Molecular mechanism of treatment of pneumonia in children with Mori cortex - Lycii cortex based on network pharmacology

Fu-Shuang Yang, Fang Cao, Yi-Bu Kong, Yan Xu, Zhong-Tian Wang, Li-Ping Sun

1. Changchun University of Chinese Medicine, Changchun 130117, China

2. Child Care Centre, Affiliated Hospital of Changchun University of Chinese Medicine, Changchun 130021, China

Keywords:Mori Cortex Lycii Cortex Network pharmacology Pneumonia Children Mechanism prediction

ABSTRACT Objective: To study the molecular mechanism of Mori Cortex - Lycii Cortex for infantile pneumonia based on network pharmacology. Methods: TCMSP and BATMAN-TCM online prediction database were used to screen and collect the active ingredients and targets of Mori Cortex - Lycii Cortex with oral bioavailability (OB) and drug-like(DL). Predictive analysis of disease targets was conducted through PubMed, GeneCards and DrugBank databases. The component-target regulation network was constructed by using Cytoscape 3.7.2 software, and the network topology of the core target was analyzed. Finally, the Bioconductor platform and R language were used for GO function analysis and KEGG pathway enrichment analysis,and the target-key pathway network diagram was constructed. Results: A total of 43 active components, including quercetin, kaempferol, acacetin, and beta-sitosterol, were identified with 242 potential targets. There were 3271 pneumonia targets in children, among which the key targets were IL-6, AKT1, MAPK8, etc. There were 31 common targets of MMP9, TNF, AKT1 and so on. GO biological processes include the response to lipopolysaccharides, molecule of bacterial origin, metal ions, regulation of apoptotic signaling pathway, and T cell activation.The KEGG signaling pathways involved mainly include TNF, PI3K/AKT and MAPK signaling pathway.Conclusion: Mori Cortex - Lycii Cortex for quercetin, kaempferol, beta-sitosterol and acacetin element composition such as possible through the AKT1, MAPK8, IL-6 and MMP9 targets in the role of such as TNF, PI3K/AKT, MAPK signal pathways and so on several signal transduction pathways that play to the role of the treatment of children pneumonia, the results can be for the further study of Mori Cortex - Lycii Cortex for mechanisms to provide the reference for the treatment of pneumonia in children.

1. Introduction

Pneumonia is the most common respiratory disease in childhood and the main reason for children’s hospitalization. It is mostly caused by single or mixed infections of respiratory viruses, bacteria,and atypical microorganisms [1]. The basic pathological changes are bronchial walls and alveoli Inflammation is mainly manifested clinically by fever, cough, shortness of breath, and fine wet rales in the lungs [2]. Because its incidence rate is the first among children's respiratory diseases, the disease has been listed as one of the key children's diseases in my country [3]. Child pneumonia is equivalent to the pneumonia cough in Chinese medicine. It is caused by feeling the six evils. Modern physicians generally believe that phlegm and heat are the pathological products, and lung qi depression is the key to its pathogenesis [4].

A drug pair means that the two drugs are compatible,interdependent and restrict each other, so as to play a synergistic effect to increase efficiency and reduce toxicity. The Mori Cortex-Lycii Cortex drug pair is an important drug combination of Xiebaisan in Qian Yi's "Straight Jue of Pediatric Medicine Syndrome" in Song Dynasty. Mori Cortex is sweet and cold, enters the lungs to divide the qi, relieves the evil heat in the lungs, and relieves the lungs and relieves asthma. Lycii Cortex is sweet and cold, it enters blood circulation, clears heat and cools blood, clears lungs and heats. Compatibility of the two drugs, qi and blood shuangqing,enhance the effect of clearing lung heat, reducing lung fire, reducing lung qi, expelling phlegm and coughing, and relieving asthma [5].Modern pharmacological studies have shown that the main chemical components of Mori Cortex are flavonoids, including Mulberrin,Sanggenon AP, Moracenin C, D, etc., which have anti-inflammatory,antitussive, Expectorant and anti-asthmatic effects [6]. The components of Lycii Cortex are alkaloids, anthraquinones, organic acids and their esters, mainly including Kukoamine A, Emodin, and linoleum Linoleic acid, etc., have antipyretic, antibacterial, antiinflammatory and immune regulation effects [7]. Clinical studies have also found that the prescription containing Mori Cortex and Lycii Cortex has a good therapeutic effect on childhood pneumonia[8,9], but its specific material basis and mechanism of action are still unclear.

