Abstract：
Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future.
摘要：
雷公藤甲素（TP）是一种主要的生物活性化合物，来源于雷公藤。F.(TwHF)以其药用特性而闻名，但它也表现出潜在的毒性作用。它已被证明会引起严重的男性生殖毒性，然而这背后的确切机制仍不清楚，这限制了其广泛的临床应用。本研究旨在探讨TP致小鼠睾丸损伤和精子生成功能障碍的机制。使用小鼠模型和精母细胞来源的细胞系GC-2spd。在本研究中,发现TP通过促进ROS的形成，表现出明显的睾丸微结构损伤和精子发生缺陷，包括浓度降低和形态异常，MDA产生和抑制GSH水平，谷胱甘肽过氧化物酶4（GPX4）在体内表达。此外，Ferrostatin-1(FER-1),一种铁凋亡抑制剂，被发现显著减少脂质过氧化的积累，减轻睾丸微结构损伤，增强小鼠的生精功能。此外,细胞活力显著下降，塌陷的线粒体膜电位,在体外观察到升高的DNA损伤。FER-1预处理可以逆转上述现象，表明铁凋亡参与了TP介导的精子发生功能障碍。机械上,TP可以通过触发GPX4的K63连接的多泛素化来增强GPX4泛素降解，从而刺激精母细胞的铁凋亡。功能上,GPX4缺失加剧了GC-2细胞的铁凋亡并加剧了DNA损伤，而GPX4过表达减轻TP诱导的铁细胞凋亡。总的来说,这些发现首次表明，通过促进GPX4K63连接的聚泛素化，铁凋亡在TP诱导的睾丸损伤和生精功能障碍中的重要作用，有望为与TP相关的男性生殖损伤提供潜在的治疗途径。此外,本研究也为今后改进TP或TwHF的临床应用提供了理论基础。
