铁过载诱导断奶仔猪肠道损伤的机制研究

Intestinal functions in piglets are not well developed after weaning, excessive iron in feed can interact with intestine directly and cause intestinal damage on piglets. Previous studies in iron toxicity have shown that the major toxic effect of iron is the result of the exposure of cellular macromolecules to ROS, meanwhile, a significant part of iron toxicity may involve the activation of specific signaling pathway. While, the precise mechanism of iron induced intestinal damage of piglets is still unknown. On the basis of previous studies, feeding trial, cell monolayer model experiment and damage evaluation are designed in current project to study the effects of iron induced intestinal damage on piglets and a newborn swine intestinal epithelial cell line (IPEC-1). Iron metabolism pattern, ROS generation, oxidative stress and sphingolipid signaling pathway activation level are determined in vivo to investigate the mechanism of iron induced intestinal damage on piglets. Redox active iron, ROS and mitochondrial membrane potential are tracked and located under the circumstances of ROS and mitochondrial permeability transition inhibition by fluorescent to study the mechanism of ROS mediated damage on intestinal epithelial cells. Under RNA interference and protein inhibition, protein expression and phosphorylation level of kinases are detected to study the toxicity function of sphingolipid signaling pathway in IPEC-1. These studies will provide us with more details about the mechanism of iron induced intestinal damage on piglets and guide us to use iron scientifically in animal husbandry, also help us find desirable therapeutic methods for iron overload induced diseases.

断奶仔猪的肠道功能尚不成熟，日粮中过量铁易造成断奶仔猪肠道损伤。前期研究发现过量铁能够催化活性氧（ROS）生成并攻击细胞大分子物质，同时可能通过特定的信号通路来发挥毒性，然而过量铁造成断奶仔猪肠道损伤的机制尚无定论。本项目在前期研究基础上，以断奶仔猪及其肠上皮细胞为对象，拟通过饲养试验、细胞单层模型及损伤评估，研究过量铁对肠道的损伤效应；测定机体铁代谢、ROS、氧化应激及鞘脂信号通路激活水平，探讨过量铁在体水平的肠道损伤机制。以仔猪肠上皮细胞为模型，结合ROS及线粒体通透性抑制，荧光示踪和定位胞内毒性铁离子、ROS及线粒体膜电位，研究ROS介导肠上皮细胞损伤的机制；同时针对鞘脂信号通路关键激酶，进行蛋白抑制及RNA干扰，研究蛋白表达及其磷酸化水平，探讨鞘脂信号通路介导肠上皮细胞损伤的机理。通过上述研究，揭示过量铁诱导断奶仔猪肠道损伤的可能机制与通路，为畜牧生产科学应用铁元素提供理论依据。

断奶仔猪的肠道功能尚不成熟，日粮中过量铁易造成断奶仔猪肠道损伤。本项目在前期研究基础上，以断奶仔猪、SD大鼠及多种细胞为对象，通过饲养试验、灌胃模型、细胞模拟进行损伤评估，研究过量铁对机体的损伤效应。在断奶仔猪中，高剂量铁饲喂会改变断奶仔猪肝脏铁代谢，产生自由基，降低抗氧化酶活性，激活Nrf2信号通路；高剂量铁导致断奶仔猪肠绒毛造成损伤，诱导氧化应激，降低铁吸收蛋白，破坏肠道屏障，改变肠道微生物结构。在SD大鼠中，灌胃过量铁能够导致显著的肠道和肝脏损伤，并出现组织病理学改变，线粒体膨胀和细胞死亡等现象；过量铁能够破坏肠道屏障功能，改变肠道菌群结构，后肠段损伤和通透性的增加对整个肠道健康存在不利影响。在细胞模型中，FAC诱导的慢性铁过载导致HT-1080细胞铁死亡，鞘脂信号通路可能参与到铁死亡过程当中，同时筛选到了一个有效的铁死亡抑制剂GSK2334470；Fe-8HQ诱导的急性胞内铁过载能够部分激活依赖性死亡，最终诱导不可调控的细胞死亡，酚类化合物能够有效地抑制该细胞死亡。通过上述研究，揭示过量铁诱导断奶仔猪肠道损伤的可能机制与通路，为畜牧生产科学应用铁元素提供理论依据。