Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening

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,3D4/31 macrophages,and jejunal explants were verified using RT-qPCR.Augmentation of the antibacterial activity of porcine 3D4/31 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 pBD3 mRNA expression,showing a minimum 10-fold increase in IPEC-J2,3D4/31 cells,and jejunal explants.Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants,but not 3D4/31 cells.In addition to pBD3,other porcine HDP genes such as pBD2,PG1-5,and pEP2C were induced to different magnitudes by xanthohumol,deoxyshikonin,isorhapontigenin,and calycosin,although clear gene-and cell 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 3D4/31 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.