Zhang Lei, Qin Zhiqiang, Li Ran, Wang Shangqian, Wang Wei, Tang Min, Zhang Wei
Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210009, China.
Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210009, China.
Environ Toxicol Pharmacol. 2019 Nov;72:103236. doi: 10.1016/j.etap.2019.103236. Epub 2019 Aug 1.
Di-N-butylphthalate (DBP) have given rise to more and more attention due to its unique endocrine toxicity to male reproductive system. Our previous studies have demonstrated antioxidative Nrf2 (nuclear factor erythroid related factor 2) pathway play a vital role in DBP induced oxidative stress injury. ANXA5 (annexin A5), which is highly expressed in testicular Leydig and Sertoli cells, was found upregulated after DBP stimulation. Mouse Leydig and Sertoli cells were exposed to different concentration of DBP for 24 h to examine the ROS (Reactive oxygen species), MDA (Malondialdehyde), SOD (superoxide dismutase) level and ANXA5, Nrf2, NQO1 (NAD(P)H-quinone oxidoreductase 1), HO-1 (heme oxygenase 1) and ERK/P-ERK protein expression by DHE (Dihydroethidium) staining, ELISA (enzyme-linked immunosorbent assay) and Western blot respectively. Firstly, the oxidative stress injury induced by DBP was re-validated. Then, we confirmed the change of Nrf2 pathway and ANXA5 level after DBP exposure to testicular cells. Additionally, overexpressed ANXA5 could activate Nrf2/HO-1/NQO1 antioxidant pathway and significantly attenuate DBP-induced oxidative stress. Ultimately, we demonstrated ANXA5 could increase ERK phosphorylated level and the activated role of ANXA5 on ERK/Nrf2 pathway could be reversed by ERK inhibitor. Overall, this study illuminated that ANXA5 could defend testicle Leydig and Sertoli cells against DBP-induced oxidative stress injury through ERK/Nrf2 pathway.
邻苯二甲酸二丁酯（DBP）因其对雄性生殖系统独特的内分泌毒性而受到越来越多的关注。我们之前的研究表明，抗氧化的Nrf2（核因子红细胞相关因子2）通路在DBP诱导的氧化应激损伤中起关键作用。在睾丸间质细胞和支持细胞中高表达的膜联蛋白A5（ANXA5），在DBP刺激后被发现上调。将小鼠睾丸间质细胞和支持细胞暴露于不同浓度的DBP中24小时，分别通过二氢乙锭（DHE）染色、酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法检测活性氧（ROS）、丙二醛（MDA）、超氧化物歧化酶（SOD）水平以及ANXA5、Nrf2、NAD（P）H-醌氧化还原酶1（NQO1）、血红素加氧酶1（HO-1）和细胞外信号调节激酶/磷酸化细胞外信号调节激酶（ERK/P-ERK）蛋白表达。首先，重新验证了DBP诱导的氧化应激损伤。然后，我们证实了DBP暴露于睾丸细胞后Nrf2通路和ANXA5水平的变化。此外，过表达的ANXA5可激活Nrf2/HO-1/NQO1抗氧化通路，并显著减轻DBP诱导的氧化应激。最终，我们证明ANXA5可增加ERK磷酸化水平，并且ERK抑制剂可逆转ANXA5对ERK/Nrf2通路的激活作用。总体而言，本研究表明ANXA5可通过ERK/Nrf2通路保护睾丸间质细胞和支持细胞免受DBP诱导的氧化应激损伤。