Network pharmacology is an emerging science that integrates molecular biology, bioinformatics, pharmacology, and multiple network computing platforms. It explains the relationship between drugs and diseases at multiple levels from the macro to the micro level. It uses multiple computers The software and data platform visualize the mechanism of action, that is, intuitively clarify the molecular mechanism of Chinese medicine on multiple molecules,multiple targets, and multiple pathways of diseases [10,11]. Therefore,this study uses network pharmacology as a research method to construct a multi-dimensional network of "components-targetspathways-disease", and explores the potential molecular mechanism of Mori Cortex-Lycii Cortex medicine on the treatment of childhood pneumonia. The clinical application of Mori Cortex-Lycii Cortex and the clinical and basic research of childhood pneumonia provide reference.

2. Materials and methods

2.1 Database and software

The pharmacology database and analysis platform of Chinese medicine system (TCMSP, http://tcmspw.com/tcmsp.php). The bioinformatics analysis tool of the molecular mechanism of Chinese medicine (BATMAN-TCM, http://bionet.ncpsb.org/batman- tcm/).PubMed database (http://www.ncbi.nlm.nih.gov).

GeneCards database (http://www.genecards.org/). DrugBank database (http://www.drugbank.ca/). Unitprot database (http://www.Unitprot.org/). Bioconductor platform (http: //www.bioconductor.org/). Cytoscape 3.7.2 software (https://cytoscape.org/). R language 3.5.2 software (http://www.r-project.org/). Perl 3.6. 0 software(https://www.perl.org/). Venn diagram drawing platform (http://bioinformatics.psb.ugent.be/webtools/Venn/). STRING platform(http://string-db. org/).

2.2 Screening of ingredients and corresponding targets in drugs

In the online prediction databases of TCMSP and BATMANTCM, respectively, the chemical composition was retrieved with the keywords of "Mori Cortex" and "Lycii Cortex", and the oral bioavailability (OB) ≥30% and met the drug-like properties(DL) ≥0.18 active ingredient, and search for the target protein corresponding to the active ingredient from TCMSP, and determine the gene abbreviation of the target protein through the Unitprot database.

2.3 Prediction of childhood pneumonia targets

Use "infantile pneumonia", "children pneumonia" and "Pneumonia in Children" as keywords to search the GEO database in GeneCards and PubMed to collect the related genes of childhood pneumonia. At the same time, according to the clinical guidelines to understand the marketed drugs for the treatment of children's pneumonia, collect the target points of the relevant drugs in the DrugBank Database, and finally integrate the above search results to remove the weight, and establish a predictive target database of children's pneumonia.

2.4 Active ingredients-network construction of key targets

Use the Venn diagram online drawing tool to match and map the target points of the active ingredients of traditional Chinese medicine and the disease targets, and draw the Venn diagram to obtain the common gene of the two, namely the Mori Cortex-Lycii Cortex active ingredient, the potential target of pneumonia treatment. Cytoscape 3.7.2 was used to construct an "active ingredient-key target"regulatory network to visualize the mechanism of action of Mori Cortex-Lycii Cortex in the treatment of childhood pneumonia. The type of node is the common target of active ingredient and Chinese medicine-disease.

2.5 Protein interaction network construction and network topology analysis

Upload the common target of Mori Cortex-Lycii Cortex and disease to STRING platform to build a protein interaction network (PPI),input protein interaction genes into the "BisoGenet" plug-in of Cytoscape 3.7.2 software, select "Organism" "Homo sapiens",select "Gene" for the mapping range, select "DIP, BIOGRID,HPRD, INTACT, MINT, BIND" for the database source, and set the remaining parameters as defaults to further analyze the interaction relationship; combine the interactions obtained by the "BisoGenet"plugin The active network uses the CytoNCA plug-in to analyze the network topology. First, the degree (DC) is used as the filter condition for analysis, and the obtained network is analyzed again with the Betweenness (BC) as the filter condition, and finally the PPI core network is obtained.

2.6 GO function analysis and KEGG pathway enrichment analysis

Use R language and Bioconductor platform to perform GO function analysis and KEGG pathway enrichment analysis on the obtained intersection genes. The results of GO enrichment analysis should include three aspects: biological process (BP), cellular component(CC) and molecular function (MF). For biological processes with a threshold value of P＜0.001 [12] and pathways are arranged in descending order according to the number of enriched genes, and the biological processes and pathways with the highest number of enriched genes are selected as the main discussion objects of this study.

