基于线粒体凋亡途径探讨“秩边透水道”针法对弱精子症小鼠生殖功能的影响

郝健亨; 常博雅; 任佳; 高珍; 张艳林; 王海军; 冀来喜

doi:10.13703/j.0255-2930.20231103-k0005

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基于线粒体凋亡途径探讨“秩边透水道”针法对弱精子症小鼠生殖功能的影响

机制探讨 | 更新时间：2025-01-09

- 基于线粒体凋亡途径探讨“秩边透水道”针法对弱精子症小鼠生殖功能的影响
- Effect of "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture on reproductive function in mice with asthenozoospermia based on mitochondrial apoptosis
- 中国针灸 2025年45卷第1期页码：71-81
- 作者机构：
  
  1.成都中医药大学针灸推拿学院，四川　成都　610075
  2.山西中医药大学第二临床学院，晋中　030619
- 作者简介：
  
  △郝健亨，成都中医药大学博士研究生。E-mail：651194582@qq.com
  ✉冀来喜，教授。E-mail：jlx@sxtcm.edu.cn
- 基金信息：
  
  国家自然科学基金资助项目(82074549);山西省中医药管理局项目(2023ZYYB048);山西中医药大学博士科研启动基金项目(2023BK17)
- DOI：10.13703/j.0255-2930.20231103-k0005
  中图分类号：
- 收稿：2023-11-03，
  
  网络首发：2024-10-06，
  
  纸质出版：2025-01-12
- 稿件说明：
移动端阅览
郝健亨, 常博雅, 任佳, 等. 基于线粒体凋亡途径探讨“秩边透水道”针法对弱精子症小鼠生殖功能的影响[J]. 中国针灸, 2025,45(1):71-81.

HAO Jianheng, CHANG Boya, REN Jia, et al. Effect of "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture on reproductive function in mice with asthenozoospermia based on mitochondrial apoptosis[J]. Chinese Acupuncture & Moxibustion, 2025, 45(1): 71-81.
郝健亨, 常博雅, 任佳, 等. 基于线粒体凋亡途径探讨“秩边透水道”针法对弱精子症小鼠生殖功能的影响[J]. 中国针灸, 2025,45(1):71-81. DOI： 10.13703/j.0255-2930.20231103-k0005.

HAO Jianheng, CHANG Boya, REN Jia, et al. Effect of "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture on reproductive function in mice with asthenozoospermia based on mitochondrial apoptosis[J]. Chinese Acupuncture & Moxibustion, 2025, 45(1): 71-81. DOI： 10.13703/j.0255-2930.20231103-k0005.

摘要

目的：

观察“秩边透水道”针法对弱精子症小鼠睾丸组织线粒体凋亡途径中关键调控因子的影响，探究其对生殖功能保护作用的可能机制。

方法：

将30只C57BL/6雄性小鼠随机分成空白组、模型组和针刺组，各10只。模型组和针刺组小鼠

给予环磷酰胺（30 mg·kg

-1

·d

-1

）腹腔注射7 d制备弱精子症模型。造模成功后针刺组小鼠予“秩边透水道”针法干预，每日1次，每次20 min，共干预2周。观察小鼠一般情况，并记录小鼠造模前、造模后和干预后体质量。干预后，记录小鼠睾丸质量并计算睾丸系数；检测小鼠精子质量；ELISA法检测血清睾酮（T）、卵泡刺激素（FSH）、黄体生成素（LH）含量；HE染色法观察睾丸组织形态；透射电镜观察睾丸组织线粒体超微结构；JC-1染色检测睾丸组织线粒体膜电位水平；TUNEL染色法观察睾丸组织凋亡细胞情况；实时荧光定量PCR法和Western blot法分别检测睾丸组织B淋巴细胞瘤-2（Bcl-2）、Bcl-2关联X的蛋白（Bax）、细胞色素c（Cyt C）、凋亡蛋白酶活化因子1（Apaf-1）、半胱氨酸蛋白酶（Caspase）9、Caspase-3 mRNA和蛋白表达；免疫组化法检测睾丸组织剪切的Caspase-3（Cleaved Caspase-3）阳性表达。

