The Flavanone, Naringenin, Modifies Antioxidant and Steroidogenic Enzyme Activity in a Rat Model of Letrozole-Induced Polycystic Ovary Syndrome.

BACKGROUND Worldwide, polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder that affects women of reproductive age. Naringenin is a natural flavanone, derived from grapefruit. The aims of this study were to investigate the effects of naringenin on the antioxidant and steroidogenic enzyme activity in a rat model of letrozole-induced PCOS. MATERIAL AND METHODS The induction of PCOS was undertaken by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. There were four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. Estradiol, testosterone, and steroidogenic enzyme activities, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activity were determined in the four treatment groups, and histology was performed on the rat ovarian tissue. Serum glucose levels were measured with a glucometer. RESULTS Naringenin treatment in a rat model of PCOS significantly increased the levels of the reactive oxygen species (ROS) scavenging enzymes CAT, SOD, and GPX (p<0.05), and prevented weight increase. Naringenin treatment resulted in a significant reduction in serum glucose levels (p<0.05), normalized estradiol and testosterone levels steroidogenic enzyme activity, and maintained the normal anatomy of the ovaries. CONCLUSIONS Naringenin treatment, in a rat model of PCOS, demonstrated antioxidant and steroidogenic enzyme activity.

Background

Polycystic ovary syndrome (PCOS) is one of the more common endocrine disorders and affects approximately 4–10% of women of reproductive age [1,2]. PCOS involves hyperandrogenism, insensitivity to insulin, and oligo-ovulation [3]. Previously published studies during the past few decades have shown that PCOS is associated with an increased risk of type 2 diabetes mellitus [4]. Also, it has been shown that although women with mild forms of PCOS can have hyperandrogenism and may have normal ovulation, they are at an increased risk of developing chronic PCOS in the future [5]. In women with PCOS, there is increased activity of androgens, which leads to altered gonadotropin-triggered estrogen and progesterone synthesis in the ovarian follicle [6].

Because PCOS is associated with metabolic abnormalities that include insulin insensitivity associated with type 2 diabetes mellitus, current treatment strategies include insulin sensitizers, including metformin [7]. However, drugs such as metformin can be associated with severe side effects that adversely affect the standard of the life of patients [8]. Therefore, there is a need to develop alternative treatment strategies for women with PCOS, which is why increasing studies have also been conducted using natural plant-based products for the treatment and management of PCOS. Of all the plant-derived metabolites, flavonoids and flavanones constitute a large and diverse group of plant secondary metabolites that have been shown to have pharmacological potential [9]. Naringenin is an important flavanone that can be derived from grapefruit, but is widely present in the plant kingdom and has been isolated from several plant species [10,11]. Also, naringenin has been shown to have hypoglycemic activity in rat models of type 2 diabetes [12].

Given these previously published findings, the aims of this study were to investigate the effects of naringenin on the antioxidant and steroidogenic enzyme activity in a rat model of letrozole-induced PCOS.

Material and Methods

Ethics and study approval

The study was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (approval number NUCM/55621/2017). For the rat model studies, all international guidelines for animal studies were followed, as described previously [10]

The rat model of polycystic ovary syndrome (PCOS) and the animal treatment groups

Twenty-eight Sprague-Dawley virgin female rats weighing approximately 85 gm were purchased from the Nanjing University of Chinese Medicine. The animals were given free access to a dry pellet diet, and water was freely available. The animals were maintained in well-ventilated rooms with a controlled environment that included a 12-hour light and dark cycle and a temperature of 26±2°C. Cytological analysis of the vagina was performed on a daily basis to confirm the four-day ovarian cycle. Naringenin (98%) was obtained from Sigma-Aldrich (St Louis, MO USA).

The induction of polycystic ovary syndrome (PCOS) was performed by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. The rat model PCOS included four different treatment groups: Group I received 1% of carboxymethyl cellulose (CMC); Group II received 1% of CMC plus naringenin 20 mg/kg; Group III received letrozole only; Group IV received letrozole plus naringenin 20 mg/kg. In the naringenin-treated groups, naringenin was given once daily for seven days. The animals were then sacrificed at the end of the study and the ovaries were removed for further examination, with one ovary fixed in Bouin’s solution (Sigma-Aldrich, St Louis, MO USA) and the second ovary was assayed for steroidogenic enzyme activity.

Determination of the oral glucose tolerance (OGT) and steroid hormone concentrations

The rats were fasted for 12 hours before the end of the study and then given glucose at a dose of 300 mg/kg and the oral glucose test was performed, as previously described [13]. Peripheral blood samples were collected from the tail vein at 0, 30, 60, and 120 minute time intervals to estimate the glucose levels, using a glucometer. The testosterone and estradiol levels were measured, as previously described [13].

