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 Table of Contents  
Year : 2018  |  Volume : 2  |  Issue : 1  |  Page : 21-29

Baicalein Ameliorates Chronic Stress-Mediated Ovarian Dysfunction by Upregulating the Expression of Gamma-Aminobutyric Acid B2 Receptor

1 Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China
3 Obstetrics and Gynecology Hospital; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai 200011, China

Date of Submission07-Feb-2018
Date of Web Publication21-May-2018

Correspondence Address:
Yu Kang
Obstetrics and Gynecology Hospital, Fudan University, No. 419 Fangxie Road, Shanghai 200011
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.232876

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Background: The aim of this study is to assess the effect of baicalein on chronic stress-mediated ovarian dysfunction in a mouse model.
Methods: Forty female C57BL/6 mice were randomly divided into four groups as follows: the normal saline group (control, n = 10), the daily stress group (daily stress, n = 10), the baicalein group (baicalein, n = 10), and the daily stress + baicalein group (daily stress + baicalein, n = 10). For the daily stress model, we used a restricted stress model. Baicalein (10 mg/kg) was administered by gavage every day, and control mice received normal saline equivalently. Biopsy specimens were harvested after 4 weeks. Measurement of norepinephrine (NE) in serum was performed to assess the psychological stress level of the mice. In addition, histological changes of the uterus and ovaries and the levels of anti-Müllerian hormone (AMH) in serum were assessed to evaluate changes in ovarian function. To detect the underlying mechanisms of the amelioration of baicalein in chronic stress-mediated ovarian dysfunction, immunohistochemical methods, and quantitative real-time polymerase chain reaction were applied to determine the expression of gamma-aminobutyric acid (GABA) receptors.
Results: Compared with values in the control group, serum NE concentrations were significantly increased (P < 0.001), AMH concentrations were markedly decreased (P < 0.01), the thickness of the endometrium was clearly reduced, and the percentage of atretic follicles was significantly increased in the daily stress group (P < 0.001), indicating that the chronic stress model was successfully established. In contrast, compared with values in the daily stress group, serum NE concentrations were significantly reduced (P < 0.001), AMH concentrations were significantly enhanced (P < 0.05), the thickness of the endometrium was clearly increased, and the percentage of atretic follicles was significantly reduced (P < 0.001) in the daily stress + baicalein group, indicating that baicalein clearly attenuated the ovarian dysfunction mediated by chronic stress. Moreover, the expression of the GABAB2receptor in the daily stress group was significantly reduced (P < 0.01). In contrast, treatment with baicalein resulted in increased expression of the GABAB2receptor (P < 0.01).
Conclusions: Treatment with baicalein ameliorates the enhancing effect of chronic stress on ovarian dysfunction, and the mechanism can be attributed, in part, to the increased expression of the GABAB2receptor.

Keywords: Baicalein; Chronic Stress; Gamma-Aminobutyric Acid B2 Receptor; Ovarian Dysfunction

How to cite this article:
Li K, Li J, Xu J, Zhang L, Liu QY, Huang YK, Zhang ZG, Kang Y, Xu CJ. Baicalein Ameliorates Chronic Stress-Mediated Ovarian Dysfunction by Upregulating the Expression of Gamma-Aminobutyric Acid B2 Receptor. Reprod Dev Med 2018;2:21-9

How to cite this URL:
Li K, Li J, Xu J, Zhang L, Liu QY, Huang YK, Zhang ZG, Kang Y, Xu CJ. Baicalein Ameliorates Chronic Stress-Mediated Ovarian Dysfunction by Upregulating the Expression of Gamma-Aminobutyric Acid B2 Receptor. Reprod Dev Med [serial online] 2018 [cited 2021 May 11];2:21-9. Available from: https://www.repdevmed.org/text.asp?2018/2/1/21/232876

