Network Pharmacology Analysis, Molecular Docking, and In Vitro Verification Reveal the Action Mechanism of Prunella vulgaris L. in Treating Breast Cancer

Background Prunella vulgaris L. is effective in the treatment of breast cancer (BRCA); however, the underlying mechanism is still unclear. The aim of this study was to elucidate the mechanism of treatment of BRCA by P. vulgaris using network pharmacology and molecular docking technology, and to verify the experimental results using human BRCA MDA-MB-231 cells. Methods Active components and action targets of P. vulgaris were determined using the TCMSP™, SwissTarget Prediction™, and TargetNet™ databases. GeneCards™ and OMIM™ provided BRCA targets. After obtaining common targets, a protein-protein interaction (PPI) network was constructed using the STRING™ database, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using the Xiantao™ academic database. Cytoscape™ was used to construct “single drug-disease-component-target” and “single drug-disease-component-target-pathway” networks. The Human Protein Atlas™ was used to determine protein expression levels in BRCA cell lines. AutoDock tools™ were used to carry out molecular docking for the first 10 targets of quercetin and the PPI network. Finally, the abovementioned results were verified using cell experiments. Results We obtained 11 active components, 198 targets, and 179 common targets, including DUOX2, MET, TOP2A, and ERBB3. The results of KEGG pathway analysis screened 188 related signaling pathways and indicated the potential key role of PI3K-Akt and MAPK signaling pathways in the antibreast cancer process of P. vulgaris. The results of molecular docking showed that the first 10 targets of quercetin interacted well with the protein network. Cell experiments showed that quercetin effectively inhibited the proliferation of MDA-MB-231 cells by regulating apoptosis and cell cycle, which may be partly related to the MAPK signaling pathway. Conclusion Synergistic effects of multiple components, targets, and pathways on the anti-BRCA activity of P. vulgaris could provide a theoretical basis for further study on its complex anti-BRCA mechanism.


Introduction
In recent years, the incidence rate of breast cancer (BRCA) has been increasing worldwide, at a rate of 3% per year [1]. erefore, BRCA is becoming the cancer with the fastest growing incidence rate among women [2]. Simultaneously, the age of onset of BRCA is also gradually decreasing, causing a great psychological burden on the patients, making patients and their families bear great economic and life pressure, and seriously reducing people's happiness levels [3]. According to the International Cancer Institute of WHO, the incidence rate of BRCA worldwide was 24.2% and the mortality rate was 15.0% by 2018 [4]. At present, the treatment of BRCA mainly involves a combination of surgery, chemotherapy, and radiotherapy. However, the abovementioned methods present several limitations. e treatment of breast disease affects the health of other systems and leads to complications such as slow wound recovery, changes in body shape, liver and kidney injury, changes in metabolism, and severe alopecia. BRCA adversely affects women physically and mentally [5,6]. erefore, treatments with few adverse reactions and wide applications need to be developed. Traditional Chinese medicine treatment presents great potential owing to its characteristics of small adverse reactions and a good curative effect. e occurrence, development, invasion, and metastasis of BRCA is a complex process. Traditional Chinese medicine is an important way to treat BRCA, regardless of the stage. e dried ear of Prunella vulgaris L, a perennial herb in the family Lamiaceae, has a medicinal and edible homology. It has a medicinal history of thousands of years and was first mentioned in the Shennong herbal classic. Its Chinese name means "grass will wither after the summer solstice." Prunella vulgaris is bitter, spicy, and cold, and it acts along the liver and gallbladder meridians. It can clear the liver and dissipate fire, clear eyesight, dissipate nodules, and disperse swelling [7], pain, photophobia, dizziness, vertigo, gall, BRCA, hypertension, lymphatic tuberculosis, infiltrative pulmonary tuberculosis, simple goiter, mumps, acute icteric infectious hepatitis, and other diseases with a good clinical therapeutic effect [8][9][10][11]. Modern research shows that the water, alcohol, and ethyl acetate extracts of P. vulgaris have various pharmacological effects, including antitumor, antibacterial, anti-inflammatory, immunosuppressive, free radical scavenging, antioxidative, and antiviral properties [12][13][14]. With the gradual deepening of research on P. vulgaris and its extract, its related pharmacological effects are being gradually clarified, and clinical research on it is becoming increasingly extensive.
Network pharmacology is a recently introduced analytical method. It conducts "multitarget and multipathway" analysis of drugs and diseases by integrating various databases and constructs a "drug active ingredient disease" model to visually analyze the studied action mechanism and comprehensively predict the targets [15].
ough P. vulgaris has been shown to be clinically effective in the treatment of BRCA in a vast number of studies, its underlying mechanism is unclear.
is study aimed to predict the potential components, targets, and molecular pathways of P. vulgaris against BRCA based on network pharmacology. Furthermore, we used molecular docking and cell experiments to verify and provide a theoretical basis for the potential mechanism ( Figure 1).

