MAPK-Runx2信号通路介导汞暴露损伤斑马鱼脊柱发育的机制研究

Mercury (Hg) is a non-essential and highly toxic heavy metal pollutant with carcinogenicity, teratogenicity and mutagenicity, which can produce wide toxic effects on vertebrates such as fish. Our previous study found that Hg exposure could inhibit spinal development and induce spinal deformity in zebrafish, which might be mediated by MAPK-Runx2 signaling pathway. However, the specific mechanism of action is not yet clear. Taking zebrafish as the research object, the present project is to: 1) investigate the development and histomorphological changes of the spine under Hg exposure, obtaining the phenotype of the spine development damaged by Hg; 2) establish an in vitro culture model of zebrafish osteoblasts to detect the protein expression and phosphorylation level of MAPK signaling pathways such as ERK1/2, JNK and p38 after Hg exposure, identifying the MAPK signaling pathway involved in Hg-induced impairment of zebrafish spinal development; 3) analyze the protein interactions of ERK, JNK and p38 with the osteogenic key transcription factor Runx2 under Hg exposure by using co-immunoprecipitation and yeast two-hybrid techniques, revealing the mechanism of Hg impairing spinal development in zebrafish mediated by MAPK-Runx2 signaling pathway. This study not only provides a scientific basis for the study of teratogenic mechanisms and the related control strategies of heavy metal pollution in fish, but also has important reference value for the prevention and treatment of human bone diseases induced by heavy metal exposure.

汞是一种非必需、高毒性、具有“三致”作用的重金属污染物，可对鱼类等脊椎动物产生广泛的毒性效应。我们前期研究发现，汞暴露能抑制斑马鱼的脊柱发育并诱导脊柱畸形，分析推测MAPK-Runx2信号通路可能介导了该过程，但具体的作用机制尚不清楚。本项目以斑马鱼为研究对象，观察汞暴露下其脊柱发育情况和组织形态学变化，获得汞损伤鱼类脊柱发育的表型；建立斑马鱼成骨细胞体外培养模型，检测汞暴露下ERK1/2、JNK、p38等MAPK信号通路的蛋白表达及磷酸化水平，明确参与汞损伤斑马鱼脊柱发育的MAPK信号传导途径；采用免疫共沉淀和酵母双杂交技术分析汞暴露下ERK、JNK、p38与成骨关键转录因子Runx2的蛋白相互作用，揭示MAPK-Runx2信号通路介导汞损伤斑马鱼脊柱发育的机制。本研究不仅能为鱼类重金属污染的致畸机理及防治策略研究提供科学依据，而且对重金属暴露诱导的人类骨骼疾病的防治也有重要的参考价值。

汞（Hg）是一种非必需、易富集、高毒性，具有“三致”作用的全球性重金属污染物，能损伤脊椎动物的脊柱发育甚至导致形态畸形。本项目以模式动物斑马鱼为研究对象，结合在体和离体手段，研究了汞暴露损伤鱼类脊柱发育的机制。研究发现：1）汞暴露组斑马鱼仔鱼的椎体、神经弧、血管弧发育较对照组迟缓，部分椎体形态异常，表明汞暴露抑制了斑马鱼的脊柱生长发育以及矿化过程，进而可能导致其脊柱畸形；2）汞暴露导致斑马鱼仔鱼体内成骨重要物质钙、Ⅰ型胶原蛋白和骨钙素的含量减少，并下调了成骨细胞分化与矿化关键基因（runx2、col1α1、oc和opn）的表达水平，表明汞可能抑制了成骨细胞的早期发育以及细胞外基质的成熟和矿化；3）汞孵育上调了原代培养的斑马鱼成骨细胞的ERK1/2、JNK、p38 MAPK的磷酸化水平，且下调了成骨关键转录因子Runx2的蛋白表达水平，提示ERK1/2、JNK、p38 MAPK-Runx2信号通路可能介导了汞损伤斑马鱼的脊柱发育；4）使用特异性抑制剂分别阻断ERK1/2、JNK、p38 MAPK信号通路后，汞孵育对成骨细胞Runx2蛋白表达水平的抑制效应消失，表明MAPK-Runx2信号通路介导了汞暴露损伤斑马鱼的脊柱发育。因此，本研究表明，汞暴露通过激活ERK1/2、JNK、p38 三条MAPK信号通路的级联反应，下调成骨关键转录因子Runx2的表达，从而抑制成骨细胞的分化增殖，进而损伤斑马鱼的脊柱发育并诱导脊柱畸形。本研究可以为鱼类重金属污染的致畸机理及防治策略研究提供思路，也对重金属暴露诱导的人类骨骼疾病的防治具有一定的参考价值。