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Abstract

Objective

To identify the principal constituents of Nourishing Yin Formula, analyze its active substances and effective targets for treating DOR, and explore its potential mechanism of action.

Methods

Results

After screening, the primary chemical constituents of Nourishing Yin Formula for treating DOR were identified, including progesterone, beta-sitosterol, 17-Beta-Estradiol, quercetin, kaempferol, etc Among the 52 critical targets for Nourishing Yin Formula in DOR treatment, the core targets were AKT1, INS, IL6, TNF, EGFR, ESR1, MYC, CTNNB1, NOS3, ERBB2, EDN1, and ESR2.

Conclusions

The potential mechanism of Nourishing Yin Formula in the treatment of DOR may involve the herbal compound's impact on AKT1, INS, IL6, TNF, and EGFR pathways. It regulates ovarian granulosa cell proliferation and apoptosis, and restores ovarian blood supply.

Keywords

nourishing yin formula network pharmacological study diminished ovarian reserve follicular development treatment

Introduction

Diminished ovarian reserve (DOR) is the term used to describe a decrease in follicles within the ovarian cortex, a diminished ability for the ovary to produce eggs, and a reduction in the quality of the follicles, which results in decreased sex hormone production and reduced fertility in women. It is characterized by reduced menstrual flow, prolonged periods, dripping vaginal bleeding, and prolonged infertility, which further leads to premature ovarian failure and adversely affects the quality of life of women.^1,2 In recent years, DOR in women has become a hot topic of global concern due to the increasing pressure of life and the deteriorating living environment. It has a negative impact on women's fertility, significantly increases medical costs and physical and psychological burdens, and negatively impacts their quality of life.^3,4 Relevant studies have shown that DOR remains a challenging clinical problem in reproductive medicine, and finding reasonable and effective treatment options is particularly important for improving ovarian function, enhancing women's quality of life, and reducing their infertility.

DOR has few clinical options at present, and hormone replacement therapy is the most common treatment for DOR in Western medicine. In addition to its adverse effects such as increased risk of stroke, breast cancer, endometrial cancer, and other diseases, its long-term therapeutic effect is not ideal. However, TCM treatment for DOR restores ovulation, establishes natural cycles, has fewer side effects, and possesses a good prospect in terms of safety, effectiveness and low toxicity.^5,6 Based on our extensive experience in treating DOR with TCM, we have found that sequential therapy with nourishing Yin and tonifying Yang can enhance follicle development, improve ovum quality, establish a natural cycle, and then significantly improve ovarian growth and development.^7–10 Based on the “adjustment of menstrual cycle rhythm” by a TCM master Professor Xia Guicheng, the sequential therapy of nourishing Yin and tonifying Yang formula has been proposed by Professor Tan Yong of Nanjing University of Chinese Medicine, and it simplifies the classification of “four, five, and seven phases” of the menstrual cycle into two phases of post-menstruation and pre-menstruation. By sequentially treating reproductive endocrine diseases such as DOR with nourishing Yin and tonifying Yang formulas, the method has shown satisfactory clinical results.

This therapy utilizes the Nourishing Yin Formula, which consists of Angelica Sinensis 10 g, Cornus Officinalis 6 g, Radix Paeoniae Alba 10 g, Semen Cuscutae 12 g, Rehmanniae Radix Praeparata 10 g, and Placenta Hominis 10 g, while the tonifying Yang formula consists of Morinda officinalis 10 g, Dipsacus asper 15 g, Psoralea corylifolia 10 g, Epimedium 10 g, Codonopsis pilosula 10 g, and Chinese yam 15 g. The postmenstrual period is the material foundation of the menstrual cycle. When Nourishing Yin Formula is used during this time, the eggs are nourished, follicles are developed, and a foundation for ovulation is laid. During the post-menstrual period, herbs of nourishing Yin and Blood such as Angelica Sinensis, Cornu Cervi Pantotrichum, Radix Paeoniae Alba and Rehmannia Glutinosa are used in the treatment. In spite of this, the mechanism of action of Nourishing Yin Formula for DOR is still unclear and requires further study. In this study, a network pharmacological method was used to investigate the mechanism of action and target of action of Nourishing Yin Formula for the treatment of DOR, in order to provide a theoretical basis for the clinical application of the formula.

