Skip to main content

System-level investigation of anti-obesity effects and the potential pathways of Cordyceps militaris in ovariectomized rats

Abstract

Background

Cordyceps species have been used as tonics to enhance energy, stamina, and libido in traditional Asian medicine for more than 1600 years, indicating their potential for improving reproductive hormone disorders and energy metabolic diseases. Among Cordyceps, Cordyceps militaris has been reported to prevent metabolic syndromes including obesity and benefit the reproductive hormone system, suggesting that Cordyceps militaris can also regulate obesity induced by the menopause. We investigated the effectiveness of Cordyceps militaris extraction (CME) on menopausal obesity and its mechanisms.

Methods

We applied an approach combining in vivo, in vitro, and in silico methods. Ovariectomized rats were administrated CME, and their body weight, area of adipocytes, liver and uterus weight, and lipid levels were measured. Next, after the exposure of MCF-7 human breast cancer cells to CME, cell proliferation and the phosphorylation of estrogen receptor and mitogen-activated protein kinases (MAPK) were measured. Finally, network pharmacological methods were applied to predict the anti-obesity mechanisms of CME.

Results

CME prevented overweight, fat accumulation, liver hypertrophy, and lowered triglyceride levels, some of which were improved in a dose-dependent manner. In MCF-7 cell lines, CME showed not only estrogen receptor agonistic activity through an increase in cell proliferation and the phosphorylation of estrogen receptors, but also phosphorylation of extracellular-signal-regulated kinase and p38. In the network pharmacological analysis, bioactive compounds of CME such as cordycepin, adenine, and guanosine were predicted to interact with non-overlapping genes. The targeted genes were related to the insulin signaling pathway, insulin resistance, the MARK signaling pathway, the PI3K–Akt signaling pathway, and the estrogen signaling pathway.

Conclusions

These results suggest that CME has anti-obesity effects in menopause and estrogenic agonistic activity. Compounds in CME have the potential to regulate obesity-related and menopause-related pathways. This study will contribute to developing the understanding of anti-obesity effects and mechanisms of Cordyceps militaris.

Peer Review reports

Background

Menopause leads to various physical and mental symptoms, such as daily hot flashes, a lack of energy, back pain, breast pain, anxiety, and poor memory [1]. Menopause is also considered to be related to obesity [1], because estrogen plays an important role in the energy balance and metabolism of adipose tissue and other organs [2]. Indeed, the prevalence rate of overweight in women over the age of 45 is higher than that in men, whereas the trend is reversed among young adults, suggesting that obesity coincides with menopause in women [3]. There is a report showing the interaction between reduced estrogen secretion and obesity in menopausal women, implying that estrogen deficiency in menopausal women contributes to obesity [4]. Obesity is believed to increase the risk of type 2 diabetes mellitus, cardiovascular diseases, and osteoarthritis [5]. Thus, the World Health Organization has classified obesity as a preventable cause of death [6]. However, the prevalence of obesity has increased to the point that nearly one-third of the world’s population was classified as overweight or obese in 2019 [3], and the prevalence of adult obesity and severe obesity will continue to increase [7, 8].

Hormone replacement therapy (HRT), also known as menopausal hormone therapy or postmenopausal hormone therapy, is used to treat various symptoms involved in menopause [9], and it has been reported that HRT can alleviate obesity and its related symptoms [10,11,12]. Many women, however, avoid HRT due to its adverse effects and choose non-hormonal therapies [13, 14]. As an alternative, natural products have been studied as effective and safe drugs for relieving postmenopausal diseases and symptoms [15,16,17,18]. Systematic reviews have shown that some natural products significantly inhibit weight gain in animals and humans without severe adverse events or mortality [19, 20]. In addition, it has been reported that herbal extracts proliferate estrogen receptor (ER)-positive MCF-7 cells, and increase the mRNA expression of estrogen-related genes [21,22,23], suggesting that herbal compounds can improve estrogen-deficiency-related menopause symptoms through their estrogenic effects.

The fungal genus Cordyceps is parasitic on Lepidopteron insects, and the combination of the fungi and dead insect has been used as a drug in traditional Asian medicine. Cordyceps species are traditionally called ‘winter worm summer grass’ in east Asian countries because they are parasitic on living insects in winter and grow out of dead underground pupae in summer. Traditionally, Cordyceps has been regarded as a tonic that improves energy, stamina, and libido, exhibiting a potential relationship to sex hormones and energy metabolism [24]. Among Cordyceps species, Cordyceps militaris was reported to dramatically decrease liver weight and fat deposition and improve lipid levels, suggesting that Cordyceps militaris can have a favorable role in regulating obesity [25]. Moreover, it was shown that Cordyceps militaris improves reproductive hormone concentration [26] and alleviates osteoporosis, which results from reproductive hormone deficiency [27, 28]. Therefore, Cordyceps militaris is expected to be able to regulate obesity induced by menopause. However, the therapeutic effect of Cordyceps militaris in postmenopausal obesity has not been studied thus far.

Network pharmacology is an effective method to investigate the system-level mechanism of drugs that combine multiple compounds [29]. It has been successfully used in contributing to discovering the potential mechanisms of natural products containing multiple compounds [18, 30,31,32]. It is expected that Cordyceps militaris regulates various pathways which affect the development of obesity and complications via its bioactive compounds; therefore, employing network pharmacological analysis can be effective for investigating the potential anti-obesity mechanism of Cordyceps militaris.

Here, we investigated whether Cordyceps militaris could alleviate menopause-induced obesity by combining in vivo, in vitro, and in silico methods. To explore whether Cordyceps militaris could relieve menopause-induced obesity, we observed the anti-obesity effects of CME in ovariectomized rats. To estimate whether Cordyceps militaris mitigates down-regulated estrogen receptor alpha (ERα) induced by reproductive hormone deficiency in menopause, we measured the cell proliferation and phosphorylation of ERα on MCF-7 cell lines. Finally, we investigated the underlying mechanism of Cordyceps militaris by analyzing the potential active compounds and pathways related to obesity and menopause.

Methods

Extraction of Cordyceps militaris

As described in our previous study [33], CME was provided by Dong-A Pharmaceutical (Yongin, Korea). Briefly, CME was extracted in a 50% ethanol (v/v) solution in water from Cordyceps militaris cultured in brown rice and concentrated under low pressure. The extract was freeze-dried for the in vitro and in vivo experiments.

In vivo experiment

Animal model

Female Sprague Dawley rats (\(n=64\), 7 weeks old), which were either ovariectomized or sham-operated, were provided by DooYeol Biotech (Seoul, Korea). The rats were housed in a restricted access rodent facility with up to three rats per polycarbonate cage. The rats were given seven days to acclimate before being ovariectomized (or sham-operated), and maintained on a 12-h light/dark cycle, 20–23 °C laboratory temperature, and 45–55% relative humidity. All methods were performed in accordance with the related guidelines [34] and approved by the Panel on Laboratory Animal Care of Gachon University (GIACUC-R2017037). The ARRIVE guidelines [35] were adhered to throughout this study.

