Network Pharmacology-Based Approach to Investigate the Mechanisms of Piwei-Peiyuan Decoction in the Treatment of Gastric Carcinoma

Purpose Piwei-Peiyuan Decoction is a traditional Chinese medicine decoction, which has shown promising treatment in gastric carcinoma. However, the molecular mechanisms of gastric carcinoma (GC) have not been systematically revealed yet. In this work, a network pharmacology research was conducted to analyze mechanisms of Piwei-Peiyuan Decoction, aiming to provide a basis for the development and application of new drugs in treatment of GC. In this study, a network pharmacology approach was used to predict targets, construct network maps and analyze relevant signaling pathways. We discovered active chemical ingredients and their targets in the Piwei-Peiyuan Decoction by the TCMSP and ADME database. Then we found main targets of GC by GeneCards (https://www.genecards.org), OMIM (http://www.omim.org) and DRUGBANK (https://go.drugbank.com) databases. According to the information of ingredients and GC, a PPI network was performed by the String (https://string-db.org) database. And a targets-pathways interaction network was constructed by Cytoscape (version 3.7.2), a bioinformatics software used for data visualization and integration.


Background
As a type of malignancy that origin from epithelial cells of gastric mucosa. Almost 95% of GC is adenocarcinoma. GC is a malignancy with the highest morbidity and mortality in China after lung cancer, breast cancer and colon cancer, its 5-year survival rate is only 29%. Although the morbidity of GC in China has declined in recent years, the mortality has not decreased signi cantly 1 2 . The morbidity of GC still ranks second and fth among all malignancy among men and women, and the mortality ranks third and second in China 3 4 . At present, surgery is main treatment of GC. Perioperative chemotherapy and radiotherapy are main treatments to improve the surgical resection rate 5 6 . However, since the high recurrence rate after surgery and the complications of other treatments, the nal prognosis of patients is poor 7 8 .
Traditional Chinese medicine has been widely used in clinical treatment of cancer in Asian countries, especially China and Japan 9 . As many clinical trials on comprehensive cancer treatment continue to be conducted, we have found that TCM not only relieves the discomfort caused by surgery and other treatments, but also alleviates the clinical symptoms of patients and improves their immune function and postoperative survival rate [10][11][12] . The results of a Meta-analysis on TCM treatment improves the survival rate of GC patients in Taiwan 13 showed that the median survival time of patients who received TCM treatment was longer than that of patients who did not receive TCM treatment, suggesting that TCM treatment improves the medium overall survival rate of GC patients. TCM has its unique advantages in treating cancer, which can effectively inhibit tumor spread and metastasis, reduce toxicity of chemotherapy drugs and enhance immunity. For thousands of years, TCM has been widely used to treat various diseases because of its multi-target, low price and few side effects 14 . TCM also helps to improve the quality of life among GC patients 15  Based on genomic, proteomic and pharmacological information a "disease-gene" biological network and a "drugs-targets" biological network can be constructed through a network pharmacology approach. After analysis, we can understand the mechanisms of drugs in treatment of diseases systematically and comprehensively 16 17 . Through the network pharmacology method, the main targets of Piwei-Peiyuan Decoction can be identi ed, which is helpful to further investigate the mechanisms of Piwei-Peiyuan Decoction in treating GC and provide an important theoretical basis for its clinical treatment.

Predicting Targets of Piwei-Peiyuan Decoction
To obtain the targets of Piwei-Peiyuan Decoction, TCMSP database (https://tcmspw.com/tcmsp.php) were used. TCMSP 18 contains 499 Chinese herbal medicines registered in the Chinese Pharmacopoeia, including information of 29,384 ingredients, 3,311 targets and 837 related diseases. TCM are mostly oral medication, according to oral bioavailability (OB) and drug-likeness point (DL), active ingredients and related targets can be selected 19 20 . We took OB≥30% and DL≥0.18 as a standard, discovered the active ingredients and related targets. Then the names of those identi ed targets were sent to UNIPROT database (http://www.uniprot.org) for normalization.

Collecting Targets Related to Gastric Carcinoma
To obtain the disease-related gene, Genecards database (https://www.genecards.org) and OMIM database (http://www.omim.org) were used. GeneCards 21 is a comprehensive and authoritative database of human gene information that has been widely used for nearly 26 years. Its information is automatically discovered and integrated from more than 80 digital sources, including more than 73,000 human genes. We set "Gastric Carcinoma" as keywords, selected targets with hit scores greater than median (1.55) and deleted the repeated targets. Finally, GC-related targets were obtained.

