水母毒素肾脏毒性蛋白分离及其通过损伤线粒体引起肾小管上皮细胞死亡的作用机制

Jellyfish sting is one of the most common injuries caused by marine creatures. Jellyfish venom at a high dose leads to cardiorespiratory arrest and rapid death of rats, while at lower dose it causes systemic multi-organ dysfunction which manifests as the delayed jellyfish envenomation syndrome (DJES) and obvious renal injury in particular, where large amounts of vacuoles and hyaline substance emerge in the lumen of the distal tubules accompanied by necrosis of the renal tubular epithelial cells. These results suggested that renal injury may be the primary reason for rats' delayed death induced by jellyfish venom. In this research project, we plan to separate and identify the nephrotoxic component of the venom from the jellyfish Cyanea capillata via the method of liquid chromatography combining with activity monitoring at first. Then, an in-depth analysis on the injury mechanism of nephrotoxic protein will be carried out, where the exploration is going to focus on the roles of mitochondrial damage and reactive oxygen accumulation in the apoptosis and necrosis of the renal tubular epithelial cells, and the research results will also be verified by drug intervention. On the other hand, iTRAQ protein quantification will be adopted to construct a differential proteome of the mitochondria from the renal tubular epithelial cells after the nephrotoxic protein treatment so as to scan the differentially expressed proteins and the related metabolic pathway in mitochondrial damage, so the targets of the nephrotoxic protein in mitochondria may eventually be sought out. Our project is expected to clarify the mechanism of tubular epithelial cell death caused by the nephrotoxic protein via mitochondrial damage in order to provide instructions for the protection and medical treatment of DJES, as well as to obtain the nephrotoxic protein from jellyfish venom and define the material basis of renal injury in DJES.

水母蜇伤是最常见的海洋生物伤。水母毒素在高剂量时可致大鼠急性循环功能衰竭，快速死亡；较低剂量时造成多器官功能损伤，表现为延迟毒性综合征，尤以肾脏损伤最为明显，肾小管腔内出现大量空泡及透明质样物质，肾小管上皮细胞脱落坏死，表明肾脏损伤可能是水母毒素致大鼠延迟死亡的重要原因。拟以发形霞水母毒素为样品，通过液相色谱定向分离纯化其肾脏毒性蛋白；深入分析肾脏毒性蛋白的作用机理，重点探索线粒体损伤和活性氧堆积在肾小管上皮细胞死亡中的作用，并通过药物干预试验加以验证；再利用iTRAQ蛋白定量技术构建肾脏毒性组分作用后线粒体差异蛋白质组，筛查毒素作用后线粒体的差异表达蛋白及其相关代谢通路，寻找毒素蛋白在线粒体上的作用靶点。本课题将阐明水母毒素通过损伤肾小管上皮细胞线粒体发挥肾脏毒性效应的基本机制，为水母蜇伤延迟毒性综合征的防治提供指导；也有望获取水母毒素肾脏毒性蛋白组分，明确水母毒素损伤肾脏的物质基础。

有毒水母是常见的伤人海洋生物，水母毒素在高剂量时可致大鼠急性循环功能衰竭、快速死亡；较低剂量时造成多器官功能损伤，表现为延迟毒性综合征，尤以肾脏损伤最为明显。本课题以我国东南海域常见有毒水母发形霞水母为研究对象，采用梯度离心和短时振荡刺激法，建立了提取高活性水母毒液的新方法，体外检测试验发现水母毒液含有金属蛋白酶活性成分，结合前期机制研究和水母触手组织转录组分析结果，表明金属蛋白酶类是水母毒素肾脏毒性的主要因子。接下来利用pET24a原核表达质粒构建了发形霞水母来源虾红素样金属蛋白酶CALP1的重组表达载体，转入E.coli Rosetta (DE3)表达菌中进行诱导表达，重组蛋白经鉴定与目标蛋白分子一致。然后分析了水母毒素损伤肾脏的途径与效应。利用MTT法检测不同浓度水母毒素不同作用时间对NRK-52E细胞活力的影响，采用Annexin V-FITC/PI双染，流式细胞仪检测毒素作用后细胞凋亡与坏死情况，利用荧光探针DCFH-DA标记技术观察水母毒素作用后细胞内ROS水平，用TBA比色法检测细胞MDA水平，Rhodamine 123染色检测线粒体膜电位的变化。结果发现水母毒素对NRK-52E细胞活力的影响具有剂量和时间依赖性，毒素作用后细胞形态改变，凋亡和坏死细胞增多，且NRK-52E细胞内活性氧与MDA水平升高、线粒体膜电位降低。在整体动物水平，水母毒素作用后大鼠肾脏组织观察到大量凋亡细胞及细胞碎片，其Caspase-3的表达显著升高。最后筛选了致命性水母蜇伤综合征的药物干预方案。金属蛋白酶抑制剂BB-94和膜保护剂聚乙二醇（PEG4000-6000）合用可有效对抗水母毒素肝、肾毒性；低分子右旋糖酐Dextran-40单用也可以拮抗水母毒素的毒性作用，有效保护肾脏等重要脏器。本课题阐明了水母毒素损伤肾脏的物质基础及其毒性效应的作用机制，筛选的药物干预方案可望为水母蜇伤延迟毒性综合征的防治提供具体指导。