Data Sources and Research Methods

Collection of Chemical Components and Target Prediction of Nourishing Yin Formula

As part of the Traditional Chinese Medicine Database and Analysis Platform¹¹ (TCMSP, https://tcmsp-e.com/), all active ingredients of Angelica Sinensis, Cornus Officinalis, Radix Paeoniae Alba, Semen Cuscutae, Rehmanniae Radix Praeparata, and Placenta Hominis were assessed for oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. They were retrieved and screened for active ingredients. We calibrated the collated targets using Uniprot (https://www.uniprot.org/) data, excluded non-human genes, and removed invalid duplicate targets. For drugs that could not be retrieved from TCMSP, candidate genes were obtained using the BATMAN-TCM database (http://bionet.ncpsb.org.cn/batman-tcm/) to obtain plausible proteins. The collated targets were calibrated by Uniprot (https:// www.uniprot.org/) data, non-human genes were excluded, and invalid duplicate targets were removed to obtain standardized gene names.

Acquisition of Targets Associated with DOR

Through the use of the keywords “decreased ovarian reserve” and “diminished ovarian reserve” in GeneCards (https://www.genecards.org/) and OMIM (https://www.omim.org/), we were able to obtain the disease-related targets by combining all the targets in an Excel spreadsheet. Using the Uniprot database, the gene information of disease targets was obtained by excluding duplicate genes.

Prediction Results of Drug-Disease Targets

Drug-component targets were mapped to disease targets, and Veen plots were made to determine which genes intersected. As a next step, the network of drug-components-targets was constructed using Cytoscape 3.7.2 software.

Network Construction of Target Protein Interactions

To further investigate protein-protein interactions (PPI) in the treatment of DOR with the Nourishing Yin Formula, the drug-interacting genes were uploaded to the String (https://string-db.org/) database for PPI. To ensure the credibility of this study, the species was set to “Homosapiens” with a minimum interaction score of 0.7. The remaining parameters were kept as default settings. The results were stored in TSV format and imported into Cytoscape 3.7.2, which analyzed the network (Cytoscape→Tools→Networkanalyzer→Networkanalysis→Analyzenetwork), and then saved. Degree was reflected in the size of the node and the color response: the larger the node, the greater the value of Degree. Edge thickness reflected the size of the Combinescore: the thicker the edge, the higher the Combinescore. The core targets were selected to make the PPI graph.

GO Enrichment Analysis and KEGG Pathway Analysis

DAVID (https://david.ncifcrf.gov/summary.jsp) was used to upload drug-disease intersection genes. The gene identifier was set to OFFICIAL_GENE_SYMBOL and the species was set to HomoSapiens. The gene function of DAVID6.8GO was used to annotate the role of target proteins in the gene function of Nourishing Yin Formula for the treatment of DOR from three perspectives, namely biological process (BP), cellular component (CC), and molecular function (MF). The KEGG pathway enrichment analysis in the signaling pathway was performed to elucidate the targets of Nourishing Yin Formula for the treatment of DOR. There were 20 KEGG pathway entries associated with DOR and the top 10 GO functions of BP, CC, and MF were selected as the main gene function enrichment processes and signaling pathways of Nourishing Yin Formula for the treatment of DOR, and its mechanism of action was predicted.

Molecular Docking

In general, the higher the Degree value in a network, the more significant it is. Thus, the protein with the highest Degree value in the PPI network plays an important role in the treatment of DOR with drugs. To verify the interactions between drug active ingredients and key targets, AutoDock Vina (1.1.2) was used to perform molecular docking. It consists of the following steps: (1) download the compound in mol2 format from TCMSP official website, pour it into Chembio3D for energy minimization, then import AutodockTools-1.5.6 to add hydrogen, calculate charge, assign charge, set rotatable keys and save it as “pdbqt” format; (2) download key target proteins from PDB (http://www.rcsb.org/) database (human-derived proteins are preferred, those with high structural similarity between the original ligand and the active ingredient to be docked are preferred, and those with high resolution are selected); (3) pour the proteins into PyMoL (2.3.0) to remove the original ligands and water molecules, and then import the proteins into AutoDocktools (v1.5.6) for hydrogenation, charge calculation, charge assignment, specifying the atom type and saving in “pdbqt” format; (4) use the original protein ligand as the center of the docking box, or in the absence of the original ligand, use the area near the reported key amino acid residues as the docking area, and set the size of the grid box to 60 × 60 × 60 (the spacing of each lattice point 0.375 Å), and the rest parameters set as default; and (5) perform interaction pattern analysis using PyMOL and Ligplot.

