解偶联蛋白2对早期断奶仔猪肠道活性氧调控和肠道发育的作用及其分子机制解析

Early weaning stress of piglets is always a tough problem in pig industry and animal nutriology. Our previous work has shown that reactive oxygen species (ROS) in the piglets at 3 days post weaning was significantly increased and negatively correlated with intestinal structure and function. Uncoupling protein 2 (UCP2), a member of the mitochondrial inner membrane carrier family, is implicated in the control of ROS production and energy metabolism. There have not been related reports on UCP2 expression pattern in the intestine of post weaning piglets and effects on intestinal development and ROS production in weaning piglets up to now. This project aims to explore the distribution and expression pattern of UCP2 in the small intestine of weaning piglets using immunohistochemistry, real-time RT-PCR and Western-blot. Construct recombinant plasmid containing over-expressed UCP2 gene or siRNA mediated silencing of the UCP2 gene, and transfect to the intestinal porcine epithelial cell. In order to further discussion of the molecular mechanisms of UCP2 on the intestinal development from tissue, molecule and cell level, the effect of different UCP2 gene expression level (over expression, normal expression, silence and suppression expression of UCP2 gene) on proliferation, apoptosis and energy metabolism in the damaged intestinal epithelial cells induced by hydrogen peroxide were studied using transmission electron microscopy, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL). The results will be very significant for researching and developing new functional feed for relief of weaning stress to sustaining intestinal health using UCP2 gene regulation in piglets and rich the connotation of mechanisms of weaning stress, and provide reference for study of animal physiology metabolism and pathological injury mechanisms.

早期断奶应激一直是困扰养猪业的一个难题，也是动物营养学界亟待解决的命题之一。针对本课题组前期研究结果表明，断奶后仔猪体内产生大量活性氧，并与肠黏膜结构和功能呈显著负相关，解偶联蛋白2（UCP2）具有调控活性氧产生和能量代谢的功能，而有关UCP2如何调控断奶仔猪肠道活性氧产生及与肠道发育的关系目前并不明确。本研究应用免疫组化、荧光定量RT-PCR、Western-blot、基因重组和RNA干扰等技术，分析断奶前后仔猪肠道UCP2的分布和表达规律及其与活性氧和肠道发育的关系，研究调控UCP2基因表达对应激状态下猪肠上皮细胞增殖、凋亡与能量代谢的作用，深入探讨UCP2对仔猪肠道活性氧调控和肠道发育的关系及其分子机制，研究成果对通过调控UCP2表达途径，研发新型功能性饲料维持肠道健康以缓解仔猪断奶应激具有重要的指导意义，丰富断奶应激理论的内涵，为今后研究动物生理和病理损伤机制提供科学依据。

早期断奶是集约化养猪生产的关键技术，也是仔猪出生后最大的应激因素，断奶应激成为当前养猪业亟待解决的难题之一。解偶联蛋白2（UCP2）是位于线粒体内膜上的载体蛋白家族成员，具有调控ROS产生和能量代谢的功能。本项目首先研究了断奶应激对仔猪肠道发育、抗氧化能力、菌群结构的影响，揭示肠道不同部位UCP2的分布和表达规律，分析UCP2与肠道ROS含量的关系，并研究N-乙酰半胱氨酸（NAC）对断奶仔猪肠道损伤修复与UCP2表达的调控作用。结果表明，断奶造成仔猪肠道产生大量ROS、抗氧化酶活降低、肠黏膜严重受损，有益菌数量下降。免疫组化及荧光实时定量PCR结果显示，UCP2在不同肠段均有分布，断奶后UCP2表达水平上升，与ROS水平及肠道发育呈现显著正相关关系。NAC可一定程度缓解断奶应激损伤。利用体外细胞培养技术，成功构建了H2O2诱导的猪肠上皮细胞氧化应激模型；利用分子生物学手段，成功构建猪UCP2过表达慢病毒载体及UCP2过表达肠上皮细胞系。分别研究了氧化应激对猪UCP2正常表达肠上皮细胞、UCP2过表达肠上皮细胞、UCP2抑制表达肠上皮细胞的增殖、凋亡及UCP2表达的影响。结果表明，H2O2诱导的氧化应激可抑制细胞增殖、上调促凋亡基因、下调凋亡抑制基因，诱导UCP2 mRNA的表达。UCP2过表达可缓解氧化应激对细胞的损伤，与过氧化氢作用正常肠上皮细胞相比，细胞活力和抗氧化酶活力升高、胞内总自由基水平下降，凋亡抑制基因表达上调、促凋亡基因表达下调。而利用京尼平抑制UCP2表达后，氧化应激对细胞的损伤加剧，表现为细胞活力显著下降、上调促凋亡基因、下调凋亡抑制基因。本研究首次阐明了断奶后仔猪肠道UCP2分布与表达规律，明确了机体UCP2表达水平与氧化应激对机体的损伤程度密切相关，提高机体UCP2表达水平可一定程度缓解氧化损伤。这一研究发现对通过调控UCP2表达水平、研发新型功能性饲料添加剂以缓解仔猪断奶应激提供了理论依据。