Therapeutic Effects of Modified Gengnianchun Formula on Stress-Induced Diminished Ovarian Reserve Based on Experimental Approaches and Network Pharmacology

Abstract

Aim

To verify the effects of modified Gengnianchun formula (MGNC), a traditional Chinese medicine, on a stressed diminished ovarian reserve (DOR) animal model and predict the underlying mechanisms through network pharmacology strategies.

Methods

Sexually mature female C57BL/6 mice were allocated to five groups, abbreviated as the control (C) group, stress manipulated model (M) group, stress with normal saline gavage (N) group, stress with low-dose MGNC gavage (L) group, and stress with high-dose MGNC gavage (H) group. Body weight and the estrous cycle were monitored during the stress and gavage process. Serum stress hormones and reproductive hormones were evaluated by ELISA. Ovarian follicle counts were calculated, and ovarian follicle-stimulating hormone receptor (FSHR) and anti-Müllerian hormone (AMH) expression were assessed by Western blotting and immunohistochemistry. Network pharmacology strategies included active compound screening, drug and disease target analysis, gene ontology analysis, pathway analysis, and visualization of results.

Results

MGNC treatment significantly decreased serum corticosterone (CORT) and follicle-stimulating hormone (FSH) levels and increased testosterone (T) levels in the H group compared with the M and N groups. Primordial and preantral follicle counts and ovarian AMH and FSHR expression were significantly increased in the H group compared to those in the M and N groups. Through pharmacokinetic screening, we found 244 active compounds in MGNC. A total of 186 candidate intersection target genes of disease and MGNC were further screened to construct the interaction network. Gene ontology and KEGG pathway enrichment analysis ultimately unveiled a series of key targets that mainly mediated the effects of MGNC on DOR induced by chronic stress. The PI3K-Akt pathway may serve as the critical pathway underlying this therapeutic mechanism.

Conclusion

MGNC is a promising formula to treat DOR induced by chronic stress, and the PI3K-Akt pathway may play an essential role in this effect.

Keywords:

• modified Gengnianchun formula
• diminished ovarian reserve
• stress
• follicle development
• network pharmacology
• target gene

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Therapeutic Effects of Modified Gengnianchun Formula on Stress-Induced Diminished Ovarian Reserve Based on Experimental Approaches and Network Pharmacology [Erratum]

Acknowledgment

This work was supported by the National Natural Science Foundation of China (grant no. 81273956) and the Science and Technology Commission of Shanghai Municipality (grant no. 18401932000, no. 15401931800). The authors would also like to thank Mr. Junhao Gao for his generous support of the coding work in this study, and all the colleagues who have contributed to this research are appreciated.

Abbreviations

MGNC, Modified Gengnianchun; GNC, Gengnianchun; TCM, Traditional Chinese Medicine; DOR, Diminished ovarian reserve; CORT, Corticosterone; FSH, Follicle-stimulating hormone; FSHR, FSH receptor; LH, Luteinizing hormone; AMH, Anti-Mmüllerian hormone; E2, Estradiol; T, Testosterone; CUS, Chronic Unpredictable stress; H&E, Hematoxylin and eosin; TBST, Tris-buffered saline tween; OB, Oral bioavailability; DL, Drug likeness; ADME, Absorption, distribution, metabolism, and excretion; NS, Normal saline; PPI, Protein–protein interaction; GO, Gene ontology; BP, Biological process; CC, Cellular components; MF, Molecular functions; KEGG, Kyoto encyclopedia of genes and genomes; PI3K, Phosphatidylinositol-4,5-bisphosphate 3-kinase; AKT, Protein kinase B; AGE, Advanced glycation end products; RAGE, Receptor of advanced glycation end products; PTEN, Phosphatase and tensin homolog; GPCR, G protein-binding receptor; NF-κB, Nuclear factor kappa b; POI, Primary ovarian insufficiency; FOR, Functional ovarian reserve; TOR, Total ovarian reserve; VCD, 4-vinylcyclohexene diepoxide; APP, Amyloid beta precursor protein; IL1B, Interleukin 1 beta; IL2, Interleukin 2; IL6, Interleukin 6; TNF, Tumor necrosis factor; INFG, Interferon gamma; MAPK, Mitogen-activated protein kinase 1; CASP3, Caspase 3; BAX, BCL2 associated x, apoptosis regulator; PARP1, Poly (adp-ribose) polymerase 1; SOD1, Superoxide dismutase 1; CREB, CAMP response element binding protein; BDNF, Brain-derived neurotrophic factor.

Ethics Approval

All experimental protocols were carried out according to the National Institutes of Health (NIH) guidelines. The research was approved by the Institutional Animal Care and Use Committee of Animal Ethics of the School of Traditional Chinese Pharmacy, China Pharmaceutical University.

Disclosure

The authors report no conflicts of interest in this work.