Experiment Validation

The rats were divided into three groups: normal group, model group, and beta-sitosterol group, with 10 rats in each group. The model group and the beta-sitosterol group constructed the DOR rat models. We chose 3-month-old female SD rats, dissolved deoxyethylcyclohexene (VCD) in sesame oil and injected VCD 80 mg/kg intraperitoneally once a day for 15 consecutive days. Except for the normal group, all other rats were intraperitoneally injected with VCD 80 mg/kg. The normal group was intraperitoneally injected with equal volume of sesame oil once a day for 15 consecutive days. Starting from the 16th day, rats in the beta-sitosterol group were given 150 mg/kg daily, once a day, for a total of 15 days. The normal group and the model group were given 10 g/kg physiological saline by gavage once a day for a total of 15 days. After 15 days of administration, the rats were euthanized, and ovarian tissues were taken by laparotomy to detect the following indicators.

① Ovarian morphology

We made ovarian paraffin sections with thickness of 5 μm and used the HE staining kit to observe the morphological changes of rat ovarian tissues under a microscope. Ovarian tissues of rats were first immersed into the dehydrator, and dehydrated with gradient alcohol, followed by wax dipping and tissue embedding in the embedding machine. Then, microtome knives were then installed on the holder of the microtome, and the sliced paraffin sections were gently laid on the water surface of the water bath. The paraffin sections floated in the warm water and unfolded naturally and smoothly after the heating. Then, the slices were temporarily dried at the room temperature and baked in a 40 °C constant temperature oven for 0.5–2 h. The sections were dewaxed by treatment with Xylene I and xylene II (Xylene I is the same substance as xylene II. Xylene I means first using xylene to dewax the slices by one container, and xylene II means second using xylene to dewax the slices by another container), hydrated by gradient alcohol, stained with Hematoxylin, rinsed with tap water, differentiated with the differentiation fluid, immersed in the tap water, stained with eosin, washed with tap water and dehydrated with the gradient alcohol, followed by transparent treatment with xylene I and xylene II and neutral gum sealing. The sections were dried in an oven at 60 °C and observed under a microscope. Finally, we randomly selected the visual fields and took photos (200X).

② PCR detection of gene levels

We used PCR to detect PI3 K/AKT signaling pathway factors and INS in rat oocytes, including the expression of PI3 K, Akt1, mTOR and INS. We used a glass grinder to grind ovarian tissues, extracted RNAs from the tissues and measured RNA concentration. After reverse transcription into cDNA, GAPDH was used as the internal reference to detect RNA expression. The Ct value was obtained by amplifying the curve, using 2^{− ΔΔ Ct} method for relative quantitative analysis.

Statistical Analysis

SPSS 24.0 software was used for data processing. Differences between multiple groups were examined with one-way analysis of variance for those that complied with a homogeneity of variance post hoc test. Non-parametric test was adopted for those that did not comply with homogeneity of variance. P < 0.05 was considered to indicate a statistically significant difference.

Research Results

Prediction of Active Ingredients and Targets of Nourishing Yin Formula

A total of 2 components of Angelica sinensis, 14 components of Cornus officinalis, 7 components of Paeonia lactiflora, and 10 components of Cuscutae were collected after screening by TCMSP with OB ≥ 30%, DL ≥ 0.18 and excluding invalid components. When Rehmanniae Radix Praeparata and Placenta Hominis failed to incorporate components after being screened by TCMSP, 4 and 15 components were obtained by BATMAN-TCM database under the condition of a Scorecutoff > 20, respectively. From all drug components, 45 components were identified.

Targets Associated with Decreased Ovarian Reserve Function

The keywords “decreased ovarian reserve” and “diminished ovarian reserve” were searched in GeneCards and DisGeNET databases, and after deduplication 349 and 246 targets were obtained, respectively. The obtained genes were corrected by Uniprot database. Based on the intersection of the drug targets with the DOR target genes, 52 intersection target genes were identified as interactive target genes of drug treatment for DOR (Figure 1).

Figure 1.

Drug-disease venn diagram.

Drug-Component-Target Prediction Results

Based on the drug-component-target data, a “network.xlsx” file and a “type.xlsx” file were created and imported into Cytoscape 3.7.2 for mapping. The Degree network contains 87 nodes and 225 edges, with the top five components being progesterone, beta-sitosterol, 17-beta-estradiol, quercetin, and kaempferol (Figures 2 and 3) (Sorted by degree value from front to back).

