羟基蛋氨酸锌对镉致仔猪肠道屏障功能损伤的作用及机制研究

The zinc oxide in feed grade is often found to be contaminated by cadmium, due to its limited technology in preparing and separating zinc oxide from cadmium. However, the excess intake of cadmium will change the intestinal permeability and damage the intestinal epithelial cells. If there is no viable method to remove the cadmium from the feed, the approach to decrease the damage and accumulation of cadmium to the human and animal will be particularly important. Many studies showed that zinc and methionine, as the important trace element and essential amino acid for animals, has the ability of reducing the toxicity of cadmium on animals. While methionine hydroxy analog chelated zinc (MH-Zn), one kind of new-type feed additives, provides not only zinc but also methionine to the body. Moreover, it shows higher feed conversion efficiency, less zinc emission when compared to inorganic zinc. However, the effectiveness of MH-Zn on reparing the cadmium-induced barrier dysfunction of intestinal on piglets is still a pending question. Therefore, this project will study the effect of MH-Zn on the cadmium-induced intestinal barrier dysfunction and intestinal epithelial cell injury by employing 38 days old piglets and IPEC-J2 cell lines to set up the animal models and cell models,separately. Besides, the pattern of dietary MH-Zn regulating NF-κB signaling pathway will also be studied to reveal the mechanism of the protection of gut from cadmium. And the results of this study will provide a theoretical basis for the prevention of cadmium-induced intestinal injury and the efficient use of trace elements.

饲料级氧化锌在制备过程中极难去除重金属镉，因而造成我国断奶仔猪发生了不同程度的镉中毒现象。而食用过量的镉元素会对肠道通透性、肠上皮细胞造成损伤。在无可行方法去除饲料中镉的情况下，如何减少镉对机体的损伤和蓄积尤为重要。研究表明，锌和蛋氨酸均可有效降低镉的致毒性。而新型饲料添加剂羟基蛋氨酸络（螯）合锌（MH-Zn）能给机体同时补充锌和蛋氨酸。更重要的是，其与无机锌相比，饲料转化率更高，锌排放量更少。而关于MH-Zn对镉致仔猪肠道屏障功能损伤的作用有待研究。据此，本项目拟以38日龄仔猪为试验对象，同时建立动物模型和IPEC-J2细胞模型，研究MH-Zn对镉引起的仔猪肠道屏障功能损伤及肠细胞损伤的作用，及其对NF-κB信号通路的调控作用，阐明MH-Zn对镉损伤肠道的作用机理，为缓解或保护镉暴露造成的仔猪肠道损伤以及高效利用微量元素提供理论依据。

镉与微量元素常伴生于自然界中，由于在微量元素添加剂的制备过程中镉较难被去除，因此，我国生猪常面临不同程度的镉暴露。当生猪发生镉中毒时，如何减少镉的蓄积和对机体的损伤尤为重要。本研究主要探讨了新型饲料添加剂羟基蛋氨酸锌（MH-Zn）对仔猪的镉代谢和肠道镉损伤的修复作用。动物试验结果表明，MH-Zn可显著减少镉蓄积，且随着MH-Zn的添加量升高，镉的蓄积逐渐减少；MH-Zn显著缓解了镉导致的肝、肾脂肪变性和颗粒变性；MH-Zn显著减少了镉引起的肠上皮细胞凋亡及肠粘膜屏障功能的损伤。然而，MH-Zn未能减少肠道对镉的吸收，反之，随着MH-Zn添加量的升高，肠道对镉的吸收逐渐增加。为了进一步探讨MH-Zn的作用机制，本团队分析了肠道微量元素和氨基酸转运载体的表达和血液游离氨基酸的含量。结果显示，MH-Zn显著提高了小肠微量元素转运载体DMT1，ZIP8,TRPV6的mRNA表达，由于镉和锌竞争相同的转运载体，且镉对转运载体的亲和力高于锌，因此MH-Zn在促进微量元素转运载体表达的同时，也促进了肠道对镉的吸收。另一方面，MH-Zn促进了部分小肠氨基酸转运载体的表达，提高了8种血清游离氨基酸的含量，表明MH-Zn促进了氨基酸的吸收与转运。细胞试验选用IPEC-J2细胞系建立Cd中毒模型，结果显示，镉（30 mg/kg）引起了细胞凋亡，随着硫酸锌和羟基蛋氨酸锌的添加量增加，细胞凋亡的数量减少。对NF-κB信号通路进行分析，结果显示，与正常细胞比，镉(30mg/kg)可激活 NF-κB信号通路，导致凋亡发生。加入60 mg/kg和90 mg/kg的硫酸锌后， NF-κB及通路上游相关蛋白的磷酸化水平显著降低，但90 mg/kg硫酸锌有激活NF-κB型号通路的趋势。将60 mg/kg和90 mg/kg的MHZn与镉同时处理细胞, NF-κB信号通路同样被抑制，且90mg/kg的效果优于60mg/kg。综上所述，MH-Zn虽然促进了镉的吸收，但也可能加速了镉的代谢并以尿的形式排出体外，从而减少了仔猪体内的镉蓄积及造成的脏器损伤，这一系列作用可能与氨基酸代谢、锌代谢密切相关。据此，本研究为仔猪镉中毒的临床治疗和氨基酸螯合锌的合理使用提供了理论基础和实践依据。