基于SIRT1和HDAC7研究烟酸调控仔猪肠道上皮细胞抗菌肽表达的机制

Antimicrobial peptides (AMPs) are key factors in the resistance of pathogens invasion in intestinal epithelium of piglets. It is a difficulty to effectively improve the expression of AMPs but not to cause intestinal inflammation. Previous studies showed that histone deacetylase (HDAC) inhibitors promote AMPs expression by regulating histone modifications without affecting pro-inflammatory cytokines in intestinal epithelial cells. Our previous studies found that niacin (vitamin B3) increased AMPs expression in the intestinal epithelium of weaned piglets, associated with activating SIRT1 and inhibiting HDAC7. However, the mechanism of SIRT1 and HDAC7 in the regulation of intestinal AMPs expression by niacin is still unclear. Therefore, this study uses weanling piglets and intestinal porcine epithelial cells (IPEC-1) to determine the effect of niacin on AMPs expression and the resistance of E.coli K88 infection in the intestine of piglets; using CRISPR/Cas9, activators, inhibitors and other techniques to study the interaction of SIRT1 and HDAC7 in the regulation of AMPs expression by niacin in intestinal epithelial cells of piglets. Combined with chromatin immunoprecipitation (ChIP) to detect changes in histone modification in the promoter region of AMPs, the mechanisms of SIRT1 and HDAC7-mediated histone modification in the regulation of AMPs expression by niacin are explored. The aim of this study is to provide a theoretical basis for nutritional regulation of AMPs expression and improvement of intestinal immunity in weaned piglets.

抗菌肽是仔猪肠道上皮抵抗病原菌入侵的关键因子。如何有效提升抗菌肽表达且不引发肠道炎症成为了研究难点。研究表明，组蛋白去乙酰化酶(HDAC)抑制剂调节启动子区域组蛋白修饰方式，精准提升肠道抗菌肽表达。本课题组研究发现，烟酸（维生素B3）提高断奶仔猪肠道上皮抗菌肽表达，且与激活SIRT1和抑制HDAC7相关，但其作用机制尚不清楚。因此，本项目拟利用大肠杆菌K88感染断奶仔猪和猪肠上皮细胞，首先明确烟酸对仔猪肠道上皮抗菌肽表达及其抗细菌感染的影响；并通过敲除、激活SIRT1以及过表达、抑制HDAC7，揭示SIRT1和HDAC7在烟酸调控仔猪肠道上皮细胞抗菌肽表达中的作用及互作关系；进一步结合染色质免疫沉淀（ChIP）分析抗菌肽启动子区域组蛋白修饰变化，阐明SIRT1和HDAC7介导烟酸调控抗菌肽表达的组蛋白修饰机理。以期为营养精准调控肠道抗菌肽表达、提升断奶仔猪肠道免疫力提供理论依据。

仔猪在断奶时期是致病性大肠杆菌的易感群体，感染会引起腹泻、肠道损伤及炎症反应，严重者甚至死亡，影响了养殖的经济效益。因此，寻找一种安全高效的营养防控手段至关重要。本项目的研究结果表明：烟酸可以缓解仔猪大肠杆菌K88感染引起的腹泻、肠道损伤和炎症反应，其机制一方面主要通过提高血清免疫球蛋白（IgG、IgM）的水平，下调血清中促炎细胞因子（IL-6、IL-8和TNF-α）的水平以及肠道促炎因子mRNA的相对表达水平，同时调节肠道菌群变化从而缓解大肠杆菌感染引起的炎症反应。另一方面烟酸主要通过影响组蛋白修饰水平调控抗菌肽的表达，从而增强仔猪肠道免疫抵抗大肠杆菌感染引起的肠道损伤。研究证明：烟酸通过提高抗菌肽（pBD2、PG1-5和PR39）的表达提高自身免疫力，烟酸降低了仔猪肠道中HDAC7的蛋白表达，增加了SIRT1、pH3S10、H3K9ac和H3K27ac的的蛋白表达。在肠上皮细胞体外模型上，通过Chip-qPCR进一步揭示烟酸调控猪上皮细胞抗菌肽表达的机制。研究结果表明烟酸主要是通过增强抗菌肽（pBD2、PR39）启动子区域H3赖氨酸（H3K27ac）水平的表达对抗菌肽进行调控。本项目按照计划任务完成了预定研究目标，揭示了烟酸抗大肠杆菌K88的感染作用，并阐明了烟酸通过去乙酰化酶和组蛋白修饰调控抗菌肽表达的机制。为生产中防治由于大肠杆菌K88感染引起的肠道损伤提供了有效的营养调控手段和科学依据，进而促进仔猪健康生长，以保证养殖的安全高效发展。