Jie Wang, Xiang-Dong Zhou,2, Qi Li,2?, Pan-Hong Jia, You-Qing Zhong, Ying-Jiao Qin,Liang Li

1. Department of Respiratory Diseases, the First Affiliated Hospital of Hainan Medical University, Haikou 570102, China

2. Key Laboratory of Emergency and Trauma, Ministry of Education of Hainan Medical University, Haikou 571199, China

ABSTRACT Objective: To explore the effect of active compounds of Bletilla Striata, and to predict its mechanism of the treatment of bronchoplumonary inflammation. Methods: Retrieved active compounds of Bletilla Striata from relevant databases include TCMSP,while retrieved action targets of active compounds and bronchoplumonary inflammation. Received corresponding gene symbols of targets by querying UniProt database. Then drew the compound-target network of Bletilla Striata and action targets network between active compounds and bronchoplumonary inflammation using Cytoscape 3.2.1. Subsequently performed GO functional and KEGG enrichment analysis of key targets through relevent databases including Metascape, meanwhile, analysed Protein-protein interaction using STRING. Results: Bletilla Striata contained 29 active components and 632 corresponding targets. The more important targets included AURKA, BRAF, RAF1, MMP1 and ESR2, 213 in total. GO functional enrichment analysis resulted in 2,398 items (P ＜ 0.05), including 2096 biological process(BP) items , 133 cellular component (CC) items, 169 molecular function (MF) items. KEGG enrichment analysis resulted in 160 signal pathways items (P ＜ 0.05). There were 33 common targets between 335 action targets of bronchoplumonary inflammation and 95 more important active components of Bletilla Striata. GO functional enrichment analysis resulted in 547 items (P ＜ 0.05), including 499 BP items , 19 CC items, 29 MF items. KEGG enrichment analysis resulted in 46 signal pathways items (P ＜ 0.05), mainly involving the IL-17 signaling pathways, platelet activation, endocrine resistance, Fc epsilon RI signaling pathway, Th17 cell differentiation, etc. 6 core targets including MAPK14, MMP9, HSP90AA1, ROCK2, PRKCB and PTGS2 were obtained by PPI analysis. They were involved in IL-17 signaling pathway,platelet activation, leukocyte migration, VEGF signaling pathway and Th17 cell differentiation respectively. Conclusion: The study explains "multi-component-multi-target-multi-pathwaymulti-disease" is the characteristic of Bletilla Striata using network pharmacology, and predict the active compounds of Bletilla Striata can be used for treatment of bronchoplumonary inflammation with the effect of regulating to anti-inflammation and tissue repair and remoding.This study theoretically verified the functional utility of Bletilla Striata and provided scientific basis and reference direction for further experimental research.

Keywords:Network pharmacology Bletilla striata Bronchoplumonary inflammation Signal pathway

1. Introduction

Bronchoplumonary inflammation is an inflammatory disease of human terminal airway, alveoli and pulmonary interstitium, and it is also the most common syndrome in the general population [1].Because the respiratory tract communicates directly with the outside world, when the immune function of the host is low, pathogens and other harmful substances with strong pathogenicity including invasiveness, virulence factors ,toxins, and so as invade the airway can induce inflammatory lesions with different manifestations,and even cause significant damage to function and structure.According to the estimates of the World Health Organization, lower respiratory tract infection is the most common cause of infectious death in the world, causing nearly 35 million deaths every year.Domestic statistics show that there are 2.5 million patients with bronchoplumonary inflammation and 125 thousand deaths every year [2]. If we can effectively control the inflammatory response,regulate immune function and improve organ function damage, we can slow down the deterioration of patients with severe infection and improve the survival rate of patients [3]. At present, antibiotics are mainly used to kill pathogens to control bronchoplumonary inflammation, and less by regulating the anti-inflammatory and repair ability of the body to treat bronchoplumonary inflammation.Due to the delayed detection or drug resistance of pathogens, double infection / flora imbalance, side effects of antibiotics and other factors, bronchoplumonary inflammation is complex and difficult to treat, seriously affecting the therapeutic effect and prognosis of patients [4, 5]. How to effectively control the complex and refractory bronchoplumonary inflammation is an urgent clinical problem to be solved, so it is imperative to explore and develop safe and effective drugs against bronchoplumonary inflammation, which is the purpose of our study.

