Effects of Naringenin (NG) as an anti- proliferative and anti-apoptotic factor on ER alpha and ER beta in PCOS

doi:10.21203/rs.3.rs-4808114/v1

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Effects of Naringenin (NG) as an anti- proliferative and anti-apoptotic factor on ER alpha and ER beta in PCOS

https://doi.org/10.21203/rs.3.rs-4808114/v1

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Oxidative stress is the imbalance between production of free radicals called oxidants and the ability of to defend their harmful effectsEstrogens play a significant role in the development and function of the reproduction system. The main mediators of estrogen action are two specific high affinity receptors, the estrogen receptor α (ERα) and estrogen receptor β (ERβ), both of which are members of nuclear receptor superfamily and are necessary for the proper functioning of the hypothalamic–pituitary–ovarian axis. Furthermore, while both ERα and ERβ are expressed in the human ovary, ERβ is the main type of receptor and its activation enhances folliculogenesis and ovulation.Polycystic ovary syndrome (PCOS) is a prevailing pathological status, that is extensively observed in 80% of infertile women.

Methods and result: The current experimental study was performed on animal models. Estrogen and progesterone concentrations in the PCOS group decreased significantly compared to the control group. The level of gene expression and production of ER-a and ER-b proteins in the PCOS group decreased significantly. In the PCOS group, the number of atretic follicles increased significantly compared

the present study investigated the effects of Naringenin as an anti- proliferative and anti-apoptotic factor on ER alpha and ER beta in PCOS patients.

Polycystic ovary syndrome (PCOS) is a prevailing pathological status, that is extensively observed in 80% of infertile women. (1) It exhibits a wide spectrum of clinical manifestations such as amenorrhea or oligomenorrhea, hyperandrogenism, hirsutism, acne, dyslipidemia, and polycystic ovarian morphology and is associated with oxidative stress and metabolic factors such as inflammation, insulin resistance, obesity and diabetes.(2–5)

Oxidative stress is the imbalance between production of free radicals called oxidants and the ability of to defend their harmful effects. (6) Overproduction of free radicals such as reactive oxygen species (ROS) or failure in antioxidant defense system result in oxidative stress which is involved in several conditions. (4)

The steroidogenic function of theca cells is regulated by LH and local factors. (7) Most attention is paid to the hypersecretion of LH and insulin resistance as well as hyperinsulinemia. The oldest theory emphasized the relationship between thecal cells stimulation with LH and the consequent androgen overproduction. (8) Accordingly, increased LH and enhanced insulin levels amplify the inherent impairment of steroidogenesis in theca cells.(9–11)

In addition to hyperandrogenism symptoms, follicle stimulating (FSH) and luteinizing (LH) hormones upregulation, as well as estrogen and progesterone reduction levels have been reported in PCOS patients. (8, 11)

Estrogens play a significant role in the development and function of the reproduction system. The main mediators of estrogen action are two specific high affinity receptors, the estrogen receptor α (ERα) and estrogen receptor β (ERβ), both of which are members of nuclear receptor superfamily and are necessary for the proper functioning of the hypothalamic–pituitary–ovarian axis. Furthermore, while both ERα and ERβ are expressed in the human ovary, ERβ is the main type of receptor and its activation enhances folliculogenesis and ovulation. (12, 13)

It is reported that in rodents, ERα is expressed exclusively in theca cells, whereas ERβ is expressed especially in granulosa cells (GCs). (14) Studies in knockout mice have revealed that the absence of ERα leads to the polycystic ovary syndrome (PCOS) phenotype with elevated luteinizing hormone (LH) levels and ovaries characterized by the presence of multiple hemorrhagic and cystic follicles, while the ERβ knockout mice have abnormal follicular development with early atretic follicles and are subfertile. (15–17)

Furthermore, its demonstrated that the expression of ERβ is lower in follicles derived from women with PCOS compared with healthy women, while ERα expression is markedly increased in theca cells of polycystic ovaries, causing alteration in the ERα/ERβ ratio in PCOS and possibly abnormal follicular development. (15) Actually, ERβ knockout (βERKO) mice ovaries appear normal, exhibiting follicles at all stages of development. Meanwhile, these mice represent fewer corpora lutea, resulting in mild subfertility problems. (18)

Moreover, failed responses to exogenous gonadotropins as well as a severe deficiency in response to the LH/human chorionic gonadotropin (hCG) ovulatory stimulus have been reported in βERKO mice ovaries. (19) It’s been observed in mice lacking ERα to be insulin resistant with impaired glucose tolerance and increased white adipose tissue, indicating that abnormalities in estrogen signaling may have relevant metabolic effects. (20)