Experimental groups and drug administration

The rats were randomly divided into the following groups: sham surgery with intact ovaries group (Sham, \(n=10\)); ovariectomized model group (OVX,\(n=11\)); 17β-estradiol-treated positive group (E2, 25 μg/kg/d,\(n=10\)); CME low-dose group (37.5 mg/kg/d,\(n=11\)); CME middle-dose group (75 mg/kg/d,\(n=11\)); and CME high-dose group (150 mg/kg/d, \(n=11\)). After one week of recovery, the four medication groups were treated with corresponding medicine, whereas the Sham and OVX groups were given an equal amount of the vehicle. The dosage was 1 ml/100 g body weight. CME was administered orally, whereas 17β-estradiol was injected intraperitoneally. Mice in each group were treated for 8 weeks. Rats were euthanized with CO2 gas after fasting for 12 h after eight weeks of treatment. Tissue samples were collected and kept at − 80 °C in Eppendorf tubes before assays.

Measurement of body weight and organ weight

The body weights of rats were measured daily during the experiment using an electronic balance (AdventurerTM, Ohaus, New Jersey, USA), in accordance with KFDA recommendations. The weights of the liver and uterus were measured using the same procedure applied to measure the body weight.

Serum biochemical parameters

After eight weeks of administration, blood was obtained from the abdominal veins of rats after fasting for 12 h. Blood samples were collected into CBC bottles containing EDTA-3 K (Sewon Medical, Seoul, Korea). Serum was obtained by centrifugation at 3000 × g for 15 min. High-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, total cholesterol, and triglyceride levels were measured with Accute TBA-40FR apparatus (Toshiba Medical Systems Co., Tochigi, Japan).

Histopathology

Histological photographs of adipose tissue were examined based on the paraffin method using a light microscope. Endometrial adipose tissue was embedded in paraffin blocks after being fixed in 10% neutral buffered formalin. Sections of 5 μm were cut and mounted on glass slides. Xylene and alcohol were used to remove the paraffin. Hematoxylin and eosin (H&E) staining were used to color the sections. After dehydration with alcohol, the photographs were obtained with a light microscope. The sizes of the endometrial adipocytes were determined with ImageJ software (Version 1.45 s, NIH, Bethesda, Maryland, USA).

Dual-energy X-ray (DXA)

Body fat was determined in each rat by dual‐energy X‐ray absorptiometry after eight weeks of administration using an INALYZER scanner (MEDIKORS Inc., Seongnam, Gyeonggi, Korea). Rats were anesthetized by the inhalation of isoflurane during scanning.

Statistical analysis

Statistical significance of variation among and between groups and dose-dependency was evaluated and determined by the Kruskal–Wallis test followed by the Dunn–Bonferroni post hoc test and Jonckheere–Terpstra test using IBM SPSS Statistics (Version 25, IBM, Armonk, New York, USA), respectively. The Bonferroni correction was used to adjust for multiple comparisons. The test result was regarded as statistically significant when p < 0.05; *, **, *** in figures represent p-values of the test are below 0.05, 0.01, and 0.001, respectively.

In vitro experiment

Cells and cell culture

The ER-positive MCF-7 human breast cancer cell line was supplied by the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 cells were incubated in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Corning, Manassas, VA, USA), which included 10% fetal bovine serum (FBS; Gibco BRL, Carlsbad, MD, USA) and 1% penicillin/streptomycin solution (1000 IU/mL penicillin and 10,000 μg/mL streptomycin; Life Technologies, Waltham, MA, USA). Cultures were maintained at 37 °C in a humidified environment containing 5% CO2. Dimethyl sulfoxide (DMSO) was utilized as a solvent to dissolve the material in cell studies. In comparison to naïve cells, the final concentration of DMSO was kept below 0.1%, and there was no effect of DMSO.

E-screen assay

Before treatment, MCF-7 cells (1 × 104 cells/well) were seeded into a 48-well plate and incubated for 24 h. Vehicle (DMSO) or indicated concentrations of Cordyceps militaris were dissolved in phenol red-free RPMI (Gibco, Carlsbad, CA, USA) supplemented with 10% charcoal dextran stripped serum (Innovative Research, Novi, USA). Then, the prepared samples were added to the wells and incubated for 144 h. For the antagonistic test, the ER antagonist ICI 182,780 (Tocris Bioscience, Bristol, UK) was added to the test samples. Subsequently, Ez-Cytox solution (Daeil Lab Service, Seoul, South Korea) was added to each well and the cells were cultured for 1 h. The cell viability was then calculated from the measurement of the optical density at 450 nm using a SPARK 10 M microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Cell viability was calculated based on a ratio of 100% of the vehicle control (DMSO).

Western blot analysis

MCF-7 cells (2 × 105 cells/well) were seeded into a 6-well plate and cultured for 24 h. Vehicle (DMSO) or indicated concentrations of Cordyceps militaris were added into the wells and cultured for 24 h. After incubation, the cells were washed with phosphate-buffered saline (PBS; Welgene, Gyeonsan, Korea) and lysed with radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Inc., MA, USA). Using a Pierce BCA Protein Assay Kit (Thermo Scientific, Carlsbad, CA, USA), the protein concentrations of lysates were measured. Equal amounts of protein were mixed with 4 × NuPAGE LDS Sample Buffer (Thermo Scientific, Carlsbad, CA, USA) and boiled for 10 min at 95 °C. The proteins were separated by precast 4–15% Mini-PROTEAN TGX (Tris–Glycine. eXtended) gel (Bio-Rad, Hercules, CA, USA) and then transferred onto polyvinylidene fluoride (PVDF) transfer membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk for 2 h. Prior to hybridization with the antibody, that membranes were cut to the corresponding site for each antibody. Each membrane was incubated with specific primary antibodies to ERα, phospho-estrogen receptor α (p-ERα), extracellular-signal-regulated kinase (ERK), phospho-ERK, p38, phospho-p38, c-Jun N-terminal kinases (JNK), and phospho-JNK, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Inc., Danvers, MA, USA) to enhance the protein detection, followed by incubation with horseradish-peroxidase-conjugated secondary goat anti-rabbit antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) for 6 h at 20 ± 5 ℃. The membranes were incubated with the horseradish secondary antibody conjugated by peroxidase (Cell Signaling Technology, Inc., Danvers, MA, USA) at 20 ± 5 ℃ for 1 h after washing. The bound antibodies were visualized with Pierce ECL Western Blotting Substrate (Rockford, IL, USA) and the FUSION Solo chemiluminescence system (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Network pharmacology

