Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats

Min XIAO; Wei WANG; Wei WEI; Yan-yan ZHOU; Shu-hui CHEN; Xing-yu JIANG; Min ZHAO; Ji-gang CAO

doi:10.13422/j.cnki.syfjx.20212338

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Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats

更新时间：2021-10-29

- Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 23, Pages: 72-79(2021)
- 作者机构：
  
  湖北中医药大学，武汉 430065
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20212338
  CLC： R2-0;R289;R33;Q954.43+4
- Received：24 August 2021，
  
  Published Online：19 October 2021，
  
  Published：05 December 2021
- 稿件说明：
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萧闵,王威,魏巍等.疏肝补肾毓麟汤调节VDAC2基因甲基化影响弱精症大鼠精子线粒体功能改善精子质量[J].中国实验方剂学杂志,2021,27(23):72-79.

XIAO Min,WANG Wei,WEI Wei,et al.Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(23):72-79.
萧闵,王威,魏巍等.疏肝补肾毓麟汤调节VDAC2基因甲基化影响弱精症大鼠精子线粒体功能改善精子质量[J].中国实验方剂学杂志,2021,27(23):72-79. DOI： 10.13422/j.cnki.syfjx.20212338.

XIAO Min,WANG Wei,WEI Wei,et al.Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(23):72-79. DOI： 10.13422/j.cnki.syfjx.20212338.

摘要

目的

明确疏肝补肾毓麟汤抑制电压依赖性阴离子通道2（VDAC2）基因甲基化，通过环磷酸腺苷/蛋白激酶A（cAMP/PKA）通路，调节线粒体功能，提高精子活力的作用机制。

方法

雄性SD大鼠40只随机分成空白组、模型组、疏肝补肾毓麟汤高、低剂量组及左卡尼汀组，每组8只。采用腺嘌呤（0.05 g·kg

-1

）灌胃14 d，复制少弱精症大鼠模型；疏肝补肾毓麟汤组给予疏肝补肾毓麟汤高、低剂量（32.4，8.1 g·kg

-1

）灌胃；左卡尼汀组予以左卡尼汀（0.27 g·kg

-1

）灌胃。全自动精子分析仪检测精子活力；苏木素-伊红（HE）染色观察睾丸组织病理形态学变化；流式细胞法检测精子线粒体膜电位差；免疫荧光法检测睾丸VDAC2的表达；实时荧光定量聚合酶链式反应（Real-time PCR）检测睾丸组织VDAC2 mRNA表达；亚硫酸氢盐测序法检测睾丸组织VDAC2基因甲基化；酶联免疫吸附测定法（ELISA）检测睾丸组织cAMP表达；蛋白免疫印迹法（Western blot）检测睾丸组织PKA蛋白表达。

结果

与空白组比较，模型组精子密度及活动率均显著降低（

0.01），线粒体膜电位显著增高（

0.01），睾丸组织VDAC2的mRNA及蛋白，PKA蛋白，cAMP含量均显著降低（

0.01），VDAC2甲基化程度显著增高（

0.01）；与模型组比较，左卡尼汀组、疏肝补肾毓麟汤高、低剂量组精子密度及活动率、线粒体膜电位均显著升高（

0.01），睾丸组织VDAC2的mRNA及蛋白，PKA蛋白，cAMP含量均显著升高（

0.01），VDAC2甲基化程度显著降低（

0.01）；与左卡尼汀组比较，疏肝补肾毓麟汤低剂量组精子密度及活动率、线粒体膜电位均显著降低（

0.01），睾丸组织VDAC2的mRNA及蛋白，PKA蛋白，cAMP含量表达均显著降低（

0.01），VDAC2甲基化程度显著升高（

0.01）。

结论

疏肝补肾毓麟汤可能通过抑制VDAC2基因甲基化，增加VDAC2表达，调节cAMP/PKA通路，改变线粒体膜电位增强精子活力。

Abstract

Objective

To investigate the mechanism of Shugan Bushen Yulin decoction in inhibiting voltage-dependent anion-selective channel protein 2 （VDAC2） gene methylation， affecting sperm mitochondrial function， and improving sperm motility through the cyclic adenosine monophosphate/protein kinase A （cAMP/PKA） pathway.

Method

Forty male SD rats were randomly divided into the blank group， model group， high- and low-dose Shugan Bushen Yulin decoction groups， and L-carnitine group， with eight rats in each group. Adenine （0.05 g·kg

-1

） was administered by gavage for 14 d for inducing oligospermia and asthenospermia. Rats in the Shugan Bushen Yulin decoction groups were treated with intragastric administration of 32.4， 8.1 g·kg

-1

Shugan Bushen Yulin decoction， respectively， while those in the L-carnitine group received 0.27 g·kg

-1

L-carnitine by gavage. Following the measurement of sperm motility using an automatic sperm analyzer， the pathological changes in testicular tissue were observed by hematoxylin-eosin （HE） staining. Sperm mitochondrial membrane potential was detected by flow cytometry. The expression of VDAC2 in the testicular tissue was determined by immunofluorescence assay. Real-time polymerase chain reaction （Real-time PCR） was conducted for detecting VDAC2 mRNA expression in testicular tissue. The methylation of VDAC2 gene was examined using bisulfite sequencing. The cAMP expression in testicular tissue was detected by enzyme-linked immunosorbent assay （ELISA）， and the PKA protein expression in testicular tissue by Western blot.

Result

Compared with the blank group， the model group exhibited significantly decreased sperm density and motility （

0.01）， increased mitochondrial membrane potential （

0.01）， down-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in testicular tissue （

0.01）， and elevated VDAC2 gene methylation （

0.01）. Compared with the model group， L-carnitine and Shugan Bushen Yulin decoction at the high and low doses all remarkably increased the sperm density and motility and mitochondrial membrane potential （

0.01）， up-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue （

0.01）， and lowered the methylation of VDAC2 in testicular tissue （

0.01）. The comparison with the L-carnitine group showed that the sperm density and motility and mitochondrial membrane potential in the low-dose Shugan Bushen Yulin decoction group declined significantly （

0.01）. The VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue were significantly down-regulated （

0.01）， while the methylation of VDAC2 was significantly enhanced （

0.01）.

Conclusion

Shugan Bushen Yulint decoction may inhibit VDAC2 gene methylation， increase VDAC2 expression， regulate cAMP/PKA pathway， and change mitochondrial membrane potential to enhance the sperm motility.

关键词

Keywords

references

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