Flavonoids, the so-called phytoestrogens, are well known as natural estrogen analogues and are found in abundance in roots, flowers, fruits, and stems of plants. (21–24) 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 that exhibits antioxidant, anti-apoptotic, anti-atherogenic and metal chelating activities.(25–27)

It has been reported to cause a reduction in the activity of the steroidogenic enzymes 3b-hydroxysteroid dehydrogenase (3b-HSD) and 17b-hydroxysteroid dehydrogenase (17b-HSD) in the PCO rat model. This finding might be due to the presence of the B ring of the Naringenin molecule. (28) Among several mechanisms proposed for Nar-induced anti-proliferative effects (i.e., antioxidant activities, kinase and glucose uptake inhibition) (29–32), the ability of Nar to hamper cell proliferation via estrogen receptor (ER) binding is particularly intriguing. It is well known that different flavonoids, Nar inclusive, bind both ERα and ERβ (i.e., greater affinity to ERβ than to ERα (33, 34), thus modulating the 17b-estradiol (E2)-induced gene transcription. However, the involvement of ERα or ERβ signaling in the molecular mechanisms of Nar-induced anti-proliferative effects in human cancer cells remains to be investigated. (35)

The purpose of the present study was to investigate the effects of Naringenin as an anti- proliferative and anti-apoptotic factor on ER alpha and ER beta in PCOS patients and to determine whether there are significant differences, compared with healthy women.

Chemicals and materials

Specific commercial kits were purchased for analysis of rat testosterone (Mybiosource, USA), androstenedione (Mybiosource, USA), estrogen (Bio Vender, Czech Republic), progesterone (Crystal Chem, USA), LH (Mybiosource, USA), FSH (Bio Vender, Japan). Primary antibodies were provided for Erα, Erβ and c-Myc (Rabbit- Antimouse Erα, Erβ and c-Myc; Biocare, USA). Commercial kits for SOD and GSH-px were obtained from RANDOX reagents company (Germany). All other chemical agents were commercial products of analytical grade.

Animals, PCOS induction and experimental design

The current experimental study was performed on animal models. To conduct it, 42 mature (6–8 weeks old) female wistar rats were assigned into six groups (seven rats in each group), including control (sampled after 30 days), PCOS-induced (sampled 15 and 30 days of post PCOS induction), control with a dose of 20 mg/kg, control with a dose of 40 mg/kg, PCOS-induced with a dose of 20 mg/kg, PCOS-induced with a dose of 40 mg/kg groups. The animals were given ad libitum access to food and water, kept at room temperature (20-23oC) and normal humidity (30–50) on a 12 hr light/dark cycles. The hyperandrogenic PCOSlike condition was induced based on the previous study by Honnma et al. (21). Briefly, Testosterone-Enanthate (TE, 10 mg/kg body weight) was subcutaneously injected to 7–8 weeks rats, every morning for 21 days. The animals in the control groups and PCOS-induced were received 20 and 40mg/kg oral naringenin every morning for the corresponding length of time. All experimental protocols were approved and monitored by the Ethical Committee

Histological analyses

At the end of experiment, light anesthesia was induced to animals using 5% ketamine (40 mg/kg) in addition to 2% xylazine (5 mg/kg), intraperitoneally and then euthanized by especial CO2 device (ADACO, Iran). Next, the ovarian tissues were dissected out and fixed in 10% formalin for 72 hours. Routine sample processing was performed using ascending alcohol and the samples were then embedded in paraffin. Thereafter, serial sections were prepared by rotary microtome (Leitz Wetzlar, Germany) and stained with hematoxylin-eosin. To perform histomorphometric analyses, follicles were classified to preantral and antral types. Follicles with intact/complete layers of GCs and theca cells, ordinary cytoplasm of oocyte and intact nuclei were considered as normal/intact follicles. Follicles with GC dissociation, early antrum formation, luteinized elongated GCs were considered as atretic types. The atretic preantral and antral follicles were counted in serial sections for each sample and compared between groups.

Analyses of RNA damage

Darzynkiewicz method was contemplated to appraise the RNA damage. (23). concisely, ether alcohol was used to wash the ovaries and after that, 10 µm chunk were attain using cryostat microtome. (Huntingdom, UK). various degrees of ethanol were used to fix the chunks. subsequently, the chunks were rinsed in acetic acid (1%) and washed in distilled water. The slides were stained in acridine-orange (3–5 minutes) and next counterstained in phosphate buffer (pH = 6.85, 2 minutes). lastly, the fluorescent colors differentiation was induced by calcium chloride. The follicular cells with RNA damage were characterized with loss and/or faint red stained RNA. The normal cells were marked with bright red fluorescent RNA.

