Evaluation of the mechanism of Rujiling capsules in the treatment of hyperplasia of mammary glands based on network pharmacology and molecular docking.

OBJECTIVE: The present study aimed to elucidate the molecular network mechanism of the Rujiling capsule in the treatment of hyperplasia of mammary glands through network pharmacology and molecular docking.
MATERIALS AND METHODS: TCMSP and TCMID databases were screened for the active components and their action targets of the Rujiling capsule, whereas the disease targets of hyperplasia of mammary glands were searched in GeneCard and DisGeNET databases. Venny software was employed to identify the common targets of drugs and diseases. Cytoscape software was used to construct the network pharmacological diagram of "drug-active components-target" and the intersection targets were subjected to protein-protein interaction analysis by STRING platform and Cytoscape software. The DAVID database was exploited for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of the intersection target. After that, the key target genes with a degree value greater than the median were verified with the active components in molecular docking.
RESULTS: A total of 691 drug targets, 251 disease targets, and 108 intersection targets were obtained after retrieval and screening. Among the 686 items enriched by GO included 522 biological processes, 110 molecular functions, and 54 cellular components. At the same time, 114 signal pathways were enriched by KEGG. The results of molecular docking revealed that the docking energies of main active components and some core targets were all <-5 kcal/mol.
CONCLUSION: Henceforth, highlighted the role of the Rujiling capsule in the treatment of hyperplasia of mammary glands through multiple components, multiple targets, and multiple signal pathways.

Introduction

Hyperplasia of mammary glands reflects the highest prevalence among female breast diseases in females. Its incidence is increasing, and the age of onset is reducing gradually. The incidence is nearly more than 90% in premenopausal women over 40 years old.[1] The clinical symptoms blight about 20% of women. Statistics reveal that the canceration rate varies from 1.25% to 50%.[2] It eventually develops into breast cancer[34] if not treated timely. At present, severe hyperplasia should be appropriately resected, though there remains a high risk of relapse following surgical resection of local lesions. Western medicine primarily regulates endocrine disorders, but it often stimulates the gastrointestinal tract, and long-term use of it is likely to indulge in adverse reactions.[1] As one of the internal treatment methods of traditional Chinese medicine, Rujiling finds widespread clinical application. This pure traditional Chinese medicine preparation is known for its safety and efficacy for the management of hyperplasia of mammary glands.[5] The mechanism behind this therapeutic implication of Rujiling mostly remains unknown. The difference in the drug's action mechanism makes it suitable for various types of patients with breast hyperplasia. Therefore, elucidating the action mechanism of Rujiling can provide the foundation for the clinical treatment of breast hyperplasia. At present, there is no research on the mechanism of Rujiling in the treatment of hyperplasia of mammary glands at home and abroad. The method of network pharmacology has been successfully employed in the research of the active components and action mechanisms of traditional Chinese medicine[6] by the rapid development of bioinformatics. The present study investigates the active components and protective molecular mechanism of Rujiling with the help of network pharmacology to clarify its medicinal value and provide a sound basis for the clinical application of Rujiling in the treatment of breast hyperplasia.

Materials and Methods

Screening of active components in Rujiling capsules

The TCMSP and TCMID databases were searched for the active components of principal Chinese herbal medicinal ingredients of Rujiling capsule, including Rhizoma cyperi, Radix bupleuri, Pericarpium citri, Reticulatae viride, Radix salviae miltiorrhizae, Radix paeoniae rubra, Caulis spatholobi, Vaccaria segetalis (fried), oyster, seaweed, kelp, Herba epimedii, and dodder. The active components of the Rujiling capsule were screened in light of the rules of DL ≥0.18 and OB ≥30%.

Acquisition of corresponding targets for active components of Rujiling capsule

The active components found in 1.2 were searched in turn in the TCMSP database as keywords to obtain the corresponding target proteins. The target protein was transformed into the corresponding gene name in the UniProt database, and the regulatory target gene of the Rujiling capsule was obtained after removing nonhuman target genes and duplicate.

Target acquisition of hyperplasia of mammary glands

Mammary hyperplasia (or hyperplasia of mammary glands, atypical ductal breast hyperplasia, atypical lobular breast hyperplasia, ductal breast hyperplasia of usual type, and breast hyperplasia) was used for searching in GeneCards and DisGeNET databases to screen the related gene targets with a relevance score ≥10.

