仔猪小肠甘氨酸亚铁螯合物吸收特点及其对相关跨膜转运蛋白影响的研究

Iron (Fe) is one of essential trace elements for the growth and survival of animals. It has a long history to supply food with additional iron element, especially inorganic iron. With the benefits on absorption rate, growth, immunity and environment, iron amino acid chelate has been paid more attention in human and animal nutrition. Studies showed chelated sources of Fe have 125-185% relative availability compared with ferrous sulfate..Ferrous glycinate as a novel nutritional iron reinforcing agent, is now used in infant dairy and food. Studies showed that ferrous glycine chelate has good biological utilization rate in the rat, human compared with ferrous sulfate. Our previous study also showed compared with the inorganic iron, ferrous glycinate could improve the growth performance, immunity function of weaning piglets and chicken..The better bioavailability of iron amino acid chelate is maybe mainly due to its high efficient absorption, but there is still no consensus for its absorption mechanism. One hypothesis suggested that iron amino acid chelate is absorbed via the same pathway for ionic iron from iron salts. An alternative hypothesis speculated that iron amino acid chelate may be absorbed as an intact chelate by small intestine in the form of peptide, which means they directly enter organs of animal body without being digestion. .Most researches on the ferrous glycinate in animal focused on its bioavailability assessment. Information on absorption characteristics and mechanism are limited..Present study was designed to investigate the absorption characteristics of ferrous glycinate in piglet, and the effects of ferrous glycinate on the gene and protein expression of transmembrane transporter associated with iron absorption in vivo and in vitro. Firstly, iron deficiency model of weaned piglet will be established, and then reinforced animals with ferrous glycinate and ferrous sulfate respectively to study deposition of iron, ferritin and transferrin levels, serum iron metabolism related enzyme activity, expression of intestinal iron absorption transporter ( DMT1, Fp1 ) and peptide absorption transporter PepT1. Secondly, weaned piglets intestinal ligation by in situ perfusion model were established to study ferrous glycinate intestinal absorption characteristics and its influencing factors, and to explore the correlation of intestinal membrane transport protein content and expression. Through the establishment of in vitro intestinal epithelial cell transport model, analysis of iron uptake efficiency, intracellular DMT1, Fp1, PepT1 content and mRNA abundance were conducted to investigate Ferrous glycinate absorption characteristics, meanwhile, inhibitors of transmembrane transporter were used to study the possible pathway of ferrous glycinate transmembrane absorption.

项目在前期研究基础上，从动物机体、组织及细胞水平研究甘氨酸亚铁在仔猪小肠内的吸收特点及其对相关跨膜转运蛋白的影响。在机体水平上，通过建立断奶仔猪缺铁模型和灌胃模型，研究仔猪机体组织铁沉积、转铁蛋白含量及铁相关代谢酶活力及吸收动力学，分析甘氨酸亚铁中铁的吸收效率，重点研究甘氨酸亚铁对小肠相关跨膜转运蛋白DMT1、Fp1、PepT1含量及其基因表达的影响；在组织水平上，通过仔猪小肠原位结扎灌注模型建立，研究甘氨酸亚铁的吸收特点和影响因素，并探讨其对肠段跨膜转运蛋白的影响；在细胞水平上，通过建立仔猪小肠上皮细胞转运模型，研究仔猪小肠上皮细胞对甘氨酸亚铁的摄取效率，分析胞内相关跨膜转运蛋白水平及mRNA丰度变化，并运用膜蛋白抑制剂，探讨甘氨酸亚铁小肠跨膜转运可能途径。通过上述研究，旨在探讨甘氨酸亚铁吸收特点及关键跨膜转运蛋白参与其吸收转运的可能机制，为有机微量元素的深入研究与科学应用奠定基础。

有机铁在动物体内有更高的生物学效应，其高生物学效价可能与其高效的吸收转运机制有关。本项目在前期研究基础上，从动物机体及细胞水平研究了甘氨酸亚铁在小肠内的吸收特点及其对相关跨膜转运蛋白的影响。首先以25日龄断奶仔猪为对象，建立临界仔猪缺铁模型，试验分为耗竭期和补铁期，前腔静脉采血，通过检测分析血红蛋白含量、血清铁与血清总铁结合力等参数，确定建立临界缺铁动物模型；在此基础上，探讨了甘氨酸亚铁在缺铁仔猪模型内生物学效应及对铁相关吸收转运蛋白影响，结果表明，与硫酸亚铁相比，甘氨酸亚铁能够快速改善仔猪缺铁状态，提高仔猪生产性能，增强仔猪免疫功能和抗氧化性能，甘氨酸亚铁能够显著提高仔猪十二指肠和空肠中PEPT1 基因水平，同时肠道DMT1基因水平显著低于硫酸亚铁，提示有机和无机铁源在动物肠道内的吸收途径可能不同，可能PEPT1在仔猪肠道内甘氨酸亚铁吸收转运过程中发挥重要作用；项目进一步以SD大鼠为研究对象，研究了甘氨酸亚铁在体内吸收动力学，结果显示，甘氨酸亚铁和硫酸亚铁吸收均符合药物代谢的房室模型，再据R2值最大和AIC值最小原则确定甘氨酸亚铁和硫酸亚铁在SD大鼠体内的药代过程均符合一房室模型，两种不同铁源吸收动力学相关参数显示，相对于硫酸亚铁，甘氨酸亚铁中的铁作为铁源在SD大鼠体内其在动物体内的生物利用度更高，能够更多、更快的被动物机体吸收，并且能够更快的被动物的组织所利用；采用Caco-2细胞吸收模型研究甘氨酸亚铁螯合物的吸收机制，分别考察了浓度、转运方向、时间及温度对转运过程的影响，结果表明硫酸亚铁及甘氨酸亚铁在Caco-2细胞中可能是经过主动转运吸收，其中硫酸亚铁是通过DMT1介导主动转运吸收，而甘氨酸亚铁可能存在特定或非特定的肠道转运系统，甘氨酸亚铁在Caco-2细胞中的转运率明显高于FeSO4。项目进一步研究了活体灌胃不同铁源对SD大鼠小肠内相关转运蛋白的基因与蛋白表达的影响，结果显示，相对于硫酸亚铁，甘氨酸亚铁可降低SD大鼠肠道内DMT1基因及蛋白水平，提高FP1和PepT1基因及蛋白水平，提示有机铁和无机铁被肠道吸收利用方式存在不同，小肽转运蛋白PepT1参与了甘氨酸亚铁的吸收。