Immunohistochemical staining

Tissue slides were heated at 60˚C (25 minutes) in a hot-air oven (Venticell, Germany). Tissue chunks were next dewaxed in xylene (2 changes, each change 5 minutes) and rehydrated. Following antigen retrieval process (in 10 mM sodium citrate buffer), the immunohistochemical (IHC) staining was conducted based on the manufacturer’s protocol (Biocare, USA). Summarily, endogenous peroxidases were blocked by 0.03% hydrogen peroxide containing sodium acid. The chunks were washed lightly and thereafter, incubated with Erα (1:500), Erβ (1:600) and c-Myc (1:500) biotinylated primary antibodies in 4oC, overnight. The slides were then rinsed lightly with phosphate-buffered saline (PBS) and placed in a humidified chamber with a sufficient aplenty of streptavidin conjugated to horseradish peroxidase in PBS, containing an antimicrobial abettor, for 15 minutes. Next, DAB chromogen was used to mark target proteins. Counterstaining was conducted by hematoxylin. lastly, the chunks were dipped in ammonia (0.037 ml), rinsed in distilled water and coverslipped. The positive immunohistochemical reaction was visualized as brown.

RNA extraction and cDNA synthesis

Total RNA from isolated GCs was extracted using the Trizol Plus RNA Purification kit (Life Technologies) according to the manufacturer’s instructions. The RNA purity was confirmed using a NanoDrop 2000 (Thermo Scientific) and an A260:A280 ratio of 1.9–2.1. Total RNA (1 µg) was reverse transcribed to cDNA using the PrimeScript RT reagent Kit (Takara) and diluted with nuclease-free water to a final volume of 20 µl. The cDNAs were further diluted 1:20 with nuclease-free water for use as the DNA template for qRT-PCR.

Biomarkers of oxidative stress MDA. The MDA standard in the PCOS group increased significantly compared to the control group. In the PCOS groups that received Naringenin (NG), MDA decreased significantly compared to the PCOS group. This reduction rate was higher in the group receiving 40 mg dose compared to the group receiving 20 mg naringin, but the difference between these two groups was not significant. In the control groups of 20 mg and 40 mg of Naringenin (NG), the amount of MDA decreased compared to the normal control group, but this difference was not significant. SOD levels in the PCOS group decreased significantly compared to the control group. In the PCOS groups that received Naringenin (NG), SOD levels increased significantly compared to the PCOS group. This increase was higher in the group receiving the 40 mg dose compared to the group receiving the 20 mg dose of Naringenin (NG), but the difference between these two groups was not significant. In the control groups receiving 20 mg and 40 mg of Naringenin (NG), the amount of SOD increased compared to the normal control group, but this difference was not significant. Figure 1.

GPX values in the PCOS group decreased significantly compared to the control group. In the PCOS groups that received Naringenin (NG), GPX levels increased significantly compared to the PCOS group. This increase was higher in the group receiving the 40 mg dose compared to the group receiving the 20 mg dose of Naringenin, but the difference between these two groups was not significant. In the control groups receiving 20 mg and 40 mg of Naringenin, the amount of GPX also increased compared to the normal control group, but this difference was not significant.

TAC values in the PCOS group decreased significantly compared to the control group. In the PCOS groups that received Naringenin, the TAC values increased significantly compared to the PCOS group. This increase was higher in the group receiving the 40 mg dose compared to the group receiving the 20 mg dose of Naringenin, but the difference between these two groups was not significant. In the control groups receiving 20 mg and 40 mg of Naringenin, the amount of TAC increased compared to the normal control group, but this difference was not significant.

Testosterone concentration in the PCOS group increased significantly compared to the control group. In the PCOS groups that received Naringenin, testosterone levels decreased compared to the PCOS group, but this decrease was greater in the group that received a dose of 20 mg compared to the group that received a dose of 40 mg of Naringenin,. In the control groups receiving 20 mg and 40 mg of Naringenin, the concentration of testosterone increased compared to the normal control group, but this difference was not significant.

The concentration of Androstendion in the PCOS group increased significantly compared to the control group. In the PCOS groups that received Naringenin, Androstendione cholesterol decreased compared to the PCOS group, but this reduction was greater in the group that received a dose of 20 mg compared to the group that received a dose of 40 mg of Naringenin. In the control groups receiving 20 mg and 40 mg of Naringenin, Androstendion concentration increased significantly compared to the normal control group. Figure 2

Estrogen and progesterone concentrations in the PCOS group decreased significantly compared to the control group. In the PCOS groups that received Naringenin, the concentrations of estrogen and progesterone increased significantly compared to the PCOS group. This increase was higher in the group receiving the 40 mg dose compared to the group receiving the 20 mg dose of Naringenin, but the difference between these two groups was not significant. In the control groups receiving 20 mg and 40 mg of Naringenin, the concentrations of estrogen and progesterone increased compared to the normal control group, but this difference was not significant.