We obtained the targets of compounds in Cordyceps militaris and constructed a herb–compound–target network. The herbal compound information was retrieved from TCM-MESH (http://mesh.tcm.microbioinformatics.org) [36]. We employed a method called the quantitative estimate of drug-likeness (QED), which analyzes drug-likeness based on molecular structural properties [37] to filter out compounds that are unlikely to function as drugs when administered orally. The QED scale runs from 0 to 1; a larger QED indicates more drug-likeness. A value of 0.35 is the mean QED for oral drugs approved by the Food and Drug Administration; therefore, we used it as the cut-off value for the compounds. The potential targets of the compounds were queried from STITCH (http://stitch.embl.de/). We collated the compound–protein interactions whose combined scores were over 0.7, which is considered the high-confidence criterion for filtering prediction results. Based on the herbal compound information and compound–protein interactions, we built a herb–compound–target network connecting Cordyceps militaris to its compounds, and the compounds to their targeting proteins. Using Enrichr and Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/) databases [38, 39], gene set enrichment analysis (GSEA) was conducted to investigate the relationship between targeted proteins and KEGG pathways related to estrogenic effects and obesity. We calculated the adjusted p-values and combined scores for pathways. The logarithms of the p-values and z-scores were used to calculate combined scores (notably, the combined score is different from the combined score in the STITCH).

Results

To identify the anti-obesity effects of Cordyceps militaris in menopause and its mechanism, we used a comprehensive method combining in vivo, in vitro, and in silico approaches (Fig. 1).

Fig. 1
figure 1

Schematic diagram to summarize the methods and results of our study

CME showed anti-obesity effects in ovariectomized rats

Ovariectomized rats were treated with CME for eight weeks, and their body weights were measured daily. As a result, body weights in the high-dose CME group and positive controls were shown to be significantly lowered, and the high-dose CME group and positive controls showed similar body weight loss effects at week 4 (Fig. 2A). Meanwhile, no significant differences were found between the body weights of CME groups and controls at week 8 in the Dunn–Bonferroni post hoc tests followed by Kruskal–Wallis tests (Fig. 2B). However, CME exhibited weight loss effects in a dose-dependent manner at week 4 (p = 0.014) and week 8 (p = 0.024) in Jonckheere–Terpstra tests. This trend continued during the whole experimental period (Fig. 2C), suggesting that CME prevented overweight in ovariectomized rats. We also measured the area of white adipocytes. The area of white adipocytes in the middle-dose CME group was shown to be lowered compared with the control and sham groups (Fig. 2D). However, the hypertrophy of white adipocytes was not induced by ovariectomy, and although the area of white adipocytes decreased, it was not statistically significant (p = 0.209). The difference in the area of white adipocytes between groups is representatively shown in Fig. 2E. In Fig. 2F, it is representatively shown that rats in middle-dose and high-dose CME groups had less fat accumulation than those in the control group.

Fig. 2
figure 2

Anti-obesity effects of Cordyceps militaris extract (CME). A–C Effects of CME on body weights. Body weights after 4 weeks (A) and 8 weeks (B) are shown by the dose of CME. Dots represent individual sample weights. (C) Changes in body weights are represented by time. Lines and bands represent the means of weights of rats that belong to each group and their 95% confidence intervals, respectively. D–F Effects of CME on the accumulation of fat. (D) The mean area of white adipose tissue after 8 weeks of administration is shown by the dose of CME. All other details are the same as in (A). (E) Representative results of white adipose tissue histology. (F) Representative results of dual-energy X-ray. The red-colored region represents where fat is highly accumulated. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups

We also measured the liver and uterine weights. Liver weights in each CME group were shown to be non-significantly lower compared with the controls; however, liver weights in the CME groups were lowered in a dose-dependent manner (p < 0.001). Notably, the liver weights of the middle-dose and high-dose CME groups were close to those of the sham group (Fig. 3A). This suggests that CME prevents liver hypertrophy, which was observed in OVX rats. The uterus weight rates in the CME groups did not increase compared with those in sham and positive control groups, whereas the uterus weights of the beta-estradiol group showed a trend of increase (Figs. 3B and 3C). This suggests that CME does not act as an ER agonist for the growth of the uterus. Additionally, we measured lipid levels and found that levels of triglyceride in CME groups were significantly decreased in a dose-dependent manner (p = 0.047), although each CME group showed no significant difference compared with the control (Fig. 3D). For other lipid levels including cholesterol, HDL, and LDL, CME showed neither significant differences compared with controls nor improvements in lipid levels in a dose-dependent manner (Fig. 3E-G). Taken together, our results propose that CME improves liver hypertrophy and triglyceride levels in a dose-dependent manner.

Fig. 3
figure 3

Effects of Cordyceps militaris extract (CME) on the weights of organs and lipid levels. A–C Effects of CME on the weights of organs. Weights of the liver (A) and uterus (B) after 8 weeks of administration are shown by the dose of CME. All other details are the same as in Fig. 2. (C) Representative results of uterine morphology after 8 weeks of administration. D–G Effects of CME on lipid levels. Levels of triglyceride (D), cholesterol (E), high-density lipoprotein (F), and low-density lipoprotein (G) after 8 weeks of administration are shown by the dose of CME. All other details are the same as in Fig. 2. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups

CME interacts with ERα and MAPK in MCF-7

Next, we investigated the possibility of whether CME can mitigate the lowered estrogenic activity. We measured the estrogenic activity of CME on an ER-positive MCF-7 human breast cancer cell line through a modified Soto’s E-screen assay [40]. The MCF-7 cells were exposed to 10 and 25 μg/mL of CME for 144 h. Queens One tab. (positive control), which is an extract of Trifolium pratense that exhibits estrogenic effects in vivo in ovariectomized Sprague Dawley rats [41], increased the MCF-7 cell proliferation in a concentration-dependent manner under 25 μg/mL (Fig. 4A). The effect was completely inhibited by co-treatment with ICI 182,780, which is an ER antagonist. Therefore, the cell proliferation induced by the Queens One tab. is mediated by ERs. Similarly, the CME was also shown to increase cell proliferation in a dose-dependent manner (Fig. 4B), although the effect was limited to doses of less than 25 µg/mL. The effect was also attenuated by co-treatment with ICI 182,780, but not completely inhibited. These results imply that CME acts as a partial estrogenic agent.