In the process of consulting the Traditional Chinese Medicines Integrated Database(TCMID), we found that the Chinese herbal medicine Bletilla Striata has the functions of converging sore and hemostasis, tonifying the lung, reducing swelling and promoting muscle. In addition, Bletilla Striata can also be made into "Bletilla ointment" for topical use. What was recorded in Zhao Bingnan's Clinical experience Collection has the effect of astringent muscle formation and is used to treat burns, lower limb ulcers and so on.Some studies have shown that Bletilla gum, the extract of Bletilla Striata, can protect gastrointestinal mucosa and promote ulcer repair, promote skin wound healing, promote the immune function of mice. Bletilla gum has high safety and adhesion. It can also be used as a drug delivery carrier for other drugs. In addition, the hemostatic sponge made from Bletilla Striata Panax notoginseng gelatin was used for wound hemostasis and repair [6-18]. Many studies have shown that Bletilla Striata not only has the effects of anti-inflammation, regulating immunity, promoting wound healing and hemostasis, but also is a safe medical raw material,excellent medicinal excipients and biomedical materials with good development prospects. Unfortunately, there are few in-depth studies on the mechanism of action of it, let alone the application of Bletilla gum in the treatment of bronchoplumonary inflammation.Therefore, we want to explore the efficacy of Bletilla Striata and the mechanism of action of it on bronchoplumonary inflammation through the method of network pharmacology [19], as a preliminary exploration of the mechanism of action of it. The purpose of this study is to provide scientific basis and reference direction for further study on the mechanism of Bletilla Striata in the treatment of bronchoplumonary inflammation.

2. Materials and methods

2.1 Collect and screen the active components of Bletilla Striata and its corresponding targets

In this study, the active components of Bletilla Striata were searched based on the Traditional Chinese Medicine Systems Pharmacology (TCMSP), TCMID, and a Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN). Collect all the active components, consult PubChem database of organic small molecules after weight and get the basic information of the active components.

Search the active components of Bletilla Striata in TCMSP,DrugBank, the Binding Database (BindingDB), SuperPred and Swiss Target Prediction database. Search the abbreviation of the gene name corresponding to the target protein with Search Tool for Recurring Instances of Neighbouring Genes (STRING) and the Universal Protein Resource (UniProt).

2.2 Establishment of active ingredient-target network of Bletilla Striata

The active components and related target gene names of Bletilla Striata collected for short were introduced into Cytoscape 3.2.1 software to generate the active component-target interaction network of Bletilla Striata, and the active component-target network of Bletilla Striata was analyzed by "Analyze Network" function in the software. The closely related active components and the main action targets of Bletilla Striata were obtained, and the pharmacological mechanism of Bletilla Striata was visualized.

2.3 Collect related targets of bronchoplumonary inflammation

Using the English nouns "pulmonary/lung/airway/bronchial","inflammation/infection", "pneumonia" and "bronchiectasis" as the key words, the related targets were found through DrugBank database, Therapeutic Target Database (TTD), DisGeNET, Online Mendelian Inheritance in Man (OMIM). The abbreviation of the gene name corresponding to the target protein was queried with the help of UniProt.

2.4 Establishment of a network between targets of active components of Bletilla Striata and targets related to bronchoplumonary inflammation

After sorting out the action targets of the collected active components of Bletilla Striata and the related targets of bronchoplumonary inflammation, the interaction network between the active components of Bletilla Striata and the related targets of bronchoplumonary inflammation was generated by Cytoscape 3.2.1 software, and the Analyze Network function in the software was analyzed to predict the possible pharmacological mechanism of Bletilla Striata in bronchoplumonary inflammation.