Figure 2.

Drug-component-target diagram.

Figure 3.

Drug-component-target diagram.

Note: Rectangle indicates the target, oval the drug component, and diamond node the Chinese medicine.

Core Targets and Network Interactions

Based on the intersection of all drugs’ action targets with DOR's target genes, 52 intersecting target genes of DOR and drugs were identified as the interaction target genes of drugs. The 52 intersecting target genes were imported into the String database (https://string-db.org/) for PPI prediction, with species set as the HomoSapiens and the confidence level set at 0.4. The network file was saved in TSV format, and the TSV file was imported into Cytoscape 3.7.2 software to plot the protein interaction network, which consisted of 60 nodes and 676 edges. Topological analysis of the network was performed. The degree-value reflected the size and color of the target point, and the combinedscore value reflected the thickness of the edge. As shown in the Figure 4, the PPI network was constructed. Among them, the core targets were AKT1, INS, IL6, TNF, EGFR, ESR1, MYC, CTNNB1, NOS3, ERBB2, EDN1, ESR2. AKT1 is the most crucial target.

Figure 4.

Protein-protein interaction network diagram.

Biofunctional Enrichment Analysis

GO Enrichment Analysis

GO gene function enrichment analysis was conducted on drug-disease intersection genes using the DAVID database. 312 GO entries were screened, with P < 0.05 as the screening criterion. As a result of the Nourishing Yin Formula, 230 major biological processes (BP) have been significantly enriched for the treatment of DOR, primarily involving negative regulation of apoptosis, signal transduction, positive regulation of gene expression and cell proliferation, negative regulation of gene expression, inflammatory response, positive regulation of MAPK cascade, glucose metabolic process, response to exogenous stimuli, and positive regulation of cell migration. There were 33 entries related to cellular components (CC), including plasma membrane, cytoplasm, nucleus, cytosol, membrane, nucleoplasm, extracellular region, extracellular gap, macromolecular complex, and perinuclear region of cytoplasm. The molecular functions (MF) were discussed in 49 entries, including the binding of proteins, identical proteins, and so on. (Figures 5 and 6).

Figure 5.

Go histogram.

Figure 6.

Go bubble plot.

2 KEGG Pathway Enrichment Analysis

Pathway enrichment analysis was performed using the DAVID database, and 152 pathways were screened according to P < 0.05 for the treatment of DOR with the Nourishing Yin Formula, and the pathways associated with DOR were the HIF-1 signaling pathway, human cytomegalovirus infection, pathways in cancer, endocrine resistance, estrogen signaling pathway, and so on. (Figures 7 and 8).

Figure 7.

KEGG histogram.

Figure 8.

KEGG bubble plot. Note: The size of the circles represents the data of the genes enriched in the corresponding pathways, and progressively smaller P values are denoted from green to red.

In order to draw the bubble plot, the top 20 KEGG metabolic pathways were screened based on the P value. The horizontal axis represents the number of genes enriched to the pathway, the size of the bubble represents the number of genes enriched to the corresponding pathway, and the color shade represents the significance, demonstrating the significance of the enrichment.

Results of Molecular Docking

From the previous analysis, the first five important targets were selected for semi-flexible docking with compounds of higher degree, and binding energy (affinity) indicated how well a small molecule binds to the target. If the affinity value is less than 0, then the small molecules can bind freely to the target proteins. The smaller the value, the greater the probability of binding.

In the docking results, all small molecules entered the active center of target proteins, and the small molecule with the best docking with each protein was chosen for graphical display. Beta-sitosterol formed hydrogen bonds with Glu91 (A) and Glu95 (A) of AKT1, which had lengths of 3.08 Å and 3.08 Å, respectively. 17-Beta-Estradiol formed hydrogen bonds with Pro770 (A), Met769 (A), Gly772 (A), Lys721 (A), and Glu738 (A) of EGFR, with hydrogen bond lengths of 3.99 Å, 3.13 Å, 3.85 Å, 3.07 Å, and 2.92 Å, respectively. Hydrogen bonds were formed between progesterone and Arg182 (A) of IL6, with a length of 3.20 Å. Beta-sitosterol formed hydrogen bonds with Gly8 (D) of INS, with a hydrogen bond length of 3.18 Å. Progesterone formed hydrogen bonds with Arg98 (C) and Asn112 (A) of TNF, with hydrogen bond lengths of 3.80 Å and 3.13 Å, respectively. The docking results for all of these molecules and their surrounding amino acid residues were summarized in Table 1 and Figures 9–13.

