MAPKs/Nrf2-ARE信号途径介导钒中毒引发蛋鸡膨大部管腺细胞氧化应激的研究

Magnum is the main position responsible for the egg white (albumen) synthesis(55-65%). The cationic form of vanadium complexes with oxidation state IV (4+) and V (5+) function as modulators of cellular redox potential, regulate enzymatic phosphorylation, and exert pleiotropic effects in multiple biological systems by catalyzing the generation of reactive oxygen species (ROS). The oviduct of laying hens are constantly exposed to oxidative stress per se and the environmental co-contaminant of most concern are heavy metals, typified by vanadium (5+). It was evident that dietary vanadium supplementation at low level (3mg/kg) can impair the interior egg quality, especially the albumen height and Haugh unit (storage quality) in laying hens. However, the mechanism is not clear. The redox status of the cell activates the nuclear factor erythroid 2-related factor-2 (Nrf2), a redox-sensitive member of the cap ‘n’ collar basic leucine zipper (CNC bZip) family of transicription factors. Subsequently, Nrf2 dissociates from its cytoplasmic tethering polypeptide, Kelch-like ECH associating protein 1 (Keap1), and then translocates into the nucleus, dimerizes with a musculoaponeurotic fibrosarcoma (MAF) protein, thereafter binds to and activate ARE. Mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 MAPK transmit extracellular signals into the nucleus, and have been shown to participate in a diverse array of cellular functions such as cell growth, differentiation and apoptosis.. Treatment with cadmium, inorganic mercury or tributyltin can activate ERK, JNK and p38 MAPK, and induces the expression of c-fos and c-jun genes prior to the development of apoptosis. However, the members of the MAPK family appear to be differentially activated depending on the heavy metal and the cell type exposed. Consequently, various cellular responses may be caused by the distinct pattern of MAPKs activation. MAPKs may be one of the important cellular signal transduction pathways affected by various environmental pollutants, including heavy metals, like vanadium.. In this study, we evaluate the hypothesis in vivo and in vitro that the toxicity of vanadium to albumen quality is through oxidative stress and damage to magnum of laying hens' oviduct. And this function may through the activation of MAPKs/Nrf2-ARE signing pathway, which can be inhibited by vanadium-induced oxidative stress, by modulating the translocation of Nrf2 and therefore to down-regulate the transcriptional expression of phase 2 detoxifying enzyme (NQO-1,HO-1,GCLC) in tubular gland cells of magnum...

本研究围绕“钒的毒性是通过产生氧自由基介导MAPKs/Nrf2-ARE信号通路，导致蛋鸡输卵管膨大部氧化应激，损伤了输卵管管腺细胞结构和功能，从而影响蛋清蛋白质的分泌，导致蛋清品质下降”的科学假设，通过蛋鸡体内试验研究证实钒中毒引发鸡蛋蛋清品质的降低是通过氧化应激损伤输卵管形态和功能的完整性来实现的；然后，再通过体外培养蛋鸡输卵管膨大部管腺细胞，从细胞水平研究钒可以通过引起的管腺细胞的氧化损伤和凋亡，从而减少蛋清蛋白质的分泌；同时通过使用JNK抑制剂（SP600125）,ERK1/2抑制剂（PD98059）和p38MAPK抑制剂(SB203580),考察与钒引起的氧化应激相关的细胞信号通路，证实MAPKs/Nrf2-ARE信号通路介导钒中毒的分子机制。

饲料安全是影响动物食品安全的最主要和最直接的因素，钒虽然是动物机体必需元素，但过量的钒摄入，具有潜在的毒性，家禽尤为敏感，但其机制并不清楚，且我国饲料卫生标准中并未规定其限量，有待进一步完善。因此本研究围绕“钒的毒性是通过产生氧自由基介导MAPKs/Nrf2-ARE信号通路，导致蛋鸡输卵管膨大部氧化应激，损伤了输卵管管腺细胞结构和功能，从而影响蛋清蛋白质的分泌，导蛋清品质下降”的科学假设，通过蛋鸡试验本项目发现饲粮中钒含量达到5mg/kg以上就可以降低蛋品质，这可能通过钒引起蛋鸡输卵管膨大部氧化应激，导致输卵管膨大部结构和功能的损伤，从而降低蛋品质；进一步通过添加抗氧化剂茶多酚可以通过降低氧化应激, 并通过调控金属结合（E1C5J4），细胞增殖（F1NXO5和E1BT2）和免疫应答（F1P3B2, P21760, A2N881, F2Z4L6和P02789）相关蛋白的表达，从而缓解由钒引起蛋品质的降低；然后，再通过体外培养蛋鸡输卵管膨大部管腺细胞，从细胞水平研究钒可以通过引起的管腺细胞的氧化损伤和凋亡，从而减少蛋清蛋白质的分泌；同时通过使用JNK抑制剂，ERK1/2抑制剂和p38MAPK抑制剂,考察与钒引起的氧化应激和细胞凋亡相关的细胞信号通路，揭示了钒可以通过ROS的增多，激活MAPK/Nrf2-ARE信号通路，降低Nrf2核转位，引发输卵管膨大部氧化应激的分子机制；并进一步探明了钒可以引起Bax/Bcl-2比例增加，造成膨大部上皮细胞OMECs的线粒体膜电位去极化程度增高，引起Cyt C释放增多，招募并激活Caspase-3，造成PARP1裂解，最终导致OMECs细胞凋亡的分子机制。本课题研究通过动物试验和细胞实验相互结合的方式，从分子水平揭示了钒通过MAPK-Nrf2信号通路介导细胞氧化应激和细胞凋亡导致蛋鸡蛋品质降低的机制，为人类食品安全特别是重金属钒的安全剂量以及缓解钒的技术提供理论依据，保障了畜牧业健康可持续发展，同时也为深入认识蛋品质营养调控原理具有重要的理论和实践意义。