Objective

2

To investigate whether the protective effect of moxibustion on ovarian function in mice with premature ovarian insufficiency (POI) was associated with mitochondrial dynamics and function in ovarian granulosa cells

using a mitochondrial fission activator.

Methods

2

Eighty female ICR mice with regular estrous cycles were randomly divided into a blank group

a tripterygium glycosides group (TG group)

a moxibustion group

and a carbonyl cyanide m-chlorophenyl hydrazone plus moxibustion group (CCCP+moxibustion group)

with 20 mice in each group. Except for the blank group

POI model was established in the remained groups by intragastric administration of TG suspension for 14 consecutive days. In the moxibustion group

moxibustion was applied at bilateral "Shenshu" (BL23) or alternately at "Zhongwan" (CV12) and "Guanyuan" (CV4) 1 hour after TG administration

once every day for 14 days. The CCCP+moxibustion group received intraperitoneal injection of CCCP (2.9 mg·kg

-1

·d

-1

) after TG administration

followed by the same moxibustion protocol as the moxibustion group. Estrous cycles were recorded

and the ovarian index was calculated. Ovarian morphology was observed with HE staining

and follicle counts were conducted. Serum levels of follicle-stimulating hormone (FSH)

estradiol (E

2

)

and anti-Müllerian hormone (AMH) were measured using ELISA. Immunohistochemistry and real-time quantitative PCR were used to detect the protein and mRNA expression of mitochondrial dynamics-related factors (dynamin-related protein 1 [Drp1

]

mitochondrial fission factor [Mff

]

optic atrophy 1 [Opa1

]

and mitofusin 1 [Mfn1

]

) in the ovaries

respectively. ATP concentration in granulosa cells was determined by luciferase assay

and reactive oxygen species (ROS) fluorescence intensity was detected using DCFH-DA fluorescent probe method.

Results

2

Compared with the blank group

the mice in the TG group showed a significantly higher rate of estrous cycle disorder (

P

<

0.01)

smaller ovarian volume

lower ovarian index (

P

<

0.01)

reduced numbers of primordial

growing

and antral follicles (

P

<

0.01)

increased number of atretic follicles (

P

<

0.01)

and loose and disordered arrangement of granulosa cells; serum FSH level was elevated (

P

<

0.01)

while serum AMH and E

2

levels were reduced (

P

<

0.01). Drp1 and Mff protein staining in ovarian granulosa cells was intensified

while Opa1 and Mfn1 protein staining was lighter. The average optical densities of Drp1 and Mff and the mRNA expression of Drp1 and Mff were significantly increased (

P

<

0.01)

while the average optical densities of Opa1 and Mfn1 and the mRNA expression of Opa1 and Mfn1 were significantly decreased (

P

<

0.01). ATP concentration was reduced (

P

<

0.01)

and ROS fluorescence intensity was decreased (

P

<

0.01). Compared with the TG group

the mice in the moxibustion group exhibited lower rate of estrous cycle disorder (

P

<

0.01)

larger ovarian volume

higher ovarian index (

P

<

0.01)

increased numbers of primordial

growing

and antral follicles (

P

<

0.01)

reduced number of atretic follicles (

P

<

0.01)

and a more compact and orderly arrangement of granulosa cells; serum FSH level was decreased (

P

<

0.01)

while serum AMH and E

2

levels were increased (

P

<

0.01). Opa1 and Mfn1 protein staining was deepened

Drp1 and Mff protein staining was lightened

average optical densities and mRNA expression of Opa1 and Mfn1 were increased (

P

<

0.01

P

<

0.05)

and those of Drp1 and Mff were decreased (

P

<

0.05

P

<

0.01). ATP concentration was increased (

P

<

0.01)

and ROS fluorescence intensity was decreased (

P

<

0.01). Compared with the moxibustion group

the mice in the CCCP+moxibustion group showed an increased estrous cycle disorder rate (

P

<

0.05) and a decreased

ovarian index (

P

<

0.05). The numbers of primordial

growing

and antral follicles were reduced (

P

<

0.01

P

<

0.05)

while the number of atretic follicles was increased (

P

<

0.01). The arrangement of ovarian granulosa cells became looser and more disordered. Serum FSH level was elevated (

P

<

0.01)

while serum E

2

and AMH levels were decreased (

P

<

0.05). In the ovaries of mice in the CCCP+moxibustion group

Drp1 and Mff protein staining was intensified

whereas Opa1 and Mfn1 protein staining was lighter. The average optical densities of Drp1 and Mff

as well as the mRNA expression levels of Drp1 and Mff in granulosa cells

were increased (

P

<

0.05)

while the average optical densities of Opa1 and Mfn1 and the mRNA expression levels of Opa1 and Mfn1 in granulosa cells were decreased (

P

<

0.05

P

<

0.01). Additionally

the ATP concentration in ovarian granulosa cells was reduced (

P

<

0.01)

and the ROS fluorescence intensity was elevated (

P

<

0.01).

Conclusion

2

Moxibustion may alleviate TG-induced ovarian damage by inhibiting mitochondrial fission and promoting mitochondrial fusion

thereby improving impaired mitochondrial function; this protective effect can be blocked by CCCP.