叶黄素介导TLR4/MyD88/NF-кB信号通路缓解肉仔鸡肠道损伤的机理研究

Recently, studies in human and mouse have shown that carotenoids can improve antioxidant and immune functions, promote cell proliferation and differentiation, and increase cell tight junctional expression. Our previous studies have shown that carotenoids supplementation can enhance antioxidant capacity, reduce lipid peroxidation, and improved cytokine and apoptosis expression in hens and offspring broilers. The most significant effects of carotenoids are on the jejunum. Combined with the current literature, it is speculated that carotenoids may alleviate the intestinal damage of broilers through TLR4/MyD88/NF-кB signaling pathway. Therefore, firstly, this project determines different levels of lutein on growth performance, intestinal morphology, intestinal tight junction, cytokines, and TLR4/MyD88/NF-кB signaling pathway of broilers. Then, experiment is performed to observe whether lutein addition can relieve intestinal immune stress and intestinal injury through TLR4/MyD88/NF-кB signaling pathway in LPS-injected broilers. Finally, the primary intestinal epithelial cells of chickens were separated and siRNA technology was used to block the TLR4 and MyD88 pathways to further verify the mechanism of lutein. The project can provide theoretical and practical supports for carotenoids nutrition and nutritional regulation for broilers under stress condition.

近年来，研究表明类胡萝卜素(Car)可改善人和小鼠机体的抗氧化和免疫功能，促进细胞增殖和分化，并提高细胞紧密连接的表达。本人前期研究表明添加Car可以改善母鸡和后代肉仔鸡的抗氧化能力、细胞因子和细胞凋亡的表达，且对空肠影响最为显著。结合现有文献，推测Car可能通过TLR4/MyD88/NF-кB信号通路缓解肉仔鸡的肠道损伤。因此，本项目首先测定了不同水平的叶黄素对肉仔鸡的生长性能、肠道形态、肠道紧密连接、细胞因子、TLR4/MyD88/NF-кB信号通路的影响；然后观察日粮叶黄素的添加是否会通过TLR4/MyD88/NF-кB信号通路缓解因脂多糖(LPS)引起的肉仔鸡肠道免疫应激和肠道损伤；最后分离鸡的原代肠上皮细胞，采用siRNA技术阻断TLR4和MyD88通路开展细胞试验，进一步验证叶黄素的作用机制。本研究对于认识肉仔鸡色素的营养、肉仔鸡应激条件下的营养调控具有重要的理论和实践意义。

近年来，在人和鼠上的试验研究表明叶黄素在机体抗氧化和免疫、细胞增殖和分化调控、基因表达、信号传导和细胞间隙连接通讯等方面都有重要作用，而对鸡的研究主要集中在着色方面，对其生理功能的研究比较少。本项目通过体内饲养试验和体外细胞培养试验，首先测定了不同水平的叶黄素对肉仔鸡的生长性能、肠道形态、肠道紧密连接、促炎和抗炎细胞因子、TLR4/MyD88/NF-кB信号通路的影响；然后观察日粮叶黄素的添加是否会通过TLR4/MyD88/NF-кB信号通路缓解因脂多糖（LPS）注射引起的肉仔鸡肠道免疫应激；最后通过细胞试验进一步深入探讨叶黄素的作用机制（确定叶黄素是否可以通过TLR4/MyD88/NF-кB相关信号通路影响肉仔鸡免疫应答）。主要结果如下：.（1）饲养试验结果表明：日粮中添加40mg/kg叶黄素可显著提高LPS应激下1-26日龄黄羽肉鸡的平均日增重，改善空肠形态、盲肠微生物的组成，增强杯状细胞密度、紧密连接蛋白基因表达水平，抑制促炎细胞因子表达并增强抗炎细胞因子表达，并可能经由TLR4/MyD88/NF-κB通路来缓解肠道炎症损伤。因此，日粮中添加叶黄素能促进LPS应激下肉鸡的生长性能，通过修复空肠机械屏障、化学屏障、免疫屏障和盲肠微生物屏障功能，从而保护肠道屏障。.（2）细胞试验结果表明：20μg/mL的LPS作为建立免疫应激模型的添加量，80μmol/L的叶黄素缓解免疫应激的效果最佳。叶黄素可通过抑制TLR4/MyD88信号通路的活化缓解LPS造成的免疫应激，从而抑制促炎细胞因子并提高紧密连接蛋白基因的表达。.本项目揭示了叶黄素对正常和LPS应激肉仔鸡肠道免疫功能、紧密连接、盲肠微生物的影响及其作用机理，验证了叶黄素可以通过调控TLR4/MyD88/NF-кB信号通路影响肉仔鸡免疫应答，从而为肉仔鸡色素的营养、肉仔鸡应激条件下的营养调控提供理论和技术支持。