Ganoderma lucidum and Hypericum perforatum Exhibit Anti-Inflammatory, Antioxidant, and Anti-Androgen Effect in Rat Model of Experimental Polycystic Ovarian Syndrome

Polycystic ovarian syndrome (PCOS) is a widespread endocrinologic disorder with a rate of 5–20% in women of reproductive age; this disorder consists of hyperandrogenism, polycystic ovary, hyperandrogenemia and ovulatory dysfunction [1]. Although the connection of inflammation with PCOS is not yet clear, it is thought to be involved in its pathogenesis. Serum levels of CRP were shown to be increased significantly in women with PCOS [2]. Studies on the genetic basis of chronic low-grade inflammation in PCOS have shown that it is associated with proinflammatory genotypes [2].

Hypericum perforatum (HP) is a phytotherapy remedy which has been used for centuries in conventional medicine. It is a yellow-flowered, perennial herb broad in North and South America, Europe, and Asia. It has been recommended for use as a diuretic and for treatment of menstrual disorders. It is also used in the treatment of pathologies such as urogenital infections, diabetes mellitus, neuralgia, heart diseases, gastritis, hemorrhoids, peptic ulcers in European and Turkish traditional medicine [3]. Ganoderma lucidum polysaccharide (GLP) is a classical traditional Chinese herb used to modulate immune responses [4]. Interestingly, some studies have shown that GLP exerts significant effects in inhibiting the development of diabetes and obesity [5].

Both HP and GLP have been shown to have anti-inflammatory effects through various mechanisms. GLP alleviates the inflammation parameters such as nuclear factor-kappa B (NF-κB), cyclooxygenase 2 (COX2), NO synthase, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) [6] and protects macrophages from inflammatory stress by activating nuclear factor erythroid 2-related factor 2 (Nrf2) [7]. Ethanol component of GLP induced the cellular anti-oxidant defense capacity via stimulation of Nrf2 and hemoxygenase 1 (HO-1) [8]. GLP sterols also have anti-inflammatory efficiency and suppressed NF-κB [9]. HP includes phenolic constituents such as quercetin. Therefore, HP could stimulate the Nrf2 cytoprotective signaling pathway [10] and inhibit the NF-κB activation by TNFα [11].

The use of both herbals in menstrual irregularities and strengthening the immune system has led us to consider their use in patients with PCOS, especially in infertility. We investigated the effects of the use of two herbs on ovarian and biochemical parameters in the letrozole-induced rat PCOS model.

We investigated some biochemical parameters (serum LH, FSH, insulin, and testosterone) and oxidative stress markers that are of key importance in the pathogenesis of PCOS. Malondialdehyde (MDA) was studied as a marker of lipid peroxidation, while superoxide dismutase (SOD) and reduced glutathione (GSH) was evaluated as biomarkers of antioxidant activity. Insulin-like growth factor 1 (IGF-1) has an important role in the ovarian tissue and therefore in PCOS [12]. PCOS enhances the production of pro-inflammatory cytokines, for instance, IL-6, and TNF-α [13]. Levels of sex hormone binding globulin (SHBG) are low in the long-term effects of PCOS such as hyperandrogenism, metabolic abnormalities, subfertility, but the relationship between these markers and PCOS still remains to be discovered. The goal of our study was to define the biochemical and histopathological impact of treatment with GLP and HP on ovarian tissue in a rat model of experimental PCOS.

Forty-two Sprague-Dawley female rats, 10–12 weeks old, weighing between 340 and 360 g, were divided into 6 groups with 7 animals in each group. During adaptation and experimentation, rats were housed at 22 ± 2°C room temperature, with 12 h light and 12 h dark light cycles, with food and water add-libitum. Rats were grouped as follows: Group 1 (Control): Rats were given 3 mL of distilled water by oral gavage for 25 days. Group 2 (PCOS): Letrozole-induced PCOS group; rats received Letrozole (1 mg/kg, p.o.) with 0.9% normal saline dissolved in 1% CMC once daily for 21 consecutive days. Group 3 (PCOS + HP): Letrozole-induced PCOS group: Rats were given 50 mg/kg HP dissolved in 0.9% normal saline in a 10 mL tube. Group 4 (HP): HP oil was applied via oral drip for 1 month every day during the experiment period. Group 5 (PCOS+ GLP): Letrozole-induced PCOS group were given 200 mg/kg GLP intragastrically for 30 days. Group 6 (GLP): 200 mg/kg GLP was given intragastrically for 30 days during the experiment.