Construction of “traditional Chinese medicine-active ingredient-target” network

The Wayne diagram was drawn with the help of the Venny software. It was adopted to map the target of the Rujiling capsule to the target of the disease. After that, the network diagram of “traditional Chinese medicine-active ingredient-target” was plotted using the Cytoscape 3.8.0 software by mapping the common target to traditional Chinese medicine components and active components.

Construction of protein-protein interaction

The interaction protein of intersection targets was queried, and the protein–protein interaction of the active component target was constructed through the String database. The species was limited as “Homo sapiens” and the high credibility ≥0.7 was selected as the research scope. The network data were imported into Cytoscape3.8.0 software and the core targets and main active components were screened by topological analysis.

Gene ontology enrichment and Kyoto encyclopedia of genes and genomes pathway analysis

The gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of intersection targets were conducted using the DAVID database. The main pathway of Rujiling in the treatment of hyperplasia of mammary glands was obtained and visualized by the bar chart and bubble chart of GO enrichment and KEGG analysis drawn by the R software.

Verification of active component-core target molecular docking

The active components with a degree value over the median were used for molecular docking with the core target. ChemDraw constructed the two-dimensional structure of the compound, which was then imported into the Chem3D software to obtain a three-dimensional structure diagram and saved in mol2 format after optimization. The corresponding target proteins were achieved from the PDB database. The ligands were deleted using the PyMOL software, modified by AutoDock TOOLs for hydrogenation and dehydration, and saved in pdbqt format. All ligand and receptor files were saved in pdbqt format, and molecular docking was visualized using PyMol2.3.0 software.

Experimental Results

Active components and targets of Rujiling capsule

The databases were searched for Rujiling capsule-related 112 active components in total, whose potential targets were obtained. After correction and elimination of duplicate entry using the UniProt database, 691 potential gene targets were acquired.

Collection of targets related to breast hyperplasia

After retrieving 251 targets related to breast hyperplasia, they were intersected with 691 gene targets of active components of the Rujiling capsule, and overall 108 intersection targets were obtained [Figure 1].

Figure 1

Wayne diagram of intersection targets of active components and disease

To obtain the network diagram of “traditional Chinese medicine-active components-target,” the traditional Chinese medicine, active components, and intersection targets of Rujiling capsule were imported into Cytoscape software. In the network diagram, the blue oval represents the target, the red oval signifies the active component, whereas the diamond indicates the medicine composition. The network diagram revealed higher degrees for luteolin, kaempferol, quercetin, isoliquiritigenin, emodin, and apigenin, and the average energy was higher than the median value. It was speculated that these six components might be the principal active components of the Rujiling capsule in the management of breast hyperplasia, as illustrated in Figure 2 and Table 1.

Figure 2

Network diagram of “traditional Chinese medicine-active ingredient-target”

Table 1

Related information of the six main active compounds

MOL ID	Molecule name	MW	OB (%)	DL
MOL000422	Kaempferol	286.25	41.88	0.24
MOL000098	Quercetin	302.25	46.43	0.28
MOL001789	Isoliquiritigenin	256.27	85.32	0.15
MOL000006	Luteolin	286.25	36.16	0.25
MOL000472	Emodin	270.25	24.4	0.24
MOL000008	Apigenin	270.25	23.06	0.21

MW=Molecularweight, OB=Oral bioavailability, DL=Drug-likeness

Construction of protein-to-protein interaction network protein-protein interaction

The interaction among targets was investigated by importing all the 108 intersection targets into the STRING 3.8.0 database, and the files yielded by the STRING database were then visualized using Cytoscape software. The size of the degree value is denoted by the color of the circular node, reflecting darker color for a higher degree value. The degree values of TP53, AKT1, STAT3, EGF, VEGFA, estimated glomerular filtration rate (EGFR), INS, interleukin (IL) 6, MAPK8, MAPK1, CCND1, JUN, MAPK3, SRC, ESR1, MYC, tumor necrosis factor (TNF), and PTEN were all higher than the median, indicating that these targets were core targets of Rujiling capsule in the treatment of breast hyperplasia, as represented in Figure 3 and Table 2.

