Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats / 中国实验方剂学杂志

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^-1Shugan 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 （<italic>P</italic><0.01）， increased mitochondrial membrane potential （<italic>P</italic><0.01）， down-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in testicular tissue （<italic>P</italic><0.01）， and elevated VDAC2 gene methylation （<italic>P</italic><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 （<italic>P</italic><0.01）， up-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue （<italic>P</italic><0.01）， and lowered the methylation of VDAC2 in testicular tissue （<italic>P</italic><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 （<italic>P</italic><0.01）. The VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue were significantly down-regulated （<italic>P</italic><0.01）， while the methylation of VDAC2 was significantly enhanced （<italic>P</italic><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.