Mechanism of Action of Baohe Pills in Improving Functional Dyspepsia

: The aim of this study was to predict the mechanism of action of Baohe Pill in the treatment of functional dyspepsia using network pharmacology. Predict the main active ingredients and action targets of Baohe Pill by TCMSP platform database of Traditional Chinese Medicine Systematic Pharmacological Analysis Platform, and convert the collected drug targets into gene names using Uniprot database; obtain FD genes by GeneGards, TTD, OMIM database, and use Cytoscape 3. 9.0 to visualize the “drug-ingredient -target” network visualization; and protein-protein interaction (PPI) network by using String platform; GO and KEGG analysis of common targets by using Metascape database; molecular docking by using AutoDock software to verify the binding activity of active ingredients to target proteins. After database display analysis, 107 active ingredients, 511 potential action targets and 2560 FD disease targets of Bohol Pill were screened. By KEGG analysis, GO analysis yielded 6382 biological process entries, 1101 molecular function entries, 586 cellular component entries, and 291 pathways.


Introduction
Functional dyspepsia, as one of the clinical common gastrointestinal diseases, is a group of clinical symptoms caused by gastrointestinal dysfunction, excluding organic lesions, which mainly manifests as abdominal pain, bloating, lack of appetite, nausea, vomiting and other symptoms, and some patients also have headache, impatience, depression, insomnia, lack of concentration and other uncomfortable symptoms.In recent years, due to the increasing social pressure, irregular diet and life, the incidence of FD is increasing and has become one of the high incidences affecting the modern life.The pathogenesis of FD has not been fully understood, and it is generally associated with abnormal secretion of gastric acid and digestive enzymes, and there is no clinical cure.Chinese medicine treatment emphasizes holistic concept and evidence-based treatment, and multi-level, multi-path and multi-target treatment of FD through the combination of drugs, which has achieved better efficacy in the treatment of FD and is widely used clinically and can effectively relieve patients' symptoms such as epigastric pain, abdominal burning sensation, early satiety and postprandial fullness.Baohe Pill originated from the Danxixin Method written by Zhu Danxi in the Yuan Dynasty.It is composed of Chenpi, Lianqiao, Shanzha, Laifuzi, Shenqv, Fuling and Banxia.It can eliminate food stagnation, move Qi and harmonize the stomach, and is widely used in clinical practice to relieve FD symptoms.This study utilized network pharmacology to further explore the mechanism of action of Baohe Pill in the treatment of FD, and verified the binding of each component of Bohe pill to FD targets by molecular docking, which provides a reference for further research on the pharmacological effects of Bohe pill on FD.

Target Screening of Bohe Pills
The database of the TCM Systematic Pharmacology Technology Platform was searched for the active ingredients of seven Chinese herbal medicines, namely, Banxia, Chenpi, Fuling, Lianqiao, Laifuzi, Shanzha and Shenqu, active ingredients were initially screened according to oral bioavailability (OB)≥20% , drug sample (DL)≥0.10 [1].For those that were not recorded in the database, the corresponding compounds were obtained from BATMAN and the Chinese General Knowledge Resources Database by searching the relevant literature, and then screened by the SwissADME database, and those with high Gi Absorption and in accordance with Lipinski's five The components with high Gi Absorption and Lipinski's five-fold rule were selected as the final active ingredients [2] .And the constituent targets were collected using Swiss Target Prediction database, and those with Probability ≥ 0.2 were used as the final drug targets of the drug.After screening, species condition is set to "Human" and the collected targets were normalized to their gene names using the UniProt database.

FD Target Gene Screening
The search terms "Functional Dyspepsia" was used in GeneCards, OMIM and TTD databases, and the FD targets in the three databases were combined and de-duplicated to obtain the final FD gene targets.

To Construct the Network Diagram of "TCM -Ingredient -Target" between Bohe Pills and FD
The targets of Baohe pill and FD were imported into Venny 2.1.0separately to obtain their common targets and draw Venn diagrams.Using CytoScape 3.9.0software, the "herbal-component-target" network diagrams of Baohe Pill and FD were constructed.

PPI Network and Screening of Key Genes
The PPI network diagram of the common target was obtained with String11.0platform.Download TSV format and import Cytoscape 3.9.0software to screen core targets and perform topological analysis via Network Analyzer.

Go and KEGG Analysis
Will get common target import metscape platform, species condition is set to "Homosapiens," GO and KEGG analysis, select before 20 P < 0.01 data mapping, analysis of the biological significance of FD treatment and the possible mechanism [3] .

Molecular Docking
The top 6 drug components with strong association in Baohe pill selection and correlation in the PPI strong before six common protein targets for molecular docking.The 3D structures of drug components and target protein download structure respectively from PubChem and PDB database.AutoDock software was used to hydrogenate and root small molecules and hydrogenate and dehydrogenate large molecules [4] .Then the molecular docking was verified and the results were plotted by Pymol software.

