摘要目的 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机制.方法 20只成年孕SD鼠[10～12周龄,体重(240±20 g)]按照数字随机化法分为:正常对照组(n=10)和DEHP暴露组(750 mg·kg-1·d-1)(n=10),于妊娠期(gestationday,GD)第8.5～18.5日每日灌胃,于GD 19.5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.测量肛门生殖器距离(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,透射电镜观察阴茎组织内自噬的改变,western blot检测生殖结节(GTs)中自噬相关蛋白LC3、p62、Beclin1、Akt、mTOR、p-Akt、p-mTOR和凋亡相关蛋白cleaved-caspase3、Bax、Bcl2的表达水平,TUNEL法检测雄鼠GTs中细胞凋亡.结果 DEHP暴露后出现尿道下裂,发生率为27.58％,尿道下裂组中AGD、AGI均明显降低(P＜0.05);尿道下裂雄鼠的GTs出现腹侧尿道缝融合障碍及尿道开口异位.尿道下裂组GTs中LC3和Beclin1的表达、LC3Ⅱ/Ⅰ比值增加(P＜0.05),p62、p-Aktser473、p-mTORser2448表达明显减少(P＜0.05),Bcl2表达减少(P＜0.05),cleaved-caspase3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作为一个外源性干扰物进入机体后,致使胎鼠的GTs中PI3K/Akt/mTOR信号通路被抑制,自噬水平增加,同时促凋亡与抗凋亡系统失衡,进一步促进自噬的发生,最终使得凋亡异常增加,导致尿道下裂的发生.

abstractsObjective To explore the mechanism of hypospadias induced by di (2-ethylhexyl) phthalate (DEHP) in rats.Methods Twenty adult Sprague-Dawley (SD) rats [aged 10-12 weeks,weight (240 ± 20) g] were randomly divided into 2 groups of control (n =10) and DEHP exposure group (n =10).The animals received oil and 750 mg/kg DEHP by gavage from gestation day (GD) 8.5 to GD 18.5 respectively.Genital tubercles (GTs) were collected from male fetuses on GD 19.5.Anogenital distance (AGD) and anogenital index (AGI) of male fetuses were measured and gross imaging changes of GTs observed by scanning electron microscope (SEM) and hematoxylin eosin staining.The level of autophagy in GTs was examined by transmission electron microscope (TEM) and autophagy-related proteins (LC3,p62,Beclin1,Akt,mTOR,p-Akt,p-mTOR) and apoptotic-related proteins (cleaved-caspase 3,Bax & Bcl2) in GTs were analyzed by Western blot.And apoptosis in GTs was measured by TUNEL.Results Compared with control group,the incidence of DEHP-induced hypospadias was 27.58％ in male fetuses while AGD and AGI decreased in hypospadias group (P＜0.05).SEM and HE staining revealed abnormal fusion of urethral folds and ectopia of urethral opening.Autophagosome was found in hypospadias group,but not in control group.Compared with non-hypospadias,the protein expression of LC3 and ratio of LC3 Ⅱ/LC3 Ⅰ significantly increased in GTs of hypospadiac rats (P＜0.05) while p62 expression decreased (P＜0.05).The expressions of cleaved-caspase3 and Bax increased while Bcl2 decreased (P＜ 0.05).Apoptotic cells significantly decreased in GTs of hypospadiac rats (P＜0.05).Conclusions DEHP is absorbed by body as an exogenous disruptor and Akt/mTOR signaling pathway becomes activated in GTs,Phosphorylations of Akt and mTOR decrease and enhanced autophagy leads to an imbalance of apoptosis and anti-apoptotic system in GTs.With an abnormal increase of apoptosis,it causes the occurrence of hypospadias.