Objective

2

To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI)

and explore the mechanism of EA in improving POI.

Methods

2

Thirty-two female SD rats were randomly divided into a blank group (

n

=8) and a model making group (

n

=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg

and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group

an EA group

and an estradiol (E2) group

with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave

frequency of 2 Hz

and current intensity of 0.1 to 1 mA

20 min per treatment

once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg)

once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention

ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH)

E2

anti-Mullerian hormone (AMH)

and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy

and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62

Beclin-1

and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue.

Results

2

The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest

mostly in proestrus or metestrus

with overall ovarian atrophy

disordered structure

and decreased granulosa cells. Compared with the blank group

rats in the model group showed increased serum FSH level (

P

<

0.01)

decreased serum levels of E2

AMH

and INHB (

P

<

0.01)

decreased positive expression of p62 in ovarian granulosa cells (

P

<

0.01)

with obvious

swelling of ovarian granulosa cells

mild to moderate swelling of mitochondria

slight expansion of rough endoplasmic reticulum

and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (

P

<

0.01)

the expression of p62 protein and mRNA was decreased (

P

<

0.01)

and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (

P

<

0.01). Compared with the model group

rats in the EA group and the E2 group showed decreased serum FSH levels (

P

<

0.01)

increased levels of E2

AMH

and INHB (

P

<

0.01)

increased positive expression of p62 in ovarian granulosa cells (

P

<

0.01)

alleviated degree of ovarian granulosa cell damage

with relatively intact organelle morphology

and decreased number of autophagosomes and autolysosomes in the ovaries (

P

<

0.01); the rats also showed increased expression of p62 protein and mRNA (

P

<

0.01)

and decreased expression of Beclin-1 and LC3 protein and mRNA (

P

<

0.01) in ovarian tissue.

Conclusion

2

EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes

up-regulating p62 expression

and down-regulating Beclin-1 and LC3 expression

thus inhibiting autophagy of ovarian granulosa cells

and regulating the serum levels of FSH

E2

AMH

and INHB.