Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced autophagy of mouse Leydig TM3 cells in vitro.
Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced autophagy of mouse Leydig TM3 cells in vitro.
复制标题
氧化应激参与磷酸三邻甲苯酯诱导的体外小鼠 Leydig TM3 细胞自噬
DOI：
10.1186/s12958-016-0165-x
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发表时间：
2016-06-07
期刊：
影响因子：
--
通讯作者：
Chen J
中科院分区：
文献类型：
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作者：
Liu X;Xu L;Shen J;Wang J;Ruan W;Yu M;Chen J
三邻甲酰磷酸酯（TOCP）作为增塑剂、塑料柔软剂和阻燃剂在工业上得到了广泛的应用，除神经毒性和免疫毒性外，还对动物的雄性生殖系统有毒性作用。我们已经报道了TOCP抑制大鼠精原干细胞的生精和诱导自噬，但TOCP是否诱导小鼠间质细胞的自噬及其潜在机制尚不清楚。MTT法观察细胞活力。放射免疫法测定睾酮水平。AnnexinV-FITC/PI法观察细胞凋亡。Western blotting检测LC3、Atg5-Atg12、Beclin 1的含量。透射电镜观察自噬体。采用氧化应激试剂盒测定各组大鼠血清MDA、GSH含量、SOD、GSH- px活性、总抗氧化状态（TAS）和总氧化状态（TOS）。本研究表明，TOCP可显著抑制小鼠间质TM3细胞的活力和睾酮分泌，但对细胞凋亡无影响。而TOCP显著提高了LC3-II、LC3-II与LC3-I的比值以及自噬蛋白Atg5和Beclin 1的含量。透射电镜（TEM）显示，TOCP使细胞质自噬空泡增多，表明TOCP可诱导细胞自噬。TOCP显著诱导小鼠间质TM3细胞氧化应激。H2O2抑制细胞活力，诱导细胞自噬；而n -乙酰- l-半胱氨酸（NAC）对氧化应激的抑制作用可以恢复TOCP对细胞活力的抑制和诱导自噬。结果表明氧化应激可能参与了tocp诱导的小鼠间质TM3细胞自噬。
As a plasticizer, plastic softener, and flame-retardant, tri-ortho–cresyl phosphate (TOCP) is and has been widely used in industry and reported to have a toxic effect on the male reproductive system in animals besides neurotoxicity and immunotoxicity. We have reported that TOCP inhibits spermatogenesis and induces autophagy of rat spermatogonial stem cells, but it is still unknown whether TOCP induces autophagy of mouse Leydig cells and its potential mechanism. Cell viability was observed by MTT assay. Level of testosterone was measured by radioimmunoassay. Apoptosis was observed by AnnexinV-FITC/PI assay. The contents of LC3, Atg5-Atg12, and Beclin 1 were detected by Western blotting analysis. Autophagosomes were investigated by transmission electron microscopy. The contents of MDA and GSH and the activities of SOD, GSH-PX, total antioxidant status (TAS) and total oxidant status (TOS) were measured by oxidative stress kits. The present study shows that TOCP markedly inhibited viability and testosterone output of mouse Leydig TM3 cells but had no effect on apoptosis. However, TOCP significantly increased both LC3-II and the ratio of LC3-II to LC3-I and the contents of autophagy proteins Atg5 and Beclin 1. Transmission electron microscopy (TEM) showed that TOCP increased autophagic vacuoles of the cytoplasm, indicating that TOCP could induce autophagy of the cells. TOCP significantly induced oxidative stress of mouse Leydig TM3 cells. H2O2 also inhibited viability and induced autophagy of the cells; however, inhibition of oxidative stress by N-acetyl-L-cysteine (NAC) could rescue the inhibition of cell viability and induction of autophagy by TOCP. The results show oxidative stress might be involved in TOCP-induced autophagy of mouse Leydig TM3 cells.