Collection of Active Components and Action Targets of P. vulgaris.
e TCMSP database and analysis platform (https://tcmspw.com/tcmsp.php) were used to get selfhealing active ingredients and potential targets [16]. Taking "Prunella vulgaris L" as the retrieval word, oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 are assumed as the screening conditions, and the eligible active ingredients are collected [17]. e corresponding targets were obtained according to the screened active ingredients of Prunella subtilis. Using the UniProt database (https:// www.uniprot.org/) [18], the species was defined as "Homo sapiens," and the gene names corresponding to the targets of the active ingredients of Prunella subtilis were collected. From the PubChem database (https://pubchem.ncbi.nlm. nih.gov/) [19], in chemical structure of the linear canonical SMILES, import the component target database (Swis-sTargetPrediction, https://www.swisstargetprediction.ch/) [20], the choice of species for human Homo sapiens, and potential acquisition targets. en, using the UniProt specification TargetNet of targets, the chemical composition of the canonical SMILES number was entered into TargetNet (https://targetnet.scbdd.com/), prob >0 genes were selected, and used the UniProt specification TargetNet of targets. e targets of the three databases were merged and the duplicate targets were deleted.

Construction of a "Single-Substance
e common targets of P. vulgaris and BRCA were obtained from the Xiantao academic database (https://www.xiantao.love), and the Wayne map was drawn using the Venny plate. e "single-substance drugdisease-component-target" network was constructed using Cytoscape 3.7.1 software.

Construction of the PPI Network.
Using STRING database (https://string-db.org/), the PPI network was constructed. e target points of P. vulgaris and BRCA obtained in previous step were introduced into the STRING database, the species "Homo sapiens" was defined, and the confidence level was >0.4. e light blue line represents the interprotein interaction from the database and the purple line represents the protein intercropping verified by experiments. Graph-Pad prism 7.0 was used to display the adjacent number of the top 35 proteins in the PPI network.

Analysis of Pathways and Functional Enrichment.
To further study the molecular mechanism of P. vulgaris against BRCA, GO and KEGG pathway analyses were carried out using the Xiantao academic database (https://www.xiantao. love). e top 10 biological processes, molecular functions, cellular components, and KEGG pathways were selected (P < 0.05), and the results were visualized by GraphPad prism 7.0. en, the experimental data such as active ingredients, targets, and the top 10 KEGG pathways were imported into Cytoscape 3.7.1 software to construct a "single-substance drug-disease-component-target-pathway" network.

MTT Cell Proliferation Test.
e effect of quercetin on the proliferation of human BRCA MDA-MB-231 cells was detected using the MTT method. Logarithmically grown MDA-MB-231 cells were inoculated into 96-well plates (5 × 10 4 well −1 ), 5 multiple wells in each group. After 12 h of cell adhesion, 0, 10, 20, 30, 40, and 50 μg/mL were added, respectively. Quercetin was treated for 24 or 48 h, respectively, and the cell morphology was observed. After treatment with quercetin for 24 or 48 h, a 5 mg/mL MTT solution was added to each well and incubated in the cell incubator for 4 h. Carefully suck and discard the culture supernatant in the well, add 150 μL of dimethyl sulfoxide to each well, and oscillate at low speed in the dark at room temperature for 10 min to fully dissolve the crystal. e optical density (OD) of each well was measured by using a microplate reader at 570 nm. e relative cell viability and cell inhibition rate were calculated with reference to the OD of the blank control group.

