Yu Gao, Jian-Rong Sun, Chao Deng, Li-Qun Jia?

1.Beijing University of Chinese Medicine, Beijing 100029, China

2.China-Japan Friendship Hospital, Beijing 100029, China

Keywords:Shengjiang Xiexin decoction Chemotherapy-induced diarrhea Network pharmacology Mechanism of action Molecular docking

ABSTRACT Objective: To explore the key targets and mechanism of Shengjiang Xiexin Decoction in the treatment of chemotherapy-induced diarrhea based on network pharmacological methods.Methods: The effective components and corresponding target proteins of Shengjiang Xiexin Decoction were screened by TCMSP, and the target of chemotherapy-induced diarrhea were screened by the GeneCards. R software was used to obtain the common targets of drugs and diseases, and the “component-target-disease” network diagram was constructed by Cytoscape3.8.0 software. The string datebase was used to draw the protein interaction (PPI)network, and the Bioconductor software was used to perform GO function and KEGG pathway enrichment analysis on effective targets. Result: The result showed that 216 components were screened and 276 effective targets were screened. There were 1764 chemotherapy-induced diarrhea targets. The 173 common targets were obtained through venn diagram. The GO function analysis found 2427 items of biological process, 168 items of molecular function and 79 items of cellular component. The KEGG pathway analysis found 169 items. Conclusion:The PPI network found that STAT3、AKT1、MAPK3、JUN、MAPK1、RELA、IL6、etc. may be the key targets for Shengjiang Xiexin Decoction in treatment of chemotherapyinduced diarrhea. GO biological processes include DNA-binding transcription factor activity,cytokine receptor binding, cytokine activity, response to lipopolysaccharide, cellular response to chemical stress and so on. The KEGG pathways involved mainly include Toll-like receptor signaling pathway, TNF signaling pathway, inlfuenza A signaling pathway、hepatitise B signaling pathway and other pathways, that Play the role of anti-inflammatory and repair barrier.

1. Introduction

Chemotherapy-induced diarrhea (CID) mainly refers to diarrhea that occurs during or after the application of chemotherapy drugs such as 5-fluorouracil (5-FU) and irinotecan, and is a common clinical adverse reaction to chemotherapy, which not only seriously affects the efficacy and completion of treatment, but can even cause life-threatening symptoms such as dehydration and electrolyte disturbance in severe cases, causing great health and economic damage to oncology patients [1,2]. Literature shows that the incidence of diarrhea (grade 3-4) caused by chemotherapy in tumor patients is 10% to 20%, especially the incidence of diarrhea caused by 5-fluorouracil (5-FU) and irinotecan [3]. Irinotecan, a semi-synthetic derivative of camptothecin, is an inactive precursor drug. After liver metabolism, irinotecan enters the intestine, where it is transformed by β -glucosidase into the active product SN-38 that can damage the intestine, causing delayed diarrhea [4].At present, the conventional clinical treatment is octreotide,loperamide, smecta, etc. to inhibit intestinal peristalsis and protect intestinal mucosa. The effectiveness of Traditional Chinese medicine(TCM) as one of the prevention and treatment methods for CID has been preliminarily confirmed by preliminary clinical trials and basic experiments. Shengjiang Xiexin Decoction comes from Treatise on Febrile Diseases and is composed of Zingiber Officinale Roscoe (Shengjiang), Arum Ternatum Thunb (Banxia), Codonopsis Radix (Dangshen), Zingiberis Rhizoma (Ganjiang), Scutellariae Radix (Huangqin), Coptidis Rhizoma (Huanglian), Jujubae Fructus

(Dazao), licorice (Gancao). It is used for treating "upper abdominal distension, vomiting and burping, exhalation with acid taste of food accumulation, fullness under side, hyperactive bowel sounds in abdomen and diarrhea [5]. The cause is fluid mixed with food caused by stomach disorder. After more than 10 years of research,the preliminary clinical observation shows that Shengjiang Xiexin Decoction can significantly reduce the incidence of irinotecan-CID, especially for the high-risk population with UGT1A1 gene mutation [6-7]. Meanwhile, the mechanism was explored based on the National Natural Science Foundation project, and it was found that Shengjiang Xiexin Decoction could improve the intestinal mucosal injury induced by irinotecan by promoting intestinal cell proliferation and inhibiting intestinal cell apoptosis [8-10].

In this study, the network pharmacology research strategy was adopted to construct a multi-dimensional network of "drug components-target-action pathway" to explore the mechanism of action in treating CID, so as to realize the prediction and analysis of the mechanism of action of TCM compound and provide reference for further basic research.