2.7 Potential targets and critical pathway network construction

After obtaining the 20 KEGG pathways involved in childhood pneumonia, the common targets of Chinese medicine-disease are arranged according to the data and attribute files to form a txt format file, which is input into the Cytoscape 3.7.2 software to construct potential targets Relationship with KEGG access network.

3. Results

3.1 Screening of active ingredients of Mori Cortex-Lycii Cortex

A total of 194 chemical constituents of Mori Cortex and 37 chemical constituents of Lycii Cortex were retrieved through the online prediction database of TCMSP and BATMAN-TCM. The two parameters are oral availability (OB) ≥30% and drug-like properties(DL) ≥0.18 After further screening under limited conditions, 31 potential active ingredients of Mori Cortex and 13 potential active ingredients of Lycii Cortex were finally obtained, as shown in Table 1.

Table 1 Potential active components of Mori Cortex-Lycii Cortex

3.2 Prediction of the potential targets of anti-inflammatory effects of the Mori Cortex-Lycii Cortex components

The target protein corresponding to the above active ingredients was searched in the TCMSP database, and after removing duplicate and invalid targets, 173 targets for the active ingredients of Mori Cortex and 69 targets for the active ingredients of Lycii Cortex were obtained. Through PubMed, GeneCards and DrugBank databases, we searched the targets of childhood pneumonia, and finally got 3271 targets of childhood pneumonia. Using the online Venn diagram drawing platform to calculate and draw the Venn diagram (Figure 1), 134 common genes were obtained, of which 127 belonged to the Mori Cortex, 38 belonged to the Lycii Cortex,and the drug and disease there are 31 common targets, namely SLC6A3, ADRB2, NOS2, AR, CASP3, CASP8, FASLG, PPARG,EGFR, AKT1, VEGFA, CCND1, MMP9, IL6, SOD1, ERBB2,MYC, ICAM1, IL1B, CCL2, VCAM1, NOS3, IL2, SERPINE1,COL1A1, IL1A, MPO, CRP, CXCL10, CD40LG, MAPK8. The above suggests that Mori Cortex and Lycii Cortex have their own key targets to treat pneumonia, and they also have common key targets to coordinate their anti-pneumonia effects.

Figure 1 The Venn diagram of the matching of the target genes of pneumonia in children with the target genes of Mori Cortex-Lycii Cortex

3.3 Network construction and analysis of active ingredientskey targets

There are 24 active ingredients related to the targets of pneumonia in Mori Cortex, including Sexangularetin, Iristectorigenin(9CI),Moracin B, Moracin C, Moracin D, Moracin F, Glabrone,campest-5-en-3beta-ol, 7 -methoxy-5,4'-dihydroxyflavanonol,cyclomulberrochromene, Dimethyl (methylenedi-4,1-phenylene)biscarbamate, Moracin A, moracin O, Mairin, mulberroside C_qt,sanggenone F, sanggenone H, sanggenone M, 3,5,7- trihydroxy-2-(3-hydroxyphenyl)chromone, quercetin, beta-sitosterol, kaempferol,pelargonidin, sanguinarine. There are 11 active ingredients related to the targets of children's pneumonia in the skin of Lycii Cortex,hederagenin, beta-sitosterol, Stigmasterol, CLR , OIN, Linoleyl acetate, acacetin, scopolin, Atropine, sugiol, aurantiamide acetate,and the common active ingredient of the two is beta-sitosterol.The key targets common to active ingredients, traditional Chinese medicine and diseases are imported into Cytoscape 3.7.2 software for visual network construction (Figure 2). The network shows that the top 5 connections of active ingredients of drugs are quercetin,kaempferol, acacetin, beta-sitosterol, Iristectorigenin (9CI), and Glabrone (tied for fifth). The top 5 key target genes are PTGS2,HSP90AA1, PTGS1, AR, PRSS1. The above-mentioned components and targets are important active components and important target genes of Mori Cortex-Lycii Cortex in the treatment of childhood pneumonia, and they are of great significance in the treatment of Mori Cortex-Lycii Cortex in the treatment of childhood pneumonia.

Figure 2 Active ingredients of Traditional Chinese Medicine-the regulatory network of key disease targets

3.4 Construction of PPI network of shared protein

Upload the crossed 31 target genes to the STRING platform, and construct the PPI network according to the advanced screening criteria "high confidence (0.7)", that is, the minimum required interaction score is 0.7, and the protein interaction network diagram is obtained (Figure 3). The network diagram has 31 nodes and 171 edges, mainly related to IL6, VEGFA, AKT1, MAPK8, MMP9,IL1B, CCL2, etc.

