Objective

2

To investigate the underlying mechanism of "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture technique for improving reproductive function in mice with asthenospermia by regulating ferroptosis pathway.

Methods

2

Sixty male C57BL/6 mice were randomly divided into a blank group

a model group

an acupuncture group and a Fer-1 group

15 mice in each one. Except the blank group

the intraperitoneal injection with cyclophosphamide (50·kg

-1

·d

-1

) was administered to establish the asthenospermia model in the mice of the rest 3 groups for 5 consecutive days. In the acupuncture group

"Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture technique was operated in the mice

for 20 min each time; and in the Fer-1 group

Fer-1 solution (1 mg/kg) was injected intraperitoneally. The interventions of these two groups were

delivered once daily and for 2 consecutive weeks. The testicular wet weight was measured and the testicular coefficient was calculated. Using sperm quality detection system

the sperm quality was detected. With ELISA used

the contents of testosterone (T)

follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the serum were detected. With HE staining

testicular and epididymal morphology was observed. Immunofluorescence was used to detect the expression of reactive oxygen species (ROS) in the testes. Biochemical assay was conducted to determine the contents of malondialdehyde (MDA)

reduced glutathione (GSH)

and total iron ion (TFe) in the testicular tissue. Transmission electron microscopy was used to examine mitochondrial structure of the testis

while JC-1 staining was used to assess mitochondrial membrane potential in the testicular tissue. Fluorescence quantitative PCR and Western blot analyses were employed to measure the mRNA and protein expression of solute carrier family 7 member 11 (SLC7A11)

glutathione peroxidase 4 (GPX4)

ferritin heavy chain 1 (FTH1)

and acyl-CoA synthetase long-chain family member 4 (ACSL4) in the testicular tissue.

Results

2

Compared with the blank group

in the model group

the testicular wet weight and testicular coefficient decreased (

P

<

0.01); the sperm concentration and sperm motility reduced (

P

<

0.01)

and the contents of T

FSH

and LH decreased in the serum (

P

<

0.01); and the seminiferous tubules in the testis showed loose structure and deformed lumen

sperm cells were disorganized and the sperm numbers reduced; the tubular walls became thinner

and sperm numbers in the lumen less; the expression of ROS in testicular tissue

as well as the contents of MDA and TFe increased (

P

<

0.01)

and the content of GSH decreased (

P

<

0.01); and the numbers of mitochondria reduced

the structure of cristae was serious damaged

and mitochondrial membr

ane potential level declined (

P

<

0.01); the mRNA and protein expression of SLC7A11

GPX4

and FTH1 decreased (

P

<

0.01)

while the mRNA and protein expression of ACSL4 increased (

P

<

0.01). In comparison with the model group

the acupuncture and Fer-1 groups showed the increase of testicular wet weight and coefficient (

P

<

0.01)

sperm concentration and motility (

P

<

0.01)

and the serum contents of T

FSH

and LH (

P

<

0.01); and the improvements in testicular and epididymal histopathology; ROS expression and the contents of MDA and TFe decreased (

P

<

0.01)

and the content of GSH elevated (

P

<

0.05); the mitochondrial structure and numbers were ameliorated and mitochondrial membrane potential rose (

P

<

0.01). Besides

in comparison with the model group

the mRNA expression of SLC7A11 was higher (

P

<

0.05

P

<

0.01)

the mRNA and protein expression of GPX4 and FTH1 increased (

P

<

0.01

P

<

0.05)

and the mRNA and protein expression of ACSL4 decreased (

P

<

0.01) in the acupuncture and the Fer-1 groups; and the protein expression of SLC7A11 was higher in the Fer-1 group (

P

<

0.05).

Conclusion

2

"Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture technique may improve the reproductive capacity in the mice with asthenospermia by alleviating ferroptosis-induced cellular damage and ameliorating testicular function.