Abstract
Glycinin is a major anti-nutritional factor in soybeans and can induce growth inhibition and intestinal damage in aquatic animals. N-Acetylcysteine (NAC) possesses antioxidant and anti-inflammatory properties while promoting intestinal mucosal development. Therefore, this study aimed to elucidate the protective effects and underlying mechanisms of NAC against glycinin-induced intestinal damage in common carp (Cyprinus carpio). A total of 450 juvenile common carp (2.93 ± 0.03 g) were randomly assigned to five groups: a control group (CK), an 8.00% glycinin-damaged group (Gly), and three NAC-treated groups receiving 0.15%, 0.30% and 0.60% NAC supplementation (Gly-N1, Gly-N2, and Gly-N3). Each group contained three replicates for a 56-d feeding trial. Results showed that dietary 0.30%-0.60% NAC supplementation effectively alleviated the glycinin-induced impairments in growth performance and feed utilization, while concurrently restoring intestinal protease activity and muscle protein deposition (P < 0.05). The NAC mitigated the intestinal morphological damage induced by glycinin, including mucosal fold atrophy and microvilli shedding, and enhanced barrier integrity by upregulating the mRNA expression of tight junction proteins occludin, claudin-3, claudin-7 and zo-1, and reducing intestinal permeability (P < 0.05). Intestinal transcriptome analysis indicated NAC's ameliorative effects involving inflammation- and apoptosis-related pathways. Mechanistically, NAC exerted multi-target protection: it inhibited the MAPK/PI3K-AKT/NF-κB inflammatory network by downregulating the phosphorylation of p38, JNK, PI3K and AKT, and the expression of NF-κB p65, thereby inhibiting pro-inflammatory cytokines IL-1β and TNF-α release (P < 0.05). Concurrently, NAC activated the Nrf2 antioxidant pathway to counteract oxidative stress. Furthermore, NAC inhibited the mitochondrial apoptosis pathway by modulating Bcl-2/Bax expression and inhibiting Caspases activation, while restoring ATPase activity and membrane potential to improve intestinal mitochondrial function. In summary, dietary supplementation with 0.30%-0.60% NAC alleviated glycinin-induced intestinal damage through a multifaceted mechanism involving inhibition of inflammatory signaling, activation of antioxidant defense, and inhibition of mitochondrial apoptosis.