The effect of cinnamon on polycystic ovary syndrome in a mouse model

Sixty SPF grade female prepuberal C57BL/6 mice (21-day-old)were purchased from Beijing Vital River Laboratories animal co., LTD (license: SCXK(HU)2007–0003). All animals were raised in China Medical University Animal Center, 25 °C constant temperature (Humidity 50%), 12 h light:12 h dark cyclical alternates, with food and water available ad libitum. All procedures described here were reviewed and approved by the Ethical Committee of China Medical University. At Postnatal Day 25, these mice of comparable weights (12.89 ± 1.41 g)were randomly divided into three groups (control group, DHEA group, DHEA+cinnamon group) and treated for 20 consecutive days as followings (from the day 1 to the day 20). Control group (n = 10): The mice were injected subcutaneously daily with 0.1 ml sesame oil and 100 μL 0.5% methylcellulose given once daily using gavage needle. DHEA group (n = 25): The murine model of PCOS was developed by daily injecting DHEA (6 mg/100 g body weight dissolved in 0.1 ml of sesame oil) subcutaneously as described previously [20, 21] and 100 μL 0.5% methylcellulose given once daily using gavage needle. The DHEA+cinnamon group (n = 25): The mice were injected the same amount of DHEA while given cinnamon powder (10 mg/100 g body weight mixed in 100 μL 0.5% methylcellulose) via gavage as described [22]. After 20 days of the treatments, both reproductive and metabolic features were evaluated. The treatments were continued until the mice were killed. Throughout the whole treatment period, the animals were weighed every two days. All samples were run in triplicate.

The vaginal smears were taken daily at 9 AM from the 13 days after the first injection (Day 38 of life) for 7 consecutive days. The stages of the estrous cycle were determined daily based on vaginal cytology [23]: Vaginal cells were collected via saline lavage and then fixed with methanol and stained with methylene blue staining (0.1%). Predominant nucleated epithelial cells and some cornified epithelial cells indicated the proestrus stage; predominant cornified squamous epithelial cells indicated the estrus stage; both cornified squamous epithelial cells and leukocytes indicated the metaestrus stage; and predominant leukocytes indicated the diestrus stage.

Right after 20 days of the treatment (Day 45 of life), Intraperitoneal(IP) glucose tolerance test (IPGTT) was performed [24]. The mice were fasted overnight (16 h) before the morning of the IPGTT. The mice were injected ip with glucose (2 g/kg as a 50% glucose stock solution). Glucose levels were measured by tail vein blood sampling using a blood glucose meter (Sinocare Inc., Changsha, China) immediately before the mice were injected intraperitoneally with glucose and then at 30, 60, 90, and 120 min after administration. Data were expressed as the absolute values of blood glucose concentrations. Total area under the curve of the glucose response (AUC) was calculated using GraphPad Prism 5.0 software.

Five days after the IPGTT experiment, an insulin tolerance test (ITT) was carried out (Day 51 of life) as previously described [25]. The mice were fasted for 6 h and then injected intraperitoneally with insulin (1 IU/kg body weight, Wanbang Biopharmaceuticals, China). Blood glucose was measured immediately before insulin administration and then at 30, 60, 90, and 120 min after injection. Data were expressed as the values of blood glucose concentrations corrected for fasting glucose. Total AUC of the corrected glucose values was calculated using GraphPad Prism 5.0 software.

