The effectiveness of resveratrol in treatment of PCOS on the basis of experimental model in rats

Abstract

Purpose of the study

Polycystic ovary syndrome (PCOS) is a widespread endocrine disorder in women of reproductive age. Further research is required to justify new directions of effective targeted therapy of this condition. Resveratrol possesses anti-inflammatory, antioxidant and antidiabetic properties. The purpose of this study was to evaluate the potential effectiveness of resveratrol in PCOS based on the created model of this disease in Wistar rats.

Materials and methods

The PCOS model was created by oral administration of letrozole to female Wistar rats.. The animals received resveratrol at a dosage of 20 mg/kg and 30 mg/kg for the next 30 days. Then ovariectomy was performed for histological confirmation of the effectiveness of resveratrol in the treatment of PCOS. Regularity of estrous cycle, animal’s body mass and the level of soluble receptors for advanced glycation end products (sRAGE) in the blood of rats were also evaluated in dynamics.

Results

The study revealed that administration of resveratrol leads to dose-dependent restoration of normal morphology of ovarian tissue, normalizes regularity of estrous cycle and decreases body weight of rats with PCOS.

Conclusion

The results obtained in rats suggest that resveratrol may be a promising agent for the treatment of PCOS in women.

Keywords:

• PCOS
• glycation end products
• resveratrol
• soluble receptors for advanced glycation end products (sRAGE)

Introduction

Polycystic ovary syndrome (PCOS) is a widespread endocrine disorder among women of reproductive age, with a frequency of occurrence of 8–21% [1]. The diagnosis of this pathology is based on the Rotterdam criteria (2003): oligo-/anovulation; hyperandrogenism (clinical and/or biochemical); sonographic polycystic ovarian morphology [2]. According to US National Institutes of Health (NIH) diagnostic criteria (2012), there are 4 PCOS phenotypes: phenotype A (classic) – the presence of hyperandrogenism (HA), chronic ovulatory dysfunction (OD), sonographic signs of polycystic ovarian morphology (PCOM)–(HA/OD/PCOM); phenotype B (anovulatory) – combination of hyperandrogenism and oligo/anovulation, but without PCO (HA/OD); phenotype C (ovulatory) – the presence of hyperandrogenism and PCOM with regular ovulatory cycle (HA/PCOM); phenotype D (nonandrogenic) – chronic ovulatory dysfunction and PCOM without clinical or biochemical manifestations of hyperandrogenism (OD/PCOM) [2–4]. PCOS is associated with metabolic disorders, insulin resistance (IR), impaired glucose tolerance (IGT), diabetes mellitus (DM) and significant increase in the risk of cardiovascular diseases [5,6]. The pathogenesis of carbohydrate metabolism disorders (hyperglycemia, insulin resistance) is associated with the initiation of polyol pathway of glucose metabolism, accumulation of early and advanced glycation end products (AGEs) in tissues, which include products of non-enzymatic glycation and protein oxidation. AGEs is a heterogeneous chemical group of compounds – glycotoxins. Pathological processes of glycation lead to irreversible cross-binding of proteins, loss of protein structure and function followed by cell apoptosis. The damaging effect of AGEs is manifested both independently (receptor-independent) and by binding to the receptors of the end products of glycation (RAGE–receptor for advanced glycation end products). Recent studies have shown that AGEs are involved in the pathogenesis of metabolic disorders typical for PCOS [7].

Isoflavone resveratrol (trans-3,5,4′-trihydroxystilbene) is a natural phytoalexin that can be isolated from the peel of grapes and a number of other food products, including mulberry [8,9]. Resveratrol possesses anti-inflammatory and antidiabetic activity [10]. In addition, resveratrol is able to induce [11,12] or inhibit [13,14] apoptosis, and also helps to reduce the number of atretic follicles and inhibit transition from primary to developing follicles [15].

The aim of the study was to evaluate the potential effectiveness of resveratrol in PCOS management on the basis of created model of the disease in Wistar rats.

Materials and methods

Animals and therapy

Female Wistar rats (190–230 g) (n = 24) were housed in regulated vivarium conditions in compliance with all the rules for keeping laboratory animals (time and procedure for quarantine, labeling of all individuals, constant sanitary control, standard diet, free access to water and food, automatic lighting mode ‘day/night’). Animal care and experiments were carried out according to the basic moral and ethical principles of conducting biomedical experiments on animals [16].