Flow Cytometry.
e effect of quercetin on the cell cycle and apoptosis of human BRCA MDA-MB-231 cells was detected using flow cytometry. Logarithmically grown MDA-MB-231 cells were inoculated into 96-well plates (5 × 10 4 /well). MDA-MB-231 cells were treated with quercetin at different concentrations for 24 h, digested with trypsin, and collected. Cell cycle analysis was carried out. After the abovementioned treatment, the cells were centrifuged at 1500 rpm for 5 min. After that, 1 mL of phosphate buffered saline (PBS) was added for cleaning and centrifugation, and the supernatant was sucked out. en, 2 mL of 75% ethanol was added and fixed overnight at 4°C. e supernatant was then centrifuged and removed, and the cells were resuspended twice with PBS. Finally, a 500 mL mixture of propidium iodide (PI) and ribonuclease (RNa-seA) was prepared in each tube. After being kept away from light at 37°C for 30 min, the cell cycle was detected by flow cytometry. After the abovementioned treatment, the cell density was adjusted to 1 × 10 5 L −1 . en, 100 μL of binding buffer was added to each tube to resuspended the cells. Furthermore, 5 μL of annexin v-phycoerythrin (annexin V-PE) and 5 μL of 7-amino-actinomycin D (7-AAD) were added and mixed well. en, it was incubated at room temperature in the dark for 20 min. Finally, the cells were washed with PBS, and 500 μL of PBS was added to resuspend the cells to detect apoptosis by flow cytometry.

Western Blotting.
After the abovementioned treatment, human BRCA MDA-MB-231 cells were added to the protein lysate and lysed for 30 min on ice. e total protein concentration was measured using the bicinchoninic acid (BCA) assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate 20 μg of protein at 100 V on ice for 2 h, before transferring onto the polyvinylidene fluoride (PVDF) membrane. e PVDF membrane was transferred to skimmed milk powder containing 5%, and the membrane was shaken on a decolorization shaker for 1 h at room temperature. e membrane was removed from the blocking solution, placed in a hybridization bag, and the following specific primary antibodies were added: cleaved caspase-3 (1 : 1000), or cleaved PARP (1 : 1000), or ERBB3 (1 : 1000), or MET (1 : 1000), or p38 (1 : 1000), and GAPDH (1 : 5000). After incubation at 4°C overnight, wash with Tris buffered saline tween (TBST) on a decolorizing shaker at room temperature 3 times for 8 minutes each time. e membrane was incubated in secondary antigoat antirabbit IgG (1 : 2000) at room temperature for 1 h and washed on a decolorizing shaking table with TBST 3 times at room temperature for 8 min each time. ECL chemiluminescence developing drops were added onto the PVDF film, the film was put into a chemical imaging analyzer for exposure, and photos were taken. ImageJ 1.4.1 software was used to collect the gray value of the protein and to conduct semiquantitative analysis.

Collection of Active Components and Action Targets of P. vulgaris.
A total of 242 active components of P. vulgaris were obtained in the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Based on the screening criteria of OB ≥ 30% and DL ≥ 0.18, 11 potential active components were obtained, all of which were terpenoids and sterols. To facilitate the follow-up study, these components were numbered PL1-PL11 (Table 1). Subsequently, 791 potential targets related to P. vulgaris were obtained from the TCMSP database. e UniProt database was used to correct the target gene name and remove invalid and duplicate values. Finally, 198 targets were obtained.

Collection of Potential Targets for BRCA.
Using the GeneCards and OMMI databases, 150 and 21510 BRCA targets were obtained, respectively. Combined with the elimination of duplicate values, 15231 BRCA potential targets were obtained.

Construction of the "Single-Substance Drug-Disease-Ingredient-Target" Network.
e 198 target sites of P. vulgaris collected were matched with 15231 potential targets of BRCA. Using the Xiantao academic database, 179 common targets of P. vulgaris and BRCA were identified, and a Venn map was plotted using the Wayne chart ( Figure 2). Only eight effective components of P. vulgaris corresponding to the selected common target were found, and three effective components (PL7-9; cactus flavin-i, monoglucoside, and stigmasterol) were excluded. e active ingredients of P. vulgaris and the common target of P. vulgaris and BRCA were introduced into Cytoscape 3.7.1 software to build the single-substance drug-disease ingredient-target network ( Figure 3). e network has 188 nodes, including P. vulgaris (light red round), 8 active ingredients screened from P. vulgaris (purple triangle), and 179 P. vulgaris and common target nodes of BRCA (light green and green ellipse). Quercetin is the most active component of P. vulgaris against BRCA. It is directly related to 59 targets, such as DUOX2, MET, TOP2A, ERBB3, and the interleukin family (IL-2, IL-4, IL-6, IL-1A).