The Active Ingredients and Corresponding Targets of Baohe Pill
The components and corresponding targets of five Chinese herbal medicines were retrieved from the TCMSP, and the active ingredients and targets of Shanzha were supplemented with BATMAN, and the active ingredients of Shen Qu were supplemented by searching relevant literature, and the corresponding Canonical SMILES were retrieved from the PubChem [5] .The corresponding Canonical SMILES were downloaded from the PubChem, and the ingredients with high GI Absorption and in accordance with Lipinski's five-fold rule were used as the final active ingredients through the SwissADME database to obtain 107 active ingredients of Baohe Pill, including 9 Chenpi, 8 Fuling, 10 Laifuzi, 21Banxia, 26 Lianqiao, 5 Shenqu, and 28 Shanzha.After deleting the duplicate targets, the targets were transformed into corresponding gene names using Uniprot database, and finally 511 drug target genes were obtained.

Target Genes of FD
The keyword "Functional Dyspepsia" was used to search FD-related targets, among which 2535 genes were retrieved from GeneCards, 30 from OMIM and 1 from TTD, and 2560 FD disease genes were finally after remove duplicate genes.

The "Herbal-Component-Target" Network Diagram of Baohe Pill and FD
Venn diagram was drawn, and 253 common targets of active component and FD were obtained, as shown in Figure 1.Abbreviate traditional Chinese medicine according to the initial letter of pinyin, such as hawthorn (shan zha, SZ), and code the six ingredients in Baohe Wan as A, B, C, D, E and F respectively, as Table 1.Hawthorn and divine comedy components were completed through the BATMAN database and literature review, as shown in Table 2.The network diagram of "TCMcomponent -target" was drawn, as Figure 2. Network Analysis was used to calculate and analyze 347 nodes (87 active components associated with FD, 253 common targets, 7 drug names) and 864 edges, indicating that 87 active components acted on FD in Bohe Pills, and the lines represented the relationship between the components of Bohe Pills and FD targets.

PPI Network Map and Key Targets
Using the String database, the PPI interactions of common targets were exported with scores ≥ 0.4 and "Human sapiens", and import TSV format of results into Cytoscape 3.9.0 to create a visual PPI, see Figure 3.

GO and KEGG Enrichment Analysis
Will receive 253 common target import metscape database for GO analysis, set up P < 0.01, and select 20 data online before drawing.Enrichment analysis of BP yielded 6382 data, biological process was mainly related to related to responses to inorganic substances, hormones and xenobiotic, see Figure 4-A  The highest value of six kinds of effective ingredients, quercetin, kaempferol, luteolin, oleic acid, wogonin and naringenin, were selected by Cytoscape 3.9.0 and docked with the top 6 FD genes in the PPI.The docking results with the lowest binding energy were selected and plotted by pymol software, as Figure 5.

Discussion
In this formula, Shan Zha, Laifuzi and Shen Qu work together to eliminate food and stagnation.The three herbs together can eliminate all kinds of food and stagnation, while Ban Xia and Chen Pi work together to move Qi and eliminate stagnation.Fu Ling strengthens the spleen and dispels dampness, and Lian Qiao disperses heat and eliminates stagnation.The combination of the seven herbs works together to strengthen the spleen and stomach, eliminate food and stagnation.Through the visualization network of "drug-component-target" between Baohe pill and FD, a total of 87 drug components were found to be related to Baohe pill for the treatment of FD, among which quercetin, kaempferol, luteolin, all have anti-inflammatory, antioxidant, anti-tumor and immunoregulatory functions.They are the core compounds of Bohol Pill, mainly plant polyphenols, which can regulate inflammatory factors through RAGE/NF-κB/MAPK pathways to regulate the expression of tumor necrosis genes and inflammatory mediators.By PPI network analysis, ACTB is the main target genes of Bohol pill for FD treatment.It was found that ACTB is associated with the development of cancer, and the dysregulation of ACTB expression is associated with the occurrence of many cancers such as esophageal cancer, pancreatic cancer and gastric cancer.Using Autodock, we found that all six drug components docked with six target proteins at low binding energies, further justifying the application of Bohol pills for the treatment of FD.
In summary, the active ingredients in Baohe pill, such as quercetin, kaempferol, luteolin, oleic acid, wogonin and naringenin can alleviate inflammatory response, reduce visceral hypersensitivity, improve psychological factors such as anxiety and depression, promote gastrointestinal.This study demonstrated the mechanism of action of Baohe Pills in the treatment of FD, further proved the natural advantages of TCM in the treatment of diseases, and provided certain theoretical guidance for the subsequent experimental research.

Figure 3 :
Figure 3: Gene PPI Network of Bohe Pill and Functional Dyspepsia . Cellular component was associated with membrane raft and vesicle lumen, see Figure4-B.Molecular function was mainly connected with protein kinase activity, DNA-binging transcription factor binding, and signaling receptor activity, as 4-C.KEGG analysis showed that a total of 291 pathways, as Figure4-D.The results of KEGG enrichment showed that it was related to pathways in cancer and lipid pathway, with the most significant enrichment in the cancer pathway, it suggests that it may very important to treat FD.

Table 1 :
Basic information of active ingredients in Baohe pills

Table 2 :
Basic information of effective ingredients of Shan Zha and Shen Qu