2.5 GO and KEGG Gene enrichment Analysis and PPI Network Analysis

The predicted key targets of Bletilla Striata and the key targets of Bletilla Striata on bronchoplumonary inflammation were imported into WebGestalt and Metascape database, and the threshold FDR＜ 0.05 was set. GO functional annotation and KEGG pathway enrichment analysis were performed, and PPI analysis was carried out using STRING database to further explore the functional activity of Bletilla Striata and the mechanism of Bletilla Striata in the treatment of bronchoplumonary inflammation and possible associated signaling pathways.

3. Results

3.1 Active components of Bletilla Striata

28 Active components of Bletilla Striata were screened by TCMSP according to the standard of compound property (DL) ≥ 0.18, 28 kinds of chemical constituents in Bletilla Striata were obtained by TCMID, 6 kinds of compounds in Bletilla Striata were obtained by BATMAN, and 29 kinds of active compounds were obtained.Through the PubChem, look up the basic information such as the identifier from database of chemical (CID) and the Canonical Simplified Molecular Input Line Entry System(Canonical SMILES)one by one (Table 1).

3.2 Action targets of active components in Bletilla Striata

The action targets of each active component of Bletilla Striata were searched by TCMSP, DrugBank, The Binding, SuperPred and SwissTargetPrediction databases.

3.3 Active components and target network of Bletilla Striata

29 nodes of traditional Chinese medicine components, the main active components of Bletilla Striata were collected, and the corresponding predictive target nodes of each active component were introduced into Cytoscape 3.2.1 to construct the active componentpredictive target network of Bletilla Striata. 661 nodes, include 29 component nodes and 632 target nodes, were obtained. The interaction between components and target nodes was analyzed by"Analyze Network" function ( Figure 1 ) . The triangle represents the component node; the rectangle represents the target node, and the color difference is set according to the degree of freedom. The higher the degree of freedom is, the darker the color is. A total of 19 active components, nodes above the average degree of freedom, in Bletilla Striata were obtained by analysis. According to the degree of closeness, they were ranked as follows: BS-22, BS-02, BS-01, BS-27, BS-03, BS-23, BS-21, BS-28, BS-20, BS-04, BS-09, BS-26, BS-15, BS-17, BS-10, BS-06, BS-29, BS-18, BS-12. At the same time,it is predicted that there are 213 key targets of Bletilla Striata, which above the average degree of freedom, the top 30 in which sorted from high to low according to closeness are as follows:AURKA,BRAF, RAF1, MMP1, ESR2, PDK1, PTGS2, ESR1, HSD17B2,AKR1B10, HDAC1, CA7, PTGS1, CA14, SYK, MMP13, CDK1,CDK2、EGFR, HDAC6, MMP9, MET, CA9, ALOX5, ABL1,MTOR, PARP1, GSK3B, RPS6KB1, F10.

Figure 1 Interaction-network between active ingredients of Bletilla Striata and and relative targets

3.4 GO and KEGG enrichment analysis of key targets of Bletilla Striata

The results of GO enrichment analysis were obtained by importing 213 key targets of Bletilla Striata into WebGestalt database(Fig.2A). The height value of the bar chart represents the number of gene overlap in this annotation category. The results showed that the role of active components of Bletilla Striata was highly correlated with biological regulation, metabolic process, stress response,cellular information transmission, multicellular biological process,cellular localization, development process and the organization of cell elements in BP, more in cell membrane and nucleus in CC, and more in protein binding and ion binding in MF, the results showed that the role of active components of Bletilla Striata was highly correlated with biological regulation, metabolic process, stress response, cellular information transmission, multi-cellular biological process, cellular localization, development and cellular element organization in Bletilla Striata. Through the analysis of Metascape database, the enrichment results of GO function and KEGG pathway of the key targets of Bletilla Striata were obtained. There were 2398 GO entries, including 2096 BP entries, 133 CC entries and 169 MF entries, and 160 signal pathways(P＜0.05)were obtained by KEGG pathway enrichment screening. The enrichment results were sorted by -log10(P)value and listed in the top 20. The darker the color is, the higher the correlation enrichment degree is. The results are as follows: ① functional enrichment of GO (Fig. 2B):protein kinase activity, positive requlation of transferase activity,peptidyl-serine modification, non-membrane spanning protein tyrosine kinase activity, cellular response to nitrogen compound,protein kinase binding, transmembrane receptor protein tyrosine kinase activity, response to inorganic substance, response to wounding, cellular response to organic cyclic compound, circulatory system process, modulation of chemical synaptic transmission,cellular response to oxidative stress, requlation of peptidyl-tyrosine phosphorylation, requlation of hormone levels, positive requlation of cell migration, regulation of ion transport, multicellular organismal homeostasis, carbonate dehydratase activity, positive reaulation of cell cycle. ② enrichment of KEGG pathway(Fig. 2C): pathways in cancer, progesterone-mediated oocyte maturation, cell cycle,axon quidance, gap junction, nitrogen metabolism, estrogen signaling pathway, AGE-RAGE signaling pathway in diabetic complications, neuroactive ligand-receptor interaction, platelet activation, HIF-1 signaling pathway, IL-17 signaling pathway, NFkappa B signaling pathway, ovarian steroidogenesis, epithelial cell signaling in Helicobacter pylori infection, Alzheimer's disease, Th17 cell differentiation, Cocaine addiction, Apoptosis, steroid hormone biosynthesis.