Protein-Protein-Interaction Analysis
Protein-Protein-Interaction (PPI) is fundamental for most biological processes in a living cell and is crucial for understanding cell physiology in normal and disease states. Our PPI network mapping was performed on obtained bioactive ingredients and disease targets using STRING database (http://stringdb.org) with the species limited to "homo sapiens". A targets-pathways interaction network were constructed by Cytoscape (version 3.7.2), a bioinformatics software used for data visualization and integration 22 23 . Cytoscape is the most powerful tool when large amounts of information about the interrelationship of DNA, protein and signal pathways need to be analyzed.

GO and KEGG Pathway Enrichment
The Gene Ontology (GO) provides information for functional genomics and de nes the concepts relating to gene functions 24 . The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database that is famous for its pathway information 25 . It is a powerful database for systematic analysis of gene function, which links genomic information to biological function information. In order to investigate the biological effects of Peiwei-Peiyuan Decoction, GO analysis and KEGG pathway enrichment analysis were conducted and calculated by STRING database (http://string-db.org). The enriched GO terms and pathways having a corrected P value of less than 0.01 were selected for further analysis.

Molecular Docking
Based on the information from network maps of Piwei-Peiyuan Decoction and GC, we selected main ingredients and targets of Piwei-Peiyuan Decoction for molecular docking experiment. The threedimensional (3D) structure diagrams of these compounds of Piwei-Peiyuan Decoction were downloaded through the TCMSP database and imported into the AutodockTools1.5.6 software for hydrogenation and energy optimization, then the mol2 format les were saved. We add the charge and display rotatable keys of these compounds and the les were saved in pdbqt format. Next, the protein crystal structures corresponding to the target genes were downloaded from the PDB database, imported into PyMOL software to remove water molecules and heteromolecules, imported into AutoDockTools-1.5.6 software to add hydrogen atoms, saved to pdbqt format. Finally, the compound is used as a ligand, and the protein corresponding to the target gene is used as a receptor for molecular docking. Autodock vina 1.1.2 and PyMol software were used to analyze and interpret the results.

Ingredients and Targets Analysis
In our study, the TCMSP database was used to predicted the ingredients of Piwei-Peiyuan Decoction initially. We obtained 55 ingredients for Bai Zhu, 87 ingredients for Huang Qi, 104 ingredients for Xiang Fu, 220 ingredients for Gui Zhi, 85 ingredients for Bai Shao and 46 ingredients for Liu Jinu. Then we deleted the active ingredients without matched targets and selected the active ingredients from 6 herbs according to oral bioavailability (OB) and drug likeness (DL) values (OB≥30% and DL≥0.18). Eventually, 67 active ingredients such as quercetin, isorhamnetin, luteolin and kaempferol were discovered (Table.1), and they all have hepatoprotective, anti-in ammatory, antibacterial, analgesic and antitumor effects [26][27][28][29] .  To further uncover the potential pharmacological mechanisms of Piwei-Peiyuan Decoction against GC, pathway analysis was conducted to explore the potential pathways affected by Peiwei-Peiyuan Decoction. 190 pathways were found by KEGG database, we selected top 20 pathways and draw a bubble diagram as follows ( Figure 5). Combining the pathogenesis of GC, the pathways which have no association with GC were removed. Finally, 6 remarkable terms were found to be the related pathways in treatment of GC. Results demonstrated that "Pathway in cancer", "PI3K-Akt signaling pathway", "MAPK signaling pathway", "Ras signaling pathway", "IL-17 signaling pathway", "HIF-1alpha signaling pathway" and "TNF signal pathway" were obviously enriched. According to the KEGG analysis, We found "Pathway in cancer" is the signal pathway that contains the most targets (72 targets), the network of "Pathway in cancer" is shown below (Figure 6). These signaling pathways are closely related to cell differentiation, proliferation, apoptosis and angiogenesis, most of which play a key role in the development and progression of cancer. The molecular functions and biological processes were closely related to the occurrence and development of GC, which indicated that Piwei-Peiyuan Decoction can treat GC through multiple targets and pathways. Our research shows that Piwei-Peiyuan Decoction acts as a treatment for GC mainly through the coordinated regulation of cancer-related signal pathways. For example, PI3K-Akt signaling pathway is a key pathway that regulates cell proliferation, differentiation and metastasis during the development of cancer 34 , and activated AKT can lead to apoptosis by participating in the regulation of cellular protein expression, then ultimately inhibiting cell proliferation 35  Therefore, it is clear that IL-17 levels are closely associated with the development of GC. However, few pharmacological ndings have been reported for the treatment of GC with Piwei-Peiyuan Decoction. Therefore, more experiments should be conducted to validate our ndings in the future.

