Melatonin protects premature ovarian insufficiency induced by tripterygium glycosides: role of SIRT1

Abstract

Melatonin confers protective effects on premature ovarian insufficiency (POI) induced by tripterygium glycosides (TG) by reducing oxidative stress. Silent information regulator 1 (SIRT1) signaling is found to be associated with the physiology and pathology of ovary. We hypothesize that melatonin could protect POI via activating SIRT1 signaling. The aim of this study was to investigate the protective effect of melatonin on POI and elucidate its potential mechanisms. Mice were assigned to melatonin treatment with or without SIRT1 inhibitor Ex527 or melatonin receptor antagonist luzindole (Luz) and then subjected to POI. Melatonin conferred a protective effect by improving estrous phase, ovarian and uterus mass and index, increasing ovarian follicles, corpus luteum and anti-mullerian hormone (AMH), decreasing atresia follicles and follicle stimulating hormone (FSH). Melatonin treatment also could reduce malondialdehyde (MDA) level, MDA5, Gp91^phox, Caspase3 and Bax expression, and increase total antioxidant activity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and Bcl-2 expression by up-regulating SIRT1 signaling. However, these protective effects were blocked by Ex527 and Luz, indicating that SIRT1 signaling and melatonin receptor might be specially involved in these effects. In summary, these findings suggest that melatonin protects POI by reducing oxidative stress and apoptotic damage via activation of SIRT1 signaling in a receptor-dependent manner.

Keywords: Premature ovarian insufficiency; SIRT1 signaling pathway; apoptosis; melatonin; melatonin receptor; oxidative stress.

Figure 1

Melatonin’s protective effects on premature ovarian insufficiency. A. Melatonin didn’t change body weight of mice. B. Melatonin significantly decreased abnormal rate of estrous cycle and remarkably increased estrous frequency. C. Melatonin obviously increased ovarian and uterus mass and index. D. Melatonin apparently increased surviving follicles, primordial follicles, developing follicles and corpus luteum, and decreased atresia follicles. E. Melatonin significantly reduced ovarian homogenates AMH levels. Data were shown as mean ± SEM or as ratio. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Melatonin significantly improved oxidative stress and apoptotic damage. A. Melatonin reduced serum and ovarian homogenates MDA level and MDA5 expression in the ovaries. B. Melatonin decreased the protein and mRNA expression of Gp91^phox. C. Melatonin increased serum and ovarian homogenates TAC levels. D. Melatonin increased SOD expression. E. Melatonin increased GSH-Px expression. F. Melatonin reduced Caspase3 expression. G. Melatonin decreased Bax expression. H. Melatonin increased Bcl-2 expression. Data were shown as mean ± SEM or as ratio. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Melatonin significantly increased SIRT1 signaling pathway. A and B. Melatonin increased the protein and mRNA levels of SIRT1. C and D. Melatonin increased the protein and mRNA levels of FoxO3a. E and F. Melatonin increased the protein and mRNA levels of Nrf2. G and H. Melatonin increased the protein and mRNA levels of Ho-1. Data were shown as mean ± SEM or as ratio. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

SIRT1 inhibitor Ex527 significantly reduced SIRT1 signaling pathway. A and B. Ex527 reduced the protein and mRNA levels of SIRT1. C and D. Ex527 increased the protein and mRNA levels of FoxO3a. E and F. Ex527 reduced the protein and mRNA levels of Nrf2. G and H. Ex527 decreased the protein and mRNA levels of Ho-1. Data were shown as mean ± SEM or as ratio. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

SIRT1 inhibitor Ex527 significantly aggravated oxidative stress and apoptotic damage. A. Ex527 increased serum and ovarian homogenates MDA level and MDA5 expression in the ovaries. B. Ex527 increased the protein and mRNA expression of Gp91^phox. C. Ex527 reduced serum and ovarian homogenates TAC levels. D. Ex527 decreased SOD expression. E. Ex527 reduced GSH-Px expression. F. Ex527 increased Caspase3 expression. G. Ex527 increased Bax expression. H. Ex527 decreased Bcl-2 expression. Data were shown as mean ± SEM or as ratio. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

SIRT1 inhibitor Ex527 blocked melatonin’s protective effects on premature ovarian insufficiency. A. Ex527 didn’t change body weight of mice. B. Ex527 significantly increased abnormal rate of estrous cycle and decreased estrous frequency. C. Ex527 obviously decreased ovarian and uterus mass and index. D. Ex527 apparently reduced surviving follicles, primordial follicles, developing follicles and corpus luteum, and increased atresia follicles. E. Ex527 significantly increased serum and ovarian homogenates FSH levels and decreased serum and ovarian homogenates AMH levels. Data were shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 7

Melatonin receptor antagonist luzindole significantly suppressed SIRT1 signaling pathway. A and B. Luzindole reduced the protein and mRNA levels of SIRT1. C and D. Ex527 increased the protein and mRNA levels of FoxO3a. E and F. Ex527 reduced the protein and mRNA levels of Nrf2. G and H. Ex527 decreased the protein and mRNA levels of Ho-1. Data were shown as mean ± SEM or as ratio. *P<0.05, **P<0.01, ***P<0.001.

Figure 8

Melatonin receptor antagonist luzindole significantly aggravated oxidative stress and apoptotic damage. A. Luzindole increased serum MDA level and MDA5 expression in the ovaries. B. Luzindole increased the protein and mRNA expression of Gp91^phox. C. Luzindole reduced serum and ovarian homogenates TAC levels. D. Luzindole decreased SOD expression. E. Luzindole reduced GSH-Px expression. F. Luzindole increased Caspase3 expression. G. Luzindole increased Bax expression. H. Luzindole decreased Bcl-2 expression. Data were shown as mean ± SEM or as ratio. *P<0.05, **P<0.01, ***P<0.001.

Figure 9

Melatonin receptor antagonist luzindole blocked melatonin’s protective effects on premature ovarian insufficiency. A. Luzindole didn’t change body weight of mice. B. Luzindole significantly increased abnormal rate of estrous cycle and decreased estrous frequency. C. Luzindole obviously decreased ovarian and uterus mass and index. D. Luzindole apparently reduced surviving follicles, primordial follicles, developing follicles and corpus luteum, and increased atresia follicles. E. Luzindole significantly increased serum FSH levels and decreased serum and ovarian homogenates AMH levels. Data were shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.