机体锌稳衡在锌抗肉鸡沙门氏菌感染的作用及机理研究

Salmonella enterica serovar Typhimurium (S.Typhimurium) is a gut pathogen that causes an acute gastroenteritis characterized by inflammatory diarrhea. Food-borne diseases in humans caused by members of Salmonella remain a major public health concern worldwide and are commonly associated with the consumption of broiler chicken meat contaminated with salmonellas. It is very important to prevent the intestinal infection of chickens with salmonellas. Zinc (Zn) is an essential trace mineral with lots of important function. Zinc homeostasis in body and cell are well controlled by Zn transporter proteins (ZnT 1-10, ZIP 1-15), metallothionein (MT).Research in rat showed that Zn has nutritional immunity with the decrease of free Zn level in serum and Neutrophils to prevent the S.Typhimurium to get the necessary Zn. This was induced by Calprotectin with the secretion stimulated by Zn. There is lack of research papers about the role of Zn-homeostasis in broiler with S.Typhimurium infection. In this study, we study the Zn-homeostasis in broiler body, tissue, cells under S.Typhimurium infection by using feeding trial, the method of in situ ligated Loops in vivo, and cell culturation in vitro, while the relationship of Zn-homeostasis with the expression of Zn transporter proteins (ZnT 1-10, ZIP 1-15), metallothionein (MT) and Calprotectin mPNA．The purpose is to understand the role of Zn-homeostasis in body, tissues, and cells in Zn anti-Salmonella infection.

沙门氏菌感染不仅导致肉鸡肠道炎症、生产性能下降，而且可能污染禽肉引发人食入性疾病,预防和控制家禽沙门氏菌感染非常重要。锌是生物必需的微量元素，具有重要的生物学功能。哺乳动物主要通过锌转移蛋白ZNT(Slc30a)与ZIP（Slc39a）家族调控锌的吸收与转运来维持机体的锌稳衡，保证其正常的生理功能。在鼠上的研究表明，锌具有“营养免疫作用”。在细菌感染时，机体锌稳衡发生改变（血清锌降低与锌被重分布）以减少细菌获取锌而增殖，其机理可能与锌调控钙卫蛋白分泌而螯合细菌可利用的锌有关。目前，在肉鸡上尚未见研究报道。本研究采用动物模型和原位结扎灌注肠段法，从锌吸收、转运与重分布等方面来研究肉鸡在不同锌状态下沙门氏菌感染对机体锌稳衡状态的影响；同时体外研究沙门氏菌感染时肉鸡肠上皮细胞与中性粒细胞的锌稳衡调控，并探讨钙卫蛋白与机体锌稳衡的关系，深入阐述机体锌稳衡在锌抗肉鸡沙门氏菌感染的作用及机理。

沙门氏菌严重威胁肉鸡生产和人类健康。本项目通过肉鸡饲养试验结合沙门氏菌攻毒，研究不同锌营养状况对整体、组织和细胞锌稳衡的影响及机理，证实沙门氏菌攻毒导致肉鸡锌稳衡改变，血清Zn下降，而肝、脾等组织锌增加；肠道ZIP5、ZIP10、ZIP11、ZIP12、ZIP13与ZIP14表达下降以降低锌吸收和转运；肝脏ZIP14表达上调，ZNT1、ZNT4、ZNT5、ZNT6、ZNT8与ZNT9 的表达下调以增加锌沉积；中性粒细胞的钙卫蛋白分泌增加以抑制沙门氏菌利用锌；证实饲粮锌缺乏和高锌均降低锌稳衡调控灵敏性，高锌导致肠道ZIP6、ZIP8、ZIP11、ZnT5、ZnT7和ZnT9表达上调，ZnT2和ZnT6表达下调以抑制肠道锌摄入和促进锌转出；高锌上调肉鸡肝脏的ZIP6、ZIP9、ZnT5、ZnT 6、ZnT 7和ZnT9表达，增加肝脏锌含量，降低盲肠微生物多样性，增加致病菌丰度。通过体外细胞培养证实沙门氏菌导致中性粒细胞的钙卫蛋白和MT、ZIP3、ZIP8、ZIP10、ZIP14及ZNT10表达上调，高锌时需要上调更多ZIP和ZNT表达、降低胞内钙卫蛋白和MT表达上调程度，利于沙门氏菌存活；通过采用免疫荧光、CRISP-CASE9基因敲除、流式细胞分选技术等技术手段，证实沙门氏菌感染导致巨噬细胞游离锌增加，抑制NF-κB激活、一氧化氮合酶和NADPH氧化酶等表达，降低ROS与RNS产生，进而降低巨噬细胞对沙门氏菌的清除能力。证实沙门氏菌和球虫+产气荚膜梭菌混合攻毒对肉鸡锌稳衡的影响和机制不同，混合攻毒主要损伤肠道，影响锌的吸收，混合攻毒或沙门氏菌攻毒均引起肠道微生物区系变化；有机锌改善肠道屏障功能，在混合攻毒条件下使用有机锌效果更好；在沙门氏菌攻毒条件下，有机锌效果与无机锌无明显差异。利用本研究成果“锌营养免疫作用”，有助于推动肉鸡生产实现“限抗禁抗”和“减锌减铜”目标。