壳聚糖通过TLR4信号通路调控仔猪肠道免疫功能的研究

Chitosan is currently the only cation-containing natural polysaccharide, which has a variety of biological functions, such as improving immunity, resisting oxidation, inhibiting bacteria, promoting growth, and so on, so it is expected to be a potential green feed additive, but its mechanism of improving immunity remains unclear, which has seriously hindered its development and application. 21-day-old weaned piglets and pig intestinal epithelial cells (IPEC-1) will be chosen to establish immune stress model induced by lipopolysaccharide in this study, moreover, many methods including fluorescence quantitative PCR, western blotting, immunohistochemistry, flow cytometry and enzyme-linked immunosorbent assay, and so forth, will be implemented to explore the regulation of chitosan on intestinal immune function in piglet, cell immune function in IPEC-1,TLR4-mediated NF-κB and MAPKs signaling pathways and theirs key genes, kinases, transcription factors and downstream cytokines. Consequently, the mechanism of chitosan on attenuating intestinal immune stress, decreasing intestinal inflammatory response and improving immunity will be revealed in piglet and IPEC-1 from gene, protein, cell level and molecular signaling pathway, accordingly, the cross-regulate effects of signaling pathways in immune stress response will be verified and perfected. This research will aim at providing theoretical basis for the study of chitosan biological function, animal immune response, piglet intestinal immune, the research and development of chitosan feed additive.

壳聚糖是目前唯一含阳离子的天然多糖，具有增强免疫力、抗氧化、抑菌、促生长等多种生物学功效，可望成为一种极有潜力的绿色饲料添加剂，但其增强免疫力的作用机制仍不透彻，严重制约了其开发应用。本研究选择21日龄断奶仔猪和猪小肠上皮细胞建立LPS诱导的免疫应激模型，综合运用荧光定量 PCR、免疫印迹、免疫组化、流式细胞术和酶联免疫吸附试验等技术，探讨壳聚糖对免疫应激仔猪肠道免疫功能、猪小肠上皮细胞免疫功能、TLR4介导的NF-κB和MAPK信号通路及其关键作用基因、激酶、转录因子、下游细胞因子的调控。从基因、蛋白、细胞水平与分子信号通路角度阐释壳聚糖缓解肠道免疫应激的作用机理，揭示壳聚糖降低肠道炎症反应和提高免疫力的作用机制，进一步验证和完善免疫应激反应中信号通路的交叉调控效应。研究结果将为壳聚糖生物学功能、动物免疫反应、仔猪肠道免疫研究及壳聚糖饲料添加剂研发提供理论依据。

本研究旨在掌握日粮中添加壳寡糖对仔猪生长、腹泻、肠道炎症、氧化应激损伤的影响，通过仔猪体内试验和猪肠上皮细胞体外试验建立LPS诱导的氧化应激模型，沿着TLR4信号通路探索COS对氧化损伤和肠道炎症的作用及其机理。.1、仔猪试验.本研究采用双因素完全随机分组试验设计，选购胎次相近的28日龄健康三元杂交（杜洛克×长白×大约克）去势断奶公仔猪40头，分为4个处理组：对照组、COS组、LPS组、COS+LPS组，每个处理10个重复，每个重复1头仔猪（单栏饲养）。LPS攻击仔猪在第14、21天分别腹膜注射LPS 60、80 μg/kg•bw。预试期为7d，正试期为21d，检测仔猪生长性能、腹泻率、抗氧化指标及血常规指标，进而对空肠和回肠进行组织病理学分级，采用ELISA法检测细胞因子与免疫球蛋白含量、荧光定量PCR 检测基因mRNA表达、Western blotting 检测蛋白表达、免疫组化法检测NF-κB p65蛋白表达。结果表明COS能缓解LPS攻击引起的氧化损伤，抑制LPS刺激下的TLR4信号通路，下调NF-κB、P38、JNK受体的激活，抑制下游细胞因子活化，从而降低肠道炎症反应。.2、体外细胞试验.首先，本研究采用MTT法检测LPS（0、0.1、1、10 μg/ml）和COS（0、50、100、200、400 μg/ml）对IPEC-J2细胞作用12 h、24 h、48 h后细胞增值活性的变化。其次，试验分为对照组、LPS组、COS组、LPS+COS组，采用Western Blot法检测各组中Nrf2和HO-1蛋白的表达量。采用免疫组化的方法检测各组中P38、JNK、8-OHdG蛋白的表达量。结果表明，COS可降低P38和8-OHdG蛋白的表达，诱导Nrf2和HO-1蛋白的高表达，同时，提高抗氧化酶的活性，降低氧化产物含量，增强细胞对氧化应激的抵抗力，从而起到保护作用。