Unveiling excessive feed-sources copper-induced ileitis in chickens: Insights into tight junction damage and ROS/NLRP3/pyroptosis axis

Abstract

Copper, widely used as a growth promoter and antibacterial agent, is commonly added to livestock and poultry feed. However, this widespread use leads to its accumulation in the animals' bodies, resulting in intestinal toxicity. The specific mechanisms of copper-induced ileitis in broilers remain unclear. In this study, broilers were fed diets containing 0, 100, or 300 mg/kg CuSO₄ over a five-week period. Results showed that the high‑copper group (300 mg/kg) exhibited significant suppression of antioxidant defenses, including reduced heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), total superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels, while catalase (CAT) activity was paradoxically elevated. Notably, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex activation (NLRP3, apoptosis-associated speck-like protein containing a CARD [ASC], Caspase-1) and upregulated pyroptosis markers (Caspase-1, interleukin-1β [IL-1β]) indicated reactive oxygen species (ROS)–NLRP3 axis involvement. Concurrently, pro-inflammatory mediators (interleukin-7 [IL-7], interleukin-17 [IL-17], inducible nitric oxide synthase [iNOS], tumor necrosis factor-α [TNF-α]) were dysregulated, accompanied by diminished expression of tight junction proteins (zonula occludens-1 [ZO-1], Claudin-3, Occludin) and inhibition of the Wnt/β-catenin pathway. These findings demonstrate that copper-induced intestinal inflammation and pyroptosis are driven by ROS–NLRP3 axis activation, while simultaneously compromising mucosal barrier integrity. This study elucidates critical mechanisms of copper toxicity in broilers and highlights risks associated with excessive copper exposure.