Zi-Xing Bai, Xu-Han Cao, Cheng-Yi Sun, Yan-Jun Yang, Wei-Dong Sun, Yong-Li Dong?, Xu Wei, Li-Ping Yang

1. Wangjing Hospital of China Academy of Chinese Medical Sciences, Beijing, 100102, China 2. Beijing University of Chinese Medicine, Beijing, 100029, China

Keywords:Bi xie Tu fuling Gouty arthritis Network pharmacology Mechanism of action

ABSTRACT Objective: The mechanism of Chinese herbal medicine "Bi xie and Tu fuling" on the treatment of gouty arthritis was explored through network pharmacological methods. Methods: First, the TCMSP Chinese medicine system pharmacology database and analysis platform were used to define the bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18. Database to mine genetic targets related to gouty arthritis disease. Finally, the two genes are intersected to construct a Chinese medicine regulatory network of Chinese medicine-components-target genes-disease. The genes in the network are used to construct a protein interaction network to find core genes for analysis of GO and KEGG. Results: There were a total of 84 active ingredients in Chinese medicine Poria and Poria cocos, and 17 effective ingredients and 180 genetic targets were obtained after screening. 600 disease targets for gouty arthritis were identified, and 58 were common target genes for both. PPI network analysis revealed that IL1β, VEGFA, MAPK1, IL10, and PTGS2 may be drugs for treating gouty arthritis. Key targets. GO enrichment analysis identified 1,399 entries (P ＜0.05), of which biological processes mainly include biological stimulation, biological regulation, cell metabolism, etc; KEGG pathway enrichment analysis identified 152 signal pathways, of which signal pathways involved inflammation, metabolism, In terms of aging, mainly including the IL-4,IL-10,IL-13,IL-17signal pathway, etc. Conclusion: "Bi xie and Tu fuling" drugs have anti-inflammatory and immunological effects on gouty arthritis through multiple targets and multiple pathways. The mechanism of lowering uric acid and protecting liver and kidney is not clear. It needs molecular biology research to improve it. The mechanism of the action of the "Bi xie and Tu fuling" medicine pair provides new ideas for the clinical prescriptions.?Corresponding author: Yong-Li Dong, female.

1. Introduction

Gouty arthritis is a recurrent disease with characteristic arthritis and chronic goutstone disease as the main clinical manifestations. It is mostly related to the accumulation of monosodium urate caused by flocculation of purine metabolism and reduced uric acid excretion[1,2]. Hyperuricemia is more than 20% in China's economically developed areas, and it is more common in men[3]. According to the pathogenesis and symptoms, it is mainly divided

into four phases[4]: asymptomatic hyperuricemia phase, acute gouty arthritis attack period, gout attack intermittent period, and chronic gout stone arthritis. Western medicine treatment is guided by the idea of "open source, reduce expenditure, and alleviate symptoms". By reducing uric acid formation and promoting uric acid excretion to improve symptoms, clinical observation has found that there is a risk of renal insufficiency[5]. Traditional Chinese medicine believes that gouty arthritis belongs to the category of "biosis" and "white tiger calendar festival". The disease is located in the liver, spleen, and kidney. It adheres to detoxification and detoxification. Tongli joints are the main rules. The advantages of multi-targets and the elimination of evil and righteousness have broad prospects for future development.

Dioscorea is a perennial herbaceous plant of the family Dioscoreaceae. The taste is bitter, sweet, and flat. It belongs to the liver, kidney, and stomach meridians. Earth Poria is taken from the dried rhizomes of Liliaceae, Phyllostachys pubescens. It is sweet, light, flat, and returned to the liver and stomach meridians. It has the functions of detoxification, dampness, and joints. It is not only a cure for syphilis or limb constriction caused by syphilis taking mercury, but also a good product for dampness and turbid betting and eczema and eczema. It is currently commonly used in the treatment of gouty arthritis. Modern pharmacology believes that[6,7], the combined use of Bi xie and Tu fuling has anti-inflammatory and immuneregulating effects, can increase uric acid excretion and reduce blood uric acid levels, and can be used in the treatment of gouty arthritis, but its mechanism And the material basis has not been fully defined. Therefore, in this study, with the help of network pharmacology, the mechanism of treatment of acute gouty arthritis by Bi xie and Tu fuling was further explored, providing evidence-based evidence for clinical use, and providing theoretical basis for modernization of traditional Chinese medicine.

2.2 Target prediction for gouty arthritis disease

Using "gouty arthritis" as the search term, the OMIM database, GeneCards database, and DisGeNET database were used for gene query and target screening to obtain disease target proteins currently identified for gouty arthritis, delete duplicate gene targets, and determine final targets.

