邻苯二甲酸二-(2-乙基己)酯对血睾屏障完整性的损伤及机制

Effect of Di-(2-ethylhcxyl) phthalate exposure on blood-testis barrier integrity in rats

目的探讨邻苯二甲酸二-(2-乙基己)酯(DEHP)破坏睾丸血睾屏障完整性的机制研究。方法将出生后60 d的SD雄鼠随机分为两组,即玉米油对照组和DEHP暴露组,DEHP的用药剂量为750 mg/kg,采用灌胃的方式给药,每天1次,连续处理30 d,并于最后1次处理后麻醉处死。HE染色观察睾丸的组织病理学改变情况;Western blot检测睾丸组织中构成血睾屏障的关键连接蛋白N-cadherin、β-catenin、occludin和connexin43(Cx43)的膜蛋白表达水平;MTT检测睾丸支持细胞(Sertoli cell)的活力,活性氧检测试剂盒检测细胞中活性氧(Reactive oxygen species,ROS)生成水平;Western blot检测抗氧化应激蛋白Nrf2以及p-p38的蛋白表达水平。结果与对照组相比,DEHP暴露后的睾丸曲细精管结构紊乱,管腔中可见圆形的精子细胞,精子排列失去极性;Western blot结果表明occludin和connexin43的表达明显降低,N-cadherin和β-catenin表达明显升高(P<0.05);Sertoli细胞随着DEHP暴露浓度的增加,活力显著降低,ROS生成水平明显增加,Nrf2和p-p38的蛋白表达明显增高(P<0.05)。结论 DEHP暴露可通过氧化应激激活p38 MAPK信号通路,破坏血睾屏障完整性,并最终损伤生精功能。

Objective To investigate mechanism of di- （2-ethylhcxyl）phthalate （DEHP） exposure in causing blood-testis barrier （BTB） impairment in rats.Methods Two-months-old male SD rats were randomly divided into corn oil control group and DEHP （750 mg/kg） exposure group for daily intragastic treatment for 30 consecutive days.After the treatments the rats were examined for histomorphological changes of the testicle using HE staining and the expressions of the junction proteins Ncadherin β-catenin,occludin and connexin 43 of the BTB using Western blot.In the in vitro study,the vitality and ROS generation level in Sertoli cells exposed to different concentrations of DEHP were examined with MTT and ROS assay kits,respectively,and Nrf2 and p-p38 expressions were detected with Western blot.Results Compared with the control group,the rats with DEHP exposure showed structural damage of the seminiferous tubule and polarity loss of the spermatids.DEHP exposure caused significantly decreased expressions of occludin and connexin43 but increased expressions of N-cadherin andβ-catenin in the testicle tissues of the rats （P〈0.05）.The vitality of Sertoli cells was obviously decreased and ROS level increased significantly after exposure of the cells to increasing concentrations of DEHP,which also resulted in significantly up-regulated Nrf2 and p-p38 expressions （P〈0.05）.Conclusions DEHP exposure causes increased oxidative stress in the Sertoli cells of the testis,activates p38 MAPK signaling pathway,and results eventually in impaired spermatogenesis in rats.