波形蛋白在邻苯二甲酸二丁酯干扰类固醇激素合成中的作用

Dibutyl phthalate (DBP) is one of the important endocrine disrupting chemicals, which exerts toxicities primarily due to its disruption of steroidogenesis. At present, lots of researches on the mechanisms of DBP on steroid biosynthesis were focused on transportation of cholesterol from outer to inner membrane of mitochondria and some activities of steroidogenic enzems, such as P450 side chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3βHSD), etc. Our previous study revealed the biphasic effects on steroidogenesis of mono-butyl phthalate (MBP, active metabolite of DBP) and the disruptive site was between the activation of cAMP and P450scc in mitochondria. This course consists of two steps which are cholesterol transportation from "cholesterol pool" in cytoplasm to outer membrane of mitochondria and cross transportation from outer to inner membrane of mitochondria. Furthermore, vimentin, one of the cell skeleton proteins which is a key factor in conveying cholesterol from "cholesterol pool" in cytoplasm to outer membrane of mitochondria, was up-regulated under DBP treatment at lower doses in rat testes. These data indicated that regulation of vimentin on cholesterol from cytoplasm to outer membrane of mitochondrial maybe one of the critical points in the steroidogenesis interrupted by DBP/MBP. In this project, animal experiments will be conducted to explore the roles of vimentin in disruption of DBP on steroidogenesis in different steroidogenic organs, such as testis, ovary and adrenal gland using puberty and fetal rats as models. Data showed that the molecules or events, which regulate vimentin, include microRNA, GC-box, transforming growth factor-β, ZBP-89, nuclear factor-κB and phosphorylation, etc. Which is (are) the key factor (s) in regulating vimentin treated by MBP will be deeply explored using mouse Leydig cells, mouse adrenal cortex cells and rat granulose cells as models in vitro. Summarily, this study will offer useful scientific evidence to elucidate whether vimentin is a key target protein in the regulation of DBP/MBP on steroidogenesis and how DBB/MBP regulate vimentin in vitro and in vivo.

邻苯二甲酸二丁酯（DBP）作为重要的环境污染物，是通过影响类固醇激素合成发挥内分泌干扰效应。其干扰类固醇激素合成的研究以往多探讨对胆固醇跨线粒体膜转运及相关酶活性的影响。我们前期发现，MBP（DBP活性代谢物）影响类固醇激素合成的作用位点在cAMP被激活之后的胆固醇转运环节且具有双向调节作用，并且低剂量DBP上调波形蛋白(VP，胆固醇从胞浆转运至线粒体外膜的重要分子)表达，因此DBP/MBP可能通过VP调节胆固醇从胞浆转运至线粒体外膜干扰类固醇激素合成。本项目拟以青春期大鼠及胎鼠为动物染毒模型，探讨在不同器官(睾丸、卵巢和肾上腺)中VP对DBP调节类固醇激素合成中的作用；以睾丸Leydig细胞、肾上腺皮质细胞和卵巢颗粒细胞为体外模型，验证VP是否参与MBP调节类固醇激素的合成，明确MBP调节VP的上游分子及其调节机制。通过本项目初步阐明VP在DBP/MBP影响类固醇激素合成的作用及机制。

邻苯二甲酸二丁酯（DBP）作为重要的环境污染物，是通过影响类固醇激素合成发挥内分泌干扰效应。其干扰类固醇激素合成的研究以往多探讨对胆固醇跨线粒体膜转运及相关酶活性的影响。急性调节蛋白（StAR）为胆固醇转运的关键调节因子，核转录因子(NF-κB)作为波形蛋白（vimentin）调节因子。我们前期发现，MBP（DBP活性代谢物）影响类固醇激素合成的作用位点在cAMP和 P450scc之间的胆固醇转运环节且具有双向调节作用。此外，vimentin作为细胞骨架蛋白之一，低剂量DBP上调vimentin表达，因此DBP/MBP可能通过vimentin调节胆固醇从胞浆转运至线粒体外膜干扰类固醇激素合成。本项目拟以青春期大鼠及胎鼠为动物染毒模型，探讨在不同器官(睾丸、卵巢和肾上腺)中vimnetin、StAR、NF-κB对DBP调节类固醇激素合成中的作用；以睾丸Leydig细胞(MLTC-1)、肾上腺皮质细胞(Y1)和卵巢颗粒细胞(rGCs)为体外模型，验证vimentin是否参与MBP调节类固醇激素的合成，明确MBP调节vimentin的上游分子及其调节机制。结果发现：1. 高剂量MBP抑制MLTC-1细胞孕酮合成，StAR蛋白表达下调，GATA-4磷酸化下降，揭示了MBP通过调节StAR相关转录因子从而影响类固醇激素的合成。2. 低剂量MBP对MLTC-1和Y1细胞类固醇激素的合成具有刺激效应，NF-κB、miRNA-200c所介导的vimentin表达水平的改变参与了MBP在体外刺激类固醇激素的合成。3.青春期前雌性大鼠模型及rGCs证实NF-κB-vimentin信号通路参与了低剂量DBP/MBP刺激类固醇素合成。4. 在完成项目目标的基础上，探讨了RhoG-ELMO1-RAC1信号通路在DBP/MBP干扰支持细胞吞噬功能中的作用。.通过本项目初步阐明vimentin、StAR、NF-κB在DBP/MBP影响类固醇激素合成的作用及机制，为进一步研究生殖功能障碍提供新的方向，为更好的保护人类健康提供新的贡献。在本项目资助下已发表论文6篇（其中SCI收录论文5篇、中文核心期刊1篇），培养硕士生5名。