Table 1 Basic information of some active compounds in Bletilla Striata

Figure 2 GO and KEGG enrichment analysis of key targets of Bletilla Striata

3.5 PPI enrichment analysis of key targets of Bletilla Striata

Through the analysis of Metascape database, the PPI network of 213 key targets of Bletilla Striata was obtained(Fig. 3A). 10 groups of the most important elements of MCODE are extracted from it(Fig. 3B), and different groups are represented by different colors.Independent functional enrichment analysis of MCODE components showed that the higher the negative value of P value was, the more significant the enrichment degree was(Fig. 3C). The results showed that its biological functions were mainly concentrated in pathways in cancer, prostate cancer, PI3K-Akt signaling pathway, axon guidance,proteoglycans in cancer, neuroactive ligand-receptor interaction,steroid hormone biosynthesis, complement and coaqulation cascades.

Figure 3 PPI enrichment analysis of key targets of Bletilla Striata

3.6 Related targets of bronchoplumonary inflammation

335 related targets of bronchoplumonary inflammation were searched by DrugBank, TTD, DisGeNET and OMIM database.

3.7 The relationship between the targets of active components of Bletilla Striata and the related targets of bronchoplumonary inflammation

Find out the target of prediction corresponding to each active component of Bletilla Striata and the common target of bronchoplumonary inflammation. There are a total of 95 targets except weight(Fig. 4A). The data of these targets, 28 component nodes and 95 target nodes, were imported into Cytoscape 3.2.1 to construct the network between the active components of Bletilla Striata and the related targets of bronchoplumonary inflammation.The "Analyze Network" function was used to analyze the relationship between the component nodes and the target nodes(Fig.4B), in which the triangle represents the component node and the rectangle represents the target node, and the color difference is set according to the degree of freedom. A total of 14 active components,these nodes above the average degree of freedom, in Bletilla Striata which are closely related to bronchoplumonary inflammation are obtained. According to the degree of closeness, the order from high to low are as follows: BS-20, BS-02, BS-01, BS-16, BS-12, BS-09, BS-03, BS-23, BS-10, BS-06, BS-29, BS-17, BS-11, BS-08. At the same time, it is predicted that there are 33 key targets of Bletilla Striata acting on bronchoplumonary inflammation, these target nodes above the average degree of freedom, and the top 13 are ranked from high to low according to the degree of closeness as follows: MMP1,ESR2, PTGS2, PTGS1, MMP13, SYK, MMP9, MTOR, ALOX5,MMP2, ROCK2, MAPK14, F2.

