BNIP3/BNIP3L介导的线粒体自噬效能不足在子痫前期非可控性炎症中的作用机制研究

The failure of extravillous trophoblast invasion is one of the key point of pre-eclampsia(PE). However, the mechanism remains unclear. Our research group had previously demonstrated that early autophagy may have a protective effect on PE cell models. Further researches had found that placental trophoblast cells express BNIP3 and mitophagy exists in PE cell models at early stage of hypoxia. The expression of NLRP3 inflammasome in PE tissue model and the level of IL-1B in supernatant decrease significantly when promote autophagy, vice versa, which point out that the protective effect of mitophagy in early PE on tissue hypoxia stress is related to the decrease of uncontrolled inflammation. So we speculate that “placental trophoblast cells are in physiological hypoxia in early pregnancy, and due to mitophagy insufficiency mediated by BNIP3 / BNIP3L, the clearance of damaged mitochondrial is restricted, resulting in excessive accumulation of ROS and damage of mtDNA, and then activate the NLRP3 inflammasome, produce and release IL-1β, induce uncontrolled inflammatory response, this will lead to trophoblast dysfunction which is involved in the development of PE.” Our project plans to verify the above hypothesis in "cell - placenta - body" by applying molecular biology, subcellular proteomics and genetic engineering methods to cells, tissues and mouse models, and provide a new idea for further study of the pathogenesis of PE.

绒毛外滋养细胞侵袭力受损是子痫前期（PE）发病关键点，具体机制不清。课题组前期证实，在PE细胞模型上早期自噬可能具有保护作用。进一步发现：胎盘滋养细胞有BNIP3表达；PE细胞模型在缺氧早期存在线粒体自噬；在组织模型上促进自噬，组织中NLRP3炎性体组分表达与IL-1β水平均下降，反之则上升，提示PE早期线粒体自噬对组织低氧应激的保护作用与减轻非可控性炎症有关。因而推测“早孕期胎盘滋养细胞处于生理性低氧环境，由于BNIP3 /BNIP3L介导的线粒体自噬效能不足，受损线粒体清除受限，导致ROS过度堆积并损害mtDNA，进而激活NLRP3炎性体，产生并释放IL-1β，诱发非可控性炎症，导致滋养细胞损伤，参与PE的发生发展”。项目拟在细胞、组织及小鼠模型上应用分子生物学、亚细胞器蛋白质组学及基因工程等方法，从"细胞-胎盘组织-整体"验证上述假说，将为深入研究PE发病机制提供新思路。

子痫前期是威胁产妇和胎儿的妊娠期特有性临床疾病，以严重的高血压，蛋白尿，肝功能异常，胎儿生长受限等临床症状为显著特点。已发表文献证实，众多调控方式被证实参与子痫前期的发生和发展。同时，异常的自噬水平被证实会加重子痫前期孕妇胎盘处氧化应激反应，参与子痫前期的发生。本课题围绕异常自噬水平和滋养细胞的功能之间的关系，结合分子生物学、亚细胞器蛋白质组学、细胞生物学等实验方法进一步探究滋养细胞内自噬如何参与子痫前期的发生。(1). 汇总现有建立的不同氧化应激细胞模型，摸索细胞培养和处理方法，结合高灵敏度代谢质谱分析技术首次证实，现存各氧化应激细胞模型存在各自的代谢特点，适用于不同的研究目的，为后续细胞研究选择合适的细胞模型作为参考资料；(2).通过收集临床子痫前期组和对照组的胎盘组织提取线粒体作为研究对象，结合蛋白质印迹法和代谢质谱分析技术证实，子痫前期胎盘滋养细胞线粒体修复调控网络异常，导致损伤线粒体堆积，滋养细胞代谢信号通路紊乱和功能异常；(3).结合绒毛外滋养细胞模型证实，线粒体自噬调控分子BNIP3可通过Beclin1反馈调控细胞内巨自噬的水平，进而影响滋养细胞在氧化应激状态的生存；(4).硫氧还蛋白互作蛋白TXNIP介导的内质网应激是子痫前期胎盘处炎症反应的重要调控分子，特异性的干扰滋养细胞TXNIP的表达可以缓解氧化应激状态下的细胞炎症水平，为临床潜在干预靶点提供依据。综上，BNIP介导的线粒体自噬参与调控胎盘处线粒体的质量，影响胎盘滋养细胞的代谢活动；此外，BNIP3反馈性调控巨自噬，调控滋养细胞内质网应激，炎症反应和滋养细胞的存活，为临床潜在干预靶点提供理论依据。