锌对镉中毒肉鸡巨噬细胞损伤和免疫功能的调控作用及机制研究

Cadmium poisoning is a kind of heavy metal poisoning disease seriously threatens human and animals' health. In recent years, cadmium content in grain which exceeds standards has been continuously reported in China and this arouses wide concern among the people. Macrophages belong to the member of the body's natural immune system; cytotoxicity and immune effect of cadmium on macrophage are of great importance for the body to resist cadmium poisoning as well as secondary infection. However, no reports concerning effects of cadmium on macrophage have been found until now. Study shows that appropriate dose of zinc addition can effectively inhibit cadmium poisoning while the mechanism has not been completely understood. We found in our previous study that zinc addition could significantly promote metal responsive transcription factor1 (MTF-1) and metallothionein (MT) expression in cadmium exposed cells which possibly participate in macrophage immune function regulation. The aim of this study is to investigate the effect of cadmium on the cytotoxicity and immune function of macrophage in broiler. At the meantime, we analyze the regulation of zinc on the macrophage immune function through adding zinc to cadmium treated cells. In addition, by inhibiting MTF-1 gene expression with MTF-1 siRNA and chelating intracellular Zn2+, we are to systematically reveal the regulation mechanism of zinc on macrophage immune function. Results of this project are expected to provide references for the mechanism study and treatment of cadmium poisoning.

镉中毒是严重威胁人和动物健康的一类重金属中毒性疾病。镉对巨噬细胞的毒性损伤和免疫功能的影响对机体抵御镉中毒和继发性感染至关重要，而目前尚无此方面的研究报道。研究表明适量浓度的锌添加能有效对抗镉引起的细胞毒性，但作用机理还不清楚。申请人前期研究发现锌能显著促进镉攻毒细胞中金属反应转录因子(MTF-1)和金属硫蛋白(MT)的表达，而MTF-1和MT对巨噬细胞免疫功能的发挥具有潜在的调控作用。为此本项目拟通过体内试验和体外培养两种方式，研究镉对肉鸡巨噬细胞的毒性损伤和免疫功能的影响，并通过添加锌离子的方式观察锌对镉中毒巨噬细胞损伤和免疫功能的调控作用。另外项目分别通过对镉/锌处理巨噬细胞进行MTF-1 siRNA干扰和Zn2+螯合，系统探讨锌对镉中毒巨噬细胞损伤和免疫功能基于MTF-1功能激活和MT表达促进的调控作用机理，为镉中毒机制研究提供新的数据，同时为镉中毒的防治提供新的思路和手段。

本项目旨在研究镉对肉鸡巨噬细胞的毒性损伤和免疫功能的影响，并通过添加锌离子的方式探索锌对镉染毒巨噬细胞损伤和免疫功能的调控作用及机理。结果显示：镉攻毒造成鸡肉鸡巨噬细胞和小鼠巨噬细胞时间-浓度相关性毒性损伤，锌添加能显著降低镉染毒导致的鸡巨噬细胞凋亡、ROS过度产生和线粒体损伤。锌添加能显著减少Cd2+在鸡巨噬细胞内聚集。锌补充能显著提高镉染毒巨噬细胞中金属反应转录因子（MTF-1）和金属硫蛋白（MTs）的表达。Zn2+螯合以及MTF-1基因沉默实验证明，锌对镉染毒巨噬细胞的保护作用主要是通过动员和激活MTF-1蛋白，继而促进MTs表达来实现的。MTs通过螯合Cd2+以及提高细胞抗氧化能力来对抗镉中毒。镉攻毒造成肉鸡巨噬细胞炎性细胞因子（IL-1β、IL-6和TNF-α）基因表达紊乱，锌补充能不同程度地抑制镉染毒巨噬细胞炎性因子的异常表达。另外，项目系统研究了镉对肉鸡红细胞的毒性损伤，以及锌补充对镉染毒肉鸡红细胞的保护作用。结果表明，镉染毒肉鸡红细胞出现严重的细胞凋亡，红细胞内ROS过量聚集，抗氧化酶含量显著下降。镉染毒肉鸡红细胞出现多种畸形核。体内锌补充能有效抑制镉攻毒引起的肉鸡红细胞损伤，提高机体抗氧化酶活性，降低镉染毒红细胞凋亡率和ROS聚集。以上结果表明，镉对肉鸡巨噬细胞、红细胞均产生严重的细胞毒性，锌补充能有效拮抗镉诱发的细胞毒性。锌对镉染毒细胞的保护作用可以通过动员和激活MTF-1，进而促进细胞MTs表达来实现。项目结果为人和动物镉中毒的防治提供了新的思路和可能。