PPARγ介导的线粒体合成在子痫前期发病中的作用及调控机制研究

Preeclampsia (PE) is a life-threaten disease among the population of pregnant women in China with high morbidity and high mortality. Oxidative stress has been suggested involving the pathogenesis of PE with uncovered the molecular mechanism. PPARγ modulates mitochondrial biogenesis, and the hyper-activation of PPARγ results in rodent placental pathology. We found that PPARγ was up-regulated in the human placenta with PE, which indicated the clinical significance of PPARγ in PE. Our preliminary studies further found that 1) the increased PPARγ expression was associated with the increases in oxidative stress and mitochondrial biogenesis in the placenta from PE patients; 2) our pilot mechanistic study showed that the expression of PPARγ and its function in mitochondrial biogenesis were modulated by Rnd3; 3) the decreased RND3 expression and increased oxidative stress were observed in clinical samples with PE; 4) the in vitro experiment confirmed that Rnd3 regulates oxidative stress. Therefore, we hypothesized that PPARγ, which probably was regulated by Rnd3, modulated preeclampsia by regulating mitochondrial biogenesis and oxidative stress. We proposed to generate the clinical primary cell lines, the RUPP surgery induced PE mouse model and the conditional knock-out mouse model to investigate the role of PPARγ in PE pathogenesis. We also proposed to perform the CHIP and pull-down assays to investigate the underlying mechanism. Our studies will reveal a novel mechanism in the pathogenesis of PE, and provide new angle to develop treatment for PE.

子痫前期（PE）在我国孕产妇人群中有高发病率和高致死率。氧化应激在PE发病中起重要作用，但机制未阐明。转录因子PPARγ调控线粒体合成，其过表达导致胎盘发育异常。我们发现PPARγ在PE临床样品中上调，表明其在PE中有潜在临床意义。前期研究进一步发现1）PPARγ在PE人胎盘中的变化伴随氧化应激和线粒体数量增高；2）机制研究表明PPARγ表达和线粒体合成受Rnd3调控；3）人PE样品中RND3下调伴随氧化应激；4）体外实验中Rnd3调控活性氧簇产生。据此假设：PPARγ调控线粒体合成，影响氧化应激，参与PE发病，该机制受Rnd3调控。本研究拟采用临床原代细胞模型，RUPP手术和条件性基因敲除动物模型，结合CHIP与免疫沉淀手段，从细胞-个体-分子层面探索PPARγ介导的线粒体合成在PE发生、发展中的作用，及分子调控机制。研究结果将揭示一个新的PE调控机制，为发展新的干预手段提供新思路。

子痫前期（Preeclampsia, PE）是一种高死亡率的妊娠期特有疾病，具有高血压、蛋白尿、水肿的临床表征。报道称PE患者胎盘具有严重氧化应激和线粒体损伤，然而两者之间的分子机制尚未阐明。在本项目中，我们发现一种小G蛋白Rnd3为线粒体功能的新调控因子。RND3表达量在PE患者胎盘组织中下调了46%，在PE患者胎盘分离获得的原代滋养细胞中下调了51%。同时PE原代滋养细胞中出现了超氧化物簇（ROS）累积，和线粒体损伤。为了探讨Rnd3调控线粒体的具体机制，我们在滋养细胞系BeWo中敲低Rnd3，细胞出现了线粒体内嵴空泡，部分线粒体出现与自噬体融合降解，线粒体膜电势去极化，以及线粒体氧化呼吸链4.9倍质子泄漏。在BeWo中过表达GFP-RND3则会缓解缺氧诱导的氧化应激和细胞凋亡。分子机制研究结果发现Rnd3与PPARγ分子免疫结合，过表达Rnd3导致细胞核内的PPARγ蛋白累积。Rnd3通过对PPARγ调控影响下游的mitochondrial uncoupling protein 2 (UCP2)表达，调控线粒体氧化呼吸功能。PPARγ和UCP2的下调表达也在PE患者的胎盘组织中得到验证。最后，我们在PE患者来源的原代滋养细胞中进行了腺病毒介导的RND3过表达，进行挽救实验，实验结果显示过表达RND3后PE原代滋养细胞的ROS水平降低，凋亡减少，表明RND3对PE发生发展的调控作用。我们得出结论，Rnd3为维持胎盘线粒体功能的保护因子，通过PPARγ-UCP2发挥作用。PE患者胎盘中RND3低表达或许是导致胎盘氧化应激的关键因素，在PE发病机制中发挥作用。