Figure 4 Relationship of relative action targets between active compounds of Bletilla Striata and bronchoplumonary inflammation

3.8 GO and KEGG enrichment analysis of key targets of Bletilla Striata on bronchopulmonary inflammation

Through the analysis of Metascape database, the enrichment results of GO function and KEGG pathway of 33 key targets of Bletilla Striata on bronchoplumonary inflammation were obtained (Fig.5). There were 547 GO entries, including 499 BP entries, 19 CC entries, 29 MF entries, and 46 signal pathways were obtained by KEGG pathway enrichment screening(P＜0.05) , sorted by-log10(P)value and listed in the top 20 enrichment results, the darker the color, the higher the degree of correlation enrichment. The results are as follows: ① Functional enrichment of GO(Fig. 5A): collagen catabolic process, requlation of cytokine production, peptidylserine phosphorylation, response to peptide, reactive oxygen species metabolic process, cytokine-mediated signalinq pathway, icosanoid metabolic process, requlation of inflammatory response, cellular response to lipid, production of molecular mediator involved in inflammatory response, requlation of ion transport, leukocyte migration, regulation of interleukin-10 production, positive regulation of epithelial cell migration, collagen binding, blood circulation,cellular response to drug, protein import, ossification, activation of immune response. ② Enrichment of KEGG pathway(Fig. 5B): IL-17 signaling pathway, pathways in cancer, proteoglycans in cancer,platelet activation, endocrine resistance, serotonergic synapse, Th17 cell differentiation, Fc epsilon RI signaling pathway, neuroactive ligand-receptor interaction, Rheumatoid arthritis, HIF-1 signaling pathway, cAMP signaling pathway.

Figure 5 GO and KEGG enrichment analysis of the key targets of Bletilla Striata act on bronchoplumonary inflammation

3.9 PPI analysis of key targets of Bletilla Striata on bronchoplumonary inflammation

The PPI analysis of 33 key target proteins of Bletilla Striata acting on bronchoplumonary inflammation was carried out by STRING database(Fig. 6). The results are as follows: ① Figure 6 A shows the correlation intensity of target protein interaction, each point represents a target protein, and each edge represents the interaction between protein and protein. The more lines, the greater the correlation. 94 interaction lines were obtained after analysis, with an average value of 5.7, the enrichment P＜1×10-16. ② Figure 6 B shows the target protein network with the highest confidence level of the target protein interaction, as which lowest interaction score is more than 0.9. At the same time, 5 different colors are used to label the target proteins of several key KEGG pathways of Bletilla Striata in bronchoplumonary inflammation. The target proteins involved in more than two pathways are core proteins. MAPK14 is involved in 5 KEGG pathways, which are as follow: IL-17 signaling pathway,platelet activation, leukocyte transendothelial migration, VEGF signaling pathway, Th17 cell differentiation. MMP9 is involved in IL-17 signal pathway and leukocyte migration. HSP90AA1 participates in IL-17 signaling pathway and Th17 cell differentiation.ROCK2 participates in platelet activation and leukocyte migration.PRKCB participates in leukocyte migration and VEGF signaling pathway. PTGS2 participates in IL-17 signaling pathway and VEGF signaling pathway. ③ Figure 6 C shows the cluster analysis and the molecular interaction diagram of target proteins based on Figure 6 B. The 3 colors of red, blue and green represent the clustering of 3 kinds of target proteins, and the differential lines between the 2 target proteins refer to the molecular interaction characteristics between them. The molecular interactions between core target proteins and same cluster target proteins are as follows: MAPK14 is unspecified catalysis and reaction. MMP9 is unspecified binding, catalysis,reaction and posttranslational modification. HSP90AA1 is positive activation to ROCK2, but unspecified binding, catalysis, reaction with others. ROCK2 is unspecified catalysis and reaction. PRKCB is unspecified catalysis and reaction. PTGS2 is binding to NOS2.

Figure 6 The PPI analysis of the key targets of Bletilla Striata act on bronchoplumonary inflammation

4. Discussion

Bronchoplumonary inflammation account for a high proportion of respiratory diseases. In clinical work, when there are factors such as delayed detection of pathogens, drug resistance of pathogens, double infection / flora imbalance, side effects of antibiotics and so on, so it is difficult to be controlled effectively. In severe cases, extensive lung injury can be caused, and even irrecoverable damage will occur in the function and structure of the lung. Therefore, it is very important for the timely and effective control. Different from other traditional Chinese medicine prescriptions in the form of decoction,Bletilla Striata can be taken orally or externally. Because of its nontoxic chemical properties and sticky physical properties, Bletilla gum extracted from Bletilla Striata has become more and more valuable in local application in recent years. It has been developed as a hemostatic gel and drug carrier, in view of its anti-inflammatory,promoting tissue detachment and wound repair. It is possible to achieve a good therapeutic effect when it is applied to the local treatment of bronchoplumonary inflammation.