  Introduction Top

Premature ovarian failure (POF) occurs in females under 40 years of age with normal development of secondary sex characteristics and regular menstruation who suffer from genital atrophy and persistent amenorrhea accompanied by increased follicle-stimulating hormone (FSH) and luteinizing hormone levels, as well as decreased estrogen (E2) levels.[1] The causes of POF include immunologic factors, genetic factors, hormonal and hormone receptor abnormalities, iatrogenic factors, stress, and infection.[2] In addition, with the transformation of the medical model to the biopsychosocial model and with the increased pressure imposed on women from the family and society, the damage caused by stress from the environment, society, and psychological factors has increasingly affected female reproductive function.[3],[4] Many kinds of physical and mental stressors can cause ovarian dysfunction and may even cause POF. Epidemiological studies have shown that, compared with healthy females, females with diminished ovarian reserve (DOR) suffer from greater psychological stress, with a certain degree of anxiety, depression, or other chronic stress symptoms appearing in a subset of these patients.[5] In addition, studies have shown that chronic stress, such as anxiety and depression, can impair ovarian function by disrupting the female reproductive endocrine system, thus leading to POF.[4],[6] Ovarian reserve function is one of the indicators for measuring ovarian function, and the major characteristics of DOR are declining serum levels of the anti-Mülllerian hormone (AMH), as well as decreasing quality and quantity of follicles.[7] However, there is still a shortage of effective treatments that might alleviate the effects of chronic stress in regard to ovarian dysfunction.

Traditional Chinese medicine could play an important role in the treatment of diminished ovarian function. Baicalein is a common Chinese herbal medicine that exhibits a variety of biological functions, including antianxiety and antidepression effects.[8],[9] Whether baicalein can alleviate ovarian dysfunction caused by chronic stress is the main focus of this study. Prior studies have shown that the antianxiety effect of baicalein on mice may work through the combined benzodiazepine binding site of gamma-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter that exists extensively in brain tissue.[10] In addition, GABA is also highly expressed and appears to be functional in ovarian and fallopian-tube tissue.[11] A specific binding site for GABA has been identified in ovarian tissue, and this site can affect the hypothalamic-pituitary-ovarian (HPO) axis, regulating the function of the female reproductive system.[11] As such, another focus of this study was to determine whether chronic stress could affect ovarian function by downregulating the expression of GABAB2 receptor and whether baicalein could ameliorate the effects of chronic stress on the GABAB2 receptor. In this study, we adopted a well-characterized model of chronic stress to investigate whether baicalein could ameliorate impaired ovarian function in mice caused by chronic stress by upregulating the expression of the GABAB2 receptor in ovarian tissue, providing experimental evidence for clinical treatment of POF.

  Methods Top

Drugs, reagents, and instruments

Baicalein (trihydroxyflavone) was obtained from Sigma-Aldrich (USA, 92081-50 mg). Immunohistochemical reagents, including xylene (analytical pure), hematin, and 30% hydrogen peroxide solution were obtained from Shanghai Chemical Reagent Co., (China). Absolute ethyl alcohol (AR, 99.5%) and methyl alcohol (AR, 99.5%) were obtained from Shanghai Zhenxing Chemical Factory (China). DAB staining kits were obtained from Thermo Fisher Scientific (USA). Rhamsan gum was obtained from Sangon Biotech (Shanghai, China). Norepinephrine (NE) (A2756), sodium 1-octane sulfonate (SOS), and dihydroxy benzamide (DHBA) were obtained from Sigma-Aldrich (USA). The AMH enzyme-linked immunosorbent assay (ELISA) kit (DEIA-BJ2337) was obtained from Creative Diagnostics (USA). Anti-GABA AE antibody (orb157001) was obtained from Biorbyt Co., (UK), and anti-GABA B receptor 1 (ab55051), and anti-GABA B receptor 2 (ab52248) antibodies were obtained from Abcam (UK).

Waters high-performance liquid chromatography (HPLC) system and high-speed centrifuges were purchased from Hitachi (Japan), a high-speed cryogenic centrifuge was purchased from Eppendorf (Germany), and an upright and inverted fluorescence microscope was purchased from Olympus (Japan). An automatic tissue dewatering machine, tissue paraffin embedding materials, and paraffin tissue histotome were purchased from ThermoFisher Scientific (USA).

