葡萄籽原花青素激活Nrf2/ARE 信号通路缓解玉米赤霉烯酮所致雄性仔猪生殖毒性效应的研究

Zearalenone (ZEN) can seriously damage the male reproductive system. The previous work of our group confirmed that the natural antioxidant grape seed proanthocyanidins (PC) could alleviate the oxidative damage and apoptosis of ZEN induced testicular tissue in mice. Recent studies have shown that Nrf2/ARE signaling pathway plays an important role in antagonizing the oxidative damage and apoptosis of tissue cells caused by exogenous chemicals. We speculate that PC inhibits the apoptosis and oxidative damage of ZEN induced testis in piglets by activating this pathway.Therefore, our project aims to take the Leydig cells of male piglets as the research object, Nrf2 gene in Leydig cells will be silenced or overexpressed, the oxidative damage and apoptosis related factors, and the expression of related genes and proteins in Nrf2/ARE signal pathways and apoptosis signal pathways in Leydig cells of male piglets by using qRT-PCR, Western blotting,Flow cytometry and confocal microscopic imaging methods when adding ZEN, add PC PC+ZEN to the cells, and animal experiments in vivo will be carried to confirm our scientific hypothesis. At the same time, we studied the molecular mechanism of PC activated Nrf2/ARE signaling pathway in Leydig cells. Our study can provide a theoretical basis for the future application of PC in future.

玉米赤霉烯酮（ZEN）可严重损害公畜生殖系统。本课题组前期工作证实，天然抗氧化剂葡萄籽原花青素（PC）可缓解ZEN致小鼠睾丸组织氧化损伤和凋亡。新近研究表明：Nrf2/ARE信号通路在拮抗外源化学物质引起组织细胞氧化损伤和凋亡中起到重要作用。我们推测：PC抑制ZEN致仔猪睾丸组织凋亡和氧化损伤是通过激活该通路来实现的。因此，本项目拟以雄性仔猪睾丸间质细胞为研究对象，对该细胞Nrf2基因进行基因沉默或过表达，利用qRT-PCR、免疫印迹、免疫组化、流式细胞术、共聚焦显微镜等技术检测添加PC、ZEN、PC+ZEN对仔猪睾丸间质细胞的氧化损伤和凋亡相关指标、Nrf2/ARE信号通路、凋亡信号通路上相关基因和蛋白的表达情况，并加以动物体内试验验证，进而确证我们的科学假设。同时进行PC激活睾丸间质细胞Nrf2/ARE信号通路分子机制的研究。研究结果为今后利用PC防治ZEN生殖毒性提供理论依据。

本研究选择猪睾丸细胞（ST细胞），首先通过CCK-8法测定玉米赤霉烯酮（ZEN）作用于猪睾丸细胞的半数抑制浓度和原花青素作用于猪睾丸细胞时的最佳作用剂量。应用生物化学方法检测细胞氧化损伤相关指标，应用流式细胞术和TUNNEL荧光染色法检测细胞凋亡情况，使用qRT-PCR技术和Westren Blot技术检测细胞Nrf2相关信号通路mRNA和蛋白表达。结果表明，ZEN可诱导猪睾丸细胞发生细胞凋亡和氧化损伤，而原花青素可通过激活Nrf2信号通路抑制睾丸细胞发生的细胞凋亡和氧化损伤。进一步研究证实，原花青素可解除Nrf2与Keap1蛋白的结合，减少Keap1蛋白表达，抑制Nrf2蛋白泛素化降解从而增加Nrf2蛋白的稳定性，促进Nrf2蛋白的表达，增加Nrf2蛋白在细胞内累积并入核，与Bach1竞争并激活ARE元件，进而启动Nrf2/ARE信号通路，调控下游抗氧化酶和Ⅱ相解毒酶等相关基因，启动细胞保护作用。研究成果对养猪生产中玉米赤霉烯酮中毒防控具有指导意义，也为临床应用原花青素防控玉米赤霉烯酮引起的猪生殖机能损伤提供了理论依据。