T-2毒素诱导及DL-硒代蛋氨酸颉颃肉鸡肝脏氧化应激的分子机制研究

T-2 toxin induces oxidative stress, while Nrf2-ARE is the major pathway of anti-oxidative stress in cells, however, whether Nrf2 signaling axis mediates T2-induced oxidative stress in primary hepatocytes of broilers remains unclear. DL-SeMet is the key amino acid of the anti-oxidase (GPx) in downstream of Nrf2-ARE signaling, however, whether there is a direct or indirect relationship between DL-SeMet and Nrf2-ARE in hepatocytes are still unknown. We in this project raise the hypothesis that Nrf-ARE is the major pathway mediating the pro-oxidative stress effects by T2 toxin and anti-oxidative stress effects by DL-SeMet in primary hepatocytes of broilers. A series of experiments are conducted to substantiate this hypothesis. Firstly, to determine whether Nrf2-ARE axis has important regulatory role in T2-induced oxidative stress, the expression of Nrf2 and its downstream factors is examined following the upregulation and downregulation of Nrf2, respectively in T2-treated primary hepatocytes isolated from broilers. Next, to examine whether DL-SeMet protects against T2-induced oxidative stress, the state of oxidative stress is analyzed following the treatment of hepatocytes or animals with T2 in the presence or absence of DL-SeMet. Finally, to address whether DL-SeMet has a feedback influence on GPx and its upstream signaling Nrf-ARE, the expression of them is examined both in vitro and in vivo. The resultant data not only provide novel hints for self-regulating mechanism of cells, but also lay a scientific foundation for the development of new feed additives to broilers.

T2导致细胞发生氧化应激，而Nrf2-ARE通路是细胞抗氧化应激的主要途径，但该信号轴是否参与T2诱导的肉鸡肝细胞氧化应激尚不明确。DL-SeMet是合成处于Nrf2-ARE下游的抗氧化酶GPx的关键氨基酸，但两者是否存在直、间接联系，同样缺少研究。本项目假设“Nrf2-ARE是介导T2诱导及DL-SeMet颉颃肉鸡肝细胞氧化应激的主要途径”，拟首先通过T2构建氧化应激体外模型，分别上、下调Nrf2表达，再检测Nrf2及下游因子变化，从而确定Nrf2-ARE在T2诱导的氧化应激中所发挥的作用。接着用DL-SeMet处理T2氧化应激体内、外模型，通过分析氧化应激水平，确定DL-SeMet是否具有颉颃作用；最后分析DL-SeMet对GPx及其上游信号的调控作用，进而揭示DL-SeMet对该通路的负调节机制。研究成果将为研究细胞自我调控提供新的启示，并为研发肉鸡的新型饲料添加剂奠定理论基础。

T-2毒素（T2）可诱导细胞氧化应激，而Nrf2-ARE通路是细胞抗氧化应激的主要途径，但该信号轴是否参与T2诱导的鸡细胞氧化应激尚不明确。DL-硒代蛋氨酸（DL-SeMet）是合成Nrf2-ARE下游的抗氧化酶谷胱甘肽过氧化物酶（GPX）的关键氨基酸，但两者是否存在直、间接联系，同样缺少研究。针对上述问题，我们展开如下研究：A）用T2和HT-2毒素（HT2）构建肉鸡肝细胞氧化应激模型。B) 用DL-SeMet处理该细胞模型，检测肝细胞氧化应激水平，研究DL-SeMet的抗氧化作用。C）用T2构建氧化应激鸡成纤维细胞（DF-1细胞）模型。D) T2处理“Nrf2过表达—DF-1细胞系”和“Nrf2干扰—DF-1细胞系”，分析细胞中Nrf2-ARE通路相关因子mRNA和蛋白的表达水平，探究Nrf2-ARE在T2诱导的氧化应激中的作用。通过上述研究取得的主要结果如下：.1) T2与HT2导致肉鸡原代肝细胞活性下降，LDH漏出率显著升高；且死亡细胞数呈毒素剂量依赖性升高。.2) DL-SeMet降低T2/HT2造成的细胞毒性作用和氧化应激水平，提高抗氧化酶活性。.3) T2导致DF-1细胞损伤，诱导氧化应激，且Nrf2-ARE通路参与此过程。.4) 通过上调及下调Nrf2蛋白，明确Nrf2-ARE通路调控T2所致氧化应激，且Nrf2蛋白在细胞抗氧化过程中所发挥重要的作用。.上述研究成果与发现的意义在于确定：1) T2诱导DF-1细胞的氧化应激受Nrf2-ARE通路调控，这为研究细胞的自我调控机制提供启示；2) DL-SeMet对氧化应激的颉颃作用，为肉鸡生产养殖中新型饲料添加剂生产提供科学基础。.