At the end of the experimental procedures, all the animals underwent bilateral oophorectomy and blood samples were collected. Ovarian tissue and blood samples were used for biochemical and histopathological analysis.

Ovarian tissue samples were embedded in paraffin after alcohol, xylene and paraffin follow-up procedures were completed, and were cut into sections with a thickness of 5 micrometers using a microtome device (ThermoFisher Scientific, Cheshire, UK). Sections were stained immunohistochemically using Hemotoxylin Eosin and Masson-Trichrome and Toluidine blue and Toll-like receptor 4 (TLR-4) antibody. Stained slides were evaluated via a digital camera-attached microscope.

Sections of 5 µm thickness were prepared from the blocked ovarian tissues, deparaffinized, and primary antibody TLR-4 diluted 1/50 was applied. The gradation of staining was defined as 0: no, +0.5: very little, +1: little, +2: moderate, +3: severe [14].

Serial sections of 5 μm thickness were sampled at 100 μm intervals and from 5 different levels for follicle counting. Follicles were counted using the image J software program. Follicles were classified as primary follicle, secondary follicle, graafian follicle and atretic follicle according to the granulosa cell layer and fluid content [15].

The following parameters were used to detect ovarian tissue damage; degeneration, edema, vascular congestion, fibrosis, hemorrhage, and inflammation. Grade 0: no damage; Grade 1: <33% damage; Grade 2: 33–66% damage; and Grade 3: >66% signs of damage.

Serum levels of SHBG, IL-6, IGF-1, SOD, TNF-a, GSH, and MDA levels were quantified via appropriate ELISA kits. ELISA tests were studied on the DS2 automated ELISA instrument (Dynex Technologies Inc, Chantilly, VA, USA) in accordance with the directions of the kit manufacturer (MyBioSource, San Diego, CA, USA). Standard curves were created using the measured absorbance values and based on this curve, TNF-a (Rat TNF-alpha ELISA Kit, Cat No: MBS2507393, San Diego, CA, USA), IL-6 (Rat IL-6 ELISA Kit, Cat No: MBS355410, San Diego, CA, USA), SHBG (Rat Sex Hormone-Binding Globulin (SHBG) ELISA Kit, Cat No: MBS3808419, San Diego, CA, USA), IGF-1 (Rat IGF-1 ELISA Kit, Cat No: MBS176012, San Diego, CA, USA), SOD (Superoxide Dismutase (SOD) Rat ELISA Kit, Cat No: MBS036924, San Diego, CA, USA), MDA (Malondialdehyde (MDA) RAT ELISA Kit, Cat No: MBS268427, San Diego, CA, USA), GSH (Rat Glutathione concentrations of reagents (GSH) ELISA Kit, Cat No: MBS265966, San Diego, CA, USA) were calculated.

Lipid peroxidation was evaluated via the measurement of MDA in pursuant of the method described by researchers [16]. Kit for MDA assay uses the Double Antibody Sandwich ELISA technique. The pre-coated antibody is an anti-Rat MDA monoclonal antibody, while the detection antibody is a biotinylated polyclonal antibody. The color intensity and quantity of target analyte in the sample are positively correlated [17]. The MDA concentration in each sample was interpolated from this calibration curve plotted relating the intensity of the color.

All data were analyzed with SPSS 25.0 for the Windows package program. Whether the data showed normal distribution or not was determined by the Kolmogorov-Smirnov test. One-way ANOVA and Kruskal-Wallis tests were used to determine the discrepancy among the groups. The results are presented as mean + SD p < 0.05 was considered statistically significant.

The histology was similar in the control, GLP, and HP groups. Follicles at different developmental stages had mostly normal morphological appearance. A small number of degenerated and atretic follicles were marked. Vascular texture and mast cell density were normal in each group. Hemorrhagic findings, inflammation, edema and fibrosis were not marked. Numerous cystic follicles and high follicle degeneration findings were found in the PCOS group. Hemorrhagic findings, inflammation, edema and fibrosis were not marked.

When the PCOS+GLP and PCOS+HP groups were examined, it was determined that the cystic follicles were significantly reduced compared to the PCOS group. Hemorrhagic findings, inflammation, edema, and fibrosis were not marked.