2. Methods

2.1 Screening of active ingredients and prediction of action targets

Compounds of Zingiber Officinale Roscoe (Shengjiang), Arum Ternatum Thunb (Banxia), Codonopsis Radix (Dangshen), Zingiberis Rhizoma (Ganjiang), Scutellariae Radix (Huangqin), Coptidis Rhizoma (Huanglian), Jujubae Fructus (Dazao), licorice (Gancao)were searched through TCMSP database [11]. The screening conditions were oral bioavailability OB≥30% and drug similarity DL 0.18. The target targets of the compounds were predicted by TCMSP database.

2.2 Target search for CID

Based on the keywords "Chemotherapy induced diarrhea", human gene retrieval was performed in GeneCards database and NCBI gene database. Among them, GeneCards data were used to screen median Relevance Score to obtain targets with higher disease Relevance[12-13]. After integrating the retrieval results obtained from the two databases, the prediction target database of CID is established.

2.3 Drug-disease target screening and protein interaction network-PPI construction

The screened drug targets and CID targets of Shengjiang Xiexin Decoction were input into Venny 2.1, and the common targets obtained from the intersection were used as the prediction targets of drug action on diseases. The drug-component-target-disease network was constructed using Cytoscape 3.8.0 software.

When setting parameters, researchers set the species as "Homo sapiens" and the credibility as > 0.9. The common targets of Shengjiang Xiexin Decoction -CID were input into String database to construct PPI network, and PPI network was obtained [14].

2.4 GO enrichment analysis and KEGG enrichment analysis

GO Database (Gene Ontology) divides Gene functions into three parts: Cellular Component (CC), molecular function (MF),biological process (BP). By using GO database, we can obtain enrichment of target genes at CC, MF and BP levels, and conduct enrichment analysis of KEGG pathway for common targets of drug diseases. Reference the String database, set the correction P value <0.05, screening items. Bar charts and bubble charts are drawn using R 3.6.3 software "clusterProfiler," "EnrichPlot," and "GGplot2"packages. According to the descending order of the number of enriched genes, the biological processes and pathways with the highest number of enriched genes were selected as the main objects of this study.

2.5 Construction of potential targets and key pathway networks

After the common targets of Shengjiang Xiexin Decoction -CID were arranged according to data and attribute files in the text file(TXT), they were input into Cytoscape 3.8.0 together with 20 KEGG pathways participating in CID to build the network relationship between potential targets and KEGG pathway.

2.6 Molecular Docking

The SDF structure of the compound can be obtained from the Pubchem website based on topological parameters. SDF files are converted into PDB files using Open Babel 2.3.2 software. The receptor Protein was retrieved from the Protein Data Bank database.The software PYMOL 2.3.4 was used to conduct dehydrating and ligand removal operations on the receptor Protein, and AutoDockTools was used for hydrogenation and charge balance modification of the receptor Protein. Grid Box command under Grid program is used to open Grid Option tool to process each receptor protein. The size of ligand binding pocket is jointly determined by the number and spacing of lattice points in each direction. Therefore,adjust the number of lattice points in each direction of each protein and the center of binding pocket to the spacing of pass points.Autodock Vina 1.1.2 is used for semi-flexible docking.

3. Results

3.1 Active ingredients of Shengjiang Xiexin Decoction

OB%≥30% and DL≥0.18 were set as screening conditions, at TCMSP database is summarized to obtain: 29 compounds and 199 targets were obtained from Jujubae Fructus (Dazao). 36 compounds and 110 targets were obtained from Scutellariae Radix (Huangqin).21 compounds and 106 targets were obtained from Codonopsis Radix(Dangshen). 93 compounds and 215 targets were obtained from licorice (Gancao). 5 compounds and 43 targets were obtained from Zingiberis Rhizoma (Ganjiang). 14 compounds and 175 targets were obtained from Coptidis Rhizoma (Huanglian). 13 compounds and 93 targets were obtained from Arum Ternatum Thunb (Banxia). 5 compounds and 54 targets were obtained from Zingiber Officinale Roscoe (Shengjiang). After chemical composition was sorted out and de-weighted, a total of 229 compounds were obtained, among which the top 20 OB% components were listed as follows (Table 1).276 effective targets of the chemical constituents were obtained by reprocessing.

Table 1 Active components of Shengjiang Xiexin Decoction

3.2 Target search for CID

Human gene retrieval was performed in GeneCards database and NCBI gene database. Among them, GeneCards data were filtered

by Relevance Score to obtain targets with stronger relevance. 39 genes were retrieved from NCBI database and 1761 related genes were obtained from GeneCards database. After merging and deleting genes from the two databases, 1764 related genes were obtained, and the CID target database was established. The intersection of disease genes and drug targets is needed to obtain the potential therapeutic targets of drugs for disease.

