β-胡萝卜素对仔猪早期断奶应激的保护作用及其机理研究

Early weaning is the biggest stress of piglets, and can cause huge economic losses in the pig industry. It was a hot spot to use non-antibiotics material to improve piglet's autoimmunity and reduce the weaning stress reaction in intervention weaned stress. Based on previous studies found that β-carotene can improve the intestinal tight junction, protect the intestinal cells from inflammatory injury, and increase the secretion of IgA in the mucosa of piglets, enhance immunity of piglets, reduce the diarrhea rate, this study proposed by establishing the model of stress in vivo and in vitro and immunohistochemistry, Q-PCR, western-blot and RNAi was used to detection the changes of diarrhea rate of weaning piglets, immunoglobulins in blood and intestinal mucosa, proinflammatory cytokine. Observation of the effects of β-carotene on the expression of genes and proteins related to inflammatory response in intestinal epithelial cells. To explore the relationship between β-carotene and intestinal mucosal immunity, inflammatory response and its signaling pathway protein phosphorylation for definited the protection of β-carotene on weaning stress in piglets and explore the mechanism of protection on weaning stress from induced mucosal immune responses in order to reduce the use of antibiotics,accelerate the development of non-antibiotics feed additives, and lay a theoretical foundation of guarantee the healthy breeding piglets.

早期断奶是仔猪出生后最大的应激反应，给养猪业造成了巨大经济损失。目前利用非抗生素物质提高仔猪自身免疫力，减缓断奶应激反应，是干预断奶应激的研究热点。课题组前期研究发现β-胡萝卜素可改善肠上皮细胞紧密连接，保护肠细胞免受炎症损伤，提高仔猪肠黏膜IgA分泌量，增强新生仔猪免疫力，降低腹泻率的基础上，拟建立体内外应激模型，采用免疫组化、Q-PCR、免疫印迹、RNA干扰技术，检测β-胡萝卜素对断奶仔猪腹泻率、血液和肠黏膜中免疫球蛋白、促炎细胞因子等指标的影响；观察β-胡萝卜素对肠道上皮细胞中炎症反应相关基因及蛋白表达的调控；探索β-胡萝卜素与肠黏膜免疫、炎症反应及其信号通路蛋白磷酸化的内在联系，进而明确β-胡萝卜素对仔猪断奶应激的保护作用，解析其诱发黏膜免疫反应对断奶应激保护的作用机理，为减少抗生素的使用，加速无抗饲料添加剂开发，保障仔猪健康养殖奠定理论基础。

早期断奶是仔猪出生后最大的应激反应，给养猪业造成了巨大经济损失。目前利用非抗生素物质提高仔猪自身免疫力，减缓断奶应激反应，是干预断奶应激的研究热点。课题组前期研究发现β-胡萝卜素可改善肠上皮细胞紧密连接，保护肠细胞免受炎症损伤，提高仔猪肠黏膜IgA分泌量，增强新生仔猪免疫力，降低腹泻率的基础上，拟建立体内外应激模型，采用免疫组化、Q-PCR、免疫印迹、RNA干扰技术，检测β-胡萝卜素对断奶仔猪腹泻率、血液和肠黏膜中免疫球蛋白、促炎细胞因子等指标的影响；观察β-胡萝卜素对肠道上皮细胞中炎症反应相关基因及蛋白表达的调控；探索β-胡萝卜素与肠黏膜免疫、炎症反应及其信号通路蛋白磷酸化的内在联系，进而明确β-胡萝卜素对仔猪断奶应激的保护作用，解析其诱发黏膜免疫反应对断奶应激保护的作用机理。.研究结果表明，与未断奶仔猪相比，早期断奶能够显著降低断奶仔猪的平均日增重，显著提高仔猪血清中炎性细胞因子的含量，显著提高十二指肠、空肠、回肠中炎性细胞因子的基因表达及炎症相关信号通路的蛋白表达。同时早期断奶还会破坏仔猪小肠绒毛结构，使小肠绒毛缩短甚至断裂。β-胡萝卜素的提前干预能够缓解早期断奶仔猪的生长迟滞现象及肠道炎症反应，减轻早期断奶对肠道的结构损伤，说明β-胡萝卜素的早期干预对早期断奶的仔猪具有保护作用。体外试验证明，β-胡萝卜素能够缓解脂多糖诱导的仔猪肠道细胞炎症损伤，降低肠细胞的炎症细胞因子的表达，对肠细胞活力具有保护作用。炎症信号通路蛋白Myd88基因沉默后，β-胡萝卜素对肠细胞炎症的保护作用效果降低，说明β-胡萝卜素是通过调控TLRs-Myd88-NF-κb信号通路来缓解肠细胞炎症反应，进而发挥其对肠道的保护作用。这个机制的阐明有助于解析仔猪断奶对肠道损伤的原因，能够为早期断奶仔猪的营养调控提供理论及实践经验，为天然饲料添加剂保障仔猪健康养殖奠定理论基础。