RIPK在苯胺暴露致肝脏损伤中的作用及机制研究

There are more workers who are engaged in aniline production in China and the pathogenesis of toxic liver disease is not clear.Studies have shown that chronic liver injury is associated with cell death pathways and we have confirmed that aniline could induce hepatocyte apoptosis,but the Caspase inhibitor Z-YVAD-fmk could not completely inhibite this process.It suggests that there may be other death pathways in liver injury induced by aniline.At present, the relationship between RIPK and cell death mode is a research hotspot at home and abroad.However, the relationship between RIPK and aniline induced liver injury has not been reported in the world.We speculate that necroptosis play a role in the process of liver damage caused by aniline and it may be associated with some signal transduction pathways such as RIPK, MLKL, NF-kB. To confirm this hypothesis, we will investigate the effects of aniline on RIPK activation and related pathways through in vitro and in vivo studies. Furthermore,the mechanism and the role of RIPK in the liver damage induced by aniline will be revealed by cohort study. This subject will elucidate the pathogenesis and development mechanism of liver injury induced by aniline from a new perspective, so as to provide scientific basis for biological monitoring and prevention of aniline poisoning,and provide new ideas for prevention and treatment of liver injury caused by aniline.

我国从事苯胺生产的工人较多，其患中毒性肝病的发病机制尚不清楚。有研究证实慢性肝脏损伤与细胞的死亡方式有关，我们课题组前期的研究表明，苯胺引起肝细胞凋亡，但Caspase抑制剂Z-YVAD-fmk不能够完全抑制，这提示我们苯胺引起的肝脏损伤中可能存在其他死亡通路。目前RIPK与细胞死亡方式的关系是目前国内外研究的热点，但是RIPK与苯胺所致肝脏损伤的关系，迄今国内外尚无报道。我们推测，在苯胺致肝脏损害过程中程序性坏死也有一定作用，并可能与RIPK、MLKL、NF-kB等信号转导途径有关。为证实这一假说，我们将采用细胞水平和动物实验研究，探讨苯胺对RIPK激活及相关通路的调控作用，并通过人群队列研究来进一步揭示RIPK在苯胺致肝脏损害发生中的作用机制和地位。本课题将从新的视觉阐明苯胺致肝脏损害的发病和发展机制，从而为苯胺中毒的生物学监测与预防提供科学依据，为苯胺引起肝脏损伤的防治提供新思路。

肝脏是苯胺作用的重要靶器官之一。我们前期研究表明，RIPK1是苯胺暴露引起肝脏细胞损伤的重要分子，而且与程序性坏死密切相关，但是机制不清。RIPK1增加被认为是程序性坏死的信号，为此，本项目立项时旨在研究苯胺暴露对肝脏细胞损伤的影响，阐明RIPK1在苯胺暴露致肝脏细胞程序性坏死的作用及分子机制。通过研究我们发现：（1）对苯胺作业场所进行职业卫生学调查，揭示了苯胺作业场所职业病危害因素分布规律；（2）对苯胺作业场所进行职业风险评估，对比四种模型的评估结果，为苯胺职业风险评估和职业健康相关政策的制定提供依据；（3）苯胺暴露能显著引起肝脏中RIPK1的高表达，并且呈剂量依赖性，利用生信数据库分析RIPK1/RIPK3/MLKL相关表达基因，明确了苯胺暴露诱导肝脏中出现程序性坏死的作用机制；（4）建立干预模型，转染Si-RIPK1，结合活性氧抑制剂N-乙酰-L-半胱氨酸（NAC）进行研究，发现了敲低RIPK1以及NAC可以对苯胺造成的肝细胞损伤有保护作用，可为苯胺中毒的临床防治提供积极作用；（5）建立动物模型，探究苯胺暴露的小鼠肝脏组织中RIPK1表达，结合动物实验验证了体外实验结果；（6）对比苯胺肝脏毒性与肠道菌群模型，发现苯胺引起肝脏毒性的同时也可伴随其他器官损伤。基于以上发现，本项目明确了苯胺暴露对肝脏细胞损伤的作用特点，阐明了RIPK1介导的程序性坏死在苯胺暴露致肝脏细胞损伤的作用及分子机制，系统揭示了RIPK1和程序性坏死在苯胺肝毒性中的作用机制，为揭示苯胺暴露致肝脏损伤机理和探讨防治措施提供新的依据。