After 21 days of treatment (47 days of life), the mice were anesthetized via an intraperitoneal injection of a mixture of 100 mg/ml Ketamine and 20 mg/ml Xylazine (Sigma-Aldrich Chemical, St. Louis, Missouri, USA) prepared in injectable saline (0.1 ml/10 g body weight) [26], then underwent a retrobulbar venous plexus blood draw after overnight fasting. Then the serum concentration level of testosterone, FSH, LH, IGF-1, IGFBP-1,and insulin were measured using by mouse ELISA Kit (R&D Systems. USA for testostereon testing, and Muyuan bio-tech company, Shanghai, China for the other hormones testing), as previously described [27]. All measurements were taken according to the manufacturer’s instructions. The detection limit was 0.041 ng/mL for testosterone, 0.032 ng/ml for FSH, 0.048 ng/ml for LH, 2.05 ng/ml for IGF-1 and 0.35 ng/ml for IGFBP-1. The intra- and inter-assay variation was 5.2% and 8.6% for testosterone, 3.1% and 5.4% for FSH, 5.6% and 6.3% for LH, 4.1% and 3.9% for IGF-1, 4.7% and 4.3% for IGFBP-1.

At 47 days of life, immediately after the last blood draw, ovary samples were then rapidly removed from the animals. Ovaries from each mouse were fixed in 4% paraformaldehyde (PFA) and postfixed in 20% sucrose solution. Afterward, tissues were embedded into Tissue-Tek (O.C.T. compound; Sakura), frozen at − 80 °C overnight, and cut into sections (6-μm thickness) with a Leica Cryostat (LEICA CM1850). The ovary sections were stained with hematoxylin and eosin (H&E) according to the standard histological procedures. To examine ovarian morphology, every 12th section was mounted on a glass slide. Numbers of corpora lutea and large antral follicles were counted [28, 29]. The follicle diameter was calculated as the mean distance between opposite basal membrane portions, while the wall thickness was calculated as the sum of theca interna and granulosa cell layers. A cystic follicle is considered to be a large fluid filled cyst with an attenuated granulosa cell layer and thickened theca cell layer [30]. The count of large antral follicles was performed based on the mean diameter of the follicles greater than 300 μm. The area of the largest follicles was measured with ImageJ (National Institutes of Health) software.

Ovarian sections were prepared as described above and Immunohistochemistry was performed as described previously [31]. Sections (5 μm thickness) were deparaffinized and permeabilized with 0.5% triton X-100 in tris buffered saline (TBS) for 10 min followed by 2 washes with TBS. Endogenous peroxidase activity was quenched with 3% (vol/vol) H₂O₂ in methanol for 15 min. Antigen retrieval was performed by heating sections in 10 mM citrate buffer. Tissue sections were blocked with 1% bovine serum albumin for 20 min at 37 °C, followed by overnight incubation at room temperature with the goat anti mouse IGF-1 polyclonal antibody (1:40; AF-291-NA, R&D, USA), or anti-IGFBP-1 polyclonal antibody (1:40; AF871, R&D, USA). Nonimmune immunoglobulin G (IgG) was used as the negative control. Sections were then incubated with a polyperoxidase-conjugated rabbit anti-goat IgG (Zhongshan Golden Bridge) for 20 min at 37 °C. Immunostaining was revealed by using diaminobenzidine and counterstained with hematoxylin. Immunostaining intensity was evaluated by three independent observers not involved with the study who were asked to rate the intensity of the staining without knowing the identity of samples.

Data are presented as mean ± SEM. Statistical analyses were performed with statistical software package SPSS. A One-way analysis of variance and least significant difference Tukey’s post hoc tests were used to evaluate the hormonal differences among the groups. A two-way analysis of variance with repeated measures for time was used to test significances of IPGTT and ITT experiment. Statistical significance was set at P < 0.05.

As a result, all mice from DHEA group were completely acyclic and remained in constant estrus while control mice had normal cycle. The mice from DHEA plus cinnamon group have cycle but less than controls. Representative cyclicities of mice in three groups are showed in Fig. 1a. All of the mice (10 of 10,100%)in the control group showed normal estrous cyclity, while (20 of 25, 80%) the mice treated with DHEA displayed abnormal estrous cycles. Cinnamon treatment recover the disrupted estrous cycle induced by DHEA with a longer period (17 of 25, 68%). We did not find the difference of the weight of mice body among the three groups of mice as showed in Fig. 1b. Typical micrographs of ovarian sections of the three groups are shown in Fig. 1c. Comparing with DHEA plus Cinnamon or control group, DHEA treated mice exhibited a significant decrease of morphologically healthy large antral follicles, their identifying characteristic is a fluid-filled cavity and the oocyte lies at the edge in a mound made of granulosa epithelial cells [17]. The number of corpora lutea and oocyte in DHEA group was significantly lower than the DHEA plus Cinnamon or control group (P < 0.05). The thickness of the granulosa cell layer of large antral in DHEA group is less and the cells appear degenerated.

