Dan Li, Shu-Hong Bao

1. The Fifth People's Hospital of Shenyang Liaoning Shenyang 110000

2. Affiliated Hospital of Liaoning University of Traditional Chinese Medicine Shenyang 110032

Keywords:

ABSTRACT

1. Introduction

Breast cancer ranks seventh among the top ten malignant tumors in China and is the most common tumor in women [1]. In 2014, the overall incidence of breast cancer among women was on the rise, with 279,000 new cases and 66,000 deaths [2,3]. Its high morbidity and mortality seriously threaten women's health. At present, there are many methods of western medicine to treat breast cancer, including surgical resection, radiotherapy and chemotherapy, and endocrine therapy. Affects patients' psychological burden [4-6]. A large number of studies have shown that traditional Chinese medicine has shown advantages in the treatment of early breast cancer. Treatment of breast cancer with programs such as rectifying liver and regulating liver qi can significantly relieve clinical symptoms and enhance patients' confidence in treatment [7-9].

Modern Chinese medicine practitioners mostly take the righting and relieving the evil as the treatment criterion, and apply ginseng, Shudi, Atractylodes, and Poria to benefit Qi and strengthen the spleen. It can inhibit the development of tumors by enhancing lymphocyte proliferation, enhancing immune function, and inducing apoptosis, but Its exact antitumor mechanism has not been fully elucidated [10-12]. Because Chinese medicine contains a variety of ingredients, which can aあect the human body through a complex mechanism of action, it is diきcult to determine which component of Chinese medicine will have a therapeutic eあect on disease. Network pharmacology can comprehensively analyze the constituents of traditional Chinese medicine, dig out the common targets of drugs and diseases to analyze the biological functions and signal pathways involved in the targets, and provide theoretical basis for clinical trials [13]. In this study, the mechanism of ginseng's anti-breast cancer was analyzed through the method of network pharmacology, and the research results are reported as follows.

2. Materials and methods

2.1 Screening key chemical components

Retrieve all active ingredients of ginseng through the Chinese Medicine System Pharmacology Database Analysis Platform (TCMSP, http://lsp.nwu.edu.cn/index.php), collect a total of 190 compounds, and pass the oral bioavailability (OB) 30%, drug similarity (DL)＞ 0.18 to screen the pharmacokinetics of compounds, where OB refers to the relative amount and rate of drug absorption into the blood circulation by the body after oral administration； DL reflects specific functions in the compound The similarity between the group and the known drug is of great significance to the evaluation of the chemical constituents of traditional Chinese medicine.

2.2 Screening ginseng-breast cancer targets

The TCMSP database is used to search for target information of key chemical components of ginseng, and the human gene name corresponding to the target is queried through the uniprot database (https://www.uniprot.org/), and the Genecards database (https: // www.genecards.org/) and OMIM database (https://www.omim.org/) to map the protein target genes of breast cancer to obtain drugdisease target genes.

2.3 Network visualization of drugs-key chemical components-disease targets

In order to study the mechanism of ginseng in treating breast cancer, this study introduced the common chemical components and common protein target genes of ginseng-breast cancer into Cytoscape 3.7.2 software for visualization. The form is presented. In this network diagram, the bond compounds and targets are represented by nodes, and the interaction between nodes is represented by edges.

2.4 PPI Network ConstructionPPI

A network of protein-protein interactions. The common target genes of human participation in breast cancer were imported into the String database (https://string-db.org/Version 10.5), and the research species was selected as human (Homo sapiens). The protein interaction network was obtained and the protein relationship score was set. Is 0.7, and hides the free protein that appears. The blue line represents evidence of gene co-evolution, the red line represents evidence of gene fusion, and the yellow line represents evidence of text mining. Core genes of the PPI network.

2.5 GO (gene ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis

The Bioconductor database (http://bioconductor.org/bioc Lite.R) was used to query the gene ID of the drug-disease common target gene. Use R language to install the related installation package of Bioconductor platform, set pvalue = 0.05, qvalue = 0.05 for GO enrichment analysis and KEGG enrichment analysis, output the results and draw a barplot histogram.

3. Result

3.1 Screening of chemical constituents in ginseng

A total of 71 compounds of ginseng were obtained through the TCMSP database, and then screened by OB＞ 30% and DL＞ 0.18 criteria, 22 key compounds of ginseng were obtained, including ginsenoside, ginsengdiol, proopioid, kaempferol, and stigmasterol Etc. See Table 1.