Ovarian homogenates and steroidogenic enzyme activity

The 10% ovarian homogenate was prepared in Tris-HCl buffer (0.1 M, pH 7.8) and then centrifuged at 8,000× g for 1 hour. The supernatant was used for the determination of the protein content and steroidogenic enzyme activities. The 3β-hydroxysteroid dehydrogenase (3β-HSD) activity was measured, as previously described [14]. The nicotinamide adenine dinucleotide (NADH) standard curve was used for the determination of the enzyme activity and presented as nanomoles of NADH produced per min per mg of protein. The enzymatic activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) was determined, as previously described [15], and the enzyme activity was presented as nanomoles of NADPH oxidized/min/mg protein.

Determination of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activity

The activity of reactive oxygen species (ROS) scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in ovarian tissue, according to standard protocols. The activity of SOD was determined by the method of Kakkar et al. [16], the CAT activity was determined by the method of Whanger et al. [17], and GPX was determined by the method of Paglia and Valentine [18].

Histology of the ovaries

For histological analysis, the ovaries were fixed in the Bouin’s solution were routinely processed and embedded in paraffin wax, and 5 μm thick sections were cut onto glass slides. The ovarian tissue sections were stained histochemically with hematoxylin and eosin (H&E) and examined by light microscopy.

Statistical analysis

All experiments and measurements were performed in triplicate and the data were shown as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA), followed by Tukey’s test, was used for statistical analysis using GraphPad version 7 software. P-values at p<0.01 and p<0.05 were considered as significant.

Results

The effect of naringenin on body weight and estrous cycle in the polycystic ovary syndrome (PCOS) rat model

The continuous administration of letrozole for 21 days that resulted in the rat model of PCOS resulted in the significant increase in the body weight when compared with the control rats (Group I and II) (Figure 1). Treatment of the PCOS rats with naringenin (Group IV) resulted in a significant reduction in the body weight when compared with the rats treated with letrozole only (Group III). While the PCOS rats (Group III) showed a prolonged the di-estrous stage, the administration of naringenin (25 mg/kg) could reverse the letrozole-induced effects on the di-estrous stage, which was significantly less prolonged when compared with the PCOS rats. However, there was no difference in the duration of the di-estrous stage between Group I and Group II.

Figure 1

The effect of naringenin on the weight of the rat model of letrozole-induced polycystic ovary syndrome (PCOS) in the different treatment groups. The induction of polycystic ovary syndrome (PCOS) was performed by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. The rat model of letrozole-induced PCOS included four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% of CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. The experiments were performed in triplicate and expressed as the mean ±SD. * p<0.01 for Group III vs. Group I and II; # p<0.05 for Group IV vs. Group III.

Naringenin affected the oral glucose tolerance (OGT) levels in the rat model of PCOS

The effect of naringenin on the oral glucose tolerance (OGT) levels in the rat model of PCOS resulted in no significant difference in the plasma glucose levels between the Group I and Group II rats. However, treatment with naringenin (20 mg/kg) in the letrozole and naringenin-treated rats (Group IV) resulted in significantly reduced plasma glucose levels when compared with the rats treated with letrozole only (Group III) (Figure 2).

Figure 2

The effect of naringenin on the plasma glucose levels of the rat model of letrozole-induced polycystic ovary syndrome (PCOS) in the four different treatment groups. The induction of polycystic ovary syndrome (PCOS) was performed by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. The rat model of letrozole-induced PCOS included four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% of CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. The plasma glucose levels at 0, 30, 60, 120 minutes after administration of naringenin. The experiments were performed in triplicate and expressed as the mean ±SD. * p<0.01 for Group III vs. Group I and II. # p<0.05 for Group IV vs. Group III.

Naringenin affected the testosterone and estradiol levels in the rat model of PCOS

The results indicated that testosterone levels were significantly increased in rats treated with letrozole only (Group III). However, treatment with 20 mg/kg naringenin resulted in a significant decline in the levels of testosterone. No significant difference was found between the testosterone levels of Group I and II (Figure 3A). Similar effects were found for estradiol levels (Figure 3B).

Figure 3

The effects of naringenin on the expression of testosterone and estradiol in the rat model of letrozole-induced polycystic ovary syndrome (PCOS) in the four different treatment groups. The induction of polycystic ovary syndrome (PCOS) was performed by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. The rat model of letrozole-induced PCOS included four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% of CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. (A) Testosterone and (B) estradiol in different treatment groups. The experiments were performed in triplicate and expressed as the mean ±SD. * p<0.01 for Group III vs. Group I and II. # p<0.05 for Group IV vs. Group III.

