School of Pharmacy, Anhui University of Chinese Medicine, Anhui Province Key Laboratory of Chinese Medical Formula, Hefei, China; Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, China.
School of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, China.
J Ethnopharmacol. 2023 Feb 10;302(Pt A):115867. doi: 10.1016/j.jep.2022.115867. Epub 2022 Oct 27.
Wuzi-Yanzong-Wan (WZYZW) is a classic Chinese herbal preparation, which has a significant clinical efficacy in tonifying the kidney and benefiting the sperm, and is widely used in the treatment of oligoasthenospermia with a long history. TAp73 inhibition results in the decrease of sperm quality, but the therapeutic mechanism of WZYZW on oligoasthenospermia caused by TAp73 gene inhibition remains elusive.
The purpose of this study is to investigate whether TAp73 suppression leads to oligoasthenospermia and the application of WZYZW treatment in condition of TAp73 suppression.
C57BL/6 male mice were injected with Pifithrin-α (2.5 mg/kg) intraperitoneally for 30 days to induce TAp73 suppression model, with WZYZW at 1.0, 2.0 and 4.0 g/kg were administrated in parallel. The blood, testis and epididymis were collected, with organ coefficient calculated. Makler sperm counter was used to analyze the density, motility, survival and malformation rate of sperm. Apoptosis of sperm was analyzed by flow cytometry. Serum hormone levels were determined using ELISA. HE staining and transmission electron microscopy (TEM) were used to observe histopathological changes of testis in blood-testis barrier (BTB), ectoplasmic specialization (ES) and other cell junctions. Expressions of cell adhesion factors including TAp73, Integrin-α6, N-cadherin, Nectin-2 and Occludin were determined by RT-PCR and western blotting.
Compared to control mice, TAp73 inhibition dramatically decreased the epididymal coefficient, sperm quality, and serum testosterone (T) level, while increasing apoptosis in sperm in mice. HE staining and TEM showed that the tight junction (TJ) and apical ES structure were seriously abnormal in the testis in mice with TAp73 inhibition. Additionally, the expression of Occludin protein was elevated, while that of TAp73, Integrin-α6, N-cadherin, and Nectin-2 reduced in model mice. WZYZW treatment ameliorated testicular spermatogenic dysfunctions in TAp73 suppressed mice, restoring the decreased sperm quality, serum T level and testicular histopathological changes of TJ and ES, as well as decreasing sperm malformation rate and apoptosis. Moreover, WZYZW reversed the expressions of Occludin, TAp73, Integrin-α6, N-cadherin and Nectin-2 in TAp73 suppressed mice.
By impairing spermatogenesis and maturation, TAp73 inhibition led to oligoasthenospermia in mice. WZYZW could rescue the oligoasthenospermia associated with TAp73 inhibition via affecting the dynamic remodeling of cellular junctions in testicular tissues in mice.
五子衍宗丸（WZYZW）是一种经典的中药制剂，在补肾益精方面具有显著的临床疗效，历史上广泛用于治疗少弱精子症。TAp73 抑制导致精子质量下降，但 WZYZW 治疗 TAp73 基因抑制引起的少弱精子症的治疗机制仍不清楚。
本研究旨在探讨 TAp73 抑制是否导致少弱精子症以及 WZYZW 在 TAp73 抑制情况下的应用。
将 C57BL/6 雄性小鼠腹腔内注射 Pifithrin-α（2.5mg/kg）30 天，诱导 TAp73 抑制模型，同时平行给予 WZYZW1.0、2.0 和 4.0g/kg。收集血液、睾丸和附睾，计算脏器系数。采用 Makler 精子计数器分析精子密度、活力、存活率和畸形率。流式细胞术分析精子凋亡。酶联免疫吸附试验（ELISA）测定血清激素水平。HE 染色和透射电镜（TEM）观察血睾屏障（BTB）、外质特化（ES）等细胞连接的睾丸组织学变化。采用 RT-PCR 和 Western blot 测定细胞黏附因子 TAp73、整合素-α6、N-钙黏蛋白、Nectin-2 和 Occludin 的表达。
与对照组小鼠相比，TAp73 抑制显著降低了附睾系数、精子质量和血清睾酮（T）水平，同时增加了精子凋亡。HE 染色和 TEM 显示，TAp73 抑制小鼠睾丸 TJ 和顶 ES 结构严重异常。此外，模型小鼠 Occludin 蛋白表达升高，而 TAp73、整合素-α6、N-钙黏蛋白和 Nectin-2 表达降低。WZYZW 治疗改善了 TAp73 抑制小鼠的睾丸生精功能障碍，恢复了精子质量、血清 T 水平和 TJ、ES 的睾丸组织学变化，降低了精子畸形率和凋亡率。此外，WZYZW 逆转了 TAp73 抑制小鼠 Occludin、TAp73、整合素-α6、N-钙黏蛋白和 Nectin-2 的表达。
TAp73 抑制通过影响小鼠睾丸组织细胞连接的动态重塑，导致精子发生和成熟受损，导致少弱精子症。WZYZW 可通过影响小鼠睾丸组织细胞连接的动态重塑，挽救与 TAp73 抑制相关的少弱精子症。