We determined lower expression of TLR-4 protein in the GLP, HP, and PCOS rats when compared with the control group. High spread was marked in the PCOS + GLP and PCOS + HP groups.

The number of follicles sized 2–9 mm in the PCOS rats was significantly improved in comparison with the other groups (p < 0.05). We did not find any significant variation among the groups in the counts of corpora lutea, atretic follicle, primary follicle, secondary follicle, and graafian follicle (p > 0.05). Statistical outcomes of follicle numbers are shown in Table 1.

In the PCOS rats, follicle degeneration was significantly higher when compared with the remained groups (p < 0.05). No significant differences were observed among the groups with regard to hemorrhage, vascular congestion, inflammation findings, edema, and fibrosis (p > 0.05). Statistical outcomes of the groups are shown in Table 2.

Serum levels of LH, FSH, and insulin were similar in each group. Serum testosterone level of the PCOS group was significantly higher than the other groups (p < 0.05). Supplement of GLP and HP to the PCOS rats ended in a half reduction in testosterone levels. While IL-6 and TNF-α levels were elevated in PCOS rats (p < 0.01), SHBG levels were decreased (p < 0.01). Adding GLP and HP increased SHBG levels. Adding GLP or HP to the treatment decreased TNF-α but did not affect IL-6 levels. SOD levels were alike among groups (p > 0.05). GLP and HP caused a non-significant rise in SOD levels (p > 0.05). MDA and IGF-1 measurements were higher in PCOS rats than in other groups (p < 0.02, p < 0.02, respectively). Supplementation of GLP decreased IGF-1 and MDA levels (p < 0.05) (Table 3). HP did not show any effect on IGF-1 but MDA levels decreased significantly (p < 0.05). Serum GSH amounts were meaningfully reduced in the PCOS rats (p < 0.05). Supplementation of GLP or HP enhanced GSH levels (p < 0.05).

Polycystic ovary syndrome is a widespread endocrinological disease causing subfertility and there is no effective treatment for infertility other than IVF/ICSI. For this reason, the search for new treatments continues. However, since there is no direct use of candidate drugs and supplements in humans, experimental PCOS models have come to the fore. The most preferred of these models is configured with letrozole. PCOS may develop after exogenic application of androgen or secondary to endogenic androgen surplus [1]. In the case of increased intraovarian androgen levels, abnormal follicular maturation or acceleration in follicular atresia can be observed. Hence, intraovarian androgen surplus originating from circulating hyperandrogenemia or improper steroidogenesis can cause improper follicle growing and polycystic ovary [18].

In our study, an experimental PCOS model was created with letrozole. Letrazole 1 mg per kg was applied peroral only 1 time in a day for 21 days. Histopathological examination of the ovaries was performed for confirmation of PCOS. PCOS was diagnosed when atretic follicles and cystic follicles were detected in the absence of granulosa cell stratification [19]. Considering all the results of our study, the effectiveness of GLP and HP on chronic inflammation, gene pathologies, apoptotic mechanisms and oxidation damage, which take part in the etiology of PCOS, was investigated in an experimental PCOS model. Devi et al. [20] reported that GLP blocks androgen receptors in PCOS patients and reduces hyperandrogenic status, thus demonstrating the antiandrogenic potential of GLP.

Serum testosterone levels were markedly higher in letrozole-induced PCOS rats while SHBG levels were lower in the experimental PCOS group in comparison with the other groups. These observations show that the experimental PCOS model was successfully created. Adding GLP and HP led to a significant reduction in testosterone levels. This finding suggests that both HP and GLP reduce androgen synthesis. When HP or GLP was added to the treatment, SHBG levels approached the thresholds of the control group. In the light of these findings, we can suggest that GLP and HP treatment improves the hormonal and metabolic profile by decreasing serum testosterone and increasing SHBG levels in the PCOS model.

MDA, an indicator of oxidative stress, was markedly higher in the PCOS rats. Adding GLP or HP to the treatment reduced serum MDA levels to those of the control group. This finding is the evidence of the antioxidant effect of GLP and HP. On the other hand, the antioxidant markers GSH and SOD were found to be low in the PCOS group. Treatment with HP or GLP directly increased the synthesis of both SOD and GSH. To our way of thinking, these data are important findings of the presence of anti-inflammatory effects of GLP and HP.

HP contains several biologically active components such as hypericin, pseudohypericin, hyperforin, and adhyperforin. While the most common components hypericin and hyperphorin are known to have many biological activities, their role in inflammation is not yet understood clearly [21]. Its components rich in flavonoid (sequential hexane, ethanol, then chloroform extracts) have the most potent anti-inflammatory effects [22].

Generally, the monosaccharide composition of GLP is comparatively invariable. Various monosaccharide ingredients can show distinct bioactivities, and they are related to the triple helix structure of GLP. GLP shows antioxidant activity by decreasing impairing lipid peroxidation and MDA levels. GLP could reduce the serum thresholds of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) which are secreted in consequence of the progress of inflammation-stimulated adipocytes and macrophages [11]. We observed a similar effect; levels of TNF-α and IL-6 were significantly increased in the PCOS rats. Adding GLP or HP to the treatment resulted in decreased serum levels of both biomarkers.

Ren et al. [23] studied the neuroprotective impact of GLP contrary to the induced type I diabetes mellitus induced with streptozotocin (STZ) in mice to discover the underlying anti-inflammatory linkage. Histopathological investigation demonstrated improvement of β-cells in GLP-treated mice. GAA alleviated T1DM induced with STZ in mice by inflammatory pathways similar to our study.

Inflammatory factors increase in PCOS, causing chronic inflammation in the ovarian tissue to disrupt follicle development. Macrophages, which play a major role in the systemic inflammatory response, are also the most abundant immune system elements in the ovarian tissue and play a role in maintaining homeostasis. Data on the role of macrophages in the pathogenesis of PCOS are limited, but in the case of hyperandrogenism, macrophages in the blood and ovaries are polarized to the M1 type and proinflammatory factors such as IL-6 and TNF-α are produced when chemokine-like receptor 1 (CMKLR1)-positive M1 type macrophages enter the ovary. All these pathways cause an inflammatory response in the ovarian tissue or systemically. Macrophages induce apoptosis of granulosa cells in the presence of excess androgens [24]. In our study, the histopathological proof of ovarian injury is highly likely due to the effects of proinflammatory macrophages on the ovarian tissue.

There are various limitations about our research. First, this is an animal study, and human studies would be far more valuable. Second, oxidative pathways and potential mechanisms involved in the pathogenesis have not been studied in detail. Moreover, we did not extract biologically active ingredients and analyze their effects separately. Using the most appropriate techniques to obtain the most effective extracts for the target could help obtain better effects.

Measurement of the effects of reactive oxygen species (ROS) or oxidative damage products as well as antioxidants raises practical difficulties in studies due to the short lifespan of most ROS, variable and low steady-state levels, and constantly changing production rates affected by chemicals. Murphy et al. recently reviewed the guidelines for measuring ROS and oxidative damage. To comprehend the impact of antioxidants on oxidative damage in biomolecules, it is recommended to first conduct preliminary dose determination studies using well-defined biomarkers [25, 26]. Liquid chromatography-mass spectrometry (LC–MS) based methods enhanced to analyze the lipid peroxidation products, allowing obtaining multiple end products more sensitively with fewer samples [25].

The findings of this study and literature suggest that HP and GLP have properties to correct the metabolic, hormonal and inflammatory profiles in PCOS. Comprehensive randomized controlled clinical studies on these herbs can lead to applications in humans.

This study was planned pursuant to the guidelines for animal research of the National Institutes of Health and was performed in line with the principles of the Declaration of Helsinki. This study was approved by the Animal Research and Ethics Committee (Date: May 27, 2021, Approve No: 2021/010).

Gülin Okay, Pınar Kırıcı, Selçuk Kaplan, Nihal Mavral, Ebru Annac, and Zeynep Ece Utkan Korun declare that they have no conflicts of interest relevant to this article.

This study was not supported by any sponsor or funder.

Material preparation: Gülin Okay, Pınar Kırıcı, and Nihal Mavral; data collection: Ebru Annac, Pınar Kırıcı, and Selçuk Kaplan; statistical analysis: Nihal Mavral and Zeynep Ece Utkan Korun; Preparation of first draft and editing and finalization: Gülin Okay and Pınar Kırıcı. All authors read and approved the final version of the manuscript.

The data that support the findings of this study are available on request from the corresponding author.