Construction of Protein-Protein Interaction Network (PPI).
Twenty-five common targets of P. vulgaris and BRCA were introduced into the STRING database, and the species "Homo sapiens" was defined and screened with a confidence level >0.4. e resulting PPI network had 175 nodes, representing all prediction targets ( Figure 4). e edge line represents the relationship between the targets, and the important relationship of the represented proteins in this network can be intuitively judged by the size of the circular area. Among them, the interacting proteins with a comprehensive numerical score >0.95 include MET-DUOX2, TOP2A-ERBB3, DUOX2-TOP2A, and SCN5A-MAP2. e interaction between these proteins plays an important role in the network. GraphPad prism was used to visualize the number of top 35 adjacent targets in the network and to draw a histogram ( Figure 5). e results show that SCN5A, MAP2, DUOX2, and TOP2A are the core targets, and the number of adjacent targets is 2016, 1827, 1548, and 1531, respectively.

Pathway Enrichment Analysis.
In order to further study the mechanism of P. vulgaris against BRCA, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 179 common targets of P. vulgaris and BRCA were carried out using the Xiantao academic database. A total of 2905 GO enrichment entries was obtained, including 2573 biological process entries, 224 molecular functions, and 108 cellular components. In the biological process, P. vulgaris was mainly involved in (GO: 0007188 n � 42), lipopolysaccharide (GO: 0032496, n � 40), reaction to the bacterial source (GO: 0002237, n � 40), reaction to the metal ions (GO: 0010038, n � 39), and cell response to drugs (GO: 0035690, n � 39). P. vulgaris was mainly related to membrane raft (GO: 0045121, n � 30), membrane microdomain (GO: 0098857, n � 30), membrane region (GO: 0098589, n � 30), transcription factor complex (GO:0005667, n � 17), and plasma membrane raft (GO: 0044853, n � 15). In the process of molecular function enrichment, P. vulgaris was linked with ubiquitin-like protein ligase (GO: 0044389, n � 19), endopeptidase activity (GO: 0004175, n � 18), DNA binding transcriptional activator activity and RNA polymerase II specificity (GO: 0001228, n � 18), receptor ligand activity (GO: 0048018, n � 18), and cytokine receptor binding (GO: 0005126, n � 17), which have a great impact. According to P < 0.05, the top 10 GO enrichment entries were screened and visualized by GraphPad prism (Figure 6). Using the KEGG pathway analysis, the common constituents were gathered into 188 signaling pathways. P. vulgaris was mainly involved in the PI3K-Akt signaling pathway (hsa04151, n � 30), MAPK signaling pathway (hsa04010, n � 29), hepatitis B (hsa05161, n � 32), human cytomegalovirus infection (hsa05163, n � 31), and the signaling pathway of the diabetes in the complication of diabetes mellitus. According to P < 0.05, the top 10 pathways were screened out and visualized by GraphPad Prism (Figure 7). Finally, the results of the effective ingredients of P. vulgaris, the common target of P. vulgaris and BRCA, and the top 10 KEGG signaling pathways were imported into Evidence-Based Complementary and Alternative Medicine Cytoscape software to construct a "single-substance drugdisease-ingredient-target pathway" network ( Figure 8). e network has 199 nodes, including P. vulgaris (pale blue hexagon), BRCA (light purple octagon), 8 active ingredients of P. vulgaris (light pink hexagon), 179 P. vulgaris and BRCA common targets (light yellow diamond), and the top 10 signal pathways (light green V font). From this picture we can see quercetin (PL11). Multipurpose targets are the core components of P. vulgaris against BRCA. is network map directly reflects the mechanism of P. vulgaris against BRCA through multicomponent, multitarget, and multichannel.

Molecular Docking Analysis.
Combining the "component core" target network with the PPI network, the gene targets were sorted at the degree level, and the first ten key targets (ERBB3, INSR, MET, NCOA1, NCOA2, COL3A1, TOP2A, DUOX2, MAP2, and SCN5A) were selected for molecular docking with P. vulgaris for the treatment of BRCA. Using AutoDock Vina software, quercetin was docked with the top 10 targets in the PPI network, and the binding activity of components and targets was evaluated by the binding energy. It is generally believed that binding energy < −4.52 kcal/mol indicates that there is a certain binding activity between ligand small molecules and receptor proteins; binding energy < −5.0 kcal/mol showed that they had a good binding activity; binding energy < −7.0 kcal/ mol indicates that the ligand has a strong binding activity with the receptor [23]. Molecular docking results showed that all binding energies were greater than 4.52 kcal·mol −1 , indicating that quercetin was well docked with these targets ( Table 2). PyMOL software was used to visually analyze the molecular docking results of quercetin with DUOX2 (PDB ID: AF), ERBB3 (PDB ID: 3kex), TOP2A (PDB ID: 4r1f ), and MET (PDB ID: 2rfs) ( Figure 9). As shown in Figure 9, quercetin interacts with amino acid residue ARG-241 of TOP2A through a hydrogen bond. Two hydrogen bonds were observed between quercetin and the amino acid residue PHE-1490 of DUOX2. Quercetin forms hydrogen bonds with ARG-1208 and ASN-1209 on the key residues of the active site. Quercetin interacts with amino acid residues ASP-833 and THR-768 of ERBB3 through hydrogen bonds.