The level of gene expression and production of ER-a and ER-b proteins in the PCOS group decreased significantly compared to the control group. In the PCOS groups that received Naringenin, the gene expression levels and production of ER-a and ER-b proteins increased significantly compared to the PCOS group. This increase was higher in the group receiving the 20 mg dose compared to the group receiving the 40 mg dose of Naringenin, but the difference between these two groups was not significant. In the control groups receiving 20 mg and 40 mg of Naringenin, the level of gene expression and production of ER-a and ER-b proteins decreased significantly compared to the normal control group. Figure 3

In the PCOS group, the number of atretic follicles increased significantly compared to the control group. In the PCOS groups that received Naringenin, the number of atretic follicles decreased compared to the PCOS group, and this reduction was greater in the group receiving the 40 mg dose compared to the group receiving the 20 mg dose of Naringenin. Also, the number of atretic follicles in the control groups receiving 20 mg and 40 mg of Naringenin also decreased compared to the control group, but it was not significant.

Polycystic ovary syndrome (PCOS) is a disease that affects the ovaries of women of reproductive age. Polycystic ovary syndrome can be genetic or caused by environmental factors. While there is no one-size-fits-all cure for PCOS, lifestyle changes and an appropriate diet have been effective for most patients. The Complications of chemical drugs, lack of treatment success and increased need for unnecessary surgery in persistent cysts have caused researchers to use herbal drugs and antioxidants to improve disease symptoms in recent years.

Plants and their bioactive substances are considered as the most available compounds for the treatment of tissue damage. Flavonoid compounds such orange have antioxidant properties and destroy various free radicals.

The results of this study showed that the use of Naringenin can improve biomarkers of oxidative stress in patients with PCOS. SOD and GPX are used as measures of tissue damage and protect cells against damage caused by oxidative stress. Administering Naringenin decreased MDA levels and increased SOD, GPx, and TAC levels, which indicates an increase in the ability of cells to clear free radicals. In this study, Naringenin was also able to improve the histopathological changes of ovarian tissue. A large number of atretic follicles were observed in patients with PCOS, while the number of atretic follicles decreased in the groups receiving orange.

Polycystic ovary syndrome caused a significant increase in the concentration of testosterone and androstenedione and a decrease in the levels of estrogen and testosterone compared to the control group. After receiving Naringenin, the concentration of testosterone and Androstenedione decreased and the concentration of estrogen and progesterone increased. It has been reported that in mice with PCOS, serological changes are in the form of a decrease in FSH and an increase in LH.

Nabiuni et al. (2015) reported in their study that in mice with polycystic ovaries, serological changes are a decrease in FSH, progesterone and an increase in LH, estradiol and testosterone(36) . Zurvarra et al. (2009) observed that after treating rats with letrozole to create an animal model of PCOS, the serum level of testosterone increased in the group treated with letrozole and the level of progesterone decreased(37) .

Doldi and colleagues showed that serum and progesterone concentrations showed a significant decrease and increase respectively in PCOS samples, which is not consistent with the results of this study (38).

In this study, the expression levels of estrogen receptor α (ERα) and estrogen receptor β (ERβ), (both of which are members of the nuclear receptor family and are necessary for the proper functioning of the hypothalamus-pituitary), in the PCOS group compared to the control group. significantly decreased and administration of Naringenin significantly increased their expression.

The study by Wu et al. (2020) showed that the administration of Naringenin can regulate the levels of steroid hormones in rats with PCOS induced by letrozole(39) .

The results obtained from Rashid et al.'s study in 2023 showed that in rats with PCOS induced by letrozole, the administration of Naringenin significantly reduced the number of cysts and regulated the levels of steroid hormones (40).

Yang et al. showed in 2022 that the administration of Naringenin and murine to mice with PCOS can reduce insulin resistance and reduce endometrial hyperplasia by increasing inflammation and apoptosis (41).

Ethics approval and consent to participate: approved

Consent for publication:yes

Availability of data and material:yes

Competing interests: no conflict of interest

Funding:no funding

Authors' contributions

1-Fardad.saremi,:main idea

2-Fatemeh Parvin Sabet:writing

3-Kimia Nabiee: exprimental

4-Fatemeh Tolouei:exprimental

5-Kooshyar Kouchaki:exprimental

6-Mahya Yasami:editing

7-shaghayegh Soltani:editing

5- Mona Gorji : corresponding, main idea , editing

Data is available

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No competing interests reported.

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Effects of Naringenin (NG) as an anti- proliferative and anti-apoptotic factor on ER alpha and ER beta in PCOS

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Abstract

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Introduction

Materials and Methods

Chemicals and materials

Animals, PCOS induction and experimental design

Histological analyses

Analyses of RNA damage

Immunohistochemical staining

RNA extraction and cDNA synthesis

Result

Discussion

Declarations

References

Additional Declarations

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