增塑剂DEHP通过铁死亡途径抑制小鼠GC-1 spg精原细胞生长

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摘要目的：探究邻苯二甲酸二（2-乙基己基）酯（di-2-ethylhexyl phthalate，DEHP）损伤小鼠GC1 spg精原细胞功能的可能机制。方法：采用不同浓度DEHP（0、30、60、90、120 μmol/L）处理GC-1 spg细胞，通过CCK8实验、克隆形成实验和Transwell实验检测细胞增殖和迁移能力的变化；化学分光光度比色法检测细胞内铁离子（Fe3+）和丙二醛的相对含量；蛋白质印迹实验检测细胞内铁死亡相关蛋白胱氨酸/谷氨酸逆转运蛋白（xCT）和谷胱甘肽过氧化物酶4（GPX4）的相对表达；细胞免疫荧光染色技术检测细胞内活性氧水平以及线粒体膜电位（JC-1）的改变。使用铁死亡抑制剂Ferrostatin-1（1 μmol/L）与DEHP（90 μmol/L）联合培养GC1 spg细胞24 h，CCK8实验检测细胞的增殖能力。结果：与对照组（0 μmol/L）相比，30、60、90、120 μmol/L DEHP组细胞的增殖能力、克隆形成能力和迁移能力明显下降；丙二醛、活性氧、Fe3+（90、120 μmol/L DEHP组）含量明显升高；线粒体膜电位（60、90、120 μmol/L DEHP组）明显降低；GPX4（60、90、120 μmol/L DEHP组）和xCT蛋白表达水平明显降低。DEHP与Ferrostatin1联合培养后细胞增殖能力较DEHP单独培养明显提高。结论： DEHP能够抑制GC1 spg细胞的增殖和迁移能力，降低GPX4和xCT蛋白的表达，可能通过铁死亡途径引起细胞死亡。

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	蔺博涵
	刘伟
	王子璇
	王超
	李涛
	杨琴
	虞绮雯
	孙晓春

关键词 ：精原细胞GC-1 spg, DEHP, 铁死亡, GPX4, xCT

Abstract：Objective： To investigate the mechanism of DEHP-induced damage on mouse spermatogonial cell GC-1 spg function. Methods： GC1 spg were treated with various concentrations of DEHP (0, 30, 60, 90, 120 μmol/L). The effects of DEHP on the proliferation and migration of GC-1 spg were observed by CCK8 test, colony formation test and Transwell assay, and the relative contents of iron ion (Fe3+) and malondialdehyde (MDA) in GC-1 spg cells were determined by chemical spectrophotometry. The expression of cystine glutamate reverse transporter (xCT) and glutathione peroxidase 4 (GPX4) in GC1 spg was detected by Western blotting assay. The expression of intracellular reactive oxygen species （ROS） and the changes of mitochondrial membrane potential were detected by cellular immunofluorescence. The proliferation ability of GC-1 spg was detected by CCK8 assay after ferroptosis inhibitor Ferrostatin1 (1 μmol/L) was cotreated with DEHP (90 μmol/L) 24 h. Results： Compared with the control group (0 μmol/L), the proliferation, cloning and migration ability of cells in 30, 60, 90, 120 μmol/L DEHP groups were significantly decreased, while the contents of MDA,ROS and Fe3+ (90, 120 μmol/L DEHP group) were significantly increased. The mitochondrial membrane potential (60, 90, 120 μmol/L DEHP group) and the expression of GPX4 (60, 90, 120 μmol/L DEHP group), xCT protein were significantly decreased. The ability of cell proliferation after co-treatment of DEHP and Ferrostatin-1 was significantly higher than that of DEHP alone. Conclusion： DEHP can inhibit proliferation and migration capacity of GC-1 spg and reduce the expression of GPX4 and xCT proteins in GC-1 spg, possibly causing ferroptosis.

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收稿日期: 2022-11-10

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通讯作者: 孙晓春（通讯作者），教授，硕士生导师，E-mail： xiaochun@ujs.edu.cn

作者简介: 蔺博涵（1998—），男，硕士研究生

引用本文:

蔺博涵，刘伟，王子璇，王超，李涛，杨琴，虞绮雯，孙晓春. 增塑剂DEHP通过铁死亡途径抑制小鼠GC-1 spg精原细胞生长[J]. 江苏大学学报:医学版, 2023, 33(02): 146-151. LIN Bohan1, LIU Wei1, WANG Zixuan1, WANG Chao1, LI Tao1, YANG Qin2, YU Qiwen1, SUN Xiaochun1. Plasticizer DEHP inhibits the growth of mouse spermatogonia GC-1 spg through the ferroptosis pathway#br#. Journal of Jiangsu University(Medicine Edition), 2023, 33(02): 146-151.

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http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2023/V33/I02/146

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