3.5 Network topology analysis

In order to further explore the pharmacological mechanism of Mori Cortex-Lycii Cortex on the treatment of childhood pneumonia,the common target genes were input into the BisoGenet plugin of Cytoscape 3.7.2 software for network analysis, and then the CytoNCA plug-in was opened, and a preliminary one with 3035 nodes was obtained , PPI network with 70307 edges, with Degree value＞61 for 2 screenings to obtain a PPI network with 704 nodes and 28530 edges, and then with Betweenness value＞800 for 3 screenings, to obtain a PPI network with 121 nodes, PPI network with 2582 edges, the specific selection process is shown in Figure 4.

Figure 3 The protein interaction network of Mori Cortex-Lycii Cortex in the treatment of children with pneumonia

3.6 GO function analysis and KEGG pathway enrichment analysis results

In the GO function analysis results, the biological processes with the top 10 enriched genes of BP, MF, and CC were selected based on P＜0.001. It shows that the treatment of Mori Cortex-Lycii Cortex in the treatment of childhood pneumonia mainly involves the response to molecule of bacterial origin, regulation of apoptotic signaling pathway, T cell activation and other biological processes; Cytokine receptor binding, receptor ligand activity, protein phosphatase binding and other molecular functions; Membrane raft, extracellular matrix, vesicle lumen and cell components, see Table 2 for details.Similarly, a total of 121 signal pathways were obtained by KEGG analysis, and the top 20 signal pathways were screened out according to the P -value and the number of enriched genes. Mainly related to TNF signaling pathway, PI3K-Akt signaling pathway, Influenza A, MAPK signaling pathway, IL-17 signaling pathway and HIF-1 signal pathway, etc., Use R language software to visualize the data to obtain a bubble chart (Figure 5), where the color of the bubble represents the significance of enrichment, the redder the color, the higher the enrichment degree; the size of the bubble represents the The number of genes enriched in the pathway, the larger the bubble,the more enriched genes.

Figure 5 Visualization results of KEGG signal pathway

3.7 Potential target-critical pathway network construction results

The key signal pathways and the potential targets of the Mori Cortex-Lycii Cortex are mapped one by one to construct a network of potential targets and key pathways, as shown in Figure 6. Among them, the signal path is shown by diamond-shaped nodes, and the potential targets are shown by rectangular nodes. As can be seen from the network diagram, the key targets involved in the treatment of pneumonia in children with Mori Cortex-Lycii Cortex mainly include AKT1, IL6, CASP3, MAPK8, IL1B, etc. 24. The above targets mainly involve TNF, PI3K/AKT, Inlfuenza A , MAPK and other signal pathways.

Figure 6 Potential target-critical pathway relationship network

Table 2 Analysis of GO function of the core network of Mori Cortex-Lycii Cortex regulating childhood pneumonia

4. Discussion

Because of its effect of increasing efficacy and reducing toxicity,Traditional Chinese Medicines with two relatively fixed flavors are often used together in the clinical process of Chinese medicine,and the clinical effect is quite good. However, the mechanism of Chinese medicine on diseases is not completely clear, and there is no scientific basis that can clearly explain the mechanism of Chinese medicine. The advent of network pharmacology provides a new method and thinking for the study of the mechanism of Chinese medicine on diseases. It can link the whole to the micro,and systematically analyze the molecular mechanism from multiple levels. This is unified with the overall concept of Chinese medicine and the thought of syndrome differentiation and treatment [13]. The Mori Cortex-Lycii Cortex was derived from the Xiebaisan in the Song Dynasty "Child Medicine Syndrome Zhi Jue", the compatibility of the two drugs has the effects of clearing lung heat, relieving cough and relieving asthma, and is still widely used in modern clinical practice. Modern pharmacological studies have shown that the antiinflammatory effects of Mori Cortex-Lycii Cortex are significant[6,7], and the inflammatory response has always been throughout the entire pathological process of childhood pneumonia, and has clinical manifestations of heat, cough, phlegm, and asthma. The efficacy and indications of the Mori Cortex-Lycii Cortex are very matched.Therefore, this study adopts the method of network pharmacology to explore the specific molecular mechanism of the Mori Cortex-Lycii Cortex on childhood pneumonia from a micro perspective.