In this study, several traditional Chinese medicine and compound information platforms were used to mine the active components and action targets of Bletilla Striata, and the key targets mediated by the effect of it were analyzed by Cytoscape software. GO and KEGG enrichment analysis of these targets were carried out by using Metascape and other databases, so that the function of Bletilla Striata was specified. The results of enrichment analysis showed that it could affect the protein / ion binding ability. It acts on the functional areas of the cell membrane or nucleus, and then participates in the processes of growth and development, metabolic regulation, information transmission, stress response, cell migration,etc., and predicts the effective pathway of Bletilla Striata as follows:regulate homeostasis and cell proliferation cycle by affecting tumorrelated pathways, regulate the transmission of synaptic chemicals through axonal guidance and neuroactive ligand-receptor interaction pathway, affect hormone synthesis through estrogen signal pathway and AGE-RAGE signal pathway of diabetic complications, act on gap junctions and regulate cell migration, regulate platelet activity.It can participate in the response of the body to oxidative stress,injury and apoptosis by affecting HIF-1 signal pathway, IL-17 signal pathway and NF-kappa B signal pathway. Thus it can be seen that Bletilla Striata can play its pharmacological role through " multicomponent-multi-target-multi-pathway ", and the functional category of it is very extensive, while it has high research with application value. The results of this study provide an important basis for the application of it in the treatment of many diseases.

In this study, we further predicted the role of Bletilla Striata in bronchoplumonary inflammation, obtained the key targets of its acting on bronchoplumonary inflammation through network pharmacological analysis, and analyzed its GO function and KEGG pathway. The results show that Bletilla Striata can regulate the production of cytokines through IL-17 signal pathway and HIF-1 signal pathway, and also act on the metabolic process of reactive oxygen species. And regulate the inflammatory response by affecting the production of inflammatory mediators such as IL-10. It can affect Th17 cell differentiation, platelet activation, leukocyte and epithelial cell migration, affect collagen adhesion and degradation, and activate immune response through Fc epsilon RI signal pathway. In order to make Bletilla Striata more accurate in predicting the effect of it on bronchoplumonary inflammation, PPI analysis was carried out on 33 key target proteins of its acting on bronchoplumonary inflammation.The results show that Bletilla Striata can play a role in the treatment of bronchoplumonary inflammation by acting on these targets. It is predicted that Bletilla Striata plays a key role in several important pathways of bronchoplumonary inflammation as follows: MAPK14,MMP9, HSP90AA1, ROCK2, PRKCB, PTGS2. The molecular interaction between the 6 core target proteins and the same cluster target proteins is deeply excavated, and it is proved theoretically that Bletilla Striata can regulate the anti-inflammatory and repair ability of the body. In summary, it can be seen that it may be through the synergistic effect of multi-components, multi-targets and multipathways in the treatment of bronchoplumonary inflammation. The results of this study provide an important guidance for our follow-up verification experiments.

To sum up, this study applied the method of network pharmacology to study the chemical components, action targets, and the functional category of Bletilla Striata, the functional effect and mechanism of it in the treatment of bronchoplumonary inflammation. This paper theoretically verifies the functional effectiveness of Bletilla Striata,and verifies that it is effective in the treatment of bronchoplumonary inflammation through the synergistic effect of multi-components,multi-targets and multi-pathways. This study opened up a new direction for the treatment of bronchoplumonary inflammation,especially for complex and refractory. Since the main purpose of this study is to explore the efficacy of Bletilla Striata from the theoretical level, further experimental studies need to be carried out around pharmacodynamic evaluation, metabonomics, clinical efficacy and other aspects in the later stage. To provide a solid basis for confirming Bletilla Striata in the treatment of bronchoplumonary inflammation.