基于氧化应激的T-2毒素致GH3细胞线粒体损伤机制研究

T-2 toxin is widespread in cereals and feed, which has strong toxicity. And the toxicity of T-2 toxin is difficult to be reduced. Long-term exposure to the toxin will seriously inhibit the growth of animals, which will result in huge economic losses in livestock production. Recent studies showed that T-2 toxin could cause oxidative stress in cells, which has been regarded to be an important mechanism to explain the toxic effects of the toxin. However, to T-2 toxin, the mechanism by which way induces oxidative stress still remains unclear. Our previous studies have shown that, in GH3 cells, T-2 toxin could result in the significant decrease of mitochondrial membrane potential, the oxidative stress, and the significant increase of the mitochondrial respiratory chain enzyme complex subunits NDUFS3. As known, NDUFS3 is closely related to the production of ROS. In the present study, based on the genomic and proteomic studies, NDUFS3 will be selected to investigate its potential role in mitochondrial dysfunction, oxidative stress and the transcription factor (TFAM) and the upstream signaling pathway cAMP/PKA-CREB-PGC-1α mediating the expression of respiratory chain subunits by using various techniques, such as the chromatin immunoprecipitation technique, RNA interference, overexpression of protein and electron microscopy. Various parameters including the mitochondrial membrane potential, ATP content, morphological changes and the parameters of the oxidative stress were detected to reveal the potential role of NDUFS3. The present study will throw a new light to the prevention of T-2 toxin and the ensurance of animal health.

T-2毒素在谷物和饲料中广泛存在，具有毒性强、脱毒困难等特点，长期暴露于该毒素中将严重抑制动物生长，给畜牧业生产带来巨大经济损失。最近的研究表明，T-2毒素引起细胞产生氧化应激是其一个重要的毒性作用机制。但T-2毒素是如何诱导细胞产生氧化应激的机制尚不清楚。我们前期研究表明，T-2毒素能引起GH3细胞线粒体膜电位明显降低、氧化应激的产生、显著上调与ROS产生密切相关的线粒体呼吸链酶复合物亚基NDUFS3表达。本研究在前期基因组和蛋白组学基础上，以NDUFS3为切入点，应用染色质免疫共沉淀技术、RNA干扰、蛋白超表达、电镜等技术，并结合线粒体膜电位、ATP含量、形态变化和氧化应激指标等阐释该毒素作用下，线粒体亚基在线粒体功能障碍和氧化应激中的作用及其与调控呼吸链亚基表达的转录因子TFAM和上游信号通路cAMP/PKA-CREB-PGC-1α间的相互作用，为毒素预防和保证动物健康奠定坚实基础。

T-2毒素在谷物和饲料中广泛存在，具有毒性强、脱毒困难等特点，长期暴露于该毒素中将严重抑制动物生长，给畜牧业生产带来巨大经济损失。前期研究表明，T-2毒素能引起GH3细胞线粒体膜电位明显降低、氧化应激的产生、显著上调与ROS产生密切相关的线粒体呼吸链酶复合物亚基Ndufs3表达。本研究在前期基因组和蛋白组学基础上，以Ndufs3为切入点，揭示T-2毒素的线粒体毒性机制。研究发现，T-2毒素作用下，沉默Ndufs3能进一步显著增加超氧自由基产生，降低复合物I活性和线粒体ΔΨm，说明Ndufs3表达缺陷能够引起线粒体功能障碍，也会增加GH3细胞对T-2毒素易感性，使线粒体更容易受到T-2毒素损伤。研究表明，Tfam在调控GH3细胞中呼吸链复合物I核心亚基Ndufs3的表达起着关键性作用。T-2毒素可以显著性地增加Tfam和Ndufs3基因启动子核心位点结合。T-2毒素显著增加GH3细胞中PGC-1α、Nrf-1基因和蛋白表达水平，然而通过免疫共沉淀技术初步确定PGC-1α与Nrf-1无直接相互作用。T-2毒素可显著上调PGC-1α和Nrf-2的基因和蛋白表达水平，增加线粒体转录因子Tfam的表达，进而影响GH3细胞中氧化应激水平。干扰PGC-1α后，Ndufs3的基因表达水平显著下降。本研究还首次确定Nrf-2能够结合到线粒体转录因子Tfam、Tfb1m和Tfb2m，复合物I铁硫蛋白亚基Ndufs3和Ndufs7，等的启动子区。研究发现，T-2毒素能增加cAMP的水平，活化PKA，使其活性增加，然后PKA可以磷酸化CREB形成p-CREB，从而激活整个cAMP-PKA-CREB信号通路。阻断PKA信号通路后，T-2毒素诱导的PGC-1α和Ndufs3的基因表达增加都有显著性降低，表明，T-2毒素能够通过激活cAMP-PKA-CREB通路，进而调控PGC-1α和Ndufs3的表达。本研究将为毒素预防和保证动物健康提供科学依据。