was observed only following DEX treatment, as neither P2X4R antagonist affected the colitis-associated increase in this cytokine. By contrast, the loss of colonic occludin expression was reversed by NC-2600 and NP-1815-PX, but remained unaffected by DEX. In THP-1 cells, combined exposure to lipopolysaccharide (LPS) and adenosine triphosphate (ATP) increased IL-1β release and the expression of NLRP3, caspase-1, caspase-5, and caspase-8, whereas caspase-4 expression was unchanged. NC-2600 and NP-1815-PX prevented these LPS/ATP-induced alterations. In conclusion, these findings support the potential of selective P2X4R blockade as a therapeutic strategy for intestinal inflammation, possibly through the modulation of NLRP3 inflammasome-related signaling.