Animals and stress model

A total of 40 eight-week-old female C57BL/6 mice were purchased from East China Normal University (license NO: SCXK [Shanghai] 2016-0004). All mice were acclimatized to standard laboratory conditions (SPF, license number: SCXK [Shanghai] 2015-0011) for 7 days before inducing stress, and all experiments were approved by the East China Normal University Animal Care Commission. All mice received humane care according to the criteria outlined in the “Guide for the Care and use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. A restraint-stress apparatus was used, based on that of a prior study [Figure 1]a.[12] We then randomly assigned the mice to four groups (n = 10 per cage), and the stress procedure continued until the end of the experiment. Every day, we give the mice baicalein or normal saline as control by gavage, and after gavage, mice in the stressed groups were placed in the restraint-stress apparatus for 2 h daily (from approximately 9 a.m. to 11 a.m.). The restraint-stress apparatus could effectively immobilize the mice. In addition, the mice were deprived of food and water while immobilized. The apparatus was washed, disinfected, and dried after each use. The ovaries and uterus of each mouse were harvested for weighing and were formalin-fixed after a 4-week experimental period.
Figure 1: Effects of chronic stress and/or baicalein on the behavior, endocrine hormone levels, and organ indexes of the mice. (a) The restraint-stress apparatus. (b) Weight comparison of each group from week 1 to week 4. (c) The level of serum norepinephrine in each group, as detected by high-performance liquid chromatography. (d) Comparison of the ovarian indexes among the four groups. (e) Comparison of the uterine indexes among the four groups. *P<0.001, compared with the daily stress group.

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Stress and groups

The mice were randomly divided into four groups after being fed adaptively for 1 week: the normal saline group (control, n = 10), the daily stress group (daily stress, n = 10), the baicalein group (baicalein, n = 10), and the daily stress + baicalein group (daily stress + baicalein, n = 10). The baicalein group mice were given baicalein by gavage once a day for 4 weeks (100 μl, 10 mg/kg.day), whereas the normal saline group mice were given an equal volume of normal saline following the same procedure.

Blood and tissue collection

Blood was drawn from the eye socket veins of the mice and was centrifuged at 3,000 g for 15 min immediately after collection. The serum was then separated into tubes and frozen at −80°C. The serum AMH levels were detected by ELISA. The serum NE levels were detected by HPLC. The ovaries and uterus were obtained from each mouse and were weighed immediately after euthanasia. The ovarian index and uterine index were calculated according to the following formulas: ovarian index = ovary weight/body weight × 100% and uterine index = uterus weight/body weight × 100%.

Observation of ovary morphology

The ovary and uterus samples were fixed in 10% formalin immediately after the mice were euthanized. In addition, the samples were embedded in paraffin after at least 24 h of fixation. Serial 5-μm sections were obtained from each ovary, with every fifth slide (starting with the first slide) being stained for hematoxylin and eosin (H and E) for assessment of the ovarian function. The qualitative standards of the follicles were as follows:[13],[14] (1) Primordial follicles are located in the superficial part of the cortex with small volumes and large quantities. The central primary oocyte is surrounded by a single layer of flat granule cells in this type of follicle. (2) In primary follicles, the oocyte grows larger, the transparent belt begins to form, and the follicle is surrounded by a single layer of cuboidal granulose cells. (3) In secondary follicles, the oocyte keeps growing, the follicular antrum and cumulus oophorus take shape, the follicular antrum is full of follicular fluid, the shape of the transparent belt becomes obvious, and the follicle is surrounded by multiple layers of cuboidal granule cells without antral space. (4) Antral follicles have large follicles and a follicular antrum, the granule layer turns thin, granule cells stop proliferating, and the follicles contain an antral space. (5) In atretic follicles, the oocyte structure is fuzzy, cytoplasm pyknosis appears, the transparent belt shrinks or fractures, the follicle wall collapses, the granule cells are disordered in terms of arrangement, and cell apoptosis is increased.