The 276 drug targets screened out in 2.1 and 1764 disease targets obtained in this screening were input into Venny 2.1, a Venn diagram making software, and 173 common targets were obtained as predicted targets of drug action on diseases, as shown in Figure 1.

Figure 1 Venny map of intersection target of drug-disease

3.3 PPI network construction and visualization analysis and drug - component - disease - pathway - target network construction

The composition-disease-target network diagram was constructedon the basis of the screened ingredients, the treatment of diseases and the target of action, and the composition-disease-target network diagram was obtained by using Cytoscape 3.8.0 software (Figure 2). Such a construction could lead to a better understanding of the complex interactions between drug components and disease targets.The species was set as "Homo sapiens", and the credibility was set as > 0.9. 173 common targets of drug diseases obtained by Venn diagram were input into String database to construct PPI network.Get PPI network. There are 173 nodes and 794 edges in the network with an average degree of 9.18. PPI network diagram is exported from String website and imported into Cytoscape. The color and size of the nodes are adjusted according to the degree value. The edge between the thickness indicates a darker color and a greater degree of intensity (FIG. 3)

Figure 2 Network diagram of active components-effective targets. Purple rectangles in the network are Traditional Chinese medicines, lavender circles are active ingredients, green is the target of drugs acting on diseases, and orange rectangles are diseases.

Figure 3 PPI network diagram analyzed with cytoscape

3.4 Enrichment analysis of target functional pathways

BP, MF and CC enrichment of GO was carried out for the common targets of drug diseases, and the corrected P value was set≤0.05. By referring to String database, 79 cell components, 2427 biological processes and 168 molecular functions were enriched after screening. The main enrichment items of co-action targets in cell composition (CC) were membrane raft, membrane microregion and membrane region, etc. The main enrichment items of coaction targets in molecular function (MF) are cytokine receptor binding, DNA binding transcription factor binding, cytokine activity,etc. In biological process (BP), the main enrichment items are lipopolysaccharide response, oxidative stress response, reaction to bacteria-derived molecules, chemical stress response and so on. R

3.6.3 package was used to draw bar and bubble charts, as shown in Figs. 4 and 5.

KEGG pathway enrichment analysis was performed on the common targets of Shengjiang Xiexin Decoction -CID, and a total of 169 signal pathways were enriched. Using R 3.6.3, after installing and referencing the cluster Profiler package, bubble plot is performed,as shown in Figure 6. Specific information is shown in "KEGG enrichment table", mainly involving Toll-like receptor signaling pathway, TUMOR necrosis factor (TNF) signaling pathway, hepatitis B signaling pathway, influenza A (Inlfuenza A) pathway, small cell lung cancer related pathway and other signaling pathways. The files of Shengjiang Xiexin decoction - action component - CID - signal pathway - action target network were introduced into Cytoscape3.8.0 to make the pathway network diagram. The multi-component and multi-target action characteristics of the active ingredients of Traditional Chinese medicine in the treatment of CID were more intuitively shown, as shown in Figure 7.

Figure 4 GO enrichment analysis diagram(bar charts)

Figure 5 GO enrichment analysis(Bubble diagram)

Figure 6 KEGG enrichment analysis(bubble diagram)

Figure 7 Drugs-ingredients-disease target network diagram. Purple is the traditional Chinese medicine, pink is the compound, green is the target of traditional Chinese medicine on the disease, orange is the top 20 most significant pathways, and red is the disease, namely, chemotherapy-related diarrhea.

3.5 Core disease targets were obtained by topology analysis

Import PPI network into Cystoscape 3.8.0[15], set degree,betweenness centrality, average Shortest Path Length and Total Centrality parameters. Network Analyzer is used for topology analysis. Sorted by degree, genes with scores greater than average were selected as key targets, and a total of 58 key targets were screened out. The 20 core targets were STAT3, AKT1, MAPK3,JUN, MAPK1, RELA, IL6, MAPK14, MAPK8, APP, EGFR,VEGFA, FOS, ESR1, CCND1, CTNNB1, CXCL8, EGF, MYC and RB1. The first 20 key targets were drawn by R 3.6.3, and the abscissa was the degree value of each target, as shown in Figure 8.

Figure 8 Selection of key targets

Figure 9 Molecular docking visualization

3.6 Screening of key components of TCM compound

Cytoscape 3.8.0 was used for topological analysis of the composition-disease-target network. Rank the components by degree, and select the components with greater than the average degree value as the key components (the importance of the components increases with the degree value).