Total testosterone is increasing from control group (0.033 ± 0.009 ng/ml), to DHEA and cinnamon group (0.052 ± 0.011 ng/ml), to DHEA group (0.079 ± 0.015 ng/ml), the differences among them were significant (p < 0.05). There was an increasing tendency of serum FSH level from DHEA group (5.02 ± 0.31 ng/ml), DHEA and cinnamon group (5.81 ± 0.51 ng/ml), to control group (7.13 ± 0.74 ng/ml); and there was a decreasing trend of serum LH level from DHEA group (3.75 ± 0.57 ng/ml), DHEA and cinnamon group (1.35 ± 0.61 ng/ml), or control group (0.69 ± 0.34 ng/ml); The differences of the ration of LH to FSH among control group, DHEA group and DHEA plus cinnamon group were significant (p < 0.05). Serum insulin level was significant higher in DHEA treated mice (1.61 ± 0.31 ng/ml) than control group (0.93 ± 0.19 ng/ml), add cinnamon can attenuated DHEA effect on insulin in the mice model (1.27 ± 0.23 ng/ml) (p < 0.05). The DHEA group also have a higher serum IGF-1 level (0.35 ± 0.06 ng/ml) than control group (0.17 ± 0.04 ng/ml) or DHEA and cinnamon group (0.21 ± 0.05 ng/ml) (p < 0.05). While DHEA group have a lower IGFBP-1 level (5.5 ± 1.6 ng/ml) than control group (15.8 ± 2.1 ng/ml) or DHEA and cinnamon group (10.3 ± 2.5 ng/ml) (p < 0.05); (Fig. 2).

We performed IPGTT to investigate cinnamon effect on glucose tolerance of DHEA induced PCOS model. There were no differences of fasting glucose level among the three groups (Fig. 3a). Neither differences in 0- to 120-min AUC value existed among the tree groups (Fig. 3b). We also performed Insulin tolerance test. Blood glucose levels were corrected for fasting glucose. There were significant differences of corrected serum glucose levels among the three groups of mice after injection (P < 0.05). Correspondingly, the differences of AUC were also significant among the three groups (P < 0.05; Fig. 3c). In addition, these data suggested the impaired glucose tolerance in DHEA induced PCOS mice was likely be mitigated by cinnamon treatment.

In the normal ovary of control group, there was weak IGF-1 staining was seen in oocyte and granulosa cell of small antral follicles. Atretic follicle and the surrounding stroma in DHEA treated group showed a strong IGF-1 staining. There was a moderate IGF-1 staining in DHEA and cinnamon group (Fig. 4a). While large follicles in the ovary of control mice showed a strong IGFBP-1 staining. Atretic follicles in the ovary of DHEA treated mice have no IGFBP-1 staining. Follicles in the ovary of DHEA and cinnamon treated mice have a moderate IGFBP-1 staining (Fig. 4b).

LD, YZ, MY, and YG designed the study and prepared the manuscript. YZ and LL produced the DHEA induced PCOS mice model. XG and YC performed the hormone test and immunohistochemistry. All authors read and approved the final manuscript.

This study was funded by the National Natural Science Foundation of China (No. 81572054 and No. 81171649) to Yi Guo. The funding bodies had no role in study design, collection, analysis, and interpretation of data or in manuscript preparation.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

The study was approved by the Institutional Review Board of the First Affiliated Hospital of China Medical University, Shenyang, China.

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The authors declare that they have no competing interests.

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The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.