Immunohistochemical methods were adopted to detect the expression of GABAA and GABAB receptors in each of the different groups. (1) Fresh tissue samples were fixed in formalin following a standard procedure. (2) The tissue wax block and section preparation included the following: tissue dehydration, transparentizing, waxing, embedding, and slicing. (3) The immunohistochemical staining involved the following: tissue sections were deparaffinized, rehydrated, incubated with 0.3% hydrogen peroxide for 30 min and then blocked with 5% defatted milk. Sections were first incubated using the primary antibody for GABAB2 receptor (Rabbit, Abcam, US) at 4°C overnight with an optimal dilution, were then labeled with an HRP secondary antibody (Rabbit, ThermoFisher Scientific, USA) at room temperature (20°C–25°C) for 1 h, and then incubated with DAB substrate (Gene Tech, Shanghai, China) and counterstained with hematoxylin. All slides were observed and photographed with a microscope (Carl Zeiss, Germany). Scoring was conducted depending on the ratio of positively stained cells as follows: 0 points for 0–5%; 1 point for 6%–30%; 2 points for 31%–70%; and 3 points for >70%. Scoring was also conducted for staining intensity as follows: 0 points for no staining; 1 point for weak staining; 2 points for moderate staining; and 3 points for strong staining. The above two scores were then multiplied for statistical analysis. These scores were independently graded by two senior pathologists in a blinded manner.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from the mouse ovaries using TRIzol reagent (Takara, Japan), and reversely transcribed using the PrimeScript real-time polymerase chain reaction (RT-PCR) kit (Takara, Japan) according to the manufacturer's instructions. In addition, quantitative RT-PCR was then performed with SYBR Premix Ex Taq (Takara, Japan) on a 7500 RT-PCR system (Applied Biosystems, Inc. USA). The primer sequences used in this study were as follows: GADPH, forward 5′-ATGGAAGAAGAAATCGCCGC-3′, reverse 5′-ACACGCAGCTCGTTGTAGAA-3′; and GABAB2R, forward 5′-AAGACCCCATAGAGGACATCAA-3′, reverse 5′-GGGTGGTACGTGTCTGTGG-3′. The 2−ΔΔCt method was used to quantify the relative GABAB2R expression levels, which were normalized using GADPH expression.

Statistical methods

The experimental data are presented as the mean ± standard deviation; one-way ANOVA was adopted for comparing more than two groups of means, and shortest significant ranges were used for comparisons between two groups. The SPSS 20.0 software (IBM Statistics Inc.) was used for statistical analysis. Graphical representations were performed with GraphPad Prism 6.0 software (San Diego, CA, USA), and the Student's t-tests were used for comparisons between groups. Statistical differences were considered statistically significant at values of P < 0.05.

  Results Top

Baicalein ameliorates chronic stress-mediated behavioral, weight, neuroendocrine, and genital disorders in mice

To observe the effects of chronic stress on mice, we first established a chronic stress model, which is shown in [Figure 1]a. After being restrained daily for 1 week, the daily stress group of mice displayed higher levels of anxious behavior than the control group, which manifested as excited, agitated, and easily provoked behaviors after being released into the cages. After 3 weeks of stress, the daily stress group reacted slowly when released from the restraint box each day and became sluggish. Their fur became withered and yellow, and their weight significantly decreased [Figure 1]b. The other groups of mice acted normally, had glossy fur and gradually gained weight. From the 3rd week on, compared with the control group, the body weight of the daily stress group showed significant decline (P < 0.05) [Figure 1]b. Compared with the daily stress group, the weight of the daily stress + baicalein group showed a significant increase (P < 0.05). Notably, chronic stress resulted in reduced weights of the mice, while baicalein countered this effect to a certain extent.

