不同形态锰在肉鸡小肠中吸收的分子机制

Absorptions of trace elements in the small intestine of animals and their molecular mechanisms have been a tough issue for animal nutritionists. Based on series of novel findings from our previous studies on manganese (Mn) absorptions and utilizations of different forms of Mn in broilers, the effect of different forms of Mn on gene expressions of divalent metal transporter 1 (DMT1) , ferroportin (FPN1), and transporters for neutral, dibasic or excitatory amino acids in Caco-2 cell line, ligated duodenal loops of broilers, and the duodenum of intact broilers will be investigated; and Mn transepithelial transports of different forms of Mn in DMT1 expression-inhibited Caco-2 cell line as well as in cells cultured with single inhibotor of neutral, dibasic or excitatory amino acid transporter or their combination will be determined; and Mn absorptions of different forms of Mn in ligated duodenal loops perfused with higher ferrous iron or single inhibitor of neutral, dibasic or excitatory amino acid and their combination will be estimated, so as to elucidate whether DMT1 that has been recognized as the transporter for ionic Mn will also mediate the transepithelial transport of organic Mn, and whether FPN1 and amino acid transporters will be involed in transepithelial transports of Mn, and there will be a difference between inorganic and organic forms of Mn. As the results, this project will generate unique and new information on molecular mechanisms of absorptions of Mn in the small intestine of broilers. It will also be of great importance for alternative strategy to devalop and apply highly bioavailable new organic Mn additives to promote the growth of broilers, and minimize the environmental pollution of Mn excreted by animals.

本项目在申请人及合作单位前期肉鸡不同形态锰吸收利用系列最新研究成果的基础上，通过观测不同形态锰对体外培养Caco-2细胞、肉鸡原位结扎十二指肠和自然饲喂肉鸡十二指肠中二价金属转运蛋白1(DMT1)、膜铁转运蛋白(FPN1)、中性、碱性和酸性氨基酸转运载体mRNA和翻译蛋白水平的影响、不同形态锰在DMT1表达抑制的Caco-2细胞、添加高铁抑制剂的原位结扎十二指肠及单独或同时添加上述三类氨基酸转运载体抑制剂的Caco-2细胞和原位结扎十二指肠中的转运吸收特点和规律，分别从体外、半体外半体内和全体内研究DMT1是否参与有机锰的转运吸收及FPN1和氨基酸转运载体是否参与锰的转运吸收及不同形态锰间的差异，从而系统、深入揭示不同形态锰在肉鸡小肠中吸收的分子机制，对于肉鸡生产中科学研制和应用高效吸收利用的新型锰添加剂，减少锰排出对环境的污染，具有很重要的理论与现实指导意义。

本项目在申请人及合作单位前期肉鸡不同形态锰源吸收利用系列最新研究成果的基础上，通过观测不同形态锰对体外培养 Caco-2 细胞、肉鸡原位结扎十二指肠和自然饲喂肉鸡十二指肠中二价金属转运蛋白1(DMT1)、膜铁转运蛋白(FPN1)、中性、碱性和酸性氨基酸转运载体 mRNA 和翻译蛋白水平的影响、不同形态锰在 DMT1表达抑制的 Caco-2 细胞和添加高铁抑制剂的原位结扎十二指肠中的转运吸收特点和规律，系统、深入地研究了DMT1是否参与有机锰的吸收转运及FPN1和氨基酸转运载体是否参与锰的转运吸收及不同形态锰间的差异，取得了国内、外文献中均未见报道的创新性研究新成果：（1）不同形态锰源在体外培养的Caco-2细胞中主要以饱和载体转运的方式转运吸收，不同形态锰源在DMT1表达抑制Caco-2 细胞模型中的吸收未表现出差异，表明DMT1和FPN1参与了细胞顶膜无机和有机锰的摄取和基底膜锰的排出；（2）自然饲喂中等和强络合强度有机锰源组肉仔鸡肝门静脉血浆锰含量及十二指肠DMT1 mRNA和蛋白表达量显著高于无机硫酸锰和弱络合强度有机锰源组，这种正相关提示DMT1参与了肠黏膜细胞顶膜有机锰的摄取；添加锰显著提高十二指肠FPN1 mRNA和蛋白表达量，表明FPN1参与了肠黏膜细胞内锰由底膜的排出；（3）饲粮铁通过降低肉鸡十二指肠黏膜DMT1和FPN1的表达降低原位结扎灌注十二指肠中锰的吸收和组织中锰的沉积，表明铁与锰因共用相同的转运载体而产生拮抗作用；中等和强络合强度有机锰源在添加高铁抑制剂原位结扎灌注十二指肠中的吸收显著高于无机硫酸锰，表明中等和强络合强度有机锰源可有效抵抗高铁对锰吸收的抑制作用；（4）不同形态锰源对自然饲喂肉鸡小肠各段中性（B0AT1和LAT2）、碱性（rBAT和y+LAT1）和酸性（EAAT3）氨基酸转运载体及小肽转运载体（PepT1）mRNA表达均未产生显著影响，说明上述转运载体可能均未参与有机锰的转运吸收。以上研究新成果揭示了不同形态锰源的吸收规律及其分子机制，对于肉鸡生产中科学研制和应用高效吸收利用的新型有机锰添加剂，减少锰排出对环境的污染，具有很重要的理论与现实指导意义。