2.3 Identification of drug-disease common targets and network construction

The Venn diagram was drawn by the venny2.1 drawing software, and the drug and disease gene targets obtained in 1.1 and 1.2 were intersected to obtain the common target of "Bi xie and Tu fulingBi xie and Tu fuling" and gouty arthritis. The network drawing software Cytoscape 3.7.1 was used to construct a drug component-targetdisease network diagram to visualize the target-action relationship between the two.

2.4 Construction of protein Interaction network (PPI)

2. Materials and methods

2.1 Prediction of active ingredients and action targets of "Bi xie and Tu fuling"

Use the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) to search for the keywords:"Bi xie" and "Tu fuling" drugs to determine the active ingredients,and to absorb, distribute, metabolize, and excrete according to pharmacokinetics(ADME)index to screen active ingredients with high correlation.Definition:Oral bioavailability (OB)≥30%, drug-likeness (DL) ≥ 0.18. Uniprot database is used to determine the human gene abbreviation for each target, and finally it is possible to obtain "Bi xie and Tu fuling" Target protein present.

The common target obtained in 1.3 was imported into the STRING 11.0 system software, and the protein type was set to "homo sapiens" to obtain a protein interaction information map. In this study, the highest confidence protein parameter score was set to＞ 0.9 to ensure the reliability of the research data.

2.5 GO function enrichment analysis and KEGG pathway enrichment analysis

The purpose of gene ontology (GO) functional enrichment analysis is to analyze the role of drug target proteins in gene function. By Metascape gene annotation software, GO function enrichment analysis was performed on the proteins in the "active ingredientpotential target" network of the "Bi xie and Tu fuling" drug relatedto gout. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis aims to explore the biological processes and key signaling pathways for the treatment of gouty arthritis. KEGG signal pathway analysis was performed on the key targets through the DIVID database to obtain significantly enriched signal pathways (P ＜0.05).

Table 1Bioactive constituents of "Bi xie and Tu fuling"

3. Results

3.1 Screening of Active Ingredients in "Bi xie and Tu fuling" and Corresponding Target Protein Information

A total of 84 known active ingredients of “Bi xie and Tu fuling” were searched through the TCMSP database, of which 17 were active ingredients that met the OB and DL screening conditions.

3.2 Prediction of target of gouty arthritis disease by "Bi xie and Tu fuling" drug

Target prediction was performed on the above 17 kinds of biologically active ingredients using the TCMSP target prediction instruction. Among them, 17 target proteins were obtained by Bi xie, and 260 target proteins were obtained from Tu fuling, and 180 target proteins were obtained by removing duplicates. A total of 665 targets related to gouty arthritis were searched through the OMIM database, GeneCards database, and DisGeNET, and 600 targets were obtained after rechecking. Fifty-eight targets in gouty arthritis were obtained from Bi xie and Tu fuling Poria by venny intersection.(Figure 1)

Figure 1 Venn diagram of drug prediction target and disease target

3.3 Identification of drug-disease common targets and network construction

The obtained public target proteins were drawn and analyzed using Cytoscape 3.7.1 network drawing software to analyze the component-target network diagram of "Bi xie and Tu fuling" intervention for gouty arthritis. First, the disease and drug action relationship diagram was initially obtained through Cytoscape 3.7.1 software, including 73 nodes and 262 action relationships. Then, Cytohubba plugin of Cytoscape 3.7.1 was used to filter the complex network, and the edge relationship was removed based on the Count value. The chemical composition-drug-target-disease relationship diagram includes a total of 73 nodes, of which there are 12 active ingredient nodes and 166 target protein nodes. Calculate the interaction between each target and drugs and diseases, and distinguish them with different colors, and then adjust and beautify by style and edge modules, edit the representative graphics of molecules, drugs, targets, and diseases. The red ovals represent gouty joints. Inflammatory diseases, the green diamonds represent the “Bi xie and Tu fuling” drug pair, the yellow arrows represent the effective chemical constituents of the drug, and the purple ellipse represents the gene target. Among them, the lines of action between diseases, drugs, and targets are represented by green, blue, and red lines, respectively, and the number of lines is positively related to the effect relationship between the targets.(Figure 2)

Figure 2 Drug-Compound-Target-Disease Target Network Diagram

3.4 Construction of PPI network and cluster analysis of "Bi xie and Tu fuling" gouty arthritis targets

The database of interaction between proteins (known and predicted proteins) was searched online through the STRING 11.0 database, and a PPI network graph of "Bi xie and Tu fuling" for gouty arthritis was initially constructed. The medium confidence parameter was set to 0.7. In, export the protein interaction network diagram in PNG format, and save the text file as TSV. Import the protein interaction network map constructed by STRING 11.0 into the MCODE plug-in of Cytoscape software. Use network clustering algorithm to mine protein complexes and protein sequence and annotation information from complex protein networks. Beautify and adjust the corresponding