Figure 9.

Analysis of the interaction pattern between beta-sitosterol and AKT1 protein.

Figure 10.

Analysis of the interaction pattern of 17-Beta-Estradiol with EGFR protein.

Figure 11.

Analysis of the interaction pattern of Progesterone and IL6 protein.

Figure 12.

Analysis of the interaction pattern of beta-sitosterol and INS protein.

Figure 13.

Analysis of the interaction pattern of progesterone and TNF protein.

Table 1.

Docking Results of Core Small Molecules and Core Target Proteins.

Targets	PDB ID	compound	Affinity（kcal/mol）	Targets	PDB ID	compound	Affinity（kcal/mol）
IL6	1ALU	quercetin	−7	TNF	2E7A	quercetin	−9.1
		beta-sitosterol	−6.8			beta-sitosterol	−9
		kaempferol	−6.8			Kaempferol	−9
		Progesterone	−7.4			Progesterone	−9.2
		17-Beta-Estradiol	−7.3			17-Beta-Estradiol	−8.4
AKT1	1UNQ	quercetin	−6	EGFR	2GS2	quercetin	−7.7
		beta-sitosterol	−7.2			beta-sitosterol	−7.2
		kaempferol	−5.9			Kaempferol	−7.5
		Progesterone	−7.1			Progesterone	−8
		17-Beta-Estradiol	−6.9			17-Beta-Estradiol	−8.4
INS	8EZ0	quercetin	−6
		beta-sitosterol	−8
		kaempferol	−6.1
		Progesterone	−7.3
		17-Beta-Estradiol	−6.9

Experiment Validation

① Ovarian morphology as observed by HE staining (Figure 14)

The model group exhibited decreased ovarian volume and reduced quantities of growing follicles, indicating degraded reserve function of ovaries. However, compared with the model group, the TCM group exhibited increased ovarian volume and enhanced quantities of growing follicles, suggesting that Nourishing Yin Formula could prevent the decline of ovarian reserve function to a certain extent.

Figure 14.

He staining results of ovarian tissues of the three different groups.

Through previous analysis, we found that AKT1 and INS were the most critical target. Through molecular docking experiments, we discovered that AKT1 and INS had the highest molecular binding rate with beta-sitosterol, so we chose beta-sitosterol for the study.

② Gene expression of PI3 K/AKT signaling pathway and INS (Figure 15)

Figure 15.

The levels of ovarian genes of the three different groups. Note: (1) The normal group versus the model group, *P < 0.05; (2) The normal group versus the TCM group, ^&P < 0.05; (3) The model group versus the TCM group, ^#P < 0.05.

The levels of PI3 K, Akt1, mTOR and INS in ovaries of the normal group were significantly higher than those of the model group (P < 0.05). However, compared with the model group, the TCM group exhibited significantly elevated levels of PI3 K, Akt1, mTOR and INS (P < 0.05), thereby suggesting that Nourishing Yin Formula could significantly activate PI3 K/AKT signaling pathway and INS of rat oocytes.

③ Safety evaluation

After the experiment on three groups of rats was completed, we observed the adverse reactions of rats in each group. The results showed that no obvious adverse reactions such as rash and vomiting were observed in the three groups of rats.

Discussion

In modern medicine, hormone replacement therapy is commonly used to treat DOR, but it has numerous side effects, including an increased risk of strokes, breast cancer, blood clots, and other diseases, making it unsuitable for long-term use. Chinese medicine, on the other hand, has proven to be more effective in treating DOR, restoring normal menstrual cycles, and improving ovarian reserve function with fewer side effects. Sequential therapy with nourishing Yin and tonifying Yang formula involves using Nourishing Yin Formula during the post-menstrual period and tonifying Yang formula during the pre-menstrual period to treat DOR and other diseases in a sequential manner, demonstrating good clinical efficacy. This therapy consists of Angelica Sinensis, Cornus Officinalis, Radix Paeoniae Alba, Semen Cuscutae, Rehmanniae Radix Praeparata, and Placenta Hominis, which nourish the eggs, promote the development of follicles, improve endometrial tolerance, increase endometrial thickness, and strengthen kidney essence. Additionally, it nourishes Yin, blood, and tonifies kidney essence.

