ROS介导的Notch信号通路在DEHP致未成熟睾丸发育异常的机制研究

Abnormal testicular development in childhood will lead irreversible damage to testicular function in adult, seriously affecting the reproductive function. Our previous study found that DEHP as the most important environmental endocrine disruptors, caused immature testicular abnormalities closely associated with oxidative stress, and abnormal Notch signaling pathway involved in, but the exact mechanism is unknown. The project intends to go down with this idea "oxidative stress -impaired Nrf2 antioxidant defense system - oxidation - antioxidant imbalance -Notch pathway abnormality" , in-depth study in ROS as initiating factor in affecting the core of the antioxidant Nrf2 defense system, the regulation of the activity of the Notch signaling pathway, consequently casing obstacles to the proliferation and differentiation of spermatogonial stem cells, leading to testicular abnormalities. It clarifies the molecular mechanisms of DEHP as the cause of immature testicular abnormalities from the source. And it aims at the point that ROS damage is the initiating factor and the key procedure of DEHP causing testicular dysplasia.Investigating the combined effects of vitamin E and C in the prevention and treatment as well as potential applications of intervening DEHP-induced testicular developmental and reproductive toxicity. The result is bound to provide scientific experimental basis in developing environmental policy for the government,handling pollution, clinical prevention,intervening genital deformity and dysfunction.

幼年时期睾丸发育异常将导致成年后睾丸功能不可逆损害，严重影响生殖功能。课题组前期研究发现白色污染DEHP作为最主要的环境内分泌干扰物，致未成熟睾丸发育异常与氧化应激损伤密切相关，且涉及Notch信号通路的异常，但其具体机制不详。本项目拟沿着“氧化应激损伤—Nrf2抗氧化防御体系受损—氧化-抗氧化失衡—Notch通路异常”的思路，深入研究ROS作为“始动”因素如何影响以Nrf2为核心的抗氧化防御体系，调控Notch信号通路活性，从而引起精原干细胞增殖与分化障碍，导致睾丸发育异常。从源头上阐明DEHP致未成熟睾丸发育异常的分子机制。并针对ROS损伤是DEHP导致睾丸发育异常的“始动因素”和关键环节，探讨维生素E和C联合作用在干预DEHP所致睾丸发育和生殖毒性中的防治作用及潜在的应用价值。研究结果必将为政府制定环保政策、治理污染和临床预防、干预生殖畸形的发生及功能障碍提供科学的实验依据。

幼年时期睾丸发育异常将导致成年后睾丸功能不可逆损害，严重影响生殖功能。.本项目沿着“氧化应激损伤—Nrf2抗氧化防御体系受损—氧化-抗氧化失衡—Notch通路异常”的思路：首先通过构建DEHP暴露动物模型，验证DEHP与未成熟睾丸发育毒性损伤的相关性，明确其量效关系；进一步地，聚焦ROS研究其作为“始动因素”，影响Nrf2的表达和功能，引起细胞内氧化-抗氧化平衡，进而导致Notch信号通路异常的途径，从源头上阐明DEHP致未成熟睾丸发育毒性损伤的作用机制；最后，联合维生素干预DEHP所致氧化应激损伤，逆转Notch 信号通路的异常，深入研究维生素E、维生素C及其联合使用在清除过氧化物或自由基，而起到抗氧化或阻止氧化胁迫损伤中的作用，发挥其抗氧化功能，调节生精微环境，启动内源性生精修复，实现未成熟睾丸组织形态的恢复与生殖功能的重建。.项目研究发现，与正常对照组相比，DEHP暴露后雄性大鼠的肛门生殖指数和精子计数明显下降，大鼠睾丸曲细精管结构紊乱，上皮严重萎缩，生精细胞数量极少并且脱落，氧化应激指标（Nrf2、HO-1、NQO1、GPX1及SOD1）表达增高，Notch1及Hes1的表达水平下降；维生素E和维生素C联合使用干预后，睾丸组织中睾酮含量明显增加，睾丸曲细精管结构保存尚好，形态较单独应用一种维生素有所恢复，氧化应激指标和Notch1、Hes1的表达水平，与正常对照组之间无显著统计学差异；在体外条件下，MTT结果显示联合维生素干预后明显改善Leydig细胞活力，并降低细胞内ROS生成水平。.研究结果表明：1）生后暴露DEHP可引起雄性SD大鼠未成熟睾丸损伤，导致生殖功能障碍，其中氧化应激损伤起重要作用；2）ROS-Nrf2-Notch1信号通路异常介导的睾丸Leydig细胞氧化应激损伤在DEHP引起睾丸发育异常中起重要作用；3）联合维生素可通过抵抗ROS作用稳定Nrf2功能和Notch1信号表达，维持Leydig细胞活性，最终恢复生精功能。