The analysis results show that the Mori Cortex-Lycii Cortex mainly uses quercetin, kaempferol, acacetin, β-sitosterol and other active ingredients to intervene in childhood pneumonia. Studies have shown that quercetin can reduce the levels of serum IL-6, IL-1β and TNF-α in Staphylococcus aureus mice and down-regulate the expression levels of NF-κB, IKK and IκBα mRNA and protein in lung tissues of mice. Inhibit the inflammatory response in mice [14]. Kaempferol also has a strong anti-inflammatory effect, which is mainly related to the regulation of inflammationrelated gene expression and pro-inflammatory enzyme activity,inhibition of adhesion molecules, transcription factors and matrix metalloproteinases [15]. Acacetin can not only reduce inflammatory factors such as intercellular adhesion molecule-1, IL-6 and IL-8,but also play an anti-inflammatory effect through mechanisms such as airway hyperresponsiveness and eosinophil infiltration [16,17].β-sitosterol is an important component of phytosterols. It reduces inflammatory cell viability and phagocytosis, inhibits the production of TNF-α and NO, thereby reducing the symptoms of inflammation[18].

The protein interaction network results showed that the active ingredients of the Mori Cortex-Lycii Cortex may exert their effects through key targets such as IL-6, VEGFA, AKT1, MAPK8, MMP9,IL1B, and CCL2. In pneumonia infection, IL-6, as a non-specific inflammatory cytokine, can induce T cell differentiation, promote the release of inflammatory cytokines, and participate in the pathological process of lung inflammation [19]. MMP9 is a protease that degrades the extracellular matrix. It can damage and degrade the extracellular matrix and basement membrane of the respiratory system through the mediation of calcium or zinc ions, and make the normal structure of the respiratory system It is destroyed, and then participates in airway inflammation [20]. In addition, the analysis of the protein interaction network topology showed that the regulation process is also related to ICAM1, CASP3, EGFR, AR, CRP, etc.,fully verifying that the Mori Cortex-Lycii Cortex is a multi-molecular and multi-molecule drug for the treatment of children the mechanism of the target.

KEGG results showed that the Mori Cortex-Lycii Cortex may act on the TNF signaling pathway, PI3K-Akt signaling pathway, Influenza A, MAPK signaling pathway, IL-17 signaling pathway and HIF-1 signal pathway a therapeutic role. Studies have shown that the PI3KAkt signaling pathway plays an important role in pro-inflammatory and anti-inflammatory factors by regulating the expression of dendritic cells and macrophages [21], and experimental studies have shown that the total flavonoids and total polysaccharides of Morus alba It may play a role in reducing inflammation and preventing pneumonia by regulating the PI3K/Akt signaling pathway in RSV pneumonia mice and regulating the expression of serum IFN-γ and IL-4 [22]. The MAPK signaling pathway is mainly involved in the regulation of cell proliferation, differentiation and apoptosis, and Cheng Jian et al. [23] found that quercetin, the active component of Mori Cortex, can inhibit the expression of MAPK pathway marker proteins p38, p44/p42, and SAPK/JNK. , Thereby reducing the release of inflammatory factors, and ultimately reducing the degree of inflammatory response. The anti-inflammatory effect is closely related to the MAPK pathway. Studies have also shown that both MAPK and PI3K signal transduction pathways are involved in regulating the expression of inflammatory mediators [24]. Kaempferol can effectively inhibit the expression of the MAPK signal pathway of human monocyte THP-1 induced by LPS, which can reduce macrophages. Cell-derived chemokines, IP-10 and IL-8 and other inflammatory factors are produced, thereby effectively preventing the occurrence of inflammation [25].

In summary, this study analyzed the active ingredients, targets and signal pathways of Mori Cortex-Lycii Cortex on childhood pneumonia based on the network pharmacology method, and found that Mori Cortex-Lycii Cortex is the components of quercetin,kaempferol, β-sitosterol and acacetin are likely to act on TNF,PI3K/AKT, MAPK signaling pathways and other signal transduction pathways through targets such as AKT1, IL-6, MAPK8 and MMP9.The pathway thus exerts an anti-inflammatory effect. Therefore,Mori Cortex-Lycii Cortex realizes the pharmacological action mechanism of treating childhood pneumonia through multiple components, multiple targets, and multiple pathways, which can provide a reference for further research on its mechanism of action,but this study only it is a prediction and discussion at the level of molecular mechanism, and its specific mechanism still needs further experiments to verify.

Author's contribution: Research design, data analysis, and writing are completed by the first author; Document retrieval and image processing are completed by the second author; Research guidance,revision and correction are completed by the third and fourth authors;Text translation is completed by the fifth author; The corresponding author is responsible for reviewing the final draft of the article.