Figure 3

Protein-protein interaction network diagram of related targets of Rujiling capsule in the treatment of breast hyperplasia

Table 2

Characteristic parameters of the major core target network

Target	Degree	Betweenness centrality	Closeness centrality
TP53	64	0.149411244	0.707482993
AKT1	59	0.08161137	0.670967742
STAT3	50	0.045059144	0.626506024
EGF	49	0.03860266	0.619047619
VEGFA	48	0.027391709	0.615384615
EGFR	47	0.045472826	0.622754491
INS	46	0.046077056	0.611764706
IL6	44	0.025935971	0.604651163
MAPK8	44	0.036448057	0.615384615
MAPK1	43	0.015968206	0.597701149
CCND1	43	0.029565321	0.594285714
JUN	43	0.042270141	0.611764706
MAPK3	42	0.014737757	0.594285714
SRC	42	0.018418615	0.594285714
ESR1	39	0.034792202	0.587570621
MYC	38	0.009886295	0.577777778
TNF	37	0.007104548	0.571428571
PTEN	36	0.022126105	0.574585635

Gene ontology enrichment analysis of intersection targets

Totally, 686 items were enriched under the condition of enrichment threshold P < 0.05, which encompassed 522 biological processes, accounting for 76.09%, so they are regarded as the main process, essentially concerned with cellular response to chemical stress, gland development, response to steroid hormones, regulation of apoptotic signaling pathway, response to reactive oxygen species, etc., Besides, 110 molecular functions accounting for 16.03%, are mostly engaged in ubiquitin-like protein ligase binding, phosphatase binding, hormone receptor binding, cytokine receptor binding, and receptor-ligand activity. Furthermore, 54 cellular components, accounting for 7.87%, mainly involve membrane raft, membrane microdomain, membrane region, vesicle lumen, cytoplasmic vesicle lumen, and secretory granule lumen. These findings substantiate that Rujiling may act on breast hyperplasia by regulating multiple GO functions. As illustrated in Figure 4, R. Data software was employed to draw a bar chart of the top 10 biological processes, cellular compositions, and molecular functions of the P value.

Figure 4

Gene ontology enrichment analysis (the top 10 pathways)

Kyoto encyclopedia of genes and genomes enrichment analysis

KEGG pathway analysis screened a total of 144 signal pathways under the condition of threshold value P < 0.01, mainly concerned with Pathways in cancer, Hepatitis B, Proteoglycans in cancer, PI3K-Akt signaling pathway, and MicroRNAs in cancer. The top 20 pathways in terms of P value were opted to draw the bubble chart using R. Data software, as shown in Figure 5.

Figure 5

Analysis of Kyoto encyclopedia of genes and genomes pathways (the top 20 pathways)

Molecular docking

All 18 core targets of “traditional Chinese medicine-active ingredient-target” were docked with six main active components, with the binding energy <–5 kcal/mol designating good binding activity. In contrast, the binding energy <–7 kcal/mol indicated strong binding activity. The results claimed that the docking energies of kaempferol, quercetin, emodin, apigenin, and TP53, 6 active components with STAT3 and SRC, luteolin, quercetin, isoliquiritigenin, apigenin, and EGF, kaempferol, emodin, apigenin and JUN, luteolin, and TNF were all <–7 kcal/mol. The docking energies of isoliquiritigenin and TP53, kaempferol, quercetin, isoliquiritigenin, emodin, apigenin and AKT1, kaempferol, emodin and EGF, apigenin and EGFR; luteolin, isoliquiritigenin, apigenin, and INS were all <–5 kcal. The results of molecular docking are detailed in Table 3. To determine the binding activity between key targets and corresponding active components, the six resulting compounds with low docking energy are summarized in Figure 6.

