Paeoniflorin attenuates DHEA-induced polycystic ovary syndrome via inactivation of TGF-β1/Smads signaling pathway in vivo

Abstract

Polycystic ovarian syndrome (PCOS) is one of the most common reproductive endocrine disorders which are involved in complicated and unknown pathogenic mechanisms. Paeoniflorin (PAE) plays a significant anti-fibrotic role according to previous studies. The aim of the present study was to investigate the effect of PAE on ovarian fibrosis and its underlying mechanism in PCOS development. An animal model of PCOS was established by subcutaneous injection of 60mg/kg/d dehydroepiandrosterone (DHEA) for 35 consecutive days. Rats in PAE-L, PAE-M and PAE-H groups were administrated by gavage with PAE (20, 40, 80 mg/kg/d) for 4 weeks. Our results indicated that DHEA-induced PCOS rats showed similar phenotypes with PCOS patients. PAE could significantly block the DHEA-induced decline of ovary weight and organ coefficient, shorten the prolonged diestrus period, and regulate the irregular estrous cycle of PCOS rats. Moreover, PAE regulated reproductive hormone levels and improved ovarian fibrosis induced by DHEA. PAE treatment could also reduce the expression levels of TGF-β1 and Smad3, and increase the expression levels of Smad7 and MMP2. In conclusion, PAE significantly attenuated the ovarian fibrosis in PCOS, which could be mediated by TGF-β1/Smads signaling pathway. Herein, PAE can be used for the treatment of ovarian fibrosis in PCOS progression.

Keywords: TGF-β1/Smads signaling pathway; ovarian fibrosis; paeoniflorin; polycystic ovary syndrome.

Figure 1

Influence of PAE on body weight, ovary weight, organ coefficient and estrous cycle in PCOS rats (n=10 for each group). (A) The chemical structure of PAE. (B) Influence of PAE on body weight, ovary weight and organ coefficient in PCOS rats (mean ± SEM, n=10 for each group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SDR group. #P < 0.05, ##P< 0.01 and ###P< 0.01 vs. PCOSR group. (C) Different periods of estrous cycle (toluidine blue staining; Scale bars = 200 μm): Proestrus, Estrus, Metestrus and Diestrus. (D) Representative pictures of estrous cycle in five different groups; P, Proestrus; E, Estrus; M, Metestrus; D, Diestrus. PCOS, polycystic ovarian syndrome; SDR, normal control group; PCOSR, PCOS model group; PAE-L, PAE low-dose group (20 mg/kg/d); PAE-M, PAE middle-dose group (40 mg/kg/d); PAE-H, PAE high-dose group (80 mg/kg/d).

Figure 2

Influence of PAE on ovarian structure, follicle growth and ovarian collagen deposition in PCOS rats. Rats were executed at diestrus. (A) Representative pictures of ovarian tissues in PCOS rats (Hematoxylin and eosin staining, Scale bars = 500 μm, n=10 for each group). (B) Sirius red staining under a light microscope (Scale bars = 100 μm, n=10 for each group). (C) Proportion of the area containing collagen to the ovarian area (mean ± SEM, n=10 for each group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SDR group. #P < 0.05, ##P< 0.01 and ###P< 0.01 vs. PCOSR group. PCOS, polycystic ovarian syndrome; SDR, normal control group; PCOSR, PCOS model group; PAE-L, PAE low-dose group (20 mg/kg/d); PAE-M, PAE middle-dose group (40 mg/kg/d); PAE-H, PAE high-dose group (80 mg/kg/d). “▲” is directed in the direction of collagen fibers. “→” is directed in the direction of granular cell layers. C, corpora luteum; F, cyst-like follicles; f, cyst-like follicles.

Figure 3

Influence of PAE on the reproductive hormone levels in PCOS rats (mean ± SEM, n=10 for each group). (A–D, F) The levels of serum T, E2, LH, FSH and AMH. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SDR group. #P < 0.05, ##P< 0.01 and ###P< 0.01 vs. PCOSR group. (E) The ratio of LH to FSH. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SDR group. #P < 0.05, ##P< 0.01 and ###P< 0.01 vs. PCOSR group. PCOS, polycystic ovarian syndrome; SDR, normal control group; PCOSR, PCOS model group; PAE-L, PAE low-dose group (20 mg/kg/d); PAE-M, PAE middle-dose group (40 mg/kg/d); PAE-H, PAE high-dose group (80 mg/kg/d); T, total testosterone; E2, estradiol; LH, luteinizing hormone; FSH, follicle stimulating hormone; AMH, Anti-Mullerian Hormone.

Figure 4

Influence of PAE on the activation of TGF-β1/Smads signaling pathway in PCOS rats was evaluated by qRT-PCR (mean ± SEM, n=10 for each group). (A, B) The mRNA levels of TGF-β1 and Smad3 in the ovarian tissues of PCOSR group were significantly higher than those in the SDR group (P < 0.001). The treatment with PAE reduced the mRNA levels of TGF-β1 and Smad3 (P < 0.05). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SDR group. #P < 0.05, ##P< 0.01 and ###P< 0.01 vs. PCOSR group. (C, D) The mRNA expression levels of Smad7 and MMP2 in the ovarian tissues of PCOSR group were significantly lower than those in the SDR group (P < 0.001). The Treatment with PAE increased the mRNA expression levels of Smad7 and MMP2 (P < 0.05). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SDR group. #P < 0.05, ##P< 0.01 and ###P< 0.01 vs. PCOSR group. PCOS, polycystic ovarian syndrome; SDR, normal control group; PCOSR, PCOS model group; PAE-L, PAE low-dose group (20mg/kg/d); PAE-M, PAE middle-dose group (40mg/kg/d); PAE-H, PAE high-dose group (80mg/kg/d).

Figure 5

Influence of PAE on the activation of TGF-β1/Smads signaling pathway in PCOS rats was evaluated by western blot. The protein expressions of (A) TGF-β1, (B) p-Smad3, (C) Smad3, (D) Smad7, (E) α-SMA and (F) MMP2 were detected in ovarian tissues of PCOS rats. Same one GAPDH was used as the internal standard in (A, D, E). Same one GAPDH was used as the internal standard in (B, C, F). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SDR group. #P < 0.05, ##P< 0.01 and ###P< 0.01 vs. PCOSR group. PCOS, polycystic ovarian syndrome; SDR, normal control group; PCOSR, PCOS model group; PAE-L, PAE low-dose group (20 mg/kg/d); PAE-M, PAE middle-dose group (40 mg/kg/d); PAE-H, PAE high-dose group (80 mg/kg/d).