Naringenin affected the steroidogenic enzyme levels in the rat model of PCOS

The influence of naringenin on the steroidogenic enzyme levels were investigated, and in the rat model of PCOS treated with letrozole (Group III), there was an increase in the levels of both 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) when compared with the controls (Group I and Group II). However, treatment of the PCO rats (Group IV) with naringenin at 20 mg/kg resulted in a significant decline in both the 3β-HSD and 17β-HSD levels (Figure 4).,

Figure 4

The effect of naringenin on steroid enzyme activities in the rat model of letrozole-induced polycystic ovary syndrome (PCOS) in the four treatment groups. The induction of polycystic ovary syndrome (PCOS) was performed by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. The rat model of letrozole-induced PCOS included four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% of CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. The experiments were performed in triplicate and expressed as mean ±SD. p<0.01 for Group III vs. Group I and II. # p<0.05 for Group IV vs. Group III.

Naringenin affected the activity of the reactive oxygen species (ROS)-scavenging enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), in the rat model of PCOS

The influence of naringenin treatment in the rat model of PCOS was also investigated on the levels of the reactive oxygen species (ROS)-scavenging enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). The results showed that the activity of all three enzymes was decreased in the PCOS rats treated with letrozole (Group III) when compared with the control groups (Group I and II). However, treatment of the PCOS rats with naringenin resulted in a significant increase in the levels of SOD, CAT and GPX (Figure 5A–5C).

Figure 5

Effect of naringenin on the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in the rat model of letrozole-induced polycystic ovary syndrome (PCOS) in the four treatment groups. The induction of polycystic ovary syndrome (PCOS) was performed by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. The rat model of letrozole-induced PCOS included four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% of CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. (A) Superoxide dismutase (SOD), (B) catalase (CAT), and (C) glutathione peroxidase (GPDX) activity in rats in different groups. The experiments were performed in triplicate and expressed as the mean ± SD. * p<0.01 for Group III vs. Group I and II; # p<0.05 for Group IV vs. Group III.

Naringenin treatment had an effect on the ovarian anatomy of the ovarian tissue in the rat model of PCOS

Light microscopy of the sections of rat ovary, stained with hematoxylin and eosin (H&E), showed that the control rats (Group I and II) showed normal ovarian histology. The histology of the ovarian tissue of the letrozole-treated rats in the PCOS rat model (Group III) showed follicular cysts, a reduction of the granulosa cell layer, and hyperplasia. Treatment of the PCOS rat model with (Group IV) restored the normal ovarian morphology, as seen by light microscopy (Figure 6).

Figure 6

Photomicrographs of the light microscopy of the histopathology of the rat ovarian tissue showing the effects of naringenin on the rat model of letrozole-induced polycystic ovary syndrome (PCOS) in the four treatment groups. The rat model of letrozole-induced PCOS included four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% of CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. Hematoxylin and eosin (H&E) staining. (Magnification ×4).

Discussion

The aim of this study was to evaluate the effects of naringenin, a natural flavanone, in a letrozole-treated rat model of polycystic ovary syndrome (PCOS). The findings showed that naringenin could prevent weight gain associated with PCOS and caused a reduction in the serum glucose levels of PCOS rats. These findings were consistent with those from previously published studies that showed naringenin treatment had hypoglycemic effects in diabetic rats [19].

Also, naringenin caused a reduction in the activity of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in the PCO rat model. This finding might be due to the presence of the B ring of the naringenin molecule [20]. It has previously been reported that the B ring of flavonoids and flavanones exhibit the capacity to inhibit the 3β-HSD and 17β-HSD enzymes. [21]. Also, naringenin has been previously shown to decrease the levels of testosterone and estradiol in women with PCOS [21].

Oxidative stress has been previously reported to be one of the main characteristics of PCOS and is believed to be involved in the pathogenesis of PCOS [21]. In this study, the effects of naringenin on the reactive oxygen species (ROS)-scavenging enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), showed that naringenin could increase the levels of these enzymes in the PCOS rat model, in addition to rescuing the effects of letrozole treatment on the ovarian morphology. It has previously been reported that molecules that can scavenge ROS may be beneficial in the treatment of PCOS, and the finding that naringenin could reduce the effects of oxidative stress is also supported by the findings from previous studies [22].

A recently published study has shown that soy flavonoids have protective effects in letrozole-induced PCOS in rats [23]. Also, rutin, a key flavonoid has been reported to have protective effects on PCOS by altering several of biochemical parameters [24]. Resveratrol, a plant-derived phenol and anti-oxidant, has been reported to prevent the changes in ovarian anatomy associated with PCOS [25]. Also, a recent study on the effects of a plant-derived natural product, cinnamon, has been reported to have protective effects in women with PCOS. These results indicate the potential use of naturally-occurring compounds for use in the treatment of PCOS. However, further clinical studies are required to confirm these preliminary studies.

Conclusions

In a rat model of polycystic ovary syndrome (PCOS), naringenin treatment demonstrated antioxidant and steroidogenic enzyme activity. Further controlled clinical studies are required to determine the effects of naringenin in the treatment of PCOS.