Table 3

Binding energy between main active components and core targets

Chemical composition	TP53	AKT1	STAT3	EGF	VEGFA	EGFR	INS	IL6	MAPK8	MAPK1	CCND1	JUN	MAPK3	SRC	ESR1	MYC	TNF	PTEN
Luteolin 1	−4.3	−0.6	−7.8	−7.6	/	−3.4	−5.2	/	10.7	−0.5	/	/	/	−8.5	7.3	31.1	−8.0	/
Kaempferol 2	−7.7	−4.6	−7.6	−6.6	/	−4.8	−3.1	/	3.2	1	/	−7.6	/	−9.0	10.5	36.8	32.8	/
Quercetin 3	−7.3	−6	−8.0	−7.8	/	−3.3	−3.0	/	3.0	3.2	/	53.3	/	−9.1	10.6	46.5	32.3	/
Isoliquiritigenin 4	−6.5	−5.5	−7.2	−7.0	/	−4.1	−6.5	/	3.2	−1.2	/	−7.5	/	−7.5	0.8	5.0	25.9	/
Emodin 5	−7.7	−6.8	−7.8	−6.1	/	−4.4	1.1	/	13.1	−1.7	/	−4.7	/	−7.5	27.4	46.7	34.3	/
Apigenin 6	−7.3	−6	−7.7	−7.3	/	−5.4	−5.4	/	6	−1.3	/	−7.5	/	−8.8	7.8	29.8	31.8	/

"/ " Indicates that there is no binding energy between the corresponding active ingredient and the core target in the table

Figure 6

The optimal composite structure of the key target and active ingredients

Discussion

Clinical observation and animal experiments reported the promising effect of Rujiling capsule on breast hyperplasia.[56789] The present study revealed that the major main active components of Rujiling capsules against breast hyperplasia include luteolin, kaempferol, quercetin, isoliquiritigenin, emodin, and apigenin. The results also documented that the core targets of the Rujiling capsule against breast hyperplasia are TPp53, AKT1, STAT3, EGF, VEGFA, EGFR, INS, IL6, MAPK8, MAPK1, CCND1, JUN, MAPK3, SRC, ESR1, MYC, TNF, and PTEN. The results of molecular docking showed strong binding activity between kaempferol, quercetin, emodin, apigenin, and TP53, between the six compounds associated with STAT3 and SRC, between luteolin, quercetin, isoliquiritigenin, apigenin, and EGF, and between kaempferol, Emodin, Apigenin and JUN, and between luteolin and TNF. KEGG pathway analysis substantiated that TP53, STAT3, EGF, JUN, AKT1, and EGFR, which had strong binding activities with major compounds, are over-expressed in the pathways in cancer, while SRC and TNF are highly expressed in the pathway in proteoglycans in cancer. Over-expression of INS is mostly reported in the prostate cancer pathway. All of these are highly expressed in the cancer pathway.

The role of abnormal cell proliferation in the prognosis of both breast hyperplasia and breast cancer has been explored. The unregulated expression of the indicators related to cell proliferation and apoptosis has been demonstrated at the stage of breast hyperplasia, which evolves to breast cancer. Clinically, breast atypical hyperplasia can be regarded as the precancerous lesion of breast cancer. A correlation between breast hyperplasia and breast cancer in pathogenesis has been observed, especially in molecular regulatory pathways.[1011121314]

Seo HS et al.[3] established that a high concentration of quercetin, as a plant hormone, can induce the cleavage of polyribose polymerase by upregulating the levels of caspase-8 and caspase-3 proteins, thereby triggering exogenous apoptosis, that, in turn, inhibits the occurrence and development of breast cancer. Luteolin is reported to inhibit cell proliferation and invasion, induce cell apoptosis and cell cycle arrest, enhance drug resistance, and impede cancer cell metastasis.[1516] Lan Huangrong showed that luteolin-mediated inhibition of NF-κB/c-Myc activation could prevent telomerase activity. Furthermore, by inhibiting hTERT transcription, luteolin can hinder the proliferation, migration, and cell cycle progression of breast cancer and induce cell apoptosis.[15] Huang Sisuo reported some inhibitory effects of apigenin on the proliferation of breast proliferative cells MCF-10A.[17] The beneficial effects of kaempferol include promoting apoptosis of cancer cells, resisting cytotoxicity, inhibiting proliferation of cancer cells,[18] and protecting cells from oxidative stress damage.[192021222324] The results claimed a concentration-and time-dependent inhibition of proliferation of MCF-7 cells mediated by Kaempferol.[25] Isoliquiritigenin is a potential and effective anti-breast cancer drug that acts through multiple pathways. Many studies have validated the close association of the antibreast cancer effect of isoliquiritigenin to the inhibition of cell proliferation, the induction of cell apoptosis, the promotion of autophagy, and the inhibition of blood vessel growth in tumor tissues.[2627282930]