结果：

与空白组比较，模型组小鼠精神萎靡，毛发粗糙，饮食与活动量减少，体质量减轻（

0.01）；睾丸质量和睾丸系数下降（

0.01）；精子总数、精子活力和精子活率降低（

0.01）；血清T、FSH和LH含量降低（

0.01）；睾丸组织内部分生精小管形态异常，生精细胞数量减少，线粒体数量减少，线粒体内嵴发生断裂缺失，出现空泡现象；线粒体膜电位降低（

0.01）；睾丸组织凋亡细胞阳性率升高（

0.01）；睾丸组织Bax、Cyt C、Apaf-1、Caspase-9、Caspase-3 mRNA和蛋白表达升高（

0.01，

0.05），Bcl-2 mRNA和蛋白表达降低（

0.01），Cleaved Caspase-3平均吸光度值升高（

0.01）。与模型组比较，针刺组小鼠一般情况好转；睾丸质量和睾丸系数升高（

0.01）；精子总数、精子活力和精子活率升高（

0.01）；血清T、FSH和LH含量升高（

0.01）；睾丸组织形态和内部线粒体超微结构改善；线粒体膜电位升高（

0.01）；睾丸组织凋亡细胞阳性率降低（

0.01）；睾丸组织Bax、Cyt C、Apaf-1、Caspase-9、Caspase-3 mRNA和蛋白表达降低（

0.01，

0.05），Bcl-2 mRNA和蛋白表达升高（

0.05），Cleaved Caspase-3平均吸光度值降低（

0.01）。

结论：

“秩边透水道”针法可能通过抑制线粒体凋亡途径，减轻环磷酰胺诱导的弱精子症小鼠睾丸组织损伤，改善小鼠的生殖功能。

Abstract

Objective

To observe the effects of the "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture on key regulatory factors during mitochondrial apoptosis of testicular tissue in asthenozoospermia mice

and explore the potential mechanism of the protective effect of acupuncture on reproductive function.

Methods

Thirty C57BL/6 male mice were randomly di

vided into a blank group

a model group and an acupuncture group

10 mice in each group. In the model and the acupuncture groups

the intraperitoneal injection of cyclophosphamide (30 mg·kg

-1

·d

-1

) was delivered for 7 days to prepare the asthenozoospermia model. After the success of modeling

the modeled mice in the acupuncture group were intervened with "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture

once daily and the needles were retained for 20 min. The duration of the intervention was 2 weeks. The general condition of each mouse was observed

and the body mass was recorded before modeling

after modeling and after intervention completion. After intervention

the testicular mass was recorded and the weight coefficient was calculated

and the mouse sperm quality was examined; the serum contents of testosterone (T)

follicle stimulating hormone (FSH) and luteinizing hormone (LH) were detected using ELISA

the morphology of testicular tissue was observed using HE

the mitochondrial ultra-microstructure of testicular tissue was observed under transmission electrone microscopy

the mitochondrial membrane potential level of testicular tissue was detected using JC-1 staining

the positive rate of apoptosis cell of testicular tissue was observed using TUNEL; and the mRNA and protein expression of b-cell lymphocytoma-2 (Bcl-2)

Bcl-2 associated X protein (Bax)

cytochrome c (Cyt C)

apoptotic protease-activating factor1 (Apaf-1)

Caspase-9 and Caspase-3 of testicular tissue was detected using real-time quantitative fluorescence PCR and Western blot methods separately; and the positive expression of Cleaved Caspase-3 of the testicular tissue was detected using immunohistochemistry.

Results

Compared with the blank group

the mice were in listless spirits

had shaggy hairs

the reduced appetite and movement

and weight loss in the model group (

0.01); the testicular mass and the weight coefficient decreased (

0.01); the to

tal number of sperms

sperm motility

and sperm viability were declined (

0.01); while the levels of serum T

FSH

and LH were dropped (

0.01). The morphology of seminiferous tubules in testicular tissue was abnormal

the number of spermatogenic cells and the number of mitochondria decreased

the inner mitochondrial crest was fractured and lost

and vacuoles appeared. The level of mitochondrial membrane potential was reduced (

0.01); and the positive rate of apoptosis cell in testicular tissue increased (

0.01). The mRNA and protein expression of Bax

Cyt C

Apaf-1

Caspase-9 and Caspase-3 was elevated (

0.01

0.05)

the mRNA and protein expression of Bcl-2 was dropped (

0.01)

and the average absorbance value of Cleaved Caspase-3 increased (

0.01). When compared with the model group

in the acupuncture group

the general condition of mice was improved

the testicular mass and the weight coefficient elevated (

0.01); the total number of sperms

sperm motility

and sperm viability increased (

0.01); while the levels of serum T

FSH

and LH rose (

0.01). The pathological morphology of testicular tissue and the inner mitochondrial ultra-microstructure were ameliorated

the level of mitochondrial membrane potential was elevated (

0.01); the positive rate of apoptosis cell was reduced (

0.01). The mRNA and protein expression of Bax

Cyt C

Apaf-1

Caspase-9 and Caspase-3 was dropped (

0.01

0.05)

the mRNA and protein expression of Bcl-2 elevated (

0.05)

and the average absorbance value of Cleaved Caspase-3 declined (

0.01).

Conclusion

"Zhibian" (BL54)-toward- "Shuidao" (ST28) acupuncture may ameliorate mouse reproductive function by inhibiting mitochondrial apoptosis pathway

alleviating testicular tissue damage in the asthenospermia mice induced by cyclophosphamide.

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