The study was carried out in two stages:

The first stage was randomization of rats: all the animals were assigned to three groups–two main groups and a control group. Animals from the 1st main group received non-steroidal aromatase inhibitor letrozole at a dosage of 800 mcg/day (n = 11); rats from the second main group received letrozole at a dosage of 400 mcg/day (n = 9); control group comprised rats receiving saline solution orally (n = 4).

Laboratory animals were included in the study in the estrus phase. Prior to the experiment, all of them underwent blood sampling from peripheral part of tail to determine the level of sRAGE by enzyme immunoassay method using the RayBio Rat RAGE ELISA Kit test system, USA. Then, for a period of 2 months, the rats received solution of letrozole orally. After 60 days, the rats again underwent blood sampling to determine the level of sRAGE. Further, after appropriate preoperative preparation, the rats underwent left-side ovariectomy for histological confirmation of the effectiveness of letrozole in formation of PCOS model. Surgeries were performed under general anesthesia using zolazepam hydrochloride 100 at a dosage of 30 mg/kg intramuscularly. Surgical access site was freed from the wool cover by chemical depilation cream, containing potassium thioglycolate, after which laparotomy was performed followed by left-side ovariectomy. During the first 4 days after surgery, antibacterial drug ceftriaxone 100 mg/kg and non-steroidal anti-inflammatory drug ketoprofen 40 mg/kg were administered intramuscularly once and twice a day respectively in order to prevent postoperative complications.

The obtained material was fixated in 10% neutral buffered formalin. Samples were stained with hematoxylin-eosin, then the changes in ovarian morphology were evaluated using light microscopy.

The second stage of the study was resveratrol administration. After histological confirmation of created PCOS model, 18 rats were randomized again. As a result of randomization, three groups were formed: the first main group received resveratrol solution at a dosage of 30 mg/kg/day (n = 8); the second main group received resveratrol solution at a dosage of 20 mg/kg/day (n = 6); animals from comparison group received saline solution orally (n = 4). Resveratrol therapy was carried out for 30 days, after which all the animals underwent peripheral blood sampling from distal part of tail to determine the level of sRAGE. After this procedure the rats were removed from the experiment. During the autopsy, visual assessment of morphological changes of ovaries and right-side ovariectomy were performed. The material was fixed in 10% neutral formalin solution for subsequent histological examination.

Statistical analysis

The PSPP program was used for statistical analysis. The analysis was carried out by calculating the Mann–Whitney criterion, as well as the Wilcoxon criterion.

Results

Determination of regularity of estral cycle

Based on the evaluation of vaginal cytology smears in rats receiving non-steroidal aromatase inhibitor letrozole, irregular cycle was revealed. This finding served as confirmation of successful PCOS modeling, which is also considered as a criterion for evaluating the formation of the syndrome on the basis of literature data [17]. With the use of resveratrol, restoration of estrous cycle in Wistar rats was determined. These data were also confirmed by the results of cytological research.

Body weight

Data on the changes in body weight of rats in dynamics are presented in Table 1. The weight of rats in the group receiving letrozole was significantly higher than in the control group (p<.05), and it was also significantly higher after letrozole application than prior to start of letrozole intake (p<.05).

Table 1. The weight of rats in dynamics.

Parameter	Body weight (g)
Prior to letrozole administration	203.67 ± 12.3
Control	213.75 ± 7.5
After letrazol administration for 30 days	246.94 ± 16.82
After letrazol administration for 60 days	287.14 ± 14.8
After resveratrol administration for 30 days	239.14 ± 12.07
Comparison group	292.5 ± 6.45

During the period of resveratrol use, there was a significant weight loss in rats with PCOS. In the group of animals with PCOS (comparison group) that received saline solution, body mass did not change (Table 1).

The level of sRAGE

Data on changes in the level of sRAGE in the blood of studied rats at different stages of observation are presented in Table 2. The level of sRAGE in rats with PCOS (after using letrozole) was significantly lower than in the control group and in the main group prior to treatment (p<.05). However, in rats with PCOS significant changes in the blood level of sRAGE were not revealed similar to the animals who received resveratrol (p>.05), as well as in the rats who received saline solution (Table 2).

