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.

Keywords: Cordyceps militaris; Estrogen receptor; Menopause; Mitogen-activated protein kinase; Network pharmacology; Obesity.

Fig. 1

Schematic diagram to summarize the methods and results of our study

Fig. 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

Fig. 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

Fig. 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

Fig. 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

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