The restrictive chronic stress model imposed on these mice was previously shown to involve dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis.[15],[16] This stress model leads to an increase in serum NE levels. Therefore, NE could be used as a marker to determine whether the mice were under stress or not. Four weeks after restriction and treatment, the mice were euthanized by cervical dislocation after blood sampling from the eye socket vein. The abdominal cavities were immediately opened, and the ovaries and uterus were excised and processed for quantification, H and E staining and immunohistochemical analyses. Compared with the control group, the daily stress group showed a significant increase in NE levels (P < 0.001), suggesting that the chronic stress model had been induced successfully. In addition, after giving the mice baicalein by gavage, it was found that the concentration of NE was markedly reduced (P < 0.001) [Figure 1]c in the treated mice, showing that baicalein could weaken the impact of chronic stress on mice.

In addition, compared with values in the control group, the ovarian and uterine indexes of the daily stress group were significantly reduced (P < 0.001) [Figure 1]d and [Figure 1]e. In contrast, baicalein intervention ameliorated this effect, resulting in the ovarian and uterine indexes of the daily stress + baicalein group increasing notably (P < 0.001) [Figure 1]d and [Figure 1]e. Moreover, the difference between the two baicalein treatment groups and the control group showed no statistical significance (P > 0.05) [Figure 1]d and [Figure 1]e, indicating that the organ indexes of the two baicalein treatment groups were normal. Together, these observations indicated that chronic stress could significantly lower the ovarian and uterine indexes of mice, while treatment with baicalein could ameliorate the effects caused by chronic stress.

Baicalein ameliorates the inhibitory effect of chronic stress on follicular development

Different developmental stages of the follicles, normal morphology, a large number of corpus lutea, a larger volume of follicles and orderly granule cells could be clearly visualized on the histological slices of the ovaries from the control group mice. In contrast, in the daily stress group, the follicles were sparsely distributed in different developmental stages, with a reduced number of lutea, an increasing number of atretic follicles, and disordered granule cells. However, baicalein treatment ameliorated these defects, as characterized by less atretic follicles, more normal follicles, and more orderly granule cells than in the daily stress group.

Follicular atresia is an apoptotic process in follicles that is under the control of genes. It has been proved that when follicular development is restrained, ovarian function is damaged, resulting in a significantly increased percentage of apoptotic granulose cells.[17] Within the control group, we observed a large number of normal antral follicles with orderly granulosa cells surrounding the oocytes and the total numbers of follicles in the ovary of mouse in each mouse group had no statistical difference [Figure S1] [Additional file 1]. However, the daily stress group showed a notable decrease in the number of normal secondary follicles and antral follicles (P < 0.001) and a significant increase in the number of atretic follicles [Figure 2]a, [Figure 2]b and [Figure S2] [Additional file 2]. Baicalein treatment substantially decreased the percentage of these atretic follicles, indicating that baicalein could inhibit this growth disorder in the follicles of mice and help maintain the function of the ovary (P < 0.001) [Figure 2]a and [Figure 2]b.
Figure 2: Ovarian and uterine histological and pathological examinations. (a) Ovarian histological and pathological examinations (×40). (b) Percentage of atretic follicles in each mouse group (×40). (c) Uterine histological and pathological examination (×40, ×100, and ×200). *P<0.001, compared with the daily stress group.

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Chronic stress can damage the function of the uterus in mice. The control group mice showed a thick endometrium, a large number of uterine glands in the lamina propria, and a large and regular glandular cavity [Figure 2]c. However, the mice that underwent the chronic stress procedure displayed a thin lining of the uterus, a decreasing number of uterine glands, and a shrunken and irregular glandular cavity within the uterine glands [Figure 2]c. Treatment with baicalein appeared to ameliorate the effects of chronic stress on the endometrium of the uterus.

Baicalein ameliorates ovarian reserve function

AMH is one of the main indicators in the assessment of ovarian reserve function, in addition to being the collection and an inhibitory factor of primordial follicles, the absence of which could accelerate ovarian dysfunction. Studies have shown that the serum AMH levels of mice are significantly related to the number of primordial and growing follicles.[18] Therefore, we measured the serum AMH level of each group of mice to help evaluate ovarian function. It was shown that the serum AMH level in the daily stress group was significantly reduced (P < 0.01) [Figure 3], indicating a decline in the ovarian reserve function. In contrast, baicalein treatment was shown to restore the serum AMH level to a certain extent (P < 0.05).
Figure 3: Serum anti-Müllerian hormone levels of each mouse group, as measured using ELISA. *P<0.05, P<0.01, compared with the daily stress group.

