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Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening
Journal of Animal Science and Biotechnology ( IF 6.3 ) Pub Date : 2021-01-11 , DOI: 10.1186/s40104-020-00536-0
Jing Wang , Wentao Lyu , Wei Zhang , Yonghong Chen , Fang Luo , Yamin Wang , Haifeng Ji , Guolong Zhang

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. 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. 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. 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.

中文翻译:

使用基于细胞的高通量筛选发现能够诱导猪宿主防御肽基因表达的天然产物

饲料中的抗生素正在全球范围内逐步淘汰。迫切需要抗生素替代品来维持动物健康和生产性能。宿主防御肽(HDP)以其广谱抗微生物和免疫调节功能而闻名。增强内源性HDP的合成代表了一种有前途的抗生素替代策略,可用于疾病控制和预防。为了鉴定具有刺激内源性HDP合成能力的天然产物,我们使用新建立的称为IPEC-J2 / pBD3-luc的稳定荧光素酶报道细胞株,对1261种天然产物进行了高通量筛选。使用RT-qPCR验证了命中化合物在猪IPEC-J2肠上皮细胞,3D4 / 31巨噬细胞和空肠外植体中诱导HDP基因的能力。用四种选择的诱导HDP的化合物进一步证实了猪3D4 / 31巨噬细胞对革兰氏阴性菌(肠毒素大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)的抗菌活性。经过高通量筛选后,总共鉴定出48种天然产品,其Z值最低为2.0,在后续的剂量反应实验中,有21种化合物的萤光素酶活性增加了至少2倍。进一步发现黄腐酚和脱氧Shikonin在诱导pBD3 mRNA表达方面最有效,显示出IPEC-J2、3D4 / 31细胞和空肠外植体至少增加了10倍。在IPEC-J2细胞和空肠外植体(但不是3D4 / 31细胞)中,其他化合物(如异皂甙元和calycosin)也可将pBD3 mRNA表达提高至少10倍。除pBD3外,黄腐酚,脱氧Shikonin,异皂甙元和calycosin还诱导了其他猪HDP基因如pBD2,PG1-5和pEP2C不同程度的表达,尽管观察到明确的基因和细胞类型特异性调控模式。希望地,这四种化合物对几种代表性的炎性细胞因子基因的表达具有最小的影响。此外,当以诱导HDP的浓度使用时,这些化合物没有明显的直接抗菌活性,但显着增强了3D4 / 31巨噬细胞对革兰氏阴性和革兰氏阳性细菌的抗菌活性(P <0.05)。
更新日期:2021-01-11
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