淫羊藿配方颗粒对Pex3-/-氧化应激小鼠生精障碍的挽救作用及机制研究

Male infertility is associated with oxidative stress while antioxidants could improve sperm quality and pregnancy outcome, which is recognized by experts. However, further mechanism researches concerning the uses of various antioxidants especially formula granules which may become the ideal form of traditional Chinese medicine worldwide is deficient. The basic experimental model of the study is Pex3 knockout mice with antioxidant system disorder. Dyszoospermia, male infertility and elevated testis oxidative stress level in mice are observed through our preliminary study, which also demonstrates that epimedium granules supplementation may correct reproductive phenotype to some extent by decreasing testis oxidative stress level and inhibiting gene expression associated with aging. Later, Pex3 and p16 double knockout mice will be used in this study to further explore the mechanism of epimedium granules to spermatogenesis; the following clinical study plan, epimedium formula granules will be added in syndrome differentiation depending treatment for infertile patients with kidney-yang deficiency and high DFI. This study is to explain the molecular mechanism of Pex3-induced dyszoospermia and explore the molecular mechanism of the effect of epimedium on male fertility, especially reproductive aging; all of which could provide scientific evidence to support modern traditional Chinese medicine product development.

男性不育与氧化应激相关，抗氧化剂可提高精子质量，改善妊娠结局，已成为国际专家共识。但各种抗氧化剂，尤其是有望成为中药国际化理想产品的配方颗粒剂用于男性不育尚缺乏深入机制研究。本研究采用抗氧化系统紊乱的Pex3基因缺失小鼠为基础实验模型，初步研究发现，该小鼠生精障碍、雄性不育、睾丸组织氧化应激水平升高；补充淫羊藿配方颗粒可能通过降低睾丸组织氧化应激水平，抑制衰老基因表达而部分纠正其表型。后期将结合Pex3、p16二者双敲小鼠探索淫羊藿配方颗粒对生精障碍的挽救作用的深入机制，临床研究拟将淫羊藿配方颗粒结合使用于中医辨证施治之肾阳虚男性不育病人与精子DNA碎片指数(DFI)高的患者。本研究有助于阐述Pex3影响生精障碍的分子机制；探索淫羊藿作用于雄性生殖，尤其是生殖衰老的分子机制，为现代中药深入产品开发提供科学依据。

氧化应激（OS）是导致男性不育的主要因素之一。迄今为止，已发现口服抗氧化剂可显着提高人类精子的质量。因此，抗氧化治疗已成为国际男性不育专家的共识。在本研究中，以过氧化物酶体发生因子3(Pex3)基因敲除(KO,−/−)小鼠为模型，比较三种传统中药配方颗粒(TCM)（淫羊藿 [YYH]、菟丝子[TSZ]、和红景天[HJT])用于男性生殖功能挽救。发现YYH挽救效果最佳，并且对精子发生缺陷的Pex3-/-小鼠发挥了显著的挽救作用。同时，YYH 显着降低睾丸中的 ROS 水平，抑制生精细胞中的 DNA 氧化损伤，促进生精细胞的增殖，并抑制 Pex3-/-雄性小鼠的细胞凋亡。此外，通过建立细胞周期蛋白依赖性激酶抑制剂2A（CDKN2A,P16）-KO小鼠，探究了YYH改善精子发生异常的深入机制。具体而言，Pex3-/-敲除导致氧化应激水平显著升高，这破坏了生殖细胞DNA并进一步激活细胞衰老调节蛋白P16-CDK6的信号通路导致细胞周期停滞，最终导致精子发生过程受到影响。补充YYH通过影响P16的蛋白表达部分纠正了基因敲除小鼠的不育表型，在一定程度上改善了生殖结局。