内质网应激在纳米铜诱导暗纹东方鲀肝脏脂肪沉积中的作用及机制研究

Takifugu fasciatus is one of characteristic aquaculture species in China, but fatty liver disease of T. fasciatus is now becoming a serious issue, which has seriously affected the healthy development of T. fasciatus aquaculture. Previous studies have indicated that the presence of copper nanoparticles, a new pollutant in water environment, is recognized as one of inducing factor that contributes to the formation of fatty liver in T. fasciatus, but its mechanism is not clear. It is shown that endoplasmic reticulum stress (ERS) and its mediated signaling pathways play key roles in fat deposition in mammals. Taking this as an entry point, firstly, we mainly used RACE technology to clone the full-length of cDNA sequences related to ERS and analyzed its molecular structures and gene expression signature in tissues. Secondly, qPCR and laser-scanning confocal microscopy technologies were used to determine copper nanoparticles can induce the appearance of ERS in liver and activate its signaling pathway. Thirdly, the roles of ERS and its signaling pathway in activating the SREBP-1c pathway that plays a key role in lipid synthesis were revealed in vivo experiment. Finally, by using 4-phenylbutyric acid inhibitors to stimulate ERS in vitro experiment, we carried out further observations to figure out the situation of downstream fat deposition and the activation level of SREBP-1c, thus clarify the mechanism. This research provides a new perspective for finding the pathogen of fatty liver formation in T. fasciatus, also makes a supplement to the ecological safety data service of copper nanoparticles in aquaculture water.

暗纹东方鲀（Takifugu fasciatus）是我国特色养殖鱼类，但当前脂肪肝病严重困扰了该鱼养殖业的健康发展。前期申请人发现水体中新型污染物纳米铜是诱导暗纹东方鲀脂肪肝形成的又一因素，但其机制尚不清楚。在哺乳动物中已证实内质网应激（ERS）及其介导的信号通路在脂肪沉积中扮演关键角色。本研究以此为切入点，首先，运用RACE等技术克隆暗纹东方鲀ERS相关基因的cDNA全长，并分析其分子结构及组织表达特征；其次，运用qPCR、激光共聚焦显微等技术，确定纳米铜诱导暗纹东方鲀肝脏ERS并激活其介导的信号通路；接着，在活体试验中研究ERS及其介导的信号通路在活化脂质合成SREBP-1c通路中的作用；最后，在离体试验中通过4-PBA抑制纳米铜诱导的ERS反应，进一步探讨其下游脂肪沉积及SREBP-1c活化的机制。该研究为暗纹东方鲀脂肪肝形成病因提供新的视角，也为评价纳米铜的生态安全提供基础资料。

暗纹东方鲀（Takifugu fasciatus）是我国特色养殖鱼类，但当前脂肪肝病严重困扰了该鱼养殖业的健康发展。申请人发现水体中新型污染物纳米铜暴露是诱导暗纹东方鲀脂肪肝形成的又一因素，但其机制尚不清楚。在哺乳动物中已证实内质网应激（ERS）及其介导的信号通路在脂肪沉积中扮演关键角色。本研究以此为切入点，首先，运用RACE等技术克隆暗纹东方鲀ERS相关基因的cDNA全长，并分析其分子结构及组织表达特征；其次，运用qPCR、western blot等技术，确定纳米铜诱导暗纹东方鲀肝脏ERS并激活其介导的信号通路；接着，在活体实验中研究ERS及其介导的信号通路在活化脂质合成SREBP-1c通路中的作用；最后，在离体实验中通过4-PBA抑制剂抑制纳米铜诱导的ERS反应，进一步探讨其下游脂肪沉积及SREBP-1c活化的机制。研究结果发现，暗纹东方鲀GRP78、CRT、PERK、ATF-6α 和IRE-1α 基因在进化上高度保守。暗纹东方鲀GRP78、CRT、PERK、ATF-6α 和IRE-1α基因在肝脏中表达量较高，即肝脏是进行内质网稳态调控的重要器官。揭示了PERK-EIF2α-SREBP-1c信号通路在环境剂量纳米铜暴露诱导暗纹东方鲀肝脏脂肪沉积中起到关键作用。该研究为暗纹东方鲀脂肪肝形成病因提供新的视角，也为评价纳米铜的生态安全提供基础资料。本研究第一资助发表SCI论文6篇（5分以上2篇），申请国家发明专利3项（授权2项），培养博士生1名，硕士生5名，参加国内学术会议3次。