Nrf2/ANXA5信号通路在尿道下裂发生中的作用及机制研究

Endocrine disrupting chemicals (EDCs) has been well defined as anti-androgenic effects by interfering with production and activity of testosterone. Results from recent epidemiological studies indicate an association of phthalate esters exposure with hypospadias. Oxidative stress in Leydig cells was suggested to contribute to phthalate-induced reproductive disorders. Leydig cell is one of the most important targets of testicular toxicity caused by phthalate esters. Here we found that Annexin A5 (ANXA5) and NF-E2-related factor 2(Nrf2) were concurrently upregulated in fetal Leydig cells after embryonic exposure, and overexpression of Nrf2 significantly upregulated ANXA5 mRNA and protein expression. Moreover, bioinformatics analysis showed four binding sites of Nrf2 in ANAX5 promoter. Meantime, overexpression of ANXA5 in Leydig cells obviously reduced ROS production. All these preliminary data highly suggested that Nrf2-drived ANXA5 may play a role in Leydig cell injury and subsequent occurrence of hypospadias via modulating oxidative response. In this proposal, we will fully investigate the role of ANXA5 in protecting against EDCs-induced Leydig cell injury and hypospadias occurrence, as well as the underlying mechanisms using genetic approaches and antioxidant. The present project will not only increase our understanding on the pathogenic mechanism of DBP-induced Leydig cell injury and hypospadias occurrence but also offer new insights into the clinical therapies.

尿道下裂是一种常见小儿畸形，发病率呈上升趋势。研究表明其与环境内分泌干扰物（EDCs）的暴露与蓄积有关，EDCs可诱导机体氧化应激损伤，Leydig细胞是其重要靶细胞。我们前期研究发现，ANXA5及Nrf2在染毒后胎鼠睾丸Leydig细胞内表达上调；过表达Nrf2可显著上调ANXA5表达，明显降低细胞氧化应激水平，增加睾酮分泌；生物信息学分析显示ANXA5启动子区有四个转录因子Nrf2的结合位点。我们推测：ANXA5可能在Nrf2调控下拮抗Leydig细胞氧化应激损伤，减少尿道下裂的发生。本课题拟通过过表达和表达沉默等技术，运用抗氧化剂及基因敲除小鼠，探讨ANXA5对Leydig细胞损伤及尿道下裂发生的影响及机制；通过染色质免疫共沉淀等方法检测Nrf2对ANXA5基因的直接调控作用，阐明Nrf2/ANXA5信号通路在尿道下裂发生中的作用机制，为防治EDCs所致出生缺陷提供新线索。

尿道下裂是一种常见小儿畸形，发病率呈上升趋势。研究表明其与环境内分泌干扰物（EDCs）的暴露与蓄积有关，EDCs可诱导机体氧化应激损伤，睾丸间质是其重要靶细胞。前期研究表明，Nrf2抗氧化通路在DBP诱导的氧化应激损伤中起重要作用。ANXA5 (annexin A5)在睾丸间质和支持细胞中高表达，且在DBP刺激后表达上调。首先在体外实验中，我们再次验证了DBP可诱导睾丸细胞发生氧化应激损伤。然后，我们检测了睾丸细胞暴露于DBP后Nrf2通路关键蛋白和ANXA5水平的变化。进而发现过表达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。我们进一步证明了ANXA5可提高ERK磷酸化水平，而ERK抑制剂可以逆转ANXA5对ERK/Nrf2通路的激活作用。总之，本研究发现ANXA5可以通过ERK/Nrf2途径保护睾丸间质和支持细胞免受DBP诱导的氧化应激损伤。动物实验部分，我们利用青春期大鼠染毒模型，借助于二代测序筛选出表达差异显著的microRNA，进一步验证得到miR-506-3p在DBP染毒组明显高表达。接着，我们采用构建的agomir-506-3p注射大鼠睾丸，构建体内miRNA过表达模型。我们发现过表达miRNA-506-3p能够加重DBP染毒大鼠睾丸的氧化应激损伤，而且miR-506-3p可抑制Nrf2通路的活性，进一步荧光素酶报告基因实验表明miR-506-3p为ANXA5上游调控因子，进而发现miR-506-3p通过调控ANXA5的表达来影响Nrf2抗氧化应激通路的活性。