Potential Mechanism of Danggui Buxue Decoction in Treating Iron Deficiency Anemia Based on Network Pharmacology and Molecular Docking Technology

Wei ZHOU, Liming HUANG

1. Guizhou University of Traditional Chinese Medicine, Guiyang 550002, China; 2. The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China

Abstract [Objectives] To explore the potential mechanism of Danggui Buxue Decoction in treating the iron deficiency anemia (IDA) based on network pharmacology and molecular docking technology. [Methods] The active components and target proteins of Danggui Buxue Decoction were searched in databases such as TCMSP, OMIM, GeneCards, Drugbank, String, Metascape, etc., and the target proteins shared with IDA were screened out, and the information about the signal pathways and biological functions of these target proteins was obtained. [Results] 17 active components of Danggui Buxue Decoction and 24 potential targets for the treatment of IDA were obtained. With the aid of String database and Cytoscape software, the protein interaction network was obtained and the network topology analysis was performed. Four potential core targets with higher scores were obtained, namely F2, NOS2, NOS3, and PPARG. Using the Metascape database, GO function enrichment analysis and KEGG pathway enrichment analysis were performed on the potential targets of Danggui Buxue Decoction in the treatment of IDA, and the important biological processes, cell composition, molecular functions and signal pathways related to the target were screened through the R language. The results show that biological processes are related to positive regulation of growth, cell composition is related to membrane microdomain, and molecular functions are related to oxidoreductase activity. The signal pathways involved are mainly AGE-RAGE signal pathway, TNF signal pathway and IL-17 signal pathway. Finally, the molecular docking results confirmed that the active components of Danggui Buxue Decoction have a good binding ability with the target. [Conclusions] Danggui Buxue Decoction treats the IDA through multiple components, multiple targets, multiple signal pathways, and multiple biological functions.

Key words Danggui Buxue Decoction, Iron deficiency anemia (IDA), Network pharmacology, Molecular docking technology

1 Introduction

Iron deficiency anemia (IDA) is the final stage of iron deficiency. The imbalance between the body’s iron demand and supply leads to iron depletion (ID) in the body, followed by iron deficient erythropoiesis (IDE), which eventually leads to iron deficiency anemia. Network pharmacology[1-2]takes advantage of the "drug-gene-target-disease" interaction network to study the complex relationship between biological systems, drugs and complex diseases, and explains the action mechanism of drugs from a multi-dimensional perspective. The holistic and systematic characteristics of its research strategy and those of TCM’s theory of diagnosis and treatment of diseases from the perspective of holistic concepts and syndrome differentiation achieve the same goal with different means. In this study, through network pharmacology, we analyzed the action mechanism of Danggui Buxue Decoction in the treatment of IDA, to provide a technical support and theoretical basis for the secondary development of this prescription.

2 Data and methods

2.1 Screening of active components of Danggui Buxue DecoctionDanggui Buxue Decoction consists of two traditional Chinese medicines: ANGELICAE SINENSIS RADIX and ASTRAGALI RADIX. We separately input them into TCMSP (http://tcmspw.com/tcmsp.php) to retrieve the active components and protein targets of these two medicines. According to the principle of Absorption, Distribution, Metabolism, Excretion (ADME), we set selection conditions as the oral bioavailability (OB) of the compound ≥ 30% and the drug-like activity (DL) of the compound ≥ 0.18. After the screening of the two Chinese medicines, we used the Uniprot database (https://www.uniprot.org/) to convert all protein targets into Gene Symbol ID. Then, we selected the targets and their corresponding active components of the drug, the name of Chinese medicine and other information, imported into Cytoscape 3.7.2 software, plotted the drug-component target network diagram, to make the relationship between each component and target become clearer and more intuitive.

2.2 IDA disease targetsUsing the iron deficiency anemia as a keyword, we searched in OMIM database (https://www.omim.org/), DisGeNET database (http://www.disgenet.org/), and GeneCards database (https://www.genecards.org/) for IDA-related targets, and removed the duplicate values of the targets obtained from the three databases.

2.3 Potential targets of Danggui Buxue Decoction in the treatment of IDAUsing the VennDiagram package of the R software, we mapped the target of Danggui Buxue Decoction to the IDA disease target, and get the potential target of Danggui Buxue Decoction in the treatment of IDA, and displayed it through the Venn diagram.

