围产期奶牛乳腺细胞产生氧化损伤的分子机制研究

Oxidative stress is a major problem in the dairy farming industry, which is mainly the reduction of antioxidative function in the production and scavenging of free radicals caused by the imbalance. Therefore, it has great significance in improving the pathogenesis and treatment of transition dairy cow to clarify the causes that mammary cell of transition dairy cow is susceptible to oxidative damage and its molecular mechanism. It is clarified that oxidative stress of transition dairy cow is whether induced or not in this study. Health mammary cells of dairy cow are selected as the research object, using oxidative stressed mammary cells of dairy cows induced by H2O2, this study determine the role of Nrf2-ARE signaling pathways in the defense against oxidative stress in mammary cells, to analyze of whether there is abnormal condition of Nrf2-ARE signaling pathways between mammary cells of healthy and oxidative stressed dairy cows, and the underlying reason and mechanism for the aberrant activation of Nrf2-ARE signaling pathway in mammary cells of oxidative stressed dairy cows is clarified. This study systemly illustrates the oxidative mechanism of Nrf2-ARE signaling pathway damaged mammary cells of transition dairy cows from the mRNA level and protein level by the combination of animal experiment and cell culture, which has important significance to ensure that the dairy cows can maintain the normal function of lactation and improve production performance during transition.

氧化应激已成为困扰围产期奶牛生产中的一大问题，其主要是由于机体抗氧化功能降低导致自由基产生和清除失衡所致，阐明围产期奶牛乳腺细胞易受到氧化损伤的原因及其具体分子机制对于完善围产期氧化应激发病机理、指导临床治疗具有重要意义。本研究在明确围产期奶牛机体是否产生氧化应激的基础上，以健康奶牛乳腺细胞为研究对象，采用H2O2诱导乳腺细胞建立氧化应激模型，确定Nrf2-ARE通路在保护奶牛乳腺细胞抵抗氧化应激损伤中的作用，对比分析健康和产生氧化应激奶牛乳腺细胞中Nrf2-ARE信号通路是否存在异常及该通路在产生氧化应激奶牛乳腺细胞中产生激活障碍的原因和机制。本项目通过动物试验和细胞培养试验相结合的方式，从mRNA水平和蛋白水平方面系统阐述了Nrf2-ARE通路对围产期奶牛乳腺细胞产生氧化损伤的分子机制，对于确保奶牛在围产期内能够维持正常泌乳功和提高生产性能具有重要意义。

氧化应激已成为困扰围产期奶牛生产中的一大问题，其主要诱因是围产期奶牛能量负平衡和机体炎症而导致机体产生大量的活性氧/自由基，导致奶牛在围产期间产生以隐性乳房炎和亚临床型酮病为主的代谢病，进而降低奶牛机体健康和生产性能。因此，阐明围产期奶牛乳腺细胞易受到氧化损伤的原因及其具体分子机制对于完善奶牛围产期氧化应激发病机理和指导临床治疗具有重要意义。本研究通过测定呼和浩特周边牧场围产期间奶牛的血液指标和细胞培养实验发现：⑴围产期间隐性乳房炎奶牛自产前20天至产后20天血液中抗氧化酶活性、免疫性能和生产性能显著低于健康奶牛，而自由基含量和炎性因子含量显著高于健康奶牛；⑵在此基础上用H2O2诱导构建奶牛乳腺上皮细胞氧化损伤模型，结合H2O2对乳腺上皮细胞存活率和抗氧化指标的影响，得出当H2O2作用浓度和时间分别为600 μM和6h时，乳腺上皮细胞产生了较为明显的氧化损伤，可作为构建乳腺上皮细胞氧化损伤模型的最佳条件；⑶氧化应激条件下，NFE2L2转录活性下降，说明NFE2L2在氧化应激条件下存在激活障碍；⑷氧化应激条件下，乳腺上皮细胞中NFE2L2存在激活障碍的主要原因是ATM蛋白激酶表达出现异常，减少了NFE2L2的入核，降低了NFE2L2与ARE的结合，进而降低了NFE2L2-ARE通路下游抗氧化基因HMOX-1、NQO1、GCLC和GCLM的表达，最终导致乳腺上皮细胞产生凋亡或坏死。本项目通过体内动物试验和体外细胞培养相结合的方式，从分子水平方面系统揭示出了NFE2L2-ARE信号通路对围产期奶牛乳腺细胞产生氧化损伤的分子机制，NFE2L2-ARE信号通路是乳腺上皮细胞抵抗氧化损伤最主要的防御机制之一，可保护乳腺上皮细胞免受氧化损伤。该项目的研究结果对于确保奶牛在围产期内能够维持正常泌乳功能、提高生产性能和指导临床治疗具有重要意义，同时为围产期奶牛日粮中合理应用抗氧化剂茶多酚或番茄红素提供了前期基础。