Table 1 Active ingredients of ginseng after screening

3.2 Drug-disease target prediction

The TCMSP database was used to query the target information of eあective compounds in ginseng, and the gene ID was annotated by Uniprot. Search Gene Cards and OMIM databases for breast cancer target genes, enter "breast cancer", get a total of 2962 genes with a correlation score greater than 5, and map the target genes of human participation in breast cancer to get the common There are a total of 35 targets, and the venn diagram is drawn by R language (see Figure 1). The key compounds of ginseng corresponding to the common target were recorded, and a total of 15 key chemical components of ginseng-breast cancer were obtained. Amine, arachidonic acid, shrub polygalonone A, ginsenoside rh2, ginsenoside Rh4, jiurulinine, ginseng diol, sulprolactone, carotene, protoopiate Common target genes for ginseng-breast cancer are PGR, NR3C2, NCOA2, NCOA1, PTGS1, AKR1B1, PLAU, BCL2, CASP9, CASP3, CASP8, PRKCA, PON1, HTR3A, PRSS1, AR, PPARG, F7, RELA, IKBKB , MAPK8, CYP3A4, CYP1A1, ICAM1, SELE, VCAM1, CYP1B1, ALOX5, GSTP1, AHR, GSTM1, AKR1C3, NFKBIA, CASP1, NR3C1.

Fig 1 Ginseng-breast cancer target gene matching

3.3 Construction of Drug-Key Chemical Components-Disease Network

The drug-key compound-disease-target gene network was visualized by Cytoscape 3.7.2 software (see Figure 2).

Fig2 Ginseng Drugs-Key Chemical Components and Target Networks for Breast Cancer Action

3.4 Analysis of PPI protein interaction network

The 35 target genes common to ginseng-breast cancer were imported into the String database platform, and species were selected to study humans, to obtain protein interactions, to screen protein relationships with a score＞ 0.7, and to draw a diagram of the protein interaction network (see Figure 3). The network includes 35 nodes and 77 edges. The interaction proteins with a score of ≥0.99 are IKBKB-NFKBIA, NCOA2-AR, CASP8-CASP3, NCOA1-PPARG, NCOA2-PPARG, RELA-NFKBIA, NCOA2-NR3C1, CYP1A1- AHR, IKBKB-RELA, BCL2-CASP8, CASP8-CASP9. The interaction between these proteins is very important in the network. By calculating the number of connected nodes for each factor, the first 15 PPI core genes are found, which are MAPK8, AR, RELA, CASP8, CYP1A1, CASP3, NCOA1, NR3C1, and ICAM1. , NCOA2, PGR, AKR1C3, CYP1B1, CYP3A4, GSTP1, these are the core genes for ginseng to treat breast cancer. See Figures 3 and 4.

Fig3 PPI protein interaction network

Fig4 PPI core genes

3.5 GO function enrichment analysis

The GO enrichment analysis results showed that a total of 68 GO biological processes were obtained, and the top biological function was mainly cysteine-type endopeptidase activity involved in the apoptotic process (4 targets: CASP9 / CASP3 / CASP8 / CASP1) ； Nuclear receptor activity (5 targets PGR / AR / PPARG / AHR / NR3C1)； transcription factor activity (5 targets PGR / AR / PPARG / AHR / NR3C1)； steroid hormone receptor activity (5 targets PGR / NR3C2 / AR / PPARG / NR3C1)； Steroid binding (5 targets PGR / NR3C2 / AR / CYP3A4 / NR3C1)； Oxidoreductase activity (PTGS1 / CYP3A4 / CYP1A1 / CYP1B1 / AKR1C3)； Endopeptidase activity ( PLAU / CASP9 / CASP3 / CASP8 / PRSS1 / F7 / CASP1)； Monooxygenase activity (CYP3A4 / CYP1A1 / CYP1B1 / AKR1C3)； Oxygen binding (CYP3A4 / CYP1A1 / CYP1B1)； Steroid hydroxylase activity (CYP3A4 / CYP1A1 / CYP1B1)； Hsp90 protein binding (CYP1A1 / AHR / NR3C1)； cysteine-type endopeptidase activity (CASP9 / CASP3 / CASP8 / CASP1)； heme binding (PTGS1 / CYP3A4 / CYP1A1 / CYP1B1)； tetrapyrrole binding ( PTGS1 / CYP3A4 / CYP1A1 / CYP1B1)； glutathione binding (GSTP1 / GSTM1), as shown in Figure 5. It shows that ginseng can play a role in treating osteoporosis through a variety of biological regulation processes.

Fig5 Enrichment analysis of ginseng- breast cancer key target by GO biological process

3.6 Enrichment analysis of KEGG pathway

The KEGG pathway enrichment analysis results showed that there are 83 major pathways enriched for ginseng's key gene targets for breast cancer. The main pathways involve the TNF signaling pathway, toxoplasmosis pathway, influenza A pathway, fluid shear stress and atherosclerosis. Sclerosis pathway, prostate cancer pathway, AGE-RAGE signaling pathway in diabetic complications, hepatitis B pathway, apoptotic pathway, measles pathway, EB virus infection pathway, Legionnaires disease pathway, human immunodeficiency virus infection pathway, NF-κB Signal path, etc., as shown in Figure 6.

