麒麟丸对无精子症模型小鼠睾丸生精功能的影响

Qilin Pills promote testicular spermatogenesis in azoospermia mice

目的:探讨麒麟丸是否促进无精子症模型小鼠生精功能恢复,揭示其调控睾丸生精功能的机制。方法:取15只4周龄ICR雄性小鼠,随机分成模型组、麒麟丸低剂量组、麒麟丸高剂量组,每组5只。采用白消安建立无精子症小鼠模型。建模后麒麟丸组每天给予不同剂量麒麟丸灌胃给药[低剂量组和高剂量组分别为2 000、8 000 mg/(kg·d)],模型组予正常饮食处理,28 d后颈椎脱臼法处死小鼠。HE染色检测小鼠附睾和睾丸组织显微结构,Western印迹检测小鼠睾丸中各级生精细胞、支持细胞和间质细胞特异性标志物表达情况,免疫荧光染色检测睾丸内这些标志物的定位与表达情况。结果:模型组小鼠睾丸中各级生精细胞数量明显减少,大多数附睾管中未见精子; Johnsen评分(5. 2±0. 5)分。麒麟丸高剂量组小鼠生精小管腔内生精细胞排列紧密、层次分明,附睾管腔中见大量精子,未见非精子细胞成分; Johnsen评分(9. 4±0. 6)分。麒麟丸低剂量组小鼠睾丸中各级生精细胞数量相比较模型组有所提升,但是对比高剂量组仍明显减少,部分附睾管中可见精子; Johnsen评分(7. 6±0. 6)分。Johnsen评分模型组显著低于麒麟丸高、低剂量组(P <0. 01),而且麒麟丸高、低剂量组间也有统计学差异(P <0. 05)。Western印迹结果显示,支持细胞标志物GATA4、WT1、SCF、BMP4的表达水平模型组均显著低于麒麟丸高、低剂量组(P <0. 01),高剂量组显著高于低剂量组(P <0. 05或<0. 01);精原干细胞和未分化精原细胞标志物UCHL1、STRA8、NGN3、PLZF 3组小鼠间无显著差异;精母细胞标志物DMC1、SYCP3模型组也均显著低于麒麟丸高、低剂量组(P <0. 05或P <0. 01),高剂量组显著高于低剂量组(P <0. 05或P <0. 01)。免疫荧光染色结果显示,SYCP3的表达与Western印迹结果一致; Ki67荧光信号分布在精原细胞,其荧光信号强度模型组低于麒麟丸高、低剂量组;精子顶体标志物PNA主要分布在生精小管内,模型组小鼠没有出现明显的点状信号,麒麟丸高、低剂量组可见大量的荧光信号。结论:麒麟丸可能是通过改善睾丸中支持细胞的功能,从而促进精原细胞进入减数分裂,增加精子的生成。

Objective： To investigate the effect of Qilin Pills （QP） in facilitating the recovery of spermatogenic function in azoospermia （AS） mice and to explore its mechanism of regulating testicular spermatogenesis. Methods： Fifteen 4-week-old male mice were equally randomized into an AS model control, a low-dose QP and a high-dose QP group. The AS model was established in the mice by intraperitoneal injection of busulfan at 35 mg/kg. After modeling, the animals in the low- and high-dose QP groups were treated with Qilin Pills intragastrically at 2 000 and 8 000 mg/kg/d respectively while those in the model control group fed on a normal diet, all for 28 days. Then, all the mice were sacrificed for examination of the uhrastructures of the epididymis and testis by HE stai-ning, detection of the specific markers of spermatogenic, Sertoli and Leydig cells by Western blot, and determination of the expressions of these markers in the testis tissue by immunofluorescence assay. Results ： The number of spermatogenic cells in the testis tissue was significantly decreased in the AS model eontrols, with no spermatozoa in most of the seminiferous tubules in the epididymis （ Johnsen＇s score ： 5.2 ± 0.5）. In the high-dose QP group, spermatogenic cells were tightly arranged with distinct layers in the seminiferous tu-bules, with a large number of spermatozoa but no non-sperm ceils in the lumens of the epididymis （Johnsen＇s score ： 9.4 ± 0.6）. The number of spermatogenic cells in the testis was increased in the low-dose QP group with some spermatozoa in the seminiferous tubules as compared with that in the model control, but lower than in the high-dose group （Johnsen＇s score： 7.6 ± 0.6）. The Johnsen＇s score was significantly lower in the model control than in the high- and low-dose QP groups （P 〈 0.01 ）, and higher in the high-dose than in the low-dose QP group （P 〈 0.05）. The expressions of the specific markers of Sertoli cells SCF, BMP4, SYCP3, DMC1 and Ki67 were also remarkably lower in the model control than in the high- and low-dose QP groups （P 〈 0.01 ）, and higher in the high-dose than in the low-dose QP group （ P 〈 0.05 or P 〈 0.01 ）. No statistically significant differences were observed among the three groups of mice in the markers of spermatogonial stem cells （ SSC ） and undifferentiated SSCs UCHL1, STRA8, NGN3 and PLZF3 （ P 〉 0.05 ）. The expressions of the spermatocyte markers DMC1 and SYCP3 were markedly lower in the model control than in the high- and low-doseQPgroups （P 〈 0.05 or P 〈 0.01）, and higher in the high-dose than in the low-dose QP group （P 〈 0.05 or P 〈 0.01 ）. The Ki67 fluorescence signals were distributed in the spermatogonia, with a higher intensity in the model control than in the high- and low-dose QP groups. The acrosome marker PNA was found mainly in the seminiferous tubules, with abundant fluorescence signals in the high- and low-dose QP groups but no obvious dot signals in the model controls. Conclusion ： Qilin Pills may contribute to the meiosis of spermatogonia and promote spermatogenesis by improving the function of Sertoli cells in the testis.