大黄鱼抗氧化酶Peroxiredoxin IV调控炎症反应的机理研究

Peroxiredoxin IV (Prx IV) is a novel peroxidase. In our previous study, we have demonstrated that Prx IV in large yellow croaker regulated the pro-inflammatory responses by inhibiting NF-kB activity and protected the fish against bacterial challenge. This proposal will further study the regulation mechanism of Prx IV for inflammatory response in large yellow croaker. To understand roles of PrxIV in regulation of cellular redox conditions, changes in levels of reactive oxygen species (ROS) will be measured in vivo or in vitro under Prx IV knockdown or overexression conditions. Meanwhile, the phosphorylation levels of IκB kinase (IKK ) and inhibitor of NF-κB (IκB ), status of NF-κB activity, and expression levels of inflammatory cytokines will be detected under Prx IV knockdown or overexression conditions, which facilitates to know whether PrxIV affects IKK/IκB/NF-κB signaling pathway and inflammatory cytokine expression. To further determine how PrxIV regulates inflammatory response, the effects of PrxIV on ROS levels、IKK/IκB/NF-κB pathway and inflammatory cytokine expression will be studied in the presence of antioxidant or oxidant. Finally, the interacting proteins of Prx IV will be screened by yeast two-hybrid technique, and their functions and regulation network will also be studied. These results will not only lay foundation for clarifying regulation mechanism of inflammatory response in fish, but also provide new ideas and strategies for fish disease control.

PrxIV是一种新型抗氧化酶，前期研究中我们已证明大黄鱼PrxIV通过抑制核因子NF-κB活性来负调节炎症反应，并保护鱼体免受细菌攻击。本项目拟进一步研究大黄鱼PrxIV调控炎症反应的机理。利用活体或细胞系，在PrxIV基因沉默及过表达条件下分析活性氧（ROS）水平的变化，以了解PrxIV对细胞氧化还原状况的调控；同时研究IκB激酶IKK和IκB的磷酸化水平、NF-κB活性状况及炎症因子表达水平的变化，以查明PrxIV对IKK/IκB/NF-κB信号通路及炎症因子表达的影响；进而在抗氧化剂或氧化剂拮抗条件下研究PrxIV对ROS、IKK/IκB/NF-κB信号通路及炎症因子表达的影响，以明确PrxIV调节炎症反应的分子途径；查找PrxIV的相互作用蛋白，研究其功能及调控网络，解析PrxIV作用的分子机理。该研究结果将为阐明鱼类炎症反应调控的机理奠定基础，也为其病害防治提供新的思路和策略。

PrxIV是一种新型抗氧化酶。本项目发现大黄鱼PrxIV（LycPrxIV）不仅可下调大黄鱼头肾细胞系LYCK中过氧化氢、一氧化氮、超氧阴离子的水平，亦可下调大黄鱼脾脏及人293T细胞内的过氧化氢水平，说明其可参与不同来源体外培养细胞系以及活体组织细胞内氧化还原状况的调控；进而克隆了LycPrxIV启动子，发现其对几种病原相关分子模式的刺激不响应，但过氧化氢处理后其启动子活性显著升高。LycPrxIV可负调控核转录因子NF-κB的活性及下游炎症因子的表达，上调抑炎因子的表达。过氧化氢处理稳定表达LycPrxIV的大黄鱼头肾细胞系LYCK后，炎症因子的表达亦显著下降，而抑炎因子的表达则显著上升。因此认为大黄鱼在受到氧化还原信号如过氧化氢的刺激后，细胞内LycPrxIV启动子响应刺激活性升高，相应地LycPrxIV 表达增加，受LycPrxIV 负调节的NF-κB活性下降，导致细胞内炎症因子表达下调，抑炎因子表达上升，从而负调节炎症反应。酵母双杂交和免疫共沉淀实验进一步证明了LycPrxIV与大黄鱼过氧化物酶PrxII和硫氧还原蛋白Srx之间存在相互作用。Srx可帮助PrxIV维持在还原状态，从而保持其酶活及抗氧化活性。PrxIV天然状态下以同源二聚体形式存在，推测PrxIV和PrxII聚集在一起后，在结构上由最初的同源二聚体形成十聚体，并与Srx共同参与体内氧化还原反应。.相关研究结果已发表论文12篇，其中SCI收录期刊论文11篇，中文核心期刊论文1篇。