HMGB1在MC-LR介导的黄颡鱼肝脏损伤中的作用机制研究

Inflammation plays an important role in the liver injury caused by microcystins (MCs). According to our previous study, MC-LR induced the mRNA expression of a vital proinflammatory factor HMGB1 of yellow catfish, and the HMGB1 blockers (HMGB1 A box and Ethyl Pyruvate) attenuated MC-LR hepatotoxicity. These results suggest that HMGB1 may participate in the liver injury caused by MC-LR. However, there is no report about its effect mechanism. This study aims to monitor the liver histopathological lesion using MC-LR and HMGB1 blockers to reveal the HMGB1 effects on the liver damage caused by MC-LR, using yellow catfish as an animal model; the proliferation/apoptosis/necrosis of liver primary cells and leukocytes, leukocyte percentages, leukocyte subtype percentages, inflammatory mediator release and inflammation-related gene expression in leukocytes to identify the HMGB1 effects on the inflammation caused by MC-LR; the HMGB1 expression and location to determine the MC-LR effects on HMGB1. These results will reveal the HMGB1 effect mechanism on MC-LR mediating liver inflammatory injury at animal, cell and molecule levels, which may serve as a theoretical underpinning for applying HMGB1 blockers to alleviate MC-LR hepatotoxicity.

炎症反应在微囊藻毒素（MCs）致肝损伤中发挥重要作用。我们前期研究表明MC-LR能够诱导黄颡鱼重要促炎因子HMGB1的表达，而其阻断剂HMGB1 A box和丙酮酸乙酯都能有效缓解MC-LR的肝细胞毒性，说明HMGB1可能参与了MC-LR介导的肝损伤，但其具体作用机制未见报道。本项目拟以黄颡鱼为动物模型，使用MC-LR及HMGB1阻断剂处理，检测不同处理组肝脏组织病理学损伤程度，以确定HMGB1在MC-LR致肝损伤中的作用；检测肝脏原代细胞和白细胞增殖/凋亡/坏死，白细胞比例、白细胞亚型比例、白细胞中炎症介质释放和炎症相关基因的表达，以确定HMGB1在MC-LR介导的肝脏炎症反应中的作用；分析HMGB1的表达和定位，以确定MC-LR对HMGB1的影响。从动物、细胞、分子三个层次研究HMGB1在MC-LR介导的肝脏炎症损伤中的作用机制，从而为利用其阻断剂缓解MC-LR的肝毒性提供理论依据。

微囊藻毒素-LR（MC-LR）是水华蓝藻——铜绿微囊藻的次生代谢物，能够诱发肝脏的炎症损伤。MC-LR暴露诱导黄颡鱼重要促炎因子PfHMGB1表达上调。本研究制备并筛选了最佳阻断剂PfHMGB1中和抗体（anti-PfHMGB1），从分子、细胞和动物三个层次证明PfHMGB1在MC-LR介导的肝脏炎症损伤中发挥重要作用。分子水平：MC-LR不仅能诱导肝脏中PfHMGB1 mRNA和蛋白水平的升高，还能促进蛋白胞外释放，进入外周血。而anti-PfHMGB1则能完全中和肝脏中PfHMGB1，并减少外周血中的含量。此外，纯化的rPfHMGB1能够结合DNA，并诱导肝脏原代细胞中炎症因子的表达上调；而anti-PfHMGB1则能下调MC-LR诱导的肝脏中炎症因子的表达升高，说明MC-LR可能通过诱导PfHMGB1的表达上调和胞外释放，激活炎症因子表达。细胞水平：anti-PfHMGB1能够部分缓解高浓度的MC-LR诱导的肝脏原代细胞毒性和活性氧（ROS）的大量释放；rPfHMGB1蛋白具有浓度依赖的肝细胞毒性并能诱导ROS的产生，揭示MC-LR可能通过PfHMGB1介导的ROS释放发挥细胞毒性效应。此外，重组PfHMGB1能够诱导白细胞增殖；血涂片观察显示MC-LR显著减少了外周血中淋巴细胞和血栓细胞的比例，增多单核细胞，暗示MC-LR诱导单核细胞增加可能和PfHMGB1的胞外释放有关。动物水平：黄颡鱼肝脏对MC-LR代谢缓慢，50 μg/kg即可引发肝损伤。anti-PfHMGB1能够部分中和MC-LR诱导的肝脏中央静脉扩张，肝细胞分离，细胞核皱缩，线粒体和粗面内质网的断裂，并减少血清中总NO的含量和降低ALT/AST活性，揭示MC-LR通过PfHMGB1诱导黄颡鱼的肝脏炎症损伤。HMGB1可能有助于保护黄颡鱼免于广泛的微囊藻毒素的毒害。