2.4 Constructing a network diagram for drug-component-therapeutic targetsWe imported the information of components and Chinese medicines corresponding to potential therapeutic targets into Cytoscape software to construct a network diagram of drugs-components-therapeutic targets. Through the calculation function of Cytoscape software, we calculated the network and obtained the components and targets with higher scores.

2.5 Construction of key target protein-protein interaction (PPI) networkThe String database (http://string-db.org/) can automatically score each protein interaction information. The higher the score, the higher the confidence of the protein interaction. We imported potential therapeutic targets into the "multiple proteins" search box of the String database, set the species as "homo sapiens", and other parameters as default settings to construct a protein-protein interaction network (PPI network) between Danggui Buxue Decoction and common disease targets, and analyzed the PPI network according to relevant parameters.

2.6 GO and KEGG enrichment analysisWe imported the target of Danggui Buxue Decoction in the treatment of IDA into the Metascape database (http://metascape.org/), and set the species as homo sapiens withP<0.01 as the screening condition, and carried out gene ontology (GO) for potential therapeutic targets and Kyoto encyclopedia of genes and genomes (KEGG) functional enrichment analysis. The GO analysis includes biological process (BP), cellular component (CC), and molecular function (MF). Finally, we imported the analysis results into R language for constructing diagrams.

2.7 Molecular dockingWe imported the core targets screened by the PPI network into Uniprot for searching, set the screening conditions as human and reviewed, and obtained the corresponding protein of the target genes. We selected “Method” as the protein conformation of "X-ray" and downloaded the PDB format file of this conformation in the PDB database (https://www.rcsb.org/). Using SYBYL-X 2.1.1 software, we performed molecular docking analysis on the core targets screened by PPI and their corresponding chemical components, and obtained the Total Score value and displayed them in 3D.

3 Results and analysis

3.1 Active components of Danggui Buxue Decoction and their corresponding targetsThrough TCMSP and related literature, we obtained 17 kinds of active components in each single Chinese medicine of Danggui Buxue Decoction that meetOB≥30% andDL≥0.18. We renamed unique components of each Chinese medicine using the initials of the Chinese medicine pinyin name and Arabic numerals, as indicated in Table 1.

Table 1 Active components of Danggui Buxue Decoction

3.2 IDA disease targets and potential targets of Danggui Buxue Decoction in the treatment of IDAUsing "iron deficiency anemia" as the key word, we searched 800 targets in the OMIM database, 827 targets in the GeneCards database, and 207 targets in the Drugbank database. We removed the duplicate targets and obtained 1 432 disease targets. We imported the Danggui Buxue Decoction targets and IDA disease target into R language. With the aid of the VennDiagram program package of the software, we obtained a total of 24 targets of Danggui Buxue Decoction in the treatment of IDA, and visually displayed them in Fig.1.

Fig.1 Venn diagram for Danggui Buxue Decoction-IDA targets

3.3 Construction of target-component-drug network diagram and PPI network analysisWe imported the information of components and Chinese medicines corresponding to potential therapeutic targets into Cytoscape software to establish a network diagram of drugs-components-therapeutic targets (Fig.2). We calculated the network through the calculation function of the Cytoscape software, and obtained targets with higher scores. We imported the therapeutic target into the "multiple proteins" search box of the String database (https://string-db.org/), set the species as "homo sapiens", and other parameters as the default settings, imported the results into Cytoscape for visualization and plotted PPI interaction diagram (Fig.3). The larger the degree value, the larger the node, indicating that the interaction between the targets is closer. Through the built-in Network Analyzer of CytoScape, we analyzed the topology parameters of the network. Among them, the targets with top five degree values were PPARG (degree=26), AKT1 (degree=24), IL4, ICAM1, VCAM1, CASP3 (degree=20), NOS3 (degree=18), NOS2, STAT1 (degree=16). This indicates that PPARG, AKT1, IL4, ICAM1, VCAM1, CASP3, NOS2, STAT1 targets are the core targets of Danggui Buxue Decoction acting on the IDA-related target network and occupy an important position.