Fig. 4
figure 4

Effect of Cordyceps militaris extract (CME) on proliferation in MCF-7 cells. Relative cell proliferation ratios are shown by the dose of (A) Queens One tab, used as a positive control, and (B) CME for 24 h. All other details are the same as in Fig. 2. C Levels of estrogen receptor α (ERα) phosphorylation in MCF-7 cells, determined by Western blotting, are shown by the dose of CME. Levels of protein expression of phospho-estrogen receptor α (p-ERα) are compared with levels of ERα and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). D Levels of phosphorylation of mitogen-activated protein kinases in MCF-7 cells, determined by Western blotting, are shown by the dose of CME. Levels of protein expression of phospho-extracellular-signal-regulated kinase (ERK), p- c-Jun N-terminal kinases (JNK), p-p38 are compared with levels of Total ERK, JNK, p38. Uncropped images are shown in Supplementary Figure S1

ERα, also called estrogen receptor 1 (ESR1), is a ligand-dependent nuclear hormone receptor transcription factor. When ERα is bound with 17β-estradiol (E2), a ligand of ERα, ERα binds to specific DNA sequences called estrogen response elements with high affinity. [42]. Generally, ligands of ERα, such as E2, induce the phosphorylation of Serine 118 in ERα [43]. Thus, we performed Western blotting to investigate the activation of ERα by CME. As shown in Fig. 4C, CME increased the expression of ERα phosphorylation. The results indicate that the treatment of CME induces phosphorylation with serine residues of ERα. Also, CME increased the expression of EKR and p38 phosphorylation among MAPKs (Fig. 4D). The results indicate that the treatment of CME induces phosphorylation with ERK and p38.

Compounds in Cordyceps militaris are associated with multiple pathways

Finally, we investigated the potential pathway of CME which alleviates obesity and related symptoms induced by menopause by employing a network pharmacological approach. We constructed a herb–compound–target network which consisted of 117 nodes and 152 edges. The nodes correspond to the herb, potential bioactive compounds, or their targets. Additionally, the edges indicated the inclusiveness of compounds (between herbs and compounds) or interactions (between compounds and targets). We conducted enrichment analyses of the KEGG pathways related to obesity or estrogen with the predicted targets of compounds of Cordyceps militaris. Our results showed that these predicted targets are associated with multiple pathways, such as the insulin signaling pathway, phosphatidylinositol 3 kinase (PI3K)–Akt signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, the estrogen signaling pathway, and insulin resistance, which are related to obesity and the menopause (Table 1). This association indicates that Cordyceps militaris could alleviate obesity and obesity-related symptoms via regulating these pathways. Additionally, compounds in Cordyceps militaris were predicted to interact with mutually exclusive groups of genes related to obesity. Adenine was predicted to interact with protein kinase C iota (PRKCI), acetyl-CoA carboxylase beta (ACACB), protein kinase C zeta (PRKCZ), glycogen phosphorylase, muscle-associated (PYGM), and heat shock protein 90 alpha family class A member 1 (HSP90AA1), which are mainly associated with insulin-related pathways. Meanwhile, cordycepin was predicted to interact with caspase 3 (CASP3), interleukin 1 beta (IL1B), hepatocyte growth factor (HGF), matrix metallopeptidase 9 (MMP9), and Toll-like receptor 4 (TLR4), which are associated with signaling pathways of MAPK, PI3K–Akt, and estrogen. Guanosine was predicted to interact with MAPK3 and MAPK1, which are associated with various biological pathways. We note that predicted targets of compounds that are related to obesity in menopause are mutually exclusive (Fig. 5). In the case of the estrogen signaling pathway, the compounds were predicted to interact with mutually exclusive targets, which can affect ERs (Fig. 6). These results suggest that compounds in Cordyceps militaris could regulate estrogen-related pathways in a complementary manner.

Table 1 Enrichment analysis of KEGG pathways related to obesity or estrogen through the targets of compounds of Cordyceps militaris
Fig. 5
figure 5

Herb–compound–target network of Cordyceps militaris. Edges between the herbs and compounds represent the herbs that contain the compounds. Edges between the compounds and genes represent genes that are the predicted targets of the compounds. Genes are colored by their related pathways. Note that we only visualized compounds and genes related to the potential pathways

Fig. 6
figure 6

Estrogen signaling pathway and the targets of compounds contained in Cordyceps militaris. Colored genes represent the predicted targets of compounds in Cordyceps militaris. Red border boxes represent estrogen receptor α, whose phosphorylation is described above

Discussion

Compounds in Cordyceps militaris have been determined by several methods, including multi-column liquid chromatography, linear ion trap liquid chromatography-tandem mass spectrometry, and ion-pairing reversed-phase liquid chromatography-mass spectrometry [44,45,46]. It has been reported that adenosine monophosphate (AMP), phenylalanine, uridine, hypoxanthine, inosine, guanine, guanosine, dAMP, adenosine, adenine, cordycepin, cytosine, cytidine, uracil, hypoxanthine, 2′-deoxyguanosine, inosine, 2′-deoxyuridine, β-thymidine were detected. Among them, cordycepin is mostly considered a key component of Cordyceps species, including Cordyceps militaris [47,48,49,50]. Cordycepin binds to a number of intracellular targets, including DNA/RNA, influencing apoptosis and the cell cycle. Cordycepin typically exerts therapeutic effects on tumors by inhibiting the DNA-binding activity of activator protein-1 and NF-κB [51]  or alleviating inflammation by reducing the production of inflammatory mediators such as nitric oxide, prostaglandin E2, TNF-α, and IL-1β, and downregulating iNOS, COX-2, and TNF-α gene expression [51, 52]. The roles of guanosine and adenine in the therapeutic mechanisms of Cordyceps species are less well known. However, in the case of guanosine, it has been found to exert neuroprotective [53] and anti-inflammatory properties [54, 55], implying that it may be a potential therapeutic compound for Cordyceps species.

Hormonal changes during the menopausal period lead to various symptoms, including obesity [1, 3, 56]. Obesity not only worsens the quality of life but also constitutes a risk factor for metabolic and cardiovascular diseases [56]. Moreover, adipocytokines synthesized in adipocytes are considered to be modulators of insulin resistance and chronic inflammation [57]. Therefore, regulating obesity after menopause is an important issue in women’s health. In our study, we investigated whether Cordyceps militaris can regulate obesity induced by menopause. We found that CME prevented obesity and improved some lipid levels dose-dependently in ovariectomized rats. CME also exhibited ER agonistic effects in in vitro experiments. Through network pharmacological analysis, it was shown that the bioactive compounds in Cordyceps militaris are associated with multiple pathways which can affect obesity and its complications. Our results suggest that CME could be used for treating postmenopausal obesity in women.

Concerning white adipocytes, ovariectomies are generally believed to induce obesity, inducing the hypertrophy of white adipocytes. However, regardless of the induction of obesity, the hypertrophy of white adipocytes may not be triggered within certain experimental settings, such as postoperative terms [58]. Our results indicate that ovariectomized rats had significant increases in body weight, but no increases in white adipocyte size. Therefore, additional research is required to ascertain the relationship between ovariectomy and white adipocytes, as well as the relationship between CME and white adipocytes.

