Xiao-Jun Cai, Bai-Hua Jiang, Zhen-Hua Lu, Tao Wang, Bi-Hai Zhang, Xu-Ling Wang

Heilongjiang Academy of Traditional Chinese Medicine,Harbin 150036,China

Keywords:Danbei Yifei formula Pulmonary fibrosis Network pharmacology Protocatechuic acid Arachidonate 5-lipoxygenase

ABSTRACT Objective: To determine the pharmacodynamic material basis and mechanism of Danbei Yifei formula on pulmonary fibrosis. Methods: Starting with the clear absorbed components of Danbei Yifei formula or the potential effective components in line with the five rules of Ribinsky, the network pharmacology method and technology of traditional Chinese medicine were used to predict and analyze the action targets of Danbei Yifei formula in vivo, such as Salvia miltiorrhiza, PINBEI, Taoren, etc. On the basis of enrichment analysis, the core pathway of Danbei Yifei formula in the treatment of pulmonary fibrosis was identified, and the binding energy of drug ligand and protein target was determined through molecular docking technology simulation and verification, and its affinity and stability were evaluated. To clarify the material basis and mechanism of Danbei Yifei formula in the treatment of pulmonary fibrosis.Result: The results of network pharmacology prediction of traditional Chinese medicine showed that Danbei Yifei formula contained 72 potential pharmacodynamic components and 26 corresponding targets, including CHRM1、MAPK14、CCL2、ADRB1、PTGS1、PPARG、ALOX5、Pde3a、CHRM2、Adrb2、TNF、JUN、Adora2a、LTA4H、CYP1A2、OPRD1、CHRM3、DRD2、OPRM1、ARG1、EDNRA、Il6st、TACR1、MMP1、MMP8、Ptgs2, which were related to pulmonary fibrosis and pulmonary fibrosis Lung related diseases are highly correlated. There were 26 Go items (P ＜ 0.05) in go functional enrichment analysis, including 22 biological process (BP), 9 cellular component (CC) and 3 molecular function (MF) categories. The results of network pharmacology showed that many components, such as protocatechuic acid and aminosuccinic acid, had direct effects on known targets of pulmonary fibrosis. Conclusion: Danbei Yifei formula contains many effective components which have inhibitory effect on pulmonary fibrosis, and it may play its role through the mechanism of multi-component and multi-target synergistic effect.?Corresponding author: CAI Xiao-jun, M. D., Chief Physician.E-mail: 284011620@qq.com.

1. Introduction

Pulmonary fibrosis is a disease whose underlying pathophysiology is not well understood. It is estimated that 2.9 to 42.7 people per 100,000 live with the disease[1].The average survival time from diagnosis is two to three years, and there is no effective treatment[2].Therefore, the study of the pathogenesis of the disease for the control of the development of disease and effective drug development has a vital role.Network pharmacology is an emerging high-throughput technology in recent years. With the rapid development of network platform data sharing and the rapid popularization of computer applications, this technology has been widely used in the research of complex diseases and complex drugs.During the epidemic period, it was widely used and some good results were obtained [3-10].

For thousands of years, TCM has provided effective treatments for chronic lung diseases, including reducing clinical symptoms,improving lung function and exercising ability.At present, the study of TCM formula and monomer has been proved to be an effective method for the treatment of pulmonary fibrosis.Dan Bei Yi lung recipe is from Heilongjiang Academy of Chinese Medicine Sciences and is developed by Professor Jiang Baihua from his own experience.This recipe consists of danshen 30g, Astragalus 30g, Pingbi 20g, Radix Codonopsis 20g, Ophiopogon japonicus 20g, radix platycodonis 20g, radix platycodonis 15g, Ligusticum chuanxiong 15g, peach kernel 15g, Fructus schisandrae 15g, Fructus psoraleae 15g and earthworm 15g respectively.Its curative effect on pulmonary fibrosis is definite [11-26].Therefore, in this study, network pharmacology technology was used to study the intervention effect of Danbeit lungfang on rats with pulmonary fibrosis, so as to provide experimental basis for the clinical pathogenesis of pulmonary fibrosis and the pharmacodynamic material basis and mechanism of Danbeit Lungfang.

2. Methods

2.1 The lung ingredients of Tempe were collected

Retrieving CNKI and TCMSP (https://tcmspw.com/tcmsp.php)about Dan BeiYi lung composition information, to Dan BeiYi lung reply form for retrieving items retrieved, and map data sets, included organize related Dan BeiYi lung information of the ingredients.

2.2 Active ingredient screening

According to the conventional theoretical understanding of pharmacokinetics and pharmacodynamics, the prerequisite for drug molecules to exert their efficacy is that they can be absorbed into the blood and accumulate to a certain concentration in the body and then reach the target organ along with the blood circulation to exert their efficacy.Therefore, whether the drug molecules can be absorbed into the blood is the premise of drug effect.In this study, the international five principles of generic drugs were adopted to carry out targeted screening for thousands of components in Traditional Chinese medicine, and the chemical components in the drugs were screened according to the standard of oral availability OB≥30% and drug potency DL≥0.18.Obtain the potential potency ingredient that meets the standard.