Target Pathway Validation Analysis.
Previous KEGG pathway analysis revealed that the PI3K-Akt and MAPK pathways were crucial in the process of P. vulgaris in the treatment of BRCA. erefore, the abovementioned two pathways were further validated. e names of the two pathways were entered into the KEGG database, and the target selection points (DUOX2, ERBB3, MET, and TOP2A) were entered into the target plate. e results of further analysis are shown in Figure 10. ERBB3 and MET proteins are enriched in the cell membrane and act through the classical MAPK pathway. It is shown by cBioPortal database analysis ( Figure 11). ERBB3 and MET were found to be highly expressed in BRCA. erefore, through the final verification, the important mechanism of anti-BRCA is clear.

Quercetin Inhibits Proliferation of Human BRCA MDA-MB-231 Cells.
e effect of quercetin with different concentrations on the proliferation of the human BRCA cell line MDA-MB-231 was detected using the MTT method at 24 or 48 h (Figure 12(a)). Quercetin significantly inhibited the proliferation of human BRCA MDA-MB-231 cells in a time and dose-dependent manner compared with the blank control group. As shown in Figure 12(b), the IC 50 at 24 or 48 h is 31.99 ± 0.98 and 37.26 ± 0.97 μg/mL, respectively.

Discussion
In recent years, the incidence of BRCA has increased worldwide. Approximately 30% of the cases are metastatic BRCA [24]. Early stages of BRCA often go undetected, leading to the progression of the disease. Recent studies have shown that flavonoids and sterols are the main antitumor active components of P. vulgaris [25,26]. Kaempferol (PL2) is a polyphenolic antioxidant that inhibits proliferation and induces apoptosis of cancer cells. It has been found that the mechanism of its induced apoptosis is related to the phosphatidylinositol 3-kinase/protein kinase B (PI3k/FAK) signaling pathway [27], which can significantly inhibit the proliferation of BRCA SK-BR-3 cells, which may be related to the regulation of Notch1 and cleaved caspase-3 protein expression [28]. Soybean sterol (PL4) is widely distributed in various plants. It has been found that soybean sterol, as a representative of terpenoids from Ilex latifolia, can effectively inhibit the proliferation of MDA-MB-231 cells and promote the apoptosis of cells in MDA-MB-231 [29]. Delphinidin (PL5), as an important component of the development of BRCA, has been well known by many scholars. Studies have shown that cyclin D1, c-myc, and MMP-7 are overexpressed in BRCA [30]. Cordycepin can significantly Delphinidin can also reduce the expression level of MMP-7 in breast tumor tissues [31]. Meanwhile, cordycepin can induce apoptosis of HER-2 positive BRCA MDA-MB-453 and induce autophagy by inhibiting the AKT/mTOR pathway [32]. It can also induce G2/M cycle arrest and apoptosis of MDA-MB-453 and BT-474 cells by blocking the NFkB signaling pathway, thereby having an antiproliferative effect [33]. Luteolin (PL6), originally called 3′,4′,5,7-tetrahydroxyflavone, contains c6-c3-c6, benzene ring, oxygenated carbon ring, and a C2-C3 carbon double bond, which is an important structure for luteolin to play its biological role [34,35]. Studies have shown that luteolin inhibits the growth and proliferation of colorectal cancer cells, BRCA cells, prostate cancer cells, gastric cancer cells, and hepatoma cells, as well as affects the activation of multiple signaling pathways associated with growth and proliferation [36][37][38]. It can induce the growth inhibition of BRCA cells and reduce the methylation level of the OPCML gene. Because of its natural origin and low toxicity, it has been used as a potential safe tumor adjuvant drug [39]. Morin (PL10) can significantly reduce inflammation-mediated apoptosis induced by dihydroxymethylbutyric acid (DMBA). Morin can also effectively inhibit the downregulation of Bax and the upregulation of Bcl-2. In addition, Morin can downregulate proinflammatory markers such as COX-2, NFkB, TNF-α, IL-2, and IL-1β. Some experiments also confirmed that Morin mediated mitochondrial apoptosis is related to the release of cytochrome C and the apoptosis signaling pathway. erefore, these results further suggest that Morin may induce apoptosis of cancer cells and further prevent BRCA under the condition of DMBA induction, which suggests that Morin may be clinically used as a marker of BRCA prevention [40]. In this study, 11 active components of P. vulgaris against BRCA were screened by TCMSP and OB and DL parameters. Flavonoids and sterols were the key active components.