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Baicalein upregulates the expression of the gamma-aminobutyric acid B2 receptor in ovarian tissue

Ovarian function is mainly regulated by the HPO axis, and fluctuations in ovarian hormones can significantly affect the moods of females. However, the mechanisms involved in the abovementioned effects are not well known. Studies have shown that GABA can regulate the HPO axis, and its dysregulation can increase the risk of depression in perimenopausal women.[19],[20] Studies have also proved that GABA plays an important role in the formation of the ovum and in the maturation of oocyte.[21],[22] Therefore, we examined the expression of GABAA1, GABAB1, and GABAB2 receptors in the ovarian tissue of each group (three mice per group were chosen for this analysis) using immunohistochemical methods [Figure 4]a, [Figure 4]b, and [Figure S3a][Additional file 3], [Figure S3]b, [Figure S3]c, [Figure S3]d. While chronic stress was associated with reduced GABAB2 receptor expression (P < 0.01), the addition of baicalein restored the expression of the GABAB2 receptor (P < 0.01). No obvious difference was found in the expression levels of the other two receptors [Figure 4]a and [Figure 4]b. In addition, similar results were also observed in the mRNA levels, as revealed by quantitative RT-PCR (P < 0.05) [Figure 4]c, [Figure S4]a and [Figure S4]b[Additional file 4]. These data suggested that chronic stress resulted in significantly downregulated expression of the GABAB2 receptor in ovarian tissue, whereas baicalein could prevent this downregulation [Figure 4]c, [Figure 4]d, [Figure 4]e.
Figure 4: GABAB2 receptors play a role in stress-mediated ovarian dysfunction. (a) Representative photographs of GABAB2 receptor staining in the ovarian tissue of each mouse group (×40, ×100 and ×200, scale: 200 microns). (b) Expression of GABAB2 receptor in the ovarian tissue of each mouse group, with statistical analysis (P < 0.01). (c) mRNA expression of GABAB2 receptor in the ovarian tissue of each mouse group, with statistical analysis (P < 0.05). GABA: Gamma-aminobutyric acid. *P<0.05, P <0.01, compared with the daily stress group.

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  Discussion Top

As the main endocrine organ of females, the ovaries serve two important functions: ovulation and endocrine-related functions. Women of childbearing age in modern society are subject to anxiety, depression, and other psychological illnesses. Long-term physical and mental stress can gradually disrupt the female reproductive endocrine system, leading to diminished ovarian function and an increased incidence of POF.[23]

We adopted a restraint-stress procedure that was previously published,[12] which was different from traumatic stressors, such as electric shock or hot/cold stimulations, as this model can simulate stressful incidents that might be experienced in daily life. By placing female mice in an environment of chronic stress, we hoped to explore the effects of social and psychological factors on ovarian function. The results showed that the NE levels of the mice in the daily stress group were significantly increased (P < 0.001) compared to those of the mice in the control group, indicating that the model successfully induced a state of chronic stress.

AMH can regulate the initiative circulation (starting with the static state to being collected and entering the growing pool) and recruit circulation (growing follicles continue to develop until the preovulation stage) of ovarian follicles, the inhibition of the formation of dominant follicles, and the rapid consumption of primordial follicles.[24] Inadequate secretion of AMH can accelerate the depletion of primordial follicles and prevent the formation of dominant follicles, thus attenuating the effects of FSH on follicular development in mouse ovaries.[25] Studies have shown that AMH is produced by growing follicles, and its serum level is positively related to the number of sinus follicles.[26] In addition, the quality and quantity of follicles can reflect ovarian function. When the quality and quantity of the follicles drop below a certain threshold, ovulation is absent, and amenorrhea can occur.[27] Our observations showed that the serum AMH levels of the daily stress group were significantly reduced compared to those of the control group (P < 0.01), and the proportion of atretic follicles was significantly increased (P < 0.001). These are typical features of diminished ovarian function. The above results showed that chronic stress can inhibit ovarian function in mice.