2.3 Determination of predictive targets and target genes

In this part, database retrieval and molecular docking verification were used to predict the target of the standard components contained in Danbei lung Prescription.Providing database (https://db.idrblab.org/ttd/) and CTD database (http://ctdbase.org/), included the NCBI gene data and associated with disease known targets information data, at the same time, the database and OMIM online Mendelian genetic database (https://www.omim.org/), included the comprehensive data of genes.In this study, target prediction analysis was conducted for the screened active components based on the above database.At the same time, target proteins were introduced into UniProt (https://www.uniprot.org/) database to trace the unique target gene names of corresponding upstream target proteins, and summarize and collate them.

2.4 To construct a network relationship based on the active components, targets and diseases of Dan Beyi lung Prescription

Cytoscape3.2.1 software was used to construct the network diagram of active components and key targets of Tambex decoction for pulmonary fibrosis intervention.At the same time, the targeted sure Dan BeiYi lung side of the main active ingredients and diseases associated with pulmonary fibrosis target, will Dan BeiYi lung Function target protein in the body into Omicshare (https://www.omicshare.com/) online analysis platform to GO enrichment analysis, respectively for Gene Ontology of Molecular Function analysis (Molecular Function). Biological process analysis and cellular component analysis. Focuses on the key metabolic or biological processes associated with pulmonary disease, particularly pulmonary fibrosis.

2.5 Pulmonary fibrosis related target protein interaction analysis and ligand receptor molecular docking verification analysis

The obtained target proteins related to pulmonary fibrosis were put into the String (https://string-db.org/) website for a proteinprotein interaction analysis, so as to assist to clarify the regulatory mechanism of Dan Bei lung formula for target protein-based target genes. At the same time from the PDB protein database (http://www.rcsb.org/) to retrieve the file, obtain the target protein 3 d structure and the coordination of small molecular simulation drugs absorption in vivo process after the docking, through the Discovery Studio 2.5 visualization software to simulate the process of the above,in a standardized process of molecular docking space objective evaluation on the sites and the binding energy of the process, and using the binding energy of 5.0 kJ/mol, or less as the docking possible minimum standards.

3. Results

3.1 Screening results of active components of Tempe lung Decoction

Through TCMSP and CNKI database retrieval, the chemical components of Danbeyi lung recipe were determined, namely, salvia miltiorrhiza, Pingbei, taoren, Ground dragon, Chuanxiong, Radix platycodonis, Radix Astragali, Radix Codonopsis, Fructus Psoralae,Radix Ophiopogonis and Fructus Schisandrae.The components with OB≥30% and DL≥0.18 were screened and sorted. By excluding the same component, 72 components were obtained.Among them, 19 were salvia miltiorrhiza, 20 were Pingbei, 33 were taoren, 12 were Chuanxiong, 15 were platycodon grandiflorum,24 were Astragalus, 19 were Codonopsis, 6 were Psoraleae, 10 were Ophiopogon japonicus, and 45 were Schisandrae. Due to the overabundance of ingredients,Now place the basic information in Table 1.

Table 1 Information table of active ingredients of Tempe lung Prescription

3.2 Results of network relationship analysis of active components, targets and diseases of Dan Bei Yi Lung Prescription

There were 133 nodes and 899 edges involved in the network of active components, targets and diseases of Dan Beyi lung Recipe.The network relationship diagram of active components, targets and diseases was constructed by Cytoscape 3.2.1 software (Figure 1), wherein the network heterogeneity was 0.051, the average number of adjacent nodes was 4.319, the path length was 2.145,and the average network centrality was 0.124.This indicates that the pharmacodynamic components of Dan Bei Fei Fang may have the phenomenon that one component interacts with multiple in vivo target proteins at the same time, or the phenomenon that the target protein interacts with multiple components at the same time may exist.This shows the complexity of prescription compatibility and the intuitiveness of network analysis. Related diseases are also associated with pulmonary fibrosis, such as bronchospasm (caused by histamine), allergic airway inflammation, bronchial asthma,chronic obstructive pulmonary disease, asthma, inflammation,pulmonary fibrosis, etc.

FIGure 1 Network relationship of the components, targets and diseases of Dan Beyi lung Formula