is study also found that quercetin played an anti-BRCA role by acting on multiple targets. For the abovementioned reasons, quercetin was selected for subsequent cell experiments to study its potential mechanism. In conclusion, quercetin plays a role in antagonizing BRCA through a variety of active ingredients, reflecting the synergistic effect of multiple components of Chinese medicine.
In this study, 179 common targets of P. vulgaris and BRCA were collected. Among them, SCN5A, MAP2, and TOP2A were the core predictive targets. In humans, voltagegated sodium channels (VGSC) have nine subtypes, namely Nav1.1-Nav1.9, with different functional characteristics. Nav1.5 belongs to TTX-R VGSC and SCN5A is the genotype encoding Nav1.5. At present, studies have reported that the functional expression of SCN5A is found in many malignant tumor cells, and its increased expression and activity are closely related to the malignant biological characteristics of some tumors, which may be involved in the occurrence and development of tumors [41]. An abnormal expression of SCN5A was detected in BRCA MDA-MB-231 cells and BRCA tissues with lymph node metastasis. SCN5A may be an early tumor marker and prognostic factor in BRCA. Fraser et al. [42] showed that the expression of SCN5A was 1800 times higher in the metastatic cell line MDA-MB-231 than in that of the highly metastatic BRCA MDA-MB-231 cells, which confirmed that there was a significant correlation between the expression of SCN5A and the occurrence and development of BRCA. MAP2 includes four subtypes: high molecular weight MAP2A and MAP2B, and low molecular weight MAP2C and MAP2D. Some studies have found that paclitaxel is an antimitotic anticancer agent that is effective for solid tumors. Its antitumor activity comes from the inhibition of microtubule depolymerization and the promotion of microtubule assembly. In terms of mechanism research, it was found that paclitaxel can promote the increase of affinity between MAP2 and tubulins, affect the depolymerization and assembly of microtubules,  2016  1827  1548  1531  1466  1441  1408  1382  1342  1255  1210  1203  1159  1153  928  854  850  848  745  707  660  604  480  454  427  402  393  380  331  Evidence-Based Complementary and Alternative Medicine and exert its antitumor activity [43]. Studies have shown that MAP2 also expresses corresponding mRNAs and related peptides in pancreatic ductal adenocarcinoma. Microtubules are one of the targets of docetaxel. In vivo hydrolysis of MAP2 protein can increase the antitumor effect of docetaxel in pancreatic ductal adenocarcinoma [44]. DNA topoisomerase (TOP2A) has an important relationship with the occurrence, development, invasion, treatment, and prognosis of malignant tumors. As a DNA topoisomerase, it is essentially a protein and is closely related to tumor chemotherapeutic drug targets [45]. TOP2A is mainly distributed in the nucleus and participates in the cell cycle pathway, which is closely related to cell proliferation and apoptosis. e expression of TOP2A during cell division is specific. It begins to increase in the S phase of mitosis, reaches its maximum in the G2/M phase, and decreases after division. BRCA is often associated with a high expression of the epidermal growth factor receptor (HER2/neu), while the TOP2A encoding gene is located at HER217/neu, and TOP2A expression is correlated with HER2/neu gene expression. Some scholars have found that the expression of HER2/neu and TOP2A is related to anthracycline chemotherapy. It is proven that the expression of these two genes can provide the basis for the selection of chemotherapeutic drugs for BRCA [46]. In addition, researchers have found a correlation between the expression of the TOP2A gene and     Evidence-Based Complementary and Alternative Medicine the clinicopathological features of BRCA. e results provide the basis for anthracycline chemotherapy in the treatment of BRCA [47,48]. erefore, the expression of TOP2A can predict the effect of chemotherapy on BRCA. In summary, these targets are the important targets of P. vulgaris against BRCA, which reflect the synergistic effect of multiple targets.