As the mechanisms of the damaging effect of ovarian function remain unclear, there is no effective treatment or prevention method for ovarian damage and POF. A previous study found that the proportion of women who present with anxiety and/or depression is higher in women with ovarian dysfunction than in those without.[28] This study aimed to investigate chronic stress, one of the causes of ovarian damage, and to assess the effects of the anxiolytic compound baicalein. Traditional medications used for treating chronic stress are mostly composed of agents that exert strong central nervous inhibition and muscle relaxation effects, and long-term treatment with these drugs can lead to serious drug dependence and neural inhibition. The baicalein adopted in this study is extracted from plants and features a highly safe profile, low toxicity and cost, an ample supply, and a specific chemical formula and molecular weight. Studies have shown that baicalein has notable antianxiety and antidepression effects while being both safe and effective.[29],[30] However, whether its antianxiety effect influences ovarian function is still unknown. In this study, after administering baicalein by gavage to the daily stress group of mice, it was found that treatment with baicalein significantly improved the effects of chronic stress in mice. In addition, the ovarian function of the daily stress group remained normal, indicating that baicalein was able to ameliorate the ovarian dysfunction induced by chronic stress in the ovaries of mice by improving their stress state.

GABA and its receptors are important regulators of the development of the female reproductive system. GABA can affect the pituitary gland and gonads through the HPO axis; in addition, it is also involved in the secretion and regulation of female hormones. Estrogen can adversely affect the regulation of GABA in regard to the HPO axis, and dysregulation of GABA can increase the risk of depression in perimenopausal women.[20] Previous studies have shown that GABAA5 receptor can influence follicular development, and in patients with polycystic ovary syndrome, downregulation of this receptor has been detected.[31],[32] This study assessed the expression of a variety of GABA receptors in ovarian tissues and found that the daily stress group showed a significant reduction in GABAB2 receptor expression compared to that of the control group. In addition, previous studies have shown that the antichronic stress mechanism of baicalein most likely functions through GABA receptors.[9] The study found that the daily stress + baicalein group showed a notable increase in GABAB2 receptor expression compared to the daily stress group. No other differences were found in the expression of the other receptor subtypes.

A restraint-stress procedure was used in this study that could result in POF in mice, and we concluded that chronic stress is detrimental to ovarian function. Treating mice with baicalein significantly improved the ovarian dysfunction caused by chronic stress, thus enhancing the ovarian function in these mice. Furthermore, based on the fact that GABA and its receptors can regulate the female gonadal axis to affect ovarian function, it was not surprising that baicalein could significantly enhance the GABAB2 receptor expression downregulated by chronic stress. However, further research is needed to fully investigate how baicalein can upregulate the expression of the GABAB2 receptor to improve ovarian function in mice.

Chronic stress can inhibit and/or damage many aspects of the female reproductive and endocrine systems, resulting in very serious health complications. The most commonly prescribed medications for anxiety and depression have strong effects on the central nervous system and muscle contraction, and long-term treatment with these medications can lead to serious drug dependence and neural inhibition. Therefore, it is urgent to identify safe and effective drugs that can ameliorate the ovarian dysfunction caused by chronic stress. Baicalein, a defined chemical compound extracted from plants, has a low cost and ample supply and could provide a new and important clinical option for physicians treating ovarian dysfunction caused by chronic stress.

Financial support and sponsorship

This work was supported by the Shanghai Science & Technology Commission (18140902502), the National Key Research and Development Program of China (2016YFC1303100), the National Natural Science Foundation of China (81472423), the Shanghai Clinical and Medical Center of Key Program (2017ZZ01016), and the Shanghai Science and Technology Innovation Project of Traditional Chinese Medicine (ZYKC201701020).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]


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