Although controversial, obesity in menopause is considered to result from a deficiency of estrogen. Indeed, estrogen-based HRT in menopausal women has been reported to beneficially affect lipid levels, fasting serum glucose, insulin levels, and abdominal fat [4, 12, 59, 60]. It is well known that the ventrolateral portion of the ventral medial nucleus (VL VMN) and the arcuate in the hypothalamus control energy intake and expenditure, and a decrease in ERα in VL VMN coincides with an increase in adiposity and loss in energy expenditure [61]. Moreover, it has been reported that estrogen can regulate food intake in the hindbrain [62]. Our study showed that CME significantly increased the phosphorylation of ERα at doses of 10 µg/ml and 25 µg/ml in the MCF-7 cell, suggesting that the anti-obesity effects of CME may be based on its ERα agonistic activity. Interestingly, CME did not exert estrogenic effects on uterine development in ovariectomized rats in this study. This suggests that CME could selectively activate ERα depending on the types of tissues. There is a possibility that CME regulates food intake and energy expenditure by activating ER in the central nervous system, while not activating those in uterus. Additionally, the result that CME fostered the proliferation of MCF-7 indicates that caution should be exercised when applying CME therapy for ER-positive breast cancer. Selective action of CME is a key area to be explored further. Additionally, the network pharmacological method predicted that cordycepin would interact with matrix metallopeptidase 9 (MMP9), whose pathway is related to MAPK and ERα. Moreover, adenine and guanosine, which are the other predicted bioactive compounds of Cordyceps militaris, were predicted to be related to HSP90A1 and MAPK, respectively. The MAPK and estrogen signaling pathways are closely related. It has been demonstrated that the expression of MAPK increased ERα-induced transcriptional activation, suggesting that MAPK/ER cross-talk enhances the estrogen signaling pathway [63]. Therefore, these results imply the possibility that the compounds in CME may synergistically enhance estrogenic pathways through MAPK regulation. However, the diverse experimental settings and the limited scope of studies that explore the relationship between Cordyceps and estrogen have often provided conflicting evidence. For example, one study reported that Cordyceps militaris had antimetastatic effects by inhibiting estrogen-related receptor alpha (ERRα) [64]; on the other hand, another study reported that Cordyceps sinensis alleviated osteoporosis induced by estrogen deficiency [65]. Some studies have reported that cordycepin, one of the predicted bioactive compounds of Cordyceps militaris, inhibits the growth of MCF-7 and the phosphorylation of ERRα in ovarian carcinoma cells [64, 66]. Therefore, future studies should focus on the relationship between the bioactive compounds of Cordyceps militaris and the estrogen signaling pathway.

Obesity is related to the risk of developing insulin resistance and type 2 diabetes [67]. Adipose tissue in obesity releases higher amounts of non-esterified fatty acid, glycerol, hormones, and adipocytokines involved in the development of insulin resistance [68, 69]. Obesity, type 2 diabetes, and cardiovascular diseases share a common metabolic environment characterized by insulin resistance [68]; therefore, regulating insulin resistance and secretion could be a key mechanism to prevent the aggravation of obesity and its related metabolic diseases. Cordyceps have been reported to improve insulin resistance, insulin secretion, and glucose level [70,71,72]. In our prediction, adenine regulates insulin-related genes such as acetyl-CoA carboxylase β and glycogen phosphorylase B, whereas guanosine and cordycepin regulate the MAPK and PI3Ks-Akt signaling pathways. MAPK, PI3Ks, and Akt are known to be associated with obesity and its complications, including type 2 diabetes and non-alcoholic fatty livers [73,74,75,76,77]. These results imply that Cordyceps militaris may alleviate obesity and its complications through its compounds which synergistically regulate the signaling pathways of estrogen, insulin, MAPK, and PI3K–Akt.

Conclusions

Our study investigated the therapeutic effect of CME on obesity and its mechanisms. CME alleviated obesity and its related symptoms in ovariectomized rats. CME exhibited estrogen agonistic activity in MCF-7 cell lines. Additionally, we predicted the bioactive compounds and their potential pathways to obesity. This study contributes to developing the understanding of the anti-obesity effects and mechanism of Cordyceps militaris. We also suggest that our approach combining in vivo, in vitro, and in silico methods is a reliable strategy to explore the efficacy and mechanisms of herbal medicine.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

HRT:

Hormone Replacement Therapy

ER:

Estrogen Receptor

CME:

Cordyceps militaris Extract

OVX:

Ovariectomized Model Group

ERα:

Estrogen Receptor Alpha

HDL:

High-Density Lipoprotein

LDL:

Low-Density Lipoprotein

H&E:

Hematoxylin and Eosin

DXA:

Dual-energy X-ray

SD:

Standard Deviation

DMSO:

Dimethyl Sulfoxide

RIPA:

Radioimmunoprecipitation Assay

PVDF:

Polyvinylidene Fluoride

p-ERα:

Phospho-Estrogen Receptor α

ERK:

Extracellular-signal-regulated kinase

JNK:

Jun N-terminal Kinase

GAPDH:

Glyceraldehyde 3-Phosphate Dehydrogenase

QED:

Quantitative Estimate of Drug-likeness

GSEA:

Gene Set Enrichment Analysis

ESR1:

Estrogen Receptor 1

E2 17β:

Estradiol

MAPK:

Mitogen-Activated Protein Kinase

PRKCI:

Protein Kinase C Iota

ACACB:

Acetyl-CoA Carboxylase Beta

PRKCZ:

Protein Kinase C Zeta

PYGM:

Glycogen Phosphorylase, Muscle Associated

HSP90AA1:

Heat Shock Protein 90 Alpha Family Class A Member 1

CASP3:

Caspase 3

IL1B:

Interleukin 1 Beta

HGF:

Hepatocyte Growth Factor

MMP9:

Matrix Metallopeptidase 9

TLR4:

Toll-Like Receptor 4

VL VMN:

Ventrolateral Portion of the Ventral Medial Nucleus

ERRα:

Estrogen-Related Receptor Alpha

References

  1. Nelson HD. Menopause. The Lancet. 2008;371(9614):760–70.

    Article  Google Scholar 

  2. Kozakowski J, Gietka-Czernel M, Leszczyńska D, Majos A. Obesity in menopause - our negligence or an unfortunate inevitability? Prz Menopauzalny. 2017;16(2):61–5.

    PubMed  PubMed Central  Google Scholar 

  3. Chooi YC, Ding C, Magkos F. The epidemiology of obesity. Metabolism. 2019;92:6–10.

    CAS  PubMed  Article  Google Scholar 

  4. Lizcano F, Guzmán G. Estrogen Deficiency and the Origin of Obesity during Menopause. Biomed Res Int. 2014;2014:757461–757461.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  5. Haslam DW, James WPT. Obesity. The Lancet. 2005;366(9492):1197–209.

    Article  Google Scholar 

  6. Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet. 2006;367(9524):1747–57.

    PubMed  Article  Google Scholar 

  7. Ward ZJ, Bleich SN, Cradock AL, Barrett JL, Giles CM, Flax C, Long MW, Gortmaker SL. Projected US state-level prevalence of adult obesity and severe obesity. N Engl J Med. 2019;381(25):2440–50.

    PubMed  Article  Google Scholar 

  8. Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C, Mullany EC, Biryukov S, Abbafati C, Abera SF, et al. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the Global Burden of Disease Study 2013. The Lancet. 2014;384(9945):766–81.