Results of Molecular Docking Experiment
Molecular docking was used for veri cation of interaction between ingredient and its target gene. The results of molecular docking scores of the ingredients and target genes we selected in this study were shown in Table.2, and the optimal schematic of molecular docking was shown in Figure.7.
From the results, the lowest binding free energy of caspase-3 and kaempferol is -6.1 kcal/mol. There is one hydrogen bond between amino acid THR62 and kaempferol. The lowest binding free energy of caspase-3 and luteolin is -6.1 kcal/mol. There are hydrophobic interactions between amino acid THR62, SER251 and luteolin. In addition, the lowest binding free energy of MAPK1 and luteolin is -7.9 kcal/mol. There are hydrophobic interactions between amino acid LYS54, GLN105, MET108, ASP167 and luteolin. The lowest binding free energy of caspase-3 and quercetin is -6.0 kcal/mol. There are two hydrophobic bond forces between amino acid THR62 and quercetin. Moreover, The lowest binding free energy of MAPK1 and quercetin is -8.1 kcal/mol. There are two hydrophobic bond forces between amino acid ASP106, MET108 and quercetin. All compounds showed a compact binding pattern to the protein active pocket, and these interactions enabled the proteins to form stable complexes with all compounds. In this study, molecular docking experiment gives explanation for the way of protein-compound interactions and lays the theoretical foundation for further studies of Piwei-Periyuan Decoction in treatment of GC. Note: A higher absolute value of scores means that the binding of receptor protein and active ingredient is more stable.

Conclusion
In this study, we investigated the mechanism of Piwei-Peiyuan Decoction in treatment of GC using a network pharmacology approach. The results showed that Piwei-Peiyuan Decoction exerted its excellent pharmacological effects in treatment of GC through various pathways, such as regulation of cell cycle, promotion of tumor cell apoptosis and immunomodulation. Our study also provided a theoretical basis for the clinical application of Piwei-Peiyuan Decoction, which is of clinical value to further explore the role of TCM in prevention and treatment of cancer in the future.

Discussion
GC is one of the most common cancers in the world. At present, the mechanism of GC is not fully Ramachandran L et al 63 found that isorhamnetin can reduce the migratory and invasive properties of GC cells by regulating peroxisome proliferator-activated receptor γ (PPAR-γ), thereby inhibiting GC cell proliferation and inducing cell apoptosis. In addition, the combination of isorhamnetin and chemotherapy drugs can enhance the inhibition of tumor cell proliferation. Luteolin is a common avonoid in plants, which signi cantly inhibits cell cycle progression, proliferation, migration, invasion and promotes cell apoptosis 64 . Luteolin exerts its anti-tumor, antioxidant, and anti-in ammatory effects mainly through the regulation of signaling pathways such as Notch1, PI3K, AKT and ERK signal pathway. A study has been found that high expression of Notch1 was closely associated with low overall survival rate of GC patients 65 . However, Luteolin could inhibit GC progression by inhibiting Notch1 signaling pathway and reversing epithelial-mesenchymal transition (EMT). It suggests that luteolin may be an effective antitumor compound in treatment of GC. KEGG pathway enrichment analysis showed that the target genes of Piwei-Peiyuan Decoction for the treatment of GC were mainly involved in Pathway in cancer, PI3K-Akt signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, Ras signaling pathway, HIF-1 signaling pathway and TNF signaling pathway. In summary, it can be seen that the mechanism of Piwei-Peiyuan Decoction in treatment of GC is to inhibit cell proliferation and promote cell apoptosis by interfering with tumor-related signaling pathways, thus exerting anti-tumor effects. Besides, we declare that all data were generated in-house and that no paper mill was used.

Corresponding Author
Correspondence to Xuejun Li.

Ethics Approval
Not applicable.

Con icts of Interest
There are no con icts of interest to declare.