3.3 Enrichment and target interaction analysis results

He above predicted targets related to pulmonary fibrosis were brought into ClueGO 2.3.5 for GO enrichment analysis. A total of 26 core items were selected based on the P value of pathway less than 0.05, which described the molecular function, cellular component and biological process of genes respectively.22, the content of a biological process for twenty aldehyde biosynthesis, multicellular organism metabolism, collagen metabolism, collagen biosynthesis,phagocytosis of positive adjustment, cAMP, the biosynthesis of negative regulation, cyclase activity, the response of the ammonium ion and the regulation of phospholipase C activation of G protein coupled receptor signaling pathways, cyclase activity positive adjustment, morphine reaction response, response of ammonium ion, smooth muscle contraction, G protein coupled receptor signaling pathways, With cyclic nucleotide second messenger coupling, phospholipase C activation of G protein coupled receptor signaling pathways, cellular reaction of ammonium ion, the production of saliva, the regulation of smooth muscle contraction,adenylate cyclase inhibition of G protein coupled acetylcholine receptors signaling pathways, phospholipase C G protein coupling acetylcholine receptors signaling pathways, vasoconstriction. Cell component 1, content is axon terminal response.The molecular functions were as follows: neurotransmitter receptor activity,G-protein-conjugated amine receptor activity, phosphatidylinositol phospholipase C activity.See Figure 2 for the above process.The above processes are roughly divided into four categories, namely,G protein-coupled receptor signaling pathway and ring nucleotide second messenger coupling, icosaldehyde biosynthesis process,multicellular biological metabolism process, and G protein-coupled receptor signaling pathway activated by phospholipase C.As shown in figure 3.All enrichment analyses involved metabolic pathways and signaling pathways.As shown in Figure 3.A STRING (https://string-db.org/) database was used to analyze the interaction of target proteins in the intervention of Dambeinghi in pulmonary fibrosis.See Figure 4,Through the network visualization analysis of protein interaction, it can be known that:The core target of Pulmonary fibrosis has been confirmed as Arachidonate 5-lipoxygenase, which is reflected in the target protein group of multiple Traditional Chinese medicines of Tambayi Lung Decoction.For example, protocatechuic acid in Schisandrae, 2-aminosuccinic acid in Salvia miltiorrhiza, capsaicin and 2,3-dihydroxybutanedioic acid in Dangshen Acid, Rutin, Kaempferol, Coumarin and Quercetin all had direct effects on arachidonic acid 5-lipoxygenase, the core target of pulmonary fibrosis.In addition, other diseases related to pulmonary fibrosis, such as bronchospasm (caused by histamine),allergic airway inflammation, bronchial asthma, chronic obstructive pulmonary disease, asthma, inflammation, pulmonary fibrosis,etc., are all reflected in the interaction of component targets in Danbei lung Prescription. Therefore, it can be concluded that the combination of Dan Bei Yi lung prescription and synergistic effect of the action mechanism.

Figure 2 GO analysis bar chart of Dembeti lung square

Figure 3 GO analysis results of Tambayi fei Fang in the intervention of pulmonary fibrosis

Figure 4 In vivo interaction diagram of target proteins in the treatment of pulmonary fibrosis with Dambeyi lung Formula

4. Discussion

The results of network pharmacology showed that there were 72 active components in Danbeit Lung Prescription.Which directly related to pulmonary fibrosis disease targets contained components in fructus schisandrae protocatechuic acid, protocatechuic acid),salvia miltiorrhiza contains amino succinic acid (2 - aminosuccinic acid), codonopsis contains capsaicin (capsaicin) and 2, 3 - dihydroxy succinic acid (2, 3 - dihydroxybutanedioic acid), astragalus contains rutin (rutin), rhizoma kaempferiae phenol (kaempferol), coumarin(coumarin) and quercetin (quercetin).

At present, the clinical diagnosis and treatment methods for pulmonary fibrosis are mainly western medicine, which has the characteristics of quick effect and significant effect, but it also brings disadvantages such as easy recurrence and obvious side effects to patients.Therefore, it is urgent to find new effective drugs to treat pulmonary fibrosis.Therefore, it is urgent to find new effective drugs to treat pulmonary fibrosis.Dan Bei Yi Lung prescription is widely used in the clinical of traditional Chinese medicine, the use of network pharmacology technology research found that it contains many components have a certain effect on pulmonary fibrosis.The specific mechanism of action is described below.Arachidonate 5-lipoxygenase, also known as 5-lipoxygenase (FLAP), is a membrane-bound protein. The expression of 5-lipoxygenase produces arachidonic acid, which regulates the amount of leukotriene synthesis [27,28]. It is an important medium for inflammation and connective tissue remodeling.Recent studies have shown that some single nucleotide polymorphisms (SNPs) in the arachidonic acid 5 lipoxygenase-activating protein gene are associated with an increased risk of inflammatory diseases such as asthma and atherosclerosis[29-32].The theory of Traditional Chinese medicine holds that the lung and the skin look and feel together, therefore, the lung disease and the skin system disease have the internal connection.Therefore,the relationship between the polymorphism of arachidonic acid 5 lipoxygenase activated protein and scleroderma has been studied.The experiment hypothesized that single nucleotide polymorphisms of arachidonic acid 5 lipoxygenase-activated proteins may be associated with the risk of scleroderma and/or complications in its major organs.To address this hypothesis, seven SNPS were studied in the Scleroderma patient cohort of the European Scleroderma Trial and Study group, which have been shown to be associated with a higher risk of other inflammatory diseases [33].

Tumor necrosis factor (TNF), a pro-inflammatory cytokine,is considered to be a key mediator in the pathophysiology of pulmonary fibrosis [34].Based on its efficacy as a disease modifier in the treatment of pulmonary fibrosis, studies have used infliximab as a target to block TNF in the treatment of 1 case of sero-positive RA and pulmonary fibrosis [35].