The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, PR China; The Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 211166, PR China.
Donghai Town Community Health Service Center, Qidong County, Jiangsu Province 226253, PR China.
Toxicol In Vitro. 2017 Dec;45(Pt 1):25-30. doi: 10.1016/j.tiv.2017.07.012. Epub 2017 Jul 19.
Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP) are the most common endocrine disrupting chemicals. Many studies indicated the effects of MBP on male steroidogenesis, however, little attention have been paid on the effects of low levels of MBP on female steroidogenesis. This study was aimed to assess steroidogenesis stimulated by low-dose MBP on primary cultured ovarian granulosa cells (mGCs). Ovarian granulosa cells were isolated from ICR female mice. Hormone levels in medium were detected by ELISA, mRNA and protein expressions of vimentin, NF-κB p65 and phosphorylation of NF-κB p65 (p-p65) were assayed by qRT-PCR, western blot and immunohistochemistry, respectively. Besides, confocal immunofluorescence and electrophoretic mobility shift assay (EMSA) were used for detecting vimentin expression and activity of NF-κB p65 binding to the promoter of vimentin, respectively. Progesterone levels, mRNA and protein levels of vimentin and p-p65 in cells were increased significantly in mGCs treated by MBP at 10M. Additionally, MBP-induced steroidogenesis was blocked when vimentin protein was knocked down or activity of NF-κB was inhibited. EMSA assay showed that binding activity of NF-κB to the promoter regions of vimentin was boosted after MBP exposure. Accordingly, the results suggested that MBP could up-regulated steroidogenesis through NF-κB-vimentin signal in mGCs.
邻苯二甲酸二正丁酯（DBP）及其活性代谢物单丁基邻苯二甲酸酯（MBP）是最常见的内分泌干扰化学物质。许多研究表明 MBP 对雄性类固醇生成有影响，然而，对于低水平 MBP 对雌性类固醇生成的影响关注较少。本研究旨在评估低剂量 MBP 对原代培养的卵巢颗粒细胞（mGCs）类固醇生成的刺激作用。从 ICR 雌性小鼠中分离卵巢颗粒细胞。通过 ELISA 检测培养基中的激素水平，通过 qRT-PCR、western blot 和免疫组织化学分别检测波形蛋白、NF-κB p65 和 NF-κB p65 磷酸化（p-p65）的 mRNA 和蛋白表达，此外，还通过共聚焦免疫荧光和电泳迁移率变动分析（EMSA）检测波形蛋白的表达和 NF-κB p65 与波形蛋白启动子结合的活性。在 mGCs 中用 MBP 处理 10μM 时，孕酮水平、波形蛋白的 mRNA 和蛋白水平以及细胞中的 p-p65 明显增加。此外，当波形蛋白蛋白被敲低或 NF-κB 活性被抑制时，MBP 诱导的类固醇生成被阻断。EMSA 实验表明，MBP 暴露后 NF-κB 与波形蛋白启动子区域的结合活性增强。因此，结果表明 MBP 可以通过 mGCs 中的 NF-κB-波形蛋白信号途径上调类固醇生成。