In this study, KEGG pathway analysis showed that P. vulgaris exerts anti-BRCA effects through multiple signals, including the PI3K-Akt signaling pathway, the MAPK signaling pathway, and the AGE-RAGE signaling pathway. e PI3K/Akt signaling pathway promotes BRCA formation through a variety of cell processes, including promoting cell growth, proliferation, viability, transforming glycolysis  metabolism, increasing cell migration, and reducing cell apoptosis [49]. PI3K signaling activates Akt kinase, activated Akt can be found in BRCA collection samples of 20% to 55%, and is related to the recurrence of ER-positive BRCA [50,51]. e PI3K pathway mutation is the most common gene mutation in HR-positive BRCA and is found in over 70% of BRCA patients [52]. In recent studies on e Cancer Genome Atlas, 36% of BRCA had PIK3CA mutation [53], but PIK3CA mutation as a marker of prognosis of BRCA still has some controversies, which may be related to a small sample size [54]. Because PIK3CA encodes PI3K and catalyzes the subunit P110, it is meaningful to use inhibitors for specific PI3K subtypes in the development of PI3K inhibitors for the treatment of BRCA. In recent years, a large number of studies have shown that the level of advanced glycosylation end products (AGEs) in diabetics is significantly higher than that in the general population, and by inhibiting glycosylation, the progression of diabetic nephropathy can be effectively curbed. AGEs are a combination of receptors for AGEs (RAGE) and a series of lesions. A large number of studies have shown that the AGE-RAGE signaling pathway is a very important link in the formation and progression of diabetic nephropathy. However, its downstream mechanism is very complex and has still not been fully clarified such as NF-KB, VGEF, and TGF-β1. e relationship between the MCP-1 gene and other genes needs to be confirmed by further research.
ERBB3 plays an important role in the development of normal breast tissue. e expression level of ERBB3 is very low in normal breast tissue in the embryonic stage. rough the process of postnatal maturation, the expression of ERBB3 increases. In the second and third trimesters of pregnancy, it can be observed that the phosphorylation level increases after ERBB3 is activated, and the expression of ERBB3 also increases in the mammary duct epithelium and stroma of pregnant rats. In functionally differentiated or nontransformed breast epithelial cells, the expression and activation of ERBB3 are downregulated [55]. ERBB3 was not activated in the established normal immortalized breast cell line. About 35% of BRCA cell lines express a high level of ERBB3 compared to the nontransformed mammary cell line [56]. In human BRCA tissue samples, the expression of ERBB3 mRNA was increased by 100 times compared with normal breast tissue in 46% of the samples by RT-PCR [57]. 2D-PAGE protein analysis of four normal breast samples and four BRCA samples from humans revealed that ERBB3 was detected only in malignant tissues. Cancer tissues had higher ERBB3 expression than normal tissues in 50%-70% of BRCA cases. In 18%-29% of cases, the high expression of ERBB3 was positively correlated with metastasis, tumor size, in situ recurrence, tumor grade, and tumor recurrence. e ERBB2-ERBB3 heterodimer is considered to be the most interactive dimer form in the ERBB family. In the mouse model experiment, ERBB3 expression was also increased in   combinations, ERBB2-ERBB3 coexpression increases the level of THE vascular endothelial growth factor in the cells [61]. Notably, in the invasive tumors caused by ERBB2 and ERBB3, THE insulin-like growth factor 2 (IGF2) and insulin-like growth factor binding protein 5 (IGFBP-5) are highly expressed, and these growth factors are very important for tumor growth [62]. Cellular mesenchymal to the epithelial transition factor (MET), also known as c-Met, is the product encoding the proto-oncogene MET. Its associated signaling pathway is closely related to the prognosis and drug resistance of BRCA and is an important potential therapeutic target for triple-negative BRCA [63][64][65][66][67][68]. Numerous studies have shown that the positive rate of c-Met in BRCA tissues is significantly higher than that in BRCA tissues. Its positive expression is closely related to the degree of differentiation, infiltration, lymph node metastasis, and clinical stages. It also indicates that the progression of BRCA is related to the activation of the c-Met proto-oncogene [69].
A study on the correlation between c-Met expression and BRCA in foreign countries has also confirmed that activated c-Met can induce mouse BRCA. is kind of BRCA is similar to human basal-like BRCA. It is characterized by PR (−), HER-2 (−), and CK5 (+). e high expression of c-Met protein indicates a poor prognosis and a significant negative correlation with the survival rate of BRCA [70]. erefore, c-Met can be used as an important prognostic marker for patients with basal-like BRCA and has a significant prognostic value [71]. e MAPK signaling pathway plays an irreplaceable role in the proliferation, growth, and differentiation of breast tumor cells. It is a serine/threonine protein kinase widely found in eukaryotic cells. It can phosphorylate some target proteins and activate a series of cascade reactions to produce some biological effects in cells. e MAPK signaling pathway can transduce extracellular stimulation signals into cells and nuclei through a three-level kinase cascade: extracellular signal ⟶ MAPK kinase ⟶ MAPK kinase ⟶ MAPK.