3.7 Molecular Docking

The 21 compounds with the highest degree value were selected for docking with the top 10 key genes, and the results showed that the binding energy (affinity) was less than -5kcal /mol, indicating that the receptor and ligand could spontaneously bind, suggesting strong binding activity. AutoDock Vina 1.1.2 software was used for molecular docking of receptor protein and ligand. Affinity results were expressed in the form of Affinity, the higher the Affinity value was, the worse the binding effect was, and the lower the energy value was, the better the binding effect was. Affinity scores include hydrogen bonding, repulsion, hydrophobicity, and complex spatial effects. Select representative binding energy score and docking parameters are shown in Table 3. Hydrogen bond interactions and hydrophobic interactions can be formed between the receptor protein and the ligand small molecule, indicating that the active component has a strong affinity with the target protein, and the conformation of the complex is stable, as shown in FIG. 9A to 9J.

Table 2 Key components

Table 3 Binding capability prediction of active ingredients of Shengjiang Xiexin decoction and the key targets

4. Discussion

The clinical incidence of CID is 50%-80% [16], which has a serious impact on the treatment and quality of life of patients. Cytotoxic chemotherapeutic drugs can inhibit the proliferation of cancer cells,but also have toxicity and side effects on other tissues and organs.Effective prevention and treatment of CID is a key clinical problem to be solved urgently. Current studies have found that the mechanism of CID involves intestinal inflammatory reaction and cell apoptosis[17-18].

CID belongs to the category of " diarrhea and dysentery” in TCM. At present, the pathogenesis of CID is mainly known as cold and dampness, cold and condensation with deficiency, cold and heat. Malignant tumor patients have a long course of disease,by cancer poison invasion for a long time after the consumption of healthy, qi and blood Yin and Yang imbalance, zang-fu organs function disorders, so this is deficient. Chemotherapeutic drugs mostly harm healthy qi, and do great damage to patients, especially gastrointestinal tract, when killing tumors. Common prescription for relieving diarrhea is Shengjiang Xiexin Decoction, Shenling Baizhu Powder, Sijunzi pills, Huoxiang Zhengqi pills, etc. Diarrhea disease is located in the intestinal tract, the intestinal interstitial edema caused by irinotekan is the water storage sanjiao in TCM, so the treatment is mainly to harmonize the spleen and stomach of middle coke, with the use of warming interior for diuresis, invigorating the spleen and dampness and other treatment, Shengjiang xiexin decoction, Gancao Xiexin decoction and so on.

As one of the three Xiexin decoction in Treatise on Febrile diseases, Shengjiang Xiexin Decoction has been widely used in clinical practice with remarkable curative effect and benefits many patients. The basic etiology of CID is retention of water and moisture to produce heat, cold and heat mixed. All kinds of vomiting pantothenic acid and severe diarrhea are caused by heat ". Therefore,the treatment of radix Scutellariae and rhizoma coptidis should also focus on "dredging". Water drink is Yin evil, so to use pinellia,dried ginger and large doses of ginger warm water drink, such as"sick phlegm drink when warm medicine and it", cold fluid block qi and heat, so appear "Diarrhea after hyperactive bowel sounds in abdomen", so huangqin was used to remove heat. Combined with clinical analysis, for example, diarrhea caused by irinotecan is common in clinical hyperactivity of abdominal bowel sounds,diarrhea watery stool, which is the manifestation of heat forced water fluid, and bitter mouth and dry throat, which belongs to the liver meridian. Diarrhea accompanied by anal pendant distension, sitting on the toilet for a long time, anal pendant distension is insufficient positive qi and the performance of wet, so the Shengjiang Xiexin decotion have the effect of tonifying deficiency, which is consistent with Li Zhongzi's "sweet reliveing method" for relieving diarrhea.

Through previous clinical and experimental studies, it has been confirmed that Shengjiang Xiexin Decoction can reduce the intestinal toxicity of irinotecan, and is found to be related to inhibiting the apoptosis of intestinal mucosal cells, promoting the proliferation of intestinal stem cells and changing the metabolic enzyme activity secreted by intestinal bacteria [19]. In this study, the network pharmacology was used to analyze the active components and action targets of Shengjiang Xiexin Decoction, so as to provide a theoretical basis for exploring the mechanism of Jiangjiang Xiexin Decoction in the treatment of chemotherapy-related diarrhea.

