UCH-L1在DBP导致睾丸发育不全综合征(TDS)中的作用及机制研究

Recently, the incidence of Testicular Dysgenesis syndrome (TDS) rises every year in our country. TDS has been proven to be associated with oxidative stress caused by endocrine-disrupting chemicals (EDCs), of which DBP is the most important one. Sertoli cells are one of the main target cells of oxidative stress. In our previous study, we found that Keap1-Nrf2 pathway is involved in the regulation of oxidative stress in Sertoli cells, but the mechanism is not clear. Further studies showed that DBP treatment caused the decrease of Ubiquitin C-terminal hydrolase L1（UCH-L1）and Keap1, which mainly located in spermatogenic cells and Sertoli cells. Thus, we propose a hypothesis the decrease of UCH-L1 led to the degradation of Keap1 and activated the expression of Nrf2, alleviating the oxidative stress caused by DBP in Sertoli cells. The purpose of our research was to investigate whether UCH-L1 can regulate the expression of Keap1 and Nrf2 in Sertoli cells and the molecular mechanism at cellular level, and analyzed adenovirus transfection and small RNA interference of UCH-L1 and expression changes of Keap1, Nrf2 and downstream molecules after application of UCH-L1 inhibitors. Meanwhile, the above changes were to be verified in animal model. Our research aims to investigate the role and mechanism of UCH-L1 in oxidative stress injury of Sertoli cells, and provide new therapeutic evidence for the study of the molecular mechanism of TDS.

近年来，睾丸发育不全综合征(TDS)的发病率不断上升。研究表明TDS与邻苯二甲酸二丁酯（DBP）等环境内分泌干扰物引起的氧化应激相关，睾丸Sertoli细胞是重要靶细胞之一。我们前期发现Keap1-Nrf2通路参与调控Sertoli细胞的氧化应激损伤，但通路激活的机制尚不明确。同时DBP染毒后大鼠睾丸生精细胞和Sertoli细胞中泛素C端水解酶L1（UCH-L1）及Keap1表达水平降低，因此我们推测UCH-L1可能通过调控Keap1-Nrf2通路参与Sertoli细胞的氧化应激损伤。本课题拟从细胞水平研究UCH-L1对keap1和Nrf2表达的调控作用及具体机制，分析UCH-L1过表达、干扰表达和应用UCH-L1抑制剂后Keap1、Nrf2及下游分子表达变化，并在动物模型中验证上述改变，探讨UCH-L1在Sertoli细胞氧化应激损伤中的作用及机制，为TDS的分子机制研究提供新的方向。

邻苯二甲酸二丁酯(Di-n-butylphthalate, DBP) 作为环境内分泌干扰物 (Endocrine Disrupting Chemicals， EDCs）的一种，已被中国列入优先控制的污染物名单。研究表明 DBP 的暴露与一系列男性生殖系统的先天及后天缺陷密切相关。DBP 所致的睾丸氧化应激损伤在这些缺陷中发挥了重要的作用。课题组前期研究表明Keap1 (Kelch-like ECH-associated protein-1)/Nrf2(核转录因子E2 相关因子2, Nuclear factor erythroid 2-related factor 2) 抗氧化应激通路在DBP所致的氧化应激损伤中发挥重要的作用。本研究旨在探讨DBP导致氧化应激损伤及Nrf2抗氧化应激系统反馈性激活的进一步分子调控机制。通过对青春期SD 大鼠玉米油对照组与DBP800mg/kg 染毒组进行高通量测序, 筛选出差异表达的基因，从中筛选出差异倍数2倍以上的去泛素化酶，使用RT-qPCR以及Western Blot技术进一步验证差异基因的表达，找出受到DBP显著影响的去泛素化酶类。体外实验选用HEK-293 细胞，通过敲低过表达差异的去泛素化酶 USP15，检测 Keap1/Nrf2 通路的改变及相应的去泛素化调控机制。最终，选用小鼠睾丸生殖细胞 GC1, GC2细胞系，验证USP15对DBP所诱导的生殖细胞氧化应激损伤的调控作用。与对照组相比，DBP染毒组大鼠精子数目及成熟率显著降低，睾丸氧化应激水平显著升高，Nrf2及下游抗氧化蛋白HO-1, NQO1, GCLC 的表达显著升高。进一步高通量测序以及PCR,Western blot 筛选出四个差异表达的去泛素化酶USP15, USP1, Cyld, Tnfaip3, 敲低过表达实验发现USP15对Nrf2的泛素化降解有明显的调控作用。体外泛素化实验证明USP15能够降低Keap1的泛素化水平，从而增加Nrf2的泛素化降解。 此外在GC1, GC2细胞中构建DBP诱导的细胞氧化应激模型，发现激活的Nrf2通路以及抑制的USP15表达，进一步实验表明敲低USP15后，细胞的氧化应激水平明显降低。