| Abstract: | Background: In-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides(HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.Methods: To identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3 D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3 D4/31 macrophages against a Gram-negative bacterium(enterotoxigenic E. coli) and a Gram-positive bacterium(Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds.Results: A total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening,with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment.Xanthohumol and deoxyshikonin were further found to be the most potent in inducing p BD3 m RNA expression,showing a minimum 10-fold increase in IPEC-J2, 3 D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced p BD3 m RNA expression by at least 10-fold in both IPEC-J2 cel s and jejunal explants, but not 3 D4/31 cells. In addition to p BD3, other porcine HDP genes such as p BD2, PG1-5, and p EP2 C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene-and cel type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3 D4/31 macrophages(P 0.05) against both Gram-negative and Gram-positive bacteria.Conclusions: Our results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production. |
