Abstract
The intestine is highly vulnerable to various factors and has been proposed as a promising determinant for poultry health. Phytogenic or plant-derived feed additives can be used to help improve intestinal health. In this study, we aimed to investigate the effects of DNJ on the antioxidative parameters, including malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and inflammatory cytokines (IL-6, IL-1β, and TNF-α), in plasma and intestinal tissues using layers supplemented with or without the DNJ extract of mulberry leaves (DNJ-E) via the ELISA method. A total of 192 healthy Hy-Line Brown layers, aged 47 weeks old, were used to conduct a 56-day study. All hens were randomly separated into four groups as follows: a basal diet containing 0 mg/kg DNJ-E(CON), 50 mg/kg, 100 mg/kg, and 150 mg/kg DNJ-E. Furthermore, the potential mechanism by which DNJ influences intestinal function was also investigated in in vitro cultured intestinal epithelium cells (IEC) with quantification methods including the use of a cell counting kit-8 (CCK8), ELISA, qRT-PCR, and ROS detection. The results showed that CAT in plasma significantly increased following 50 mg/kg DNJ-E supplementation. Moreover, 50 mg/kg DNJ-E supplementation was associated with increases in T-SOD in the jejunum and ileum. However, there was no significant difference in inflammatory cytokines between groups in in vivo experiments. Subsequent in vitro IEC studies revealed that cell viability increased significantly following 5 µM and 10 µM DNJ treatments while decreasing significantly following 20 µM DNJ treatment. Antioxidative parameters improved at 5 µM and 10 µM DNJ concentrations. However, there were no ameliorative effects on antioxidant parameters observed under 20 µM DNJ treatment. The expression levels of Nrf2 mRNA increased significantly under DNJ treatment. DNJ treatment was associated with significant changes in the expression of genes of inflammatory cytokines. In conclusion, our study revealed that DNJ could improve oxidative stress and inflammation responses in the chicken intestine. These findings provide a theoretical reference for the development of functional feed additives that regulate intestinal health and lay the foundation for systematically revealing the mechanism of DNJ.