Third-grade Pharmacological Laboratory on Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang, Hubei, 443002, China; College of Medicine and Health Sciences, China Three Gorges University, Yichang, Hubei, 443002, China.
Third-grade Pharmacological Laboratory on Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang, Hubei, 443002, China; College of Basic Medical Sciences, China Three Gorges University, Yichang, Hubei, 443002, China.
J Ethnopharmacol. 2024 Jan 10;318(Pt A):116865. doi: 10.1016/j.jep.2023.116865. Epub 2023 Jul 7.
Wuzi Yanzong Prescription (WZ), a classic traditional Chinese medicine formula, has the properties of kidney nourishing and essence strengthening, and it is widely used to treat male infertility with a long history. Sertoli cells are injured with aging, resulting in testicular dysfunction, and WZ effectively rejuvenates the age-related decline of testicular function. However, whether the therapeutic effects of WZ on aging-related testicular dysfunction are dependent on the restoration of Sertoli cell function remains unclear.
In a mouse model of natural aging, we explored the protective effects of WZ and its potential mechanisms.
Fifteen-month-old C57BL/6 mice were randomized to receive either standard diet or WZ (2 and 8 g/kg) for 3 months. Meanwhile, 10 1-month-old mice were considered the adult control group and received standard diet for 3 months. The testis and epididymis were rapidly collected, and the sperm quality, testicular histology, Sertoli cell numbers, tight junction (TJ) ultrastructure, and blood-testis barrier-associated protein expression and localization were assessed.
WZ significantly increased sperm concentration and sperm viability, improved the degenerative histomorphology and elevated the seminiferous epithelium height. Furthermore, WZ increased the number of Sertoli cells, restored the ultrastructure of the Sertoli cell TJ, and upregulated the expression of TJ-associated proteins (zonula occludens-1 and Claudin11), ectoplasm specialized-associated proteins (N-Cadherin, E-Cadherin and β-Catenin), and gap junction-associated protein (connexin 43), but did not affect the expression of Occludin and cytoskeletal protein (Vimentin). In addition, WZ did not change the localization of zonula occludens-1 and β-Catenin in aged testis. Moreover, WZ increased the expression of autophagy-associated proteins (light chain 3 beta and autophagy related 5) and decreased the expression of p62, phosphorylated mammalian target of rapamycin, and phosphorylated AKT in Sertoli cells. Finally, we found that WZ attenuated mTOR complex 1 (mTORC1) activity and upregulated mTORC2 activity, as evidenced by inhibition of the expression of the regulatory-associated protein of mTOR, phosphorylated p70 S6K, and phosphorylated ribosomal protein s6 and enhancement of the expression of Rictor in the Sertoli cells of aging mice.
WZ improves the injury of Sertoli cells by restoring AKT/mTOR-mediated autophagy and the mTORC1-mTROC2 balance in Sertoli cells during aging. Our findings provide a new mechanism of WZ in the treatment of aging-induced testicular dysfunction.
五子衍宗方（WZ）是一种经典的中药方剂，具有补肾填精的功效，历史上广泛用于治疗男性不育症。随着衰老，支持细胞受损，导致睾丸功能障碍，WZ 可有效恢复与年龄相关的睾丸功能下降。然而，WZ 对衰老相关睾丸功能障碍的治疗效果是否依赖于支持细胞功能的恢复尚不清楚。
在自然衰老的小鼠模型中，我们探讨了 WZ 的保护作用及其潜在机制。
将 15 月龄的 C57BL/6 小鼠随机分为标准饮食组或 WZ（2 和 8 g/kg）治疗 3 个月。同时，10 只 1 月龄的小鼠作为成年对照组，给予标准饮食 3 个月。快速采集睾丸和附睾组织，评估精子质量、睾丸组织学、支持细胞数量、紧密连接（TJ）超微结构以及血睾屏障相关蛋白的表达和定位。
WZ 显著增加了精子浓度和精子活力，改善了退行性组织形态学，提高了生精上皮高度。此外，WZ 增加了支持细胞数量，恢复了支持细胞 TJ 的超微结构，并上调了 TJ 相关蛋白（闭锁蛋白-1 和 Claudin11）、特化细胞外基质相关蛋白（N-钙黏蛋白、E-钙黏蛋白和β-连环蛋白）和间隙连接相关蛋白（连接蛋白 43）的表达，但不影响紧密连接蛋白（Occludin）和细胞骨架蛋白（波形蛋白）的表达。此外，WZ 并未改变衰老睾丸中闭锁蛋白-1 和β-连环蛋白的定位。此外，WZ 增加了自噬相关蛋白（微管相关蛋白轻链 3β和自噬相关蛋白 5）的表达，降低了 Sertoli 细胞中 p62、磷酸化哺乳动物雷帕霉素靶蛋白（mTOR）和磷酸化 AKT 的表达，以及衰老小鼠 Sertoli 细胞中 mTOR 复合物 1（mTORC1）的表达。最后，我们发现 WZ 抑制了 mTOR 调节相关蛋白、磷酸化 p70 S6K 和磷酸化核糖体蛋白 s6 的表达，增强了 Rictor 的表达，从而抑制了 mTORC1 的活性，并上调了 mTORC2 的活性。
WZ 通过恢复 AKT/mTOR 介导的自噬和 Sertoli 细胞中 mTORC1-mTROC2 平衡来改善衰老引起的支持细胞损伤。我们的研究结果为 WZ 治疗衰老引起的睾丸功能障碍提供了一种新的机制。