Fig.2 Target-component-drug network diagram

Fig.3 Analysis diagram for Danggui Buxue Decoction-IDA target PPI network

4 Discussion

Using network pharmacology and molecular docking method, we explored the potential action mechanism of Danggui Buxue Decoction in the treatment of IDA, and found that multiple active components in Danggui Buxue Decoction can act on the same target at the same time, and a single active component can also act on multiple targets at the same time. Through the results of database, gene chip data mining and molecular docking, we found that the main active components of Danggui Buxue Decoction include β-sitosterol, stigmasterol, calycosin,etc., which may interact with F2, NOS2, NOS3, PPARG, SLC6A3, CASP3, F7, XDH, AKT1,etc., as the main target of Danggui Buxue Decoction in the treatment of IDA. The following is the analysis of each target.

F2: The main cause of IDA is chronic blood loss. In men, it is mainly a digestive system disease, while in women, it is mostly caused by excessive menstrual flow. This may be related to the body’s abnormal blood coagulation function. Prothrombin encoded by the F2 gene is a "trypsin-like" serine protease protein. In the first cascade of blood coagulation, prothrombin (coagulation factor II) is proteolytically excised to produce thrombin, and the cascade finally prevents the continued loss of blood. Thrombin in turn acts as a serine protease to convert soluble fibrinogen into insoluble fibrin and catalyze many other coagulation-related reactions. If the body’s coagulation function is abnormal, the body is at risk of bleeding at any time, which greatly increases the possibility of IDA.

NOS2: In the course of various diseases, the production of free radicals will increase. An appropriate amount of free radicals is necessary for certain physiological processes, but excessive amounts will cause oxidative stress and cell damage. Reactive nitrogen species (RNS), as a group of molecules derived from nitric oxide (NO), can destroy cell structure together with reactive oxygen species, resulting in a phenomenon similar to oxidative stress. NO is produced by a reaction catalyzed by nitric oxide synthase (NOS). NO deficiency is a main cause of endothelial dysfunction[3]. In IDA patients, endothelial dysfunction is attributed to the reduction of endogenous nitric oxide (NO) bioavailability, and NO is an effective vasodilator. This damage is mainly caused by the clearance of NO by cell-free plasma hemoglobin[4]. NO is an important regulator of vascular homeostasis and can inhibit the adhesion, aggregation and recruitment of platelets and the interaction between neutrophils and endothelium. It is the product of endothelial nitric oxide synthase (eNOS) catalyzing the two-step oxidation of L-arginine to L-citrulline, which is a biochemical reaction affected by a variety of genetic factors[5-6].

NOS3: Nitric oxide (NO) and L-citrulline are produced by oxidizing L-arginine through the catalytic activity of nitric oxide synthase (NOS)[7-8]. According to findings[9], anemia is related to severe red blood cell dysfunction and increased superoxide production, which may be related to the reduction of NO pool caused by the uncoupling of eNOS in red blood cells. eNOS is located in the red blood cell membrane and has acute and chronic effects on vascular function[10-11]. Acute and chronic endothelial dysfunction often overlaps with major risk factors such as hypertension, hyperlipidemia and diabetes, which may prevent the beneficial compensation of anemia[12]. In case of anemia, the function of red blood cells leads to changes in the properties of cell membranes, the redox state in red blood cells is disturbed, the function of nitric oxide synthase in red blood cells (RBC-eNOS) is enhanced, and the hemolysis reaction is enhanced.

PPARG: PPAR is a nutrient receptor that regulates a series of homeostatic functions through ingestion, utilization, oxidation and metabolism. It can participate in the regulation of cell growth, migration and apoptosis, and may regulate the oxidative stress, antioxidant response and inflammatory diseases of the cardiovascular system[13-15]. PPARG is a ligand-activated transcription factor that belongs to the family of peroxisome proliferation-activated receptors (PPARs). PPARG can be divided into three subtypes (PPARα, β/δ, and γ) and play an important role in systemic energy metabolism, and they jointly participate in fatty acid oxidation (FAO)[16]. The activation of PPARγ plays a crucial role in regulating the expression of eNOS in endothelial cells and increasing the bioavailability of nitric oxide (NO)[17].

Based on network pharmacology and molecular docking technology, we explored the action mechanism of Danggui Buxue Decoction in the treatment of IDA. The results show that Danggui Buxue Decoction can improve vascular endothelial function, inhibit inflammation, promote hematopoiesis, and stimulate the development of early red blood cell progenitor BFU-E through multiple components, multiple targets, and multiple pathways, so as to improve anemia symptoms and treat anemia.