The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, 211166, People's Republic of China.
The Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing, 211166, People's Republic of China.
Environ Sci Pollut Res Int. 2018 Dec;25(35):35440-35450. doi: 10.1007/s11356-018-3503-z. Epub 2018 Oct 22.
Di-n-butyl phthalate (DBP) is one of the most dominant phthalate esters and is ubiquitous in the environment. Male reproductive toxicity of DBP and its active metabolite mono-butyl phthalate (MBP) has been demonstrated in in vivo and in vitro studies. The objective of this study was to explore the roles of RhoG-ELMO1-RAC1 in phagocytosis disrupted by MBP in TM4 cells. Mouse Sertoli cell lines (TM4 cells) were maintained and treated by various levels of MBP (1, 10, and 100 μM) for 24 h. Then, cells were harvested for further experiments. Phagocytic capacity of TM4 cells was detected by flow cytometry, immunofluorescence, and oil red O staining. RAC1 activity (GTP-RAC1) was measured by RAC1 pull-down assay. Expression of mRNA and protein related to phagocytosis including ELMO1, RhoG, and RAC1 was analyzed by qRT-PCR and Western blots, respectively. MBP inhibited phagocytosis of TM4 cells and downregulated GTP-RAC1 expression and movement to membrane markedly. Furthermore, ELMO1 protein expression was downregulated in a dose-dependent manner after MBP treatments. Additionally, expression of proteins relating to phagocytosis, including RhoG and GTP-RAC1, was decreased significantly, but expression of total-RAC1 remained unchanged. GTP-RAC1 expression increased dramatically after TM4 cells were transfected with ELMO1 or RhoG plasmid, but restored under co-treatments with MBP and ELMO1/RhoG plasmid. This study suggests that MBP can reduce the phagocytosis of Sertoli cells through RhoG-ELMO1-RAC1 pathway.
邻苯二甲酸二正丁酯（DBP）是最主要的邻苯二甲酸酯之一，在环境中普遍存在。体内和体外研究表明，DBP 及其活性代谢物单丁基邻苯二甲酸酯（MBP）具有雄性生殖毒性。本研究旨在探讨 RhoG-ELMO1-RAC1 在 MBP 破坏 TM4 细胞吞噬作用中的作用。维持小鼠支持细胞系（TM4 细胞）并通过不同水平的 MBP（1、10 和 100 μM）处理 24 小时。然后，收集细胞进行进一步实验。通过流式细胞术、免疫荧光和油红 O 染色检测 TM4 细胞的吞噬能力。通过 RAC1 下拉测定法测定 RAC1 活性（GTP-RAC1）。通过 qRT-PCR 和 Western blot 分别分析与吞噬作用相关的 mRNA 和蛋白表达，包括 ELMO1、RhoG 和 RAC1。MBP 抑制 TM4 细胞的吞噬作用，并显著下调 GTP-RAC1 的表达和向膜的运动。此外，MBP 处理后 ELMO1 蛋白表达呈剂量依赖性下调。此外，与吞噬作用有关的蛋白表达，包括 RhoG 和 GTP-RAC1，显著降低，但总 RAC1 的表达保持不变。TM4 细胞转染 ELMO1 或 RhoG 质粒后，GTP-RAC1 表达显著增加，但在 MBP 和 ELMO1/RhoG 质粒共同处理下恢复。本研究表明，MBP 可通过 RhoG-ELMO1-RAC1 途径降低支持细胞的吞噬作用。