Fig 6 Enrichment analysis of KEGG metabolic pathway for ginseng-breast cancer key target

4. Discussion

Modern medicine believes that tumor factors are latent in human cells. When certain exogenous or endogenous factors occur in the body, tumor gene expression will be induced, causing the cells to mutate infinitely and eventually form tumors [14-16]. In addition to age, factors that induce tumor gene expression also include nongenetic factors, such as smoking, alcoholism, high-calorie diets, and psychosocial stress [17-19]. The theory of traditional Chinese medicine believes that "the righteousness exists in the body, and evil cannot be done" and "evil cannot hurt alone." Weakness of the righteousness is the main factor for the onset of tumors. When the righteousness in the body is insuきcient, external evils invade the body and damage the human organs, qi, and blood. Therefore, the key to the treatment of breast cancer by traditional Chinese medicine [20, 21]. Panax ginseng belongs to the family Acanthaceae, Magnolia, Umbellifera, and is mainly produced in Northeast China, North Korea, South Korea, Japan, and Eastern Russia. It was first published in Shennong's Materia Medica: "It's sweet and slightly cold, it mainly supplements the five internal organs, An spirit, fix soul, stop panic, remove evil spirits, eyesight, happy puzzle. Jiufu, light weight and longevity. One title, one ghost cover. Health valley. "Ginseng contains ginsenoside, ginsengene, β-lan Various chemical components, such as elemene, ginsynol, etc., also contain multiple vitamins, amino acids, choline, enzymes, etc. [22,23]. Various ingredients may play an anti-cancer role.

The results of this study show that key compounds in ginseng can aあect the occurrence and development of breast cancer through multiple target genes and multiple signaling pathways. A total of 22 eあective chemical constituents of breast cancer were retrieved in this study, including ginsenosides, pro-opioids, kaempferol, and stigmasterol. Ginsenoside rh2 is a sterol compound, it can aあect multiple metabolic pathways, its eきcacy is complex, it can regulate the immune function of tumor patients, and its clinical anticancer eあect can cover lung cancer, colorectal cancer, prostate cancer, etc. [24,25]. Kaempferol is a natural flavonoid that is widely present in fruits, vegetables, and Chinese herbal medicines. Studies have found that kaempferol can continue to proliferate tumor cells by inducing apoptosis, regulating the cell cycle, inhibiting neovascularization and tumor metastasis. And invasion can exert antitumor eあects by regulating oxidative stress to reflect and inhibit inflammatory factors [26,27]. Stigmasterol, also known as stigmasterol, is widely present in a variety of plants and has the ability to reduce cholesterol and reduce the incidence of cardiovascular events. In recent years, it has been found to have antitumor, anti-inflammatory and antioxidant eあects [28]. Arachidonic acid is an essential fatty acid in the human body. It is a direct precursor to the synthesis of prostaglandins, thromboxane, and leukotrienes. It has a number of physiological activities, such as esterifying cholesterol, increasing vascular elasticity, and regulating cell functions. It has important eあects in vascular diseases, diabetes and tumors [29]. In this study, 15 PPI core genes were obtained through PPI protein interaction analysis, including MAPK8, AR, RELA, CASP8, CYP1A1, CASP3, NCOA1, NR3C1, ICAM1, NCOA2, PGR, AKR1C3, CYP1B1, CYP3A4, and GSTP1, these are the Core genes for ginseng to treat breast cancer. In order to study the role of targets in gene function and signaling pathways, this study conducted GO biological function enrichment analysis and found that the above-mentioned PPI core genes can participate in the apoptosis process, transcription factor activity, and cysteine-type endopeptidase activity. Steroid hormone receptor activity, steroid binding, oxidoreductase activity, endopeptidase activity, monooxygenase activity, oxygen binding and other pathways play a role in anti-breast cancer. The results of KEGG pathway enrichment analysis showed that the ginseng target genes for breast cancer were mainly enriched in TNF signaling pathway, toxoplasmosis pathway, influenza A pathway, fluid shear stress and atherosclerosis pathway, prostate cancer pathway, AGE-RAGE signaling pathway, hepatitis B pathway, apoptotic pathway, measles pathway, Epstein-Barr virus infection pathway, Legionella disease pathway, human immunodeficiency virus infection pathway, NFκB signaling pathway, etc. in diabetes complications. These pathways are closely related to the formation and development of cancer. Domestic studies have examined the epithelial-mesenchymal transition of tumor cells and the expression of NF-κB signaling pathway-related proteins by Western blot, and found that blocking the NF-kB gene in breast cancer cells can inhibit the epithelialmesenchymalization of tumor cells Attenuates the ability of breast cancer cells to invade and migrate [30]. NF-kB is the main eあector of TNF signaling. Studies have suggested that TNFα / NF-κB signaling plays a key role in breast cancer cell migration. And TNF-α levels have great potential as prognostic cancer biomarkers [31]. M Nikseresht et al. Studied the eあect of AGE-RAGE signaling pathway on epithelial-mesenchymal transition in colorectal cancer HCT116 cells by FCM and Western blotting, and found that AGERAGE signaling pathway can aあect the expression of MMP-2 and MMP-9 and aあect tumors. Cell migration plays an inhibitory role, consistent with the enrichment results of this study [32].

In summary, this study elaborated the relationship between the main active ingredients and target genes of ginseng and breast cancer pathways through a network pharmacological method, and analyzed the complex eあects of ginseng against breast cancer with multiple components, multiple targets, and multiple pathways The mechanism lays the foundation for more in-depth experimental research and provides new ideas for studying its mechanism.