Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China.
Department of Geriatrics, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China.
Ecotoxicol Environ Saf. 2020 Apr 1;192:110201. doi: 10.1016/j.ecoenv.2020.110201. Epub 2020 Feb 3.
This study focused on the oxidative stress effect of di-n-butyl phthalate (DBP) on development of the urinary system.
We examined the mRNA expression of genital tubercle (GT) in control and DBP induced hypospadias group by Affymetrix Rat 230 2.0 Array. Real-time PCR and Western Blot were used to detect the protein and mRNA expression levels of inositol-1,4,5-triphate-receptor (IP3R) and epithelial-mesenchymal-transition (EMT)-related molecular markers, such as E-cadherin, β-Catenin, Snail, N-cadherin, in the GT of hypospadiac male rats and controls. The results of array were further confirmed in vitro. The changes of intracellular calcium concentration in urethral epithelial cells were detected by Fluo-3-AM before and after DBP treatment. The levels of reactive oxygen species (ROS) in urethral epithelial cells were measured by DCFH-DA with different concentrations of DBP (0, 1, 10, 100 μmol/L) treatment.
The mRNA expression profiles of GT in control and DBP induced hypospadias group showed high expression of IP3R and the abnormalities of EMT. Compared to the control group, the expression levels of IP3R, E-cadherin and β-Catenin increased at both the protein and mRNA levels. However the expression levels of Snail and N-cadherin decreased. The intracellular calcium concentration increased significantly after DBP treatment. The effect of DBP on urethral epithelial cells was linked to the generation of oxidative stress.
DBP can influence the development of GT through its oxidative stress effect, which significantly increases the concentration of calcium and inhibits EMT in urethral epithelial cells, and block the fusion process of urethral groove, causing the occurrence of hypospadias. This study provides a new understanding of DBP's molecular mechanisms on hypospadias and may lead to new treatment strategies for the disease.
本研究旨在探讨邻苯二甲酸二丁酯（DBP）对泌尿系统发育的氧化应激作用。
通过 Affymetrix Rat 230 2.0 基因芯片检测对照组和 DBP 诱导的尿道下裂组生殖结节（GT）的 mRNA 表达。采用实时 PCR 和 Western blot 检测 GT 中肌醇 1,4,5-三磷酸受体（IP3R）和上皮-间充质转化（EMT）相关分子标志物，如 E-钙黏蛋白、β-连环蛋白、Snail、N-钙黏蛋白的蛋白和 mRNA 表达水平。在体外进一步验证了芯片的结果。用 Fluo-3-AM 检测 DB 处理前后尿道上皮细胞内钙浓度的变化。用不同浓度的 DBP（0、1、10、100 μmol/L）处理后，用 DCFH-DA 测量尿道上皮细胞内的活性氧（ROS）水平。
对照组和 DBP 诱导的尿道下裂组 GT 的 mRNA 表达谱显示 IP3R 高表达和 EMT 异常。与对照组相比，IP3R、E-钙黏蛋白和β-连环蛋白的蛋白和 mRNA 水平均升高。然而，Snail 和 N-钙黏蛋白的表达水平下降。DBP 处理后，细胞内钙浓度显著增加。DBP 对尿道上皮细胞的作用与氧化应激的产生有关。
DBP 可通过其氧化应激作用影响 GT 的发育，显著增加钙浓度，抑制尿道上皮细胞 EMT，阻断尿道沟融合过程，导致尿道下裂的发生。本研究为 DBP 对尿道下裂的分子机制提供了新的认识，并可能为该疾病的治疗提供新的策略。
