苯基丁酸钠调控猪源宿主防御肽转录与合成的分子机制

"Antibiotic-free breeding" is about to become the final destination of animal husbandry development with the global problems like antibiotic resistance and residues becoming increasingly serious. Porcine host defense peptide, as an important innate immune defense factors, has unique bacteriostatic, bactericidal, immune regulation and other biological activities. Various pathogenic microorganisms can evade the innate immune response by reducing the expression of host defense peptides. Therefore, it is extremely important to explore a neotype of functional nutritional substance that induces expression of host defense peptide, and does not cause inflammatory response. The research plans to explore the precise control networks of porcine host defense peptide genes transcription and protein synthesis or secretion process by phenyl butyric acid sodium in porcine intestinal epithelial cells from epigenetic modifications, pathway of nuclear factor, recognition receptors, post-transcriptional control though the means of molecular nutrition, including siRNA gene knockout, miRNAs, fluorescence quantitative PCR, ELISA, western blot, and so on; At the same time, the influence of cytokines on inflammatory response was analyzed. This project establishs a sound foundation for the mechanism of host defense peptide expression regulation, and provides a new pathway and theoretical support to develop secure immune-enhancing substances and steps up the pace of "antibiotic-free breeding".

随着全球抗生素耐药、残留等问题日趋严重化，“无抗养殖”即将成为畜牧业发展的最终归宿。猪源宿主防御肽作为动物机体重要的先天免疫防御分子，具有独特的抑菌、杀菌、免疫调节等生物学活性。多种病原微生物可通过降低机体宿主防御肽的表达进而逃避机体的先天免疫反应。因此，探索新型的诱导宿主防御肽表达且不引起炎症反应的功能性营养物质，并透彻研究其调控基因转录、蛋白合成及分泌的机制极其重要。本项目利用分子营养手段，通过定量PCR、ELISA、western blot、siRNA敲除、免疫共沉淀、microRNA等技术，从表观遗传修饰、识别受体、核因子途径、转录后合成等方面研究苯基丁酸钠对猪肠道上皮细胞宿主防御肽基因转录与蛋白合成、分泌过程的精密调控网络机制，同时分析其对炎症细胞因子的影响。本项目为宿主防御肽表达调控的机制奠定坚实基础，为开发安全的免疫增强物质、加快“无抗养殖”的步伐提供全新思路和理论支撑。

我国畜牧业已经进入了“无抗养殖”的时代。但是，仔猪腹泻问题更加凸显。宿主防御肽作为动物机体重要的先天免疫防御分子，具有独特的抑菌、杀菌、免疫调节等功能。因此，探索新型的诱导宿主防御肽表达的功能性营养物质，并透彻研究其调控机制对提高动物机体免疫力极其重要。本项目研究苯基丁酸钠（PBA）对猪肠道上皮细胞IPEC J2宿主防御肽基因表达过程的精密调控网络机制，同时分析其对炎症细胞因子的影响。结果表明：.（1）PBA显著促进IPEC J2细胞内源宿主防御肽(pEP2C、pBD-1和pBD-3)的基因表达并呈现时间和剂量依赖效应。.（2）PBA显著促进TLR2/4的基因表达，并且TLR2/4显著影响pEP2C、pBD-1和pBD-3。.并通过降解IκB-α蛋白和增加NF-κB p65的磷酸化激活NF-κB通路。.（3）MAPK通路在PBA上调HDPs和细胞因子发挥重要作用：p38 MAPK抑制剂SB203580和MAPKK抑制剂PD98059显著抑制PBA对肠上皮细胞内源防御肽的上调作用。表皮生长因子抑制剂Gefitinib显著抑制PBA对肠上皮细胞IPEC J2 pEP2C、pBD-1和pBD-3和IL-8、IL-18的基因表达。细胞密度显著影响PBA对肠上皮细胞IPEC J2防御肽的基因表达，并且融合后期细胞显著增加宿主防御肽的表达。.（4）PBA显著抑制组蛋白脱乙酰酶（HDAC）活性，并且HDAC活性受到TLR2/4调控；PBA显著增加IPEC J2细胞中组蛋白H3S10的磷酸化。.（5）试验结果表明PBA增加pBD3蛋白分泌的倍数小于基因表达增加的倍数，随后microRNA-转录组联合分析结果表明，PBA作用后，多种micRNA发生上调或下调，且该变化与pBD3基因表达密切相关。荧光定量PCR结果与测序结果一致。.本项目为宿主防御肽表达调控的机制、开发安全的免疫增强物质、迈进“无抗养殖”时代提供全新思路和理论支撑。