The main chemical components in the Nourishing Yin Formula for the treatment of DOR obtained through screening are progesterone, beta-sitosterol, 17-beta-estradiol, quercetin, kaempferol, etc, suggesting that the above components may play a key role in DOR treatment. Among them, quercetin can improve ovarian reserve function, regulate sex hormone secretion, enhance follicle quality, and inhibit granulosa cell apoptosis.^12–14 Also, beta-sitosterol can elevate estradiol levels in the ovarian granulosa cells and prevent bone loss associated with low estrogen levels.^15,16 In sheep antral sinus follicles cultured in vitro, kaempferol can promote primordial follicle activation via phosphatidylinositol 3-kinase/protein kinase B signaling and reduce DNA fragmentation.¹⁷

In this study, we screened 52 key targets of Nourishing Yin Formula for DOR treatment through a network pharmacology approach, among which AKT1, INS, IL6, TNF, EGFR, ESR1, MYC, CTNNB1, NOS3, ERBB2, EDN1, and ESR2 were the core targets. Based on the results of GO enrichment analysis, the Nourishing Yin Formula for the treatment of DOR is primarily composed of negative regulation of apoptosis, signal transduction, positive regulation of gene expression, positive regulation of cell proliferation, negative regulation of gene expression, inflammatory response, positive regulation of MAPK cascade, glucose metabolic process, response to exogenous stimulation, and positive regulation of cell migration. The shared targets are distributed within the nuclear membrane, nucleus, cytoplasm and other structures, and their main functions are related to protein binding, identical protein binding, metal ion binding, ATP binding, enzyme binding, homodimerization activity, zinc ion binding, hormone activity, kinase activity, ubiquitin protein ligase binding, etc KEGG signaling pathway enrichment analysis mainly involves HIF-1 signaling pathway, human giant cell virus infection, pathways in cancer, endocrine resistance, estrogen signaling pathway, regulation of adipocyte lipolysis, proteoglycans in cancer, chemo-oncogenic-receptor activation, cAMP signaling pathway, relaxin signaling pathway, AGE-RAGE signaling pathway in diabetic complications, breast cancer, EGFR tyrosine kinase inhibitor resistance, hepatitis B, endometrial cancer, the PI3K-Akt signaling pathway, calcium signaling pathway, platinum resistance, Rap1 signaling pathway, and growth hormone synthesis, secretion and action.

According to the results of molecular docking, the core ingredients of the Nourishing Yin Formula have a high docking energy and are stable in binding to the targets AKT1, INS, IL6, TNF, and EGFR. In all stages of follicular development, the PI3 K/AKT pathway may play a regulatory role in the activation of the primordial follicle, the proliferation and differentiation of granulosa cells, and the differentiation of granulosa cells. There is evidence that PI3 K/AKT is involved in the proliferation, differentiation, and apoptosis of cells.^18,19 INS promotes follicular growth and development during the primordial follicular phase, sinus follicles, and subsequent developmental stages.²⁰ Inflammatory responses, cell proliferation, and angiogenesis are affected by IL-related pathways, which are also involved in the development of DOR. IL-6, as a multifunctional cytokine, plays a role in promoting the proliferation and differentiation of cells. Research on the immune mechanism of DOR focuses on IL-6 and related pathways. An increase in IL-6 levels may result in the activation of MAPK and PI3 K, the promotion of abnormal cell proliferation, and the premature depletion of the follicular pool.²¹ TNF signaling pathway is involved in oxidative stress, inflammatory response, cell proliferation, differentiation, and apoptosis, and plays a crucial role in signaling pathways related to ovarian function.²² EGFR pathway plays an important role in ovarian function, is involved in the proliferation and apoptosis of ovarian granulosa cells, and controls follicular growth and development.²³

In this study, through core targets and network interactions, AKT1 and INS are most likely the main target of Nourishing Yin Formula in treating DOR. Therefore, this study selected the PI3 K/AKT signaling pathway and INS for validation. Through experimental research, we found that Nourishing Yin Formula could improve ovarian reserve function by activating the PI3 K/AKT signaling pathway and INS through the main chemical component beta-sitosterol in the formula.