As a common tumor suppressor gene, P53 plays a key role in regulating apoptosis, angiogenesis, cell cycle, and genomic stability. Mutation in P53 proto-oncogene results in loss of function in the regulation of cell growth and differentiation. Overexpression of mutant P53 protein may lead to transforming cells into malignant types.[3132] Few studies have been conducted on the mutation of tumor suppressor gene P53 and overexpression of its protein in some breast cells at the stage of atypical hyperplasia. AKT1 can stimulate cell proliferation or reduce estrogen receptor-dependent effects and affect estrogen receptor phosphorylation, eventually causing endocrine disorders and breast hyperplasia.[11] STAT-3 mainly promotes cell differentiation. Enhanced STAT-3 expression accelerates cell differentiation and reduces cell apoptosis, which is an important tumorigenesis mechanism.[91011] JUN, being a proto-oncogene, is related to cell proliferation and angiogenesis. Earlier studies have reported that the expression period of JUN comes mainly before the formation of breast cancer.[333435]

Higher proliferative activity is manifested by breast epithelial cells, overexpressing EGFR. EGFR expression is commonly encountered in breast epithelial atypical hyperplasia, suggesting that EGFR overexpression may act as a marker of dysplasia. The binding of EGFR to EGF may be one of the inducers triggering the transformation of atypical hyperplasia epithelium to carcinoma. SRC has been recognized as a key molecule in tumor progression, attributed to its over-activation and oncogenic role in various cancers, including colon and breast cancer. Among them, SRC-3 plays a pivotal role in the onset of hormone-dependent breast cancer and interacts with several transcription factors, and participates in nonhormone-dependent breast cancer through a variety of signaling pathways.[36] Studies have highlighted dysregulation of the immune function of patients with breast hyperplasia, which is justified by the involvement of immune-related cytokines, such as TNF-α, in the immune regulation of the body.[37] Based on the above analysis, the present study predicted that the major active ingredients of luteolin, kaempferol, quercetin, isoliquiritigenin, emodin, and apigenin in Rujiling capsules could reliably treat breast hyperplasia by targeting cancer-related pathways, including TP53, STAT3, EGF, JUN, AKT1, EGFR, SRC, TNF, and INS.

However, this study had some limitations, such as, in molecular docking, the identification of the formulation components is an important factor that restricts the reliability index. To identify the formulation components more accurately, we need to grasp the following principles: 1 – learn more about protein composition, function and structure, the length of the protein sequence. 2 – the formulation components with complete pockets were preferred, and some protein residues were missing. 3 – the structure containing eutectic formulation components was selected, and the formulation components containing nucleic acids, peptides, coenzymes, and small molecular compounds were excluded. 4 – the formulation components structure similar to the receptor was preferred. 5 – the formulation components structure with high resolution was preferred.[3839] Grasping the above principles can make the selection of formulation components more accurate and reduce the research risk caused by improper selection of ligands. Despite these limitations, this approach can quickly and accurately describe the interaction between drugs and targets through computer simulation of molecular docking which is conducive to the development of new drugs from traditional Chinese medicines. This approach will shorten the drug development cycle.[4041]

Conclusion

Thus to summarize, with the aid of network pharmacology and molecular docking technology, the present research predicted the role of luteolin, kaempferol, quercetin, isoliquiritigenin, emodin, apigenin, and other active components of Rujiling Capsules in regulating multiple biological functions and multiple signaling pathways through TP53, STAT3, EGF, JUN, AKT1, EGFR, SRC, TNF, and INS; thus, contributing to the management of breast hyperplasia. Importantly, this study elucidated the action mechanism of the Rujiling capsule with multiple components, multiple targets, and multiple pathways. However, further biological experiments are required to unearth the active ingredients and core targets to clarify the molecular mechanism of the Rujiling capsule in the treatment of breast hyperplasia.

Financial support and sponsorship

This study was financially supported by the Foundation Project of Yunnan Provincial Universities (Part) (2017FH001-095).

Conflicts of interest

There are no conflicts of interest.