Table 2. Blood level of sRAGE in rats on different stages of the experiment.

Parameter	sRAGE level (pg/ml)
Prior to letrozole administration	710.99 ± 126.92
Control	897 ± 117.9
After letrozole administration	407.82 ± 103.56
After resveratrol administration	488.95 ± 78.04

Histological examination and counting of follicles

Ovarian tissue was stained with hematoxylin and eosin, followed by light microscopy which was used to evaluate changes in ovarian morphology such as cystic dilatation of follicles and reduction in the number of granular cells layers. Light microscopy was performed on an Olympus CX31 microscope (Olympus, Japan) at magnification ×200, ×500.

After the course of letrozole intake, morphological changes in ovarian structure were detected in 18 (90%) out of 20 samples. In two ovarian samples received from rats which were administered letrozole 400 mcg/day, morphological changes in ovarian structure were not revealed (Figure 1). In the samples obtained from the control group of animals, the presence of oocytes, an increase in number of granulosa cells layers, as well as absence of cystic changes in follicles were observed (Figure 2). On the basis of morphological investigation of samples of the ovaries received from rats with PCOS after resveratrol administration, significant reduction of cystic changes, increase in number of granule cell layers, the presence of oocytes in follicles in 87.5% of cases (n = 7) receiving resveratrol at a dosage of 30 mg/kg/day, and in 66.7% of cases in studied rats (n = 4) receiving the drug at a dosage of 20 mg/kg/day (Figure 3).

Figure 1. Cystic dilatation of follicles, reduction of granule cell layers in ovary of Wistar rats after letrozole administration. H&E staining, ×500 mcm.

Figure 2. Ovarian morphology of the rats from the control group. H&E staining, ×500 mcm.

Figure 3. Increase in number of granule cell layers, presence of oocytes in follicles in resveratrol-treated rats with PCOS. H&E staining, ×500 mcm.

Discussion

Phytoestrogen resveratrol exhibits pleiotropic properties due to its estradiol-like structure. A study was conducted to investigate the effectiveness of resveratrol in treatment of hormonal disorders associated with PCOS. These properties were investigated by Zhang et al. on the experimental model of Sprague-Dawley rats. The effect of natural phytoalexin in dosages of 40, 80, and 160 mg/kg was evaluated. Based on this study, resveratrol was shown to be effective in restoration of ovarian morphological structure in rats, increasing expression of aromatase and nesfatin-1 receptors in ovarian tissue, increasing levels of estradiol and adiponectin in blood plasma [18]. In addition, a number of studies have also demonstrated the effectiveness of resveratrol in management of PCOS, but the results of these studies were mainly aimed at evaluating antioxidant and anti-inflammatory activity of this compound [19,20].

In our study, we evaluated the effectiveness of resveratrol on the basis of PCOS model in Wistar rats in relation to carbohydrate metabolism and normalization of ovarian morphology. The results of histological study showed the dose-dependent effectiveness of resveratrol in normalization of ovarian structure, that is, increase in the number of granulosa cells layers and presence of oocytes within follicles compared with morphology of ovarian tissue of the rats with PCOS after the use of letrozole. Normalization of estrous cycle in the groups of rats with PCOS receiving resveratrol, and significant decrease in animal’s weight were recorded.

After the formation of PCOS model in rats by means of aromatase inhibitor letrozole administration, a significant decrease in blood level of sRAGE was noted. According to literature, low blood level of sRAGE leads to accumulation of AGEs, thereby inhibiting cell proliferation and enhancing apoptosis of granulosa cells [7], increasing insulin resistance [21] and contributing to the activation of inflammatory and oxidative stress in the ovaries, which is accompanied by their autoimmune damage [21].

However, contrary to our expectations, there were no significant changes in blood level of sRAGE in rats with PCOS after resveratrol therapy, which may possibly be due to the insufficient duration of the use of natural phytoalexin to produce significant changes in this compound.

Conclusion

Thus, the results obtained suggest in our animal studies that resveratrol is a promising agent for the treatment of PCOS, which may also contribute to expanding the arsenal of drugs and personifying therapy of this disease.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The experiment was carried out as exploratory research of Ministry of Science and Higher Education of the Russian Federation № АААА-А20120041390030-3.