This study verified the PI3 K/AKT signaling pathway and INS, but the workload of verification was not sufficient. Meanwhile, other pathways were not verified in this study. In subsequent work, we will verify other pathways while increasing the sample size and detection indicators.

To sum up, Nourishing Yin Formula may have a potential mechanism for treating DOR by influencing AKT1, INS, IL6, TNF, and EGFR pathways, improving follicular development, regulating hormone secretion, reducing oxidative stress and inflammation on ovarian tissues, increasing ovarian angiogenesis, and restoring ovarian blood supply, among other things. The results of the network pharmacological analysis confirm the advantages of multi-targeted treatment of DOR with Chinese herbal compounds, and provide ideas and theoretical foundations for further investigation of the specific mechanism through basic experiments. In future research, we can further verify the efficacy and mechanism of Nourishing Yin Formula in treating decreased ovarian reserve through clinical trials, animal and cell experiments.

Footnotes

Acknowledgments

Not applicable.

Abbreviated Names

Ma QW, Gao YH, Wu JF, Shen MX, Ye CS, Tan Y, Chen WJ.

Authors’ Contributions

MQW, Gao YH and Tan Y conceived and designed research. Wu JF, Shen MX, Ye CS and Chen WJ carried out the bioinformatic analysis. MQW wrote the manuscript. All authors read and approved the manuscript.

Availability of Data and Materials

The analyzed datasets generated during the study are available from the corresponding author on reasonable request.

Consent to Publish

All authors consent to publish this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

All experimental protocols involving animals were approved by the Ethics Committee of Hangzhou Ninth People,s Hospital (Hangzhou, China) (The ethics number: 2022-030).

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Zhejiang Provincial Natural Science Foundation (grant number: LQ23H270018), National Natural Science Foundation (grant number: 82405452), Zhejiang Technology Program Project of Medicine and Health Science (grant number: 2023RC071), Zhejiang Technology Program Project of Traditional Chinese Medicine (grant number: 2023ZL127); Open project of Zhenjiang Clinical Medical Research Center for gynecological diseases of traditional Chinese medicine (grant number SS202204-KFB01), Zhenjiang Social Development of Key Research Project (grant number SH2021025).

Supporting information

The primary active constituents of Nourishing Yin Formula and their putative targets for treating DOR were identified through a search of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (BATMAN-TCM), GeneCards, OMIM, and Uniprot databases. Nourishing Yin Formula may have a potential mechanism for treating DOR by influencing AKT1, INS, IL6, TNF, and EGFR pathways, improving follicular development, regulating hormone secretion, reducing oxidative stress and inflammation on ovarian tissues, increasing ovarian angiogenesis, and restoring ovarian blood supply, among other things. The results of the network pharmacological analysis confirm the advantages of multi-targeted treatment of DOR with Chinese herbal compounds, and provide ideas and theoretical foundations for further investigation of the specific mechanism through basic experiments.

Statement of Human and Animal Rights

In the manuscript, All experimental protocols were approved by the Ethics Committee of Hangzhou Ninth People,s Hospital (Hangzhou, China). Experiments on animals were demonstrated to be ethically acceptable and in accordance with institutional and national guidelines or regulations for laboratory animals.

Statement of Informed Consent

Not applicable.

ORCID iDs

Qianwen Ma

Wenjun Chen

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A Network Pharmacological Study on the Treatment of Diminished Ovarian Reserve with Nourishing Yin Formula

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Data Sources and Research Methods

Collection of Chemical Components and Target Prediction of Nourishing Yin Formula

Acquisition of Targets Associated with DOR

Prediction Results of Drug-Disease Targets

Network Construction of Target Protein Interactions

GO Enrichment Analysis and KEGG Pathway Analysis

Molecular Docking

Experiment Validation

Statistical Analysis

Research Results

Prediction of Active Ingredients and Targets of Nourishing Yin Formula

Targets Associated with Decreased Ovarian Reserve Function

Drug-Component-Target Prediction Results

Core Targets and Network Interactions

Biofunctional Enrichment Analysis

GO Enrichment Analysis

Results of Molecular Docking

Experiment Validation

Discussion

Footnotes

Acknowledgments

Abbreviated Names

Authors’ Contributions

Availability of Data and Materials

Consent to Publish

Declaration of Conflicting Interests

Ethical Approval

Funding

Supporting information

Statement of Human and Animal Rights

Statement of Informed Consent

ORCID iDs

References