is pathway is involved in the processes of cell proliferation, growth, and apoptosis, and plays an important role in the occurrence and development of a variety of tumors.   Filardo and Gper have shown that in NER-negative SKBR3 BRCA cells, estrogen can accelerate the phosphorylation of ERK1/2 and promote the proliferation of cancer cells [72]. Moreover, estrogen can activate ERK1/2 in MDA-MB-231 cells in a short time. Activation of the MAPK signaling pathway may affect the expression level and invasiveness of ER in BRCA cells. e expression of phosphorylated MAPK in normal breast tissues is significantly lower than that in BRCA tissues. Estrogen can not only promote the proliferation of BRCA cells through ER, but also activate the MAPK signaling pathway to induce tumor cell proliferation. In addition, the activation and expression of the p38-MAPK signaling pathway in lymph node-infiltrating breast tumors are more common than those in lymph node-negative breast tumors. In ER-negative BRCA MDAMB-468 and MDA-MB-231 cells in TGF-2α stimulated by serum, the level of ERK/MAPK phosphorylation and activity duration in cells were higher than those in ER-positive BRCA cell MCF-7, indicating that ERK/MAPK activation can promote the invasive growth of tumor cells. Song et al. found that in MCF-7, estrogen can produce rapid induction by binding with mer, activate SHC to form a SHC-GRB2-sos complex, activate the MAPK signal pathway, and finally affect the normal cycle of cell growth and abnormal proliferation of breast cells [73]. At the same time, researchers in our country have found that some Chinese medicine therapies may inhibit BRCA by regulating the ERK/MAPK signaling pathway. After the action of nitidine chloride on human BRCA cells, it was found that the drug could significantly reduce the expression levels of p-ERK1/2, p-JNK, and p-p38, indicating that nitidine chloride could inhibit the activity of the MAPK signaling pathway and inhibit the metastasis of breast tumors. Wang and Hu used the combination of Poria cocos triterpene and total iridoid of sepulchrysoides to observe the effect of the invasion ability of breast cancer cells [74]. Jingjing et al., in the study of Poria cocos triterpenes and ajiro total iridoid two drugs on the invasive ability of BRCA cells, found that high metastatic BRCA cells MDA-MB-231 and SK-BR-3 ERK1/2, JNK, and the p38 phosphorylation level were lower than before the combination. It is presumed that the combination of two drugs may inhibit the MAPK related signal transduction. It can hinder the invasion and metastasis of cancer cells. Other studies have confirmed that the Shugan Yishen recipe can reverse the expression of the HER-2 gene in BRCA cells and inhibit the two pathways of ERK/MAPK and p38/MAPK, thereby reversing the drug resistance of tumor cells [75,76]. e study also has some limitations. First, the public database is updated in real time, so the results of this study only reveal to a certain extent the mechanism of P. vulgaris against BRCA. Second, this study was only carried out in vitro and only used human BRCA MDA-MB-231 cells. In future, further multidimensional validation is needed. It is very meaningful to study the effect of P. vulgaris on the role of anti-BRCA through various signaling pathways. For future research, the MAPK signaling pathway can be selected, and other signaling pathways, such as the PI3K-AKT signaling pathway, can be further studied. In addition, although quercetin is the main active component of P. vulgaris, it still cannot fully represent P. vulgaris, which still needs further experimental verification in the future. In conclusion, the mechanism of P. vulgaris against BRCA should be further studied in vivo and in vitro.
In this study, the potential mechanism of P. vulgaris against BRCA was preliminarily investigated by using network pharmacology, molecular docking, and in vitro experiments.
e results showed that P. vulgaris played an important role in anti-BRCA through multicomponent, multitarget, and multisignal channels. Quercetin played an important role in this process. In addition, quercetin can effectively inhibit the proliferation of human BRCA cell MDA-MB-231 by regulating cell apoptosis and the cell cycle, which may be related to the MAPK signaling pathway. is study provides new inspiration for future experimental and clinical treatment of BRCA.
Data Availability e datasets used and/or analyzed during the study are available from the corresponding author upon request.   16 Evidence-Based Complementary and Alternative Medicine