邻苯二甲酸二-(2-乙基己基)酯通过氧化应激、凋亡诱导大鼠尿道下裂

目的 探讨邻苯二甲酸二-(2-乙基己基)酯di(2-ethylhexyl)phthalate,DEHP]诱导大鼠发生尿道下裂的发生机制.方法 20只成年孕SD大鼠10 ～ 12周龄,体质量(240 ±20)g]按完全随机分为:玉米油对照组750 mg/(kg·d),n=10]和DEHP暴露组750 mg/(kg·d),n=10],于妊娠期(gestation day,GD)第8.5 ～18.5日每日灌胃,于GD19.5时剖宫取出胎鼠,以有无睾丸分辨雌雄,并在解剖显微镜下观察有无尿道下裂的发生,将DEHP暴露组有尿道下裂的列入尿道下裂组,以玉米油对照组雄性无尿道下裂的胎鼠作为正常对照组.测量雄鼠胎鼠的肛门生殖器距离(anogenital distance,AGD)和肛门生殖指数(anogenital index,AGI),扫描电镜和HE染色观察阴茎组织形态改变情况,高效液相色谱法检测胎鼠肝脏中的维生素A、E水平,Westem blot检测阴茎组织中氧化应激相关蛋白Nrf2、HO-1、SOD1、Gpx和凋亡相关蛋白cleaved caspase-3、Bax、Bcl2的表达水平,TUNEL法检测阴茎组织中的细胞凋亡.结果玉米油对照组胎鼠无尿道下裂发生,DEHP暴露组胎鼠出现尿道下裂,发生率为27.59％.与正常对照组相比,尿道下裂组AGD、AGI均明显降低(P＜0.05);尿道下裂胎鼠阴茎出现腹侧尿道皱褶融合障碍及尿道口开口异常;此外,尿道下裂胎鼠体内维生素A、E的水平明显降低(P＜0.05);阴茎组织中Nrf2、HO-1、Gpx、SOD1和Bcl2蛋白表达水平明显下降(P＜0.05),cleaved caspase-3和Bax蛋白表达水平则明显增高(P＜0.05),细胞凋亡显著增加(P＜0.05).结论DEHP诱导SD大鼠发生尿道下裂,可能与机体氧化应激失衡、Nrf2-HO-1信号通路被抑制后阴茎组织中细胞凋亡异常增加有关.

Di (2-ethylhexyl) phthalate induces hypospadias in SD rats via apoptosis and oxidative stress

Objective To explore the mechanism of hypospadias induced by di (2-ethylhexyl)phthalate (DEHP) in rats.Methods Twenty adult pregnant SD rats (10 ～ 12 weeks old, weighting 240 ±20 g) were randomly divided into control group (n =10) and DEHP exposure group (n =10), intragastrically injected with corn oil and 750 mg/kg DEHP daily from gestation day (GD) 8.5 to GD 18.5, respectively.The fetal rats were taken out on GD 19.5.According to having testicles or not to distinguish male from female,the male fetuses were collected for detection of hypospadias by dissecting microscopy.Then anogenital distance (AGD) and anogenital index (AGI) were measured and counted.Scanning electron microscopy and hematoxylin eosin staining were used to observe the pathological changes of genital tubercles (GTs).High performance liquid chromatography (HPLC) was employed to measure the contents of vitamin A and E in liver tissue.The expression levels of oxidative stress related proteins, including nuclear factor erythroid-2 p45-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase (Gpx) and superoxide dismutase 1 (SOD1), and apoptotic related proteins (cleaved caspase-3, Bax and Bcl2) in GTs were analyzed by Western blotting.Cell apoptosis in GTs was measured by TUNEL.Results No hypospadias was observed in the control group, and its incidence was 27.59％ in the DEHP exposure group.Compared with the control group, the AGD and AGI were significantly lower in the DEHP exposure group (P ＜ 0.05).There were abnormal fusion of the urethral folds and abnormal urethral opening in the hypospadias rats.What's more, the hypospadias rats also had lower contents of vitamin A and E in the liver tissue (P ＜ 0.05), significantly decreased protein levels of Nrf2, HO-1, SOD1, Gpx and Bcl2 (P ＜ 0.05), notably enhanced cleaved caspase-3 and Bax (P ＜ 0.05), and more obvious cell apoptosis in the GTs (P ＜ 0.05).Conclusion DEHP exposure induces hypospadias in male SD rats, which may be related to the imbalance of oxidative stress, inhibition of Nrf2-HO-1 signaling pathway, and enhancement of cell apoptosis in GTs.