Cav-1/Nox1/STIM1信号通路参与百草枯心肺损害机理研究及中毒标志物筛选

Paraquat (PQ, 1, 1’-dimethyl-4, 4’-bipyridinium) is a highly toxic herbicide, which is widely used in numerous developing countries around the world. Ingestion of PQ leads to multiple organ damage especially in the heart and lung. PQ induced cardiac dysfunction and progressive pulmonary fibrosis, which is often associated with high mortality, appears as early as several hours to several weeks after PQ ingestion. However, the underlying molecular mechanisms of which remain elusive. Meanwhile, the identification of the biomarker for paraquat toxicity is important for the area of forensic toxicology and identifying a crime. Therefore, it is important to study the mechanisms and identify the biomarkers in paraquat induced organ injury. In our preliminary data, we have found that the overproduction of reactive oxygen species, calcium signaling, mitochondrial dysfunction, the blockage of cell cycle and cell death are involved in paraquat induced cardiac and pulmonary injury, however the detailed mechanisms remain to be defined. This application will address the deficits in our knowledge by: 1) establishing a functional significance of Cav-1/Nox1/STIM1 signaling pathway in paraquat induced cardiac and pulmonary injury, 2) identifying the molecular regulatory mechanism of signaling molecules in paraquat toxicity, 3) understanding the impact of cell cycle and cell death by paraquat in the heart and lung, 4) identifying the diagnostic biomarkers for paraquat toxicity. Our study will provide a better understanding on the cellular and molecular mechanism underlying paraquat induced cardiac and pulmonary injury as well as the death caused by paraquat. We anticipate the development of novel therapeutic agents targeting Cav-1/Nox1/STIM1 signaling pathway that will help reduce paraquat induced organ injury and subsequently reduce the morbidity and mortality. We will also identify the potential biomarker and development the diagnostic approaches which will be beneficial for forensic practice.

百草枯（Paraquat, PQ）是触杀型除草剂，PQ中毒危害大、死亡率高，心肺是PQ中毒损害重要的靶器官，然而PQ中毒具体机理及死亡机制仍不清楚，同时PQ中毒标志物的检测是PQ中毒的法医学评价及事故责任认定的基础，因此针对PQ中毒机理及生物标志物筛选开展研究工作具有重要意义。前期研究发现了氧化应激、钙离子信号通路、线粒体损伤、细胞周期和细胞死亡和炎症因子参与PQ引起的心肺损害，其具体机制仍需进一步阐明。本研究将从以下几个方面开展：1）明确Cav-1/Nox1/STIM1在PQ中毒中的重要作用，2）研究相关信号分子间的调控机制，3）PQ影响细胞周期和细胞死亡的分子细胞机制，4）寻找PQ中毒生物标志物。项目的实施将为阐明PQ中毒机理、中毒死亡原因和死亡机制提供可靠的理论基础，为法医毒理学研究提供新思路；针对PQ中毒后分子标志物进行筛查，为PQ中毒相关检测和毒物分析提供实践应用依据。

百草枯（Paraquat, PQ）是触杀型除草剂，PQ中毒危害大、死亡率高，心肺是PQ中毒损害重要的靶器官，然而PQ中毒具体机理及死亡机制仍不清楚，同时PQ中毒标志物的检测是PQ中毒的法医学评价及事故责任认定的基础，因此针对PQ中毒机理及生物标志物筛选开展研究工作具有重要意义。我们研究发现：1）IL-6在PQ处理的肺组织和BMDM中的表达受组蛋白乙酰化的调控，同时HAT抑制剂能够减轻PQ肺纤维化，体内、体外检测HDAC各亚型的表达发现HDAC3呈PQ剂量依赖性和时间依赖性的降低，IL-6启动子区受到组蛋白乙酰化的调节，且调节方式为HDAC抑制剂促进其转录表达，而HAT抑制剂降低其转录表达。2）进一步实验发现PQ处理16HBE后，可以诱导细胞内ROS产生并增强与STIM1激活诱导SOCE的活性，PQ处理引起细胞周期G1/S期的加速转变，并减缓了S期向G2/M期的转变速度，然后导致大量细胞停滞在S期中，引起细胞死亡。而在16HBE中敲除STIM1或抑制STIM1介导的SOCE激活可改善急性PQ中毒引起的细胞死亡，这也通过调节CyclinD1，p21，CyclinA2和CDK2的表达来调节S期细胞的积聚，揭示了STIM1在急性PQ诱导的细胞周期转换和细胞死亡中发挥着重要的作用；研究同时发现Nox1及ROS在PQ心肺损伤组织中表达明显升高，特异性Nox1抑制剂阻止PQ诱导的心肌细胞的死亡；Nox1的抑制因子Cav-1在PQ处理的小鼠心肺组织中表达明显降低。Nox1能够通过产生ROS、增加细胞内Ca2+浓度和促进IL-6和IL-1b等分子机制，促进P38和Caspase3的大量激活并可能参与PQ引起的细胞死亡同时PQ能够增加肺组织和细胞中STIM1和ORAI1表达水平，并引起了细胞周期S期阻滞。3）利用非靶向和靶向代谢组学对PQ中毒后的血清代谢物进行筛查，发现了多个显著变化的代谢物，其中3吲哚丙酸和甘氨酸、色氨酸和苏氨酸变化最为显著，通过功能学试验最终锁定3吲哚丙酸可以作为PQ诊断和干预的关键分子。4）首次建立了生物样品中PQ和敌草快DQ同时快速检测的液相色谱质谱联用法。本项目的实施为阐明PQ中毒机理、中毒死亡原因和死亡机制提供可靠的理论基础，针对PQ中毒后分子标志物进行筛查，为PQ中毒相关检测和毒物分析提供实践应用依据。