This study showed that there were 276 targets of the active components of Shengjiang Xiexin Decoction, and 173 targets related to CID. Luteolin, beta-sitosterol, ent-Epicatechin, Coniferin, catechin,quercetin, kaempferol, sitosterol and other key effective compounds.Literature shows that luteolin, a flavonoid, has anti-inflammatory effects and plays an important role in regulating intestinal immune system and treating chronic inflammatory bowel disease by reducing the volume and weight of intestinal contents, thus playing an antidiarrheal role [20-21]. Epicatechin can regulate K+ and Ca2+channels [22]. It may reduce inflammatory factors TNF-α,IL-1 and IL-6, inhibit lPS-induced cellular inflammatory response, and play an anti-inflammatory role [23]. Quercetin can significantly reduce the mRNA and protein levels of TLR2 and TLR4, reduce the protein expression level of MyD88, further reduce the release amount of inflammatory factor TNF-α, and protect rat intestinal mucosal microvascular endothelial cells from inflammatory damage [24].Through network pharmacology, this study found that Shengjiang Xiexin Decoction may play a role in treating CID mainly through the above compounds.

According to the results of protein interaction network, STAT3,AKT1, MAPK3, JUN, MAPK1, RELA, IL6, MAPK14, MAPK8,APP and EGFR are the key targets in the treatment of chemotherapyrelated diarrhea with Jiangxiexin Decoction. Studies have shown that pro-inflammatory cytokines cause damage to the intestinal mucosal barrier by activating the STAT3 signal transduction pathway,leading to diarrhea [25]. The release of inflammatory factor IL-6 leads to intestinal bacterial migration and absorption of endotoxin,aggravating the damage of intestinal mucosal barrier and aggravating diarrhea [26]. AKT1 is a key signal transduction node that regulates immunity, reduces inflammation and inhibits cancer [27].

The results of GO functional enrichment analysis were mainly related to biological processes such as DNA-binding transcription factor binding, cytokine receptor binding, lipopolysaccharide response and chemical stress response. KEGG enrichment analysis of target genes showed that toll-like receptor signaling pathway,tumor necrosis factor (TNF) signaling pathway and hepatitis B signaling pathway were highly enriched, which were closely related to CID. Toll-like receptor (TOLL-like receptor) is a kind of pattern recognition receptor, which can activate related signal pathways by recognizing pathogen-related molecular structures expressed by pathogenic microorganisms to induce immune response, so as to eliminate invasive pathogenic microorganisms [28-29]. TLR can also be expressed on the surface of intestinal epithelial cells and can recognize and bind to microorganisms and their metabolites.So far, more than 10 TLR family members have been found in mammals. Among them, the expression of TLR4 is closely related to the occurrence of irinotecan delayed diarrhea, and TLR4 damages the intestinal barrier function by promoting the expression of inflammatory factors and inhibiting the expression of INTESTINAL epithelial TJs [30]. JNK signaling pathway is mainly involved in the regulation of cell proliferation, differentiation and apoptosis.Inhibition of JNK expression promotes TJs expression in intestinal epithelium and reduces intestinal permeability [31].

Molecular docking was performed with 21 active ingredients and 10 targets, and the binding energy was less than -5kcal /mol,forming hydrogen bonds. The selected drug ingredients had high binding property with the target, which verified the reliability of using network pharmacology method to investigate the mechanism of action of Shengjiang Xiexin Decoction on CID.

In summary, this study predicted the mechanism of action of Shengjiang Xiexin Decoction in treating CID by using the method of network pharmacology and molecular docking, and found 173 common targets of drugs and diseases. The treatment of CID by Shengjiang Xiexin Decoction may involve biological processes such as DNA-binding transcription factor binding, cytokine receptor binding, lipopolysaccharide response and chemical stress response through the action of STAT3, AKT1, MAPK3, JUN, MAPK1,RELA, IL6 and other proteins. Hepatitis B signaling pathway,Inlfuenza A signaling pathway, small cell lung cancer signaling pathway, etc., and play an anti-inflammatory and repair barrier role. This study provided important reference for the further study of Shengjiang Xiexin Decoction and preliminarily explored the mechanism of drug action. However, this study is only a prediction,and it is a prediction discussion at the level of molecular mechanism.Based on the top pathway in the research results and combined with the references, basic research is carried out to further verify its specific mechanism of action.

Author’s contribution

Gao Y: The first author, wrote the paper, project implementation and other work;

Jia LQ: Corresponding author, proposed the thesis theme, designed the implementation plan, and reviewed the final draft of the thesis;Sun JR: Network pharmacology, molecular docking data analysis and collation; Deng C: Chart arrangement and article modification.