    Article  Google Scholar 

  9. Stuenkel CA, Davis SR, Gompel A, Lumsden MA, Murad MH, Pinkerton JV, Santen RJ. Treatment of Symptoms of the Menopause: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2015;100(11):3975–4011.

    CAS  PubMed  Article  Google Scholar 

  10. Schierbeck LL, Rejnmark L, Tofteng CL, Stilgren L, Eiken P, Mosekilde L, Køber L, Jensen JE. Effect of hormone replacement therapy on cardiovascular events in recently postmenopausal women: randomised trial. BMJ. 2012;345: e6409.

    PubMed  Article  CAS  Google Scholar 

  11. Grodstein F, Manson JE, Colditz GA, Willett WC, Speizer FE, Stampfer MJ. A prospective, observational study of postmenopausal hormone therapy and primary prevention of cardiovascular disease. Ann Intern Med. 2000;133(12):933–41.

    CAS  PubMed  Article  Google Scholar 

  12. Salpeter SR, Walsh JM, Ormiston TM, Greyber E, Buckley NS, Salpeter EE. Meta-analysis: effect of hormone-replacement therapy on components of the metabolic syndrome in postmenopausal women. Diabetes Obes Metab. 2006;8(5):538–54.

    CAS  PubMed  Article  Google Scholar 

  13. Chang WC, Wang JH, Ding DC. Hormone therapy in postmenopausal women associated with risk of stroke and venous thromboembolism: a population-based cohort study in Taiwan. Menopause. 2019;26(2):197–202.

    PubMed  Article  Google Scholar 

  14. Trimarco V, Rozza F, Izzo R, De Leo V, Cappelli V, Riccardi C, Di Carlo C. Effects of a new combination of nutraceuticals on postmenopausal symptoms and metabolic profile: a crossover, randomized, double-blind trial. Int J Women’s Health. 2016;8:581.

    CAS  Article  Google Scholar 

  15. Zhu X, Liew Y, Liu ZL. Chinese herbal medicine for menopausal symptoms. Cochrane Database Syst Rev. 2016;3:CD009023.

  16. Haines C, Lam P, Chung T, Cheng K, Leung P. A randomized, double-blind, placebo-controlled study of the effect of a Chinese herbal medicine preparation (Dang Gui Buxue Tang) on menopausal symptoms in Hong Kong Chinese women. Climacteric. 2008;11(3):244–51.

    CAS  PubMed  Article  Google Scholar 

  17. Kronenberg F, Fugh-Berman A. Complementary and alternative medicine for menopausal symptoms: a review of randomized, controlled trials. Ann Intern Med. 2002;137(10):805–13.

    PubMed  Article  Google Scholar 

  18. Oh JH, Baek SE, Lee WY, Baek JY, Trinh TA, Park DH, Lee HL, Kang KS, Kim CE, Yoo JE. Investigating the Systems-Level Effect of Pueraria lobata for Menopause-Related Metabolic Diseases Using an Ovariectomized Rat Model and Network Pharmacological Analysis. Biomolecules. 2019;9(11):747.

    CAS  PubMed Central  Article  Google Scholar 

  19. Payab M, Hasani-Ranjbar S, Shahbal N, Qorbani M, Aletaha A, Haghi-Aminjan H, Soltani A, Khatami F, Nikfar S, Hassani S, et al. Effect of the herbal medicines in obesity and metabolic syndrome: A systematic review and meta-analysis of clinical trials. Phytother Res. 2020;34(3):526–45.

    CAS  PubMed  Article  Google Scholar 

  20. Hasani-Ranjbar S, Nayebi N, Larijani B, Abdollahi M. A systematic review of the efficacy and safety of herbal medicines used in the treatment of obesity. World J Gastroenterol. 2009;15(25):3073–85.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Zhang K, Wang Z, Pan X, Yang J, Wu C. Antidepressant-like effects of Xiaochaihutang in perimenopausal mice. J Ethnopharmacol. 2020;248: 112318.

    CAS  PubMed  Article  Google Scholar 

  22. Lee YM, Kim JB, Bae JH, Lee JS, Kim PS, Jang HH, Kim HR. Estrogen-like activity of aqueous extract from Agrimonia pilosa Ledeb. in MCF-7 cells. BMC Complement Altern Med. 2012;12:260.

    PubMed  PubMed Central  Article  Google Scholar 

  23. Bodinet C, Freudenstein J. Influence of marketed herbal menopause preparations on MCF-7 cell proliferation. Menopause. 2004;11(3):281–9.

    PubMed  Article  Google Scholar 

  24. Panda AK, Swain KC. Traditional uses and medicinal potential of Cordyceps sinensis of Sikkim. J Ayurveda Integr Med. 2011;2(1):9–13.

    PubMed  PubMed Central  Article  Google Scholar 

  25. Kim SB, Ahn B, Kim M, Ji HJ, Shin SK, Hong IP, Kim CY, Hwang BY, Lee MK. Effect of Cordyceps militaris extract and active constituents on metabolic parameters of obesity induced by high-fat diet in C58BL/6J mice. J Ethnopharmacol. 2014;151(1):478–84.

    CAS  PubMed  Article  Google Scholar 

  26. Chang Y, Jeng KC, Huang KF, Lee YC, Hou CW, Chen KH, Cheng FY, Liao JW, Chen YS. Effect of Cordyceps militaris supplementation on sperm production, sperm motility and hormones in Sprague-Dawley rats. Am J Chin Med. 2008;36(5):849–59.

    CAS  PubMed  Article  Google Scholar 

  27. Lee HS, Kim MK, Kim Y-K, Jung EY, Park CS, Woo MJ, Lee SH, Kim JS, Suh HJ. Stimulation of osteoblastic differentiation and mineralization in MC3T3-E1 cells by antler and fermented antler using Cordyceps militaris. J Ethnopharmacol. 2011;133(2):710–7.

    PubMed  Article  Google Scholar 

  28. Kusama K, Miyagawa M, Ota K, Kuwabara N, Saeki K, Ohnishi Y, Kumaki Y, Aizawa T, Nakasone T, Okamatsu S. Cordyceps militaris Fruit Body Extract Decreases Testosterone Catabolism and Testosterone-Stimulated Prostate Hypertrophy. Nutrients. 2021;13(1):50.

    CAS  Article  Google Scholar 

  29. Zhang R, Zhu X, Bai H, Ning K. Network Pharmacology Databases for Traditional Chinese Medicine: Review and Assessment. Front Pharmacol. 2019;10:123.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  30. Li W, Yuan G, Pan Y, Wang C, Chen H. Network pharmacology studies on the bioactive compounds and action mechanisms of natural products for the treatment of diabetes mellitus: a review. Front Pharmacol. 2017;8:74.

    PubMed  PubMed Central  Google Scholar 

  31. Lee WY, Lee CY, Kim YS, Kim CE. The methodological trends of traditional herbal medicine employing network pharmacology. Biomolecules. 2019;9(8):362.

    PubMed Central  Article  CAS  Google Scholar 

  32. Zhou Z, Chen B, Chen S, Lin M, Chen Y, Jin S, Chen W, Zhang Y. Applications of Network Pharmacology in Traditional Chinese Medicine Research. Evid Based Complement Alternat Med. 2020;2020:1646905.

    PubMed  PubMed Central  Google Scholar 

  33. Lee D, Lee WY, Jung K, Kwon YS, Kim D, Hwang GS, Kim CE, Lee S, Kang KS. The Inhibitory Effect of Cordycepin on the Proliferation of MCF-7 Breast Cancer Cells, and its Mechanism: An Investigation Using Network Pharmacology-Based Analysis. Biomolecules. 2019;9(9):414.

  34. Union TEPatCotE. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Off J Eur Union. 2010;276:20.

    Google Scholar 

  35. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, Clark A, Cuthill IC, Dirnagl U. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. J Cerebral Blood Flow Metab. 2020;40(9):1769–77.

    Article  Google Scholar 

  36. Zhang R, Yu S, Bai H, Ning K. TCM-Mesh: The database and analytical system for network pharmacology analysis for TCM preparations. Sci Rep. 2017;7(1):2821.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  37. Bickerton GR, Paolini GV, Besnard J, Muresan S, Hopkins AL. Quantifying the chemical beauty of drugs. Nat Chem. 2012;4(2):90–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR. Ma’ayan A: Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013;14(1):1–14.

    Article  Google Scholar 

  40. Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect. 1995;103 Suppl 7(Suppl 7):113–22.

    CAS  PubMed  Article  Google Scholar 

  41. Burdette JE, Liu J, Lantvit D, Lim E, Booth N, Bhat KPL, Hedayat S, Van Breemen RB, Constantinou AI, Pezzuto JM, et al. Trifolium pratense (Red Clover) Exhibits Estrogenic Effects In Vivo in Ovariectomized Sprague-Dawley Rats. J Nutr. 2002;132(1):27–30.

    CAS  PubMed  Article  Google Scholar 

  42. Kumar V, Chambon P. The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell. 1988;55(1):145–56.

    CAS  PubMed  Article  Google Scholar 

  43. Joel PB, Traish AM, Lannigan DA. Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase. J Biol Chem. 1998;273(21):13317–23.

    CAS  PubMed  Article  Google Scholar 

  44. Yang FQ, Li DQ, Feng K, Hu DJ, Li SP. Determination of nucleotides, nucleosides and their transformation products in Cordyceps by ion-pairing reversed-phase liquid chromatography–mass spectrometry. J Chromatogr A. 2010;1217(34):5501–10.

    CAS  PubMed  Article  Google Scholar 

  45. Meng C, Han Q, Wang X, Liu X, Fan X, Liu R, Wang Q, Wang C. Determination and Quantitative Comparison of Nucleosides in two Cordyceps by HPLC–ESI–MS-MS. J Chromatogr Sci. 2019;57(5):426–33.

    CAS  PubMed  Article  Google Scholar 

  46. Qian Z, Li S. Analysis of Cordyceps by multi-column liquid chromatography. Acta Pharm Sin B. 2017;7(2):202–7.

    PubMed  Article  Google Scholar 

  47. Yu W-Q, Yin F, Shen N, Lin P, Xia B, Li Y-J, Guo S-D. Polysaccharide CM1 from Cordyceps militaris hinders adipocyte differentiation and alleviates hyperlipidemia in LDLR(+/−) hamsters. Lipids Health Dis. 2021;20(1):178.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Zhou X, Luo L, Dressel W, Shadier G, Krumbiegel D, Schmidtke P, Zepp F, Meyer CU. Cordycepin is an Immunoregulatory Active Ingredient of Cordyceps sinensis. Am J Chin Med. 2008;36(05):967–80.

    CAS  PubMed  Article  Google Scholar 

  49. Tuli HS, Sharma AK, Sandhu SS, Kashyap D. Cordycepin: A bioactive metabolite with therapeutic potential. Life Sci. 2013;93(23):863–9.

    CAS  PubMed  Article  Google Scholar 

  50. Cunningham KG, Manson W, Spring FS, Hutchinson SA. Cordycepin, a Metabolic Product isolated from Cultures of Cordyceps militaris (Linn.) Link. Nature. 1950;166(4231):949–949.

    CAS  PubMed  Article  Google Scholar 

  51. Jeong J-W, Jin C-Y, Park C, Han MH, Kim G-Y, Moon S-K, Kim CG, Jeong YK, Kim W-J, Lee J-D. Inhibition of migration and invasion of LNCaP human prostate carcinoma cells by cordycepin through inactivation of Akt. Int J Oncol. 2012;40(5):1697–704.

    CAS  PubMed  Google Scholar 

  52. Kim HG, Shrestha B, Lim SY, Yoon DH, Chang WC, Shin D-J, Han SK, Park SM, Park JH, Park HI. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-κB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur J Pharmacol. 2006;545(2–3):192–9.

    CAS  PubMed  Article  Google Scholar 

  53. Lanznaster D, Dal-Cim T, Piermartiri TCB, Tasca CI. Guanosine: a Neuromodulator with Therapeutic Potential in Brain Disorders. Aging Dis. 2016;7(5):657–79.

    PubMed  PubMed Central  Article  Google Scholar 

  54. Jiang S, Bendjelloul F, Ballerini P, D’Alimonte I, Nargi E, Jiang C, Huang X, Rathbone MP. Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury. Purinergic Signalling. 2007;3(4):411–21.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. Hansel G, Tonon AC, Guella FL, Pettenuzzo LF, Duarte T, Duarte MMMF, Oses JP, Achaval M, Souza DO. Guanosine protects against cortical focal ischemia. Involvement of inflammatory response. Mol Neurobiol. 2015;52(3):1791–803.

    CAS  PubMed  Article  Google Scholar 

  56. Stachowiak G, Pertyński T, Pertyńska-Marczewska M. Metabolic disorders in menopause. Prz Menopauzalny. 2015;14(1):59–64.

    PubMed  PubMed Central  Google Scholar 

  57. Rabe K, Lehrke M, Parhofer KG, Broedl UC. Adipokines and insulin resistance. Mol Med. 2008;14(11–12):741–51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Vieira Potter VJ, Strissel KJ, Xie C, Chang E, Bennett G, Defuria J, Obin MS, Greenberg AS. Adipose Tissue Inflammation and Reduced Insulin Sensitivity in Ovariectomized Mice Occurs in the Absence of Increased Adiposity. Endocrinol. 2012;153(9):4266–77.

    Article  CAS  Google Scholar 

  59. Munoz J, Derstine A, Gower BA. Fat distribution and insulin sensitivity in postmenopausal women: influence of hormone replacement. Obes Res. 2002;10(6):424–31.

    CAS  PubMed  Article  Google Scholar 

  60. Gormsen LC, Høst C, Hjerrild BE, Pedersen SB, Nielsen S, Christiansen JS, Gravholt CH. Estradiol acutely inhibits whole body lipid oxidation and attenuates lipolysis in subcutaneous adipose tissue: a randomized, placebo-controlled study in postmenopausal women. Eur J Endocrinol. 2012;167(4):543–51.

    CAS  PubMed  Article  Google Scholar 

  61. Musatov S, Chen W, Pfaff DW, Mobbs CV, Yang X-J, Clegg DJ, Kaplitt MG, Ogawa S. Silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus leads to metabolic syndrome. Proc Natl Acad Sci. 2007;104(7):2501–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Thammacharoen S, Lutz TA, Geary N, Asarian L. Hindbrain Administration of Estradiol Inhibits Feeding and Activates Estrogen Receptor-α-Expressing Cells in the Nucleus Tractus Solitarius of Ovariectomized Rats. Endocrinology. 2008;149(4):1609–17.

    CAS  PubMed  Article  Google Scholar 

  63. Atanaskova N, Keshamouni VG, Krueger JS, Schwartz JA, Miller F, Reddy KB. MAP kinase/estrogen receptor cross-talk enhances estrogen-mediated signaling and tumor growth but does not confer tamoxifen resistance. Oncogene. 2002;21(25):4000–8.

    CAS  PubMed  Article  Google Scholar 

  64. Wang CW, Hsu WH, Tai CJ. Antimetastatic effects of cordycepin mediated by the inhibition of mitochondrial activity and estrogen-related receptor α in human ovarian carcinoma cells. Oncotarget. 2017;8(2):3049–58.

    PubMed  Article  Google Scholar 

  65. Zhang DW, Wang ZL, Qi W, Zhao GY. The effects of Cordyceps sinensis phytoestrogen on estrogen deficiency-induced osteoporosis in ovariectomized rats. BMC Complement Altern Med. 2014;14:484.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  66. Lee D, Lee WY, Jung K, Kwon YS, Kim D, Hwang GS, Kim CE, Lee S, Kang KS. The Inhibitory Effect of Cordycepin on the Proliferation of MCF-7 Breast Cancer Cells, and Its Mechanism: An Investigation Using Network Pharmacology-Based Analysis. Biomolecules. 2019;9(9):414.

    CAS  PubMed Central  Article  Google Scholar 

  67. Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature. 2006;444(7121):840–6.

    CAS  PubMed  Article  Google Scholar 

  68. Shoelson SE, Lee J, Goldfine AB. Inflammation and insulin resistance. J Clin Investig. 2006;116(7):1793–801.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. Scherer PE. Adipose tissue: from lipid storage compartment to endocrine organ. Diabetes. 2006;55(6):1537–45.

    CAS  PubMed  Article  Google Scholar 

  70. Choi SB, Park CH, Choi MK, Jun DW, Park S. Improvement of insulin resistance and insulin secretion by water extracts of Cordyceps militaris, Phellinus linteus, and Paecilomyces tenuipes in 90% pancreatectomized rats. Biosci Biotechnol Biochem. 2004;68(11):2257–64.

    CAS  PubMed  Article  Google Scholar 

  71. Cheng YW, Chen YI, Tzeng CY, Chen HC, Tsai CC, Lee YC, Lin JG, Lai YK, Chang SL. Extracts of Cordyceps militaris lower blood glucose via the stimulation of cholinergic activation and insulin secretion in normal rats. Phytother Res. 2012;26(8):1173–7.

    CAS  PubMed  Article  Google Scholar 

  72. Li SP, Zhang GH, Zeng Q, Huang ZG, Wang YT, Dong TT, Tsim KW. Hypoglycemic activity of polysaccharide, with antioxidation, isolated from cultured Cordyceps mycelia. Phytomedicine. 2006;13(6):428–33.

    PubMed  Article  CAS  Google Scholar 

  73. Schultze SM, Hemmings BA, Niessen M, Tschopp O. PI3K/AKT, MAPK and AMPK signalling: protein kinases in glucose homeostasis. Expert Rev Mol Med. 2012;14: e1.

    PubMed  Article  CAS  Google Scholar 

  74. Lee BC, Lee J. Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance. Biochim Biophys Acta. 2014;1842(3):446–62.

    CAS  PubMed  Article  Google Scholar 

  75. Matsuda S, Kobayashi M, Kitagishi Y. Roles for PI3K/AKT/PTEN pathway in cell signaling of nonalcoholic fatty liver disease. International Scholarly Research Notices. 2013;2013:472432.

  76. Xiao J, Wang J, Xing F, Han T, Jiao R, Liong EC, Fung ML, So KF, Tipoe GL. Zeaxanthin dipalmitate therapeutically improves hepatic functions in an alcoholic fatty liver disease model through modulating MAPK pathway. PLoS ONE. 2014;9(4): e95214.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  77. Zhang W, Thompson BJ, Hietakangas V, Cohen SM. MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila. PLoS Genet. 2011;7(12): e1002429.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to Gachon University and Pharmaceutical Co., LTD. for providing the laboratory facilities to conduct this work.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2017R1A2B1010690). This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (2020M3A9E4103843).

Author information

Affiliations

Authors

Contributions

C.-E.K, D.K., and Y.K. designed and supervised the study. E.L. performed the animal experiments. S.L. performed the cell line experiments. D.J. performed the network pharmacological analysis. D.J. performed the statistical analysis and visualization. D.J. wrote the original draft, and K.S.-K., C.-E. K., and D.K. provided manuscript revision. All data were generated in-house. The author(s) read and approved the final manuscript.

Corresponding authors

Correspondence to Chang-Eop Kim or Daeyoung Kim.

Ethics declarations

Ethics approval and consent to participate

All methods were performed in accordance with the Directive 2010/63/EU of the European Parliament and of the counsel on the protection of animals used for scientific purposes [34]. All the research protocols were approved by the Panel on Laboratory Animal Care of Gachon University (GIACUC-R2017037). The ARRIVE guidelines [35] were adhered to throughout this study.

Consent for publication

Not Applicable.

Competing interests

The authors report no declarations of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1. Supplementary Figure S1.

Levels of estrogen receptor α (ERα) and MAPKs phosphorylation in MCF-7 cells, determined by western blotting, are shown by the dose of CME.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Jang, D., Lee, E., Lee, S. et al. System-level investigation of anti-obesity effects and the potential pathways of Cordyceps militaris in ovariectomized rats. BMC Complement Med Ther 22, 132 (2022). https://doi.org/10.1186/s12906-022-03608-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12906-022-03608-y

Keywords

  • Cordyceps militaris
  • Menopause
  • Obesity
  • Estrogen receptor
  • Mitogen-activated protein kinase
  • Network pharmacology