Melatonin protects against ovarian damage by inhibiting autophagy in granulosa cells in rats

Liu, Yan; Zhu, Xiaohe; Wu, Chunli; Lang, Yan; Zhao, Wenjie; Li, Yanmin

doi:10.1016/j.clinsp.2022.100119

Yan Liu

Conceptualization

Investigation

Writing – original draft

Department of Gynecology, Weifang People’s Hospital, Weifang, Shandong, China

Xiaohe Zhu

Data curation

Department of Obstetrics, Weifang People’s Hospital, Weifang, Shandong, China

Chunli Wu

Data curation

Department of Obstetrics, Weifang People’s Hospital, Weifang, Shandong, China

Yan Lang

Formal Analysis

Validation

Department of Obstetrics, Weifang People’s Hospital, Weifang, Shandong, China

Wenjie Zhao

Formal Analysis

Validation

Department of Reproductive Medicine, Weifang People’s Hospital, Weifang, Shandong, China

Yanmin Li ^** Corresponding author. E-mail address: yanminli66@163.com (Y. Li).

Conceptualization

Investigation

Writing – review & editing

Department of Reproductive Medicine, Weifang People’s Hospital, Weifang, Shandong, China

http://orcid.org/0000-0002-2478-5485

* Corresponding author. E-mail address: yanminli66@163.com (Y. Li).

Conflicts of interest

The authors declare no conflicts of interest.

Figures (5)
Tables (2)

Thumbnail

Fig. 1
HE staining of ovarian tissue sections of rats in each group. A1–A4 represent the MT group; B1–B4 represent the RAPA group; C1–C4 represent the 3-MA groups; D1–D4 represent the model groups; E1–E4 represent the control groups; Nos. 1–4 correspond to magnifications of 4 ×, 10 ×, 20 ×, and 40 ×, respectively.

Thumbnail

Fig. 2
Effect of MT on the PI3K/Akt/mTOR signaling pathway. (a) Expression of the PI3K/AKT/mTOR pathway-related proteins, p-PI3K, p-Akt, p-mTOR proteins, and corresponding native proteins in the control, model, 3-MA, RAPA, and MT groups were detected by immunoblotting. (b‒d) Expression of the PI3K/AKT/mTOR pathway-related proteins, p-PI3K, p-Akt, p-mTOR proteins, and the corresponding native proteins were detected by ImageJ software (three experiments were independently performed).

Thumbnail

Fig. 3
The protective effect of MT on ovarian granulosa cells in each group of rats. (a) The survival of rat ovarian granulosa cells in each group was detected by the CCK8 method. (b) The death of ovarian granulosa cells in each group was detected by the LDH method.

Thumbnail

Fig. 4
Effect of MT on autophagic apoptosis of rat ovarian granulosa cells. Flow cytometry was used to detect apoptosis in the ovarian granulosa cells of each group.

Thumbnail

Fig. 5
Effect of MT on the expression of autophagy-related proteins. (a) The expression of autophagy-associated proteins, LC3II/I and Agt5, was determined in ovarian granulosa cells by immunoblotting. (b‒c) The expression levels of autophagy-related proteins, LC3II/I and Agt5, were determined by ImageJ software (the experiments were independently performed three times).

Thumbnail

Table 1
Ovarian wet weight, ovarian index, and follicle numbers of rats in each group.

Thumbnail

Table 2
Serum AMH, INH-B, FSH, LH, E2, P, and T levels of rats in each group.

Group	n	Left ovary wet weight (mg)	Right ovary wet weight (mg)	Ovarian index (mg/g)	Primordial follicle (n)	Primary follicles (n)	Antral follicle (n)	Atretic follicles (n)
Control group	19	51.43 ± 6.92	47.67 ± 5.70	0.35 ± 0.03	9.61 ± 4.30	5.75 ± 2.33	6.51 ± 3.00	2.51 ± 1.50
Model group	19	16.43 ± 5.26^a a p compared with the control group, p < 0.05.	15.78 ± 6.78^a a p compared with the control group, p < 0.05.	0.13 ± 0.03^a a p compared with the control group, p < 0.05.	2.78 ± 1.30^a a p compared with the control group, p < 0.05.	2.33 ± 1.22^a a p compared with the control group, p < 0.05.	1.73 ± 0.83^a a p compared with the control group, p < 0.05.	8.87 ± 3.52^a a p compared with the control group, p < 0.05.
3-MA group	18	30.67 ± 7.47^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	28.88 ± 4.91^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	0.23 ± 0.02^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	6.69 ± 2.12^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	4.91 ± 2.23^b b p compared with the model group, p < 0.05.	3.97 ± 2.00^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	5.16 ± 2.64^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.
RAPA group	19	21.86 ± 6.77^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	19.44 ± 5.90^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	0.18 ± 0.03^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	4.22 ± 2.64^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	3.16 ± 1.56^a a p compared with the control group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	2.62 ± 1.58^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	7.25 ± 3.15^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.
MT group	18	29.50 ± 5.24^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with the RAPA group, p < 0.05.	28.50 ± 4.50^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with the RAPA group, p < 0.05.	0.22 ± 0.01^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with the RAPA group, p < 0.05.	6.22 ± 1.91^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with the RAPA group, p < 0.05.	4.86 ± 2.59^b b p compared with the model group, p < 0.05. ,^d d p compared with the RAPA group, p < 0.05.	3.14 ± 1.73^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with the RAPA group, p < 0.05.	4.84 ± 2.36^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with the RAPA group, p < 0.05.

Group	n	AMH (ng/mL)	INH-B (pg/mL)	FSH (IU/L)	LH (mIU/mL)	E2 (pmoL/L)	P (ng/mL)	T (ng/mL)
Control group	19	2.76 ± 0.59	29.78 ± 3.15	7.06 ± 0.47	5.01 ± 0.47	128.69 ± 0.84	2.32 ± 0.39	0.22 ± 0.03
Model group	19	0.34 ± 0.07^a a p compared with the control group, p < 0.05.	11.89 ± 2.93^a a p compared with the control group, p < 0.05.	26.06 ± 3.74^a a p compared with the control group, p < 0.05.	13.90 ± 2.68^a a p compared with the control group, p < 0.05.	40.45 ± 1.87^a a p compared with the control group, p < 0.05.	2.06 ± 0.59	0.25 ± 0.13
3-MA group	18	1.01 ± 0.10^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	21.50 ± 2.45^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	16.67 ± 1.43^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	7.88 ± 1.50^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	81.34 ± 2.70^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05.	2.25 ± 0.95	0.22 ± 0.06
RAPA group	19	0.47 ± 0.07^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	14.00 ± 2.87^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	22.10 ± 3.90^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	10.50 ± 3.48^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	62.83 ± 0.66^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^c c p compared with the 3-MA group, p < 0.05.	2.03 ± 0.53	0.29 ± 0.06
MT group	18	1.09 ± 0.06^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with RAPA group, p < 0.05.	21.12 ± 2.53^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with RAPA group, p < 0.05.	16.06 ± 2.67^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with RAPA group, p < 0.05.	7.07 ± 2.62^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with RAPA group, p < 0.05.	79.98 ± 2.72^a a p compared with the control group, p < 0.05. ,^b b p compared with the model group, p < 0.05. ,^d d p compared with RAPA group, p < 0.05.	2.21 ± 0.76	0.21 ± 0.07

[1] * Corresponding author. E-mail address: yanminli66@163.com (Y. Li).

Brasil

Brasil

Melatonin protects against ovarian damage by inhibiting autophagy in granulosa cells in rats

Abstract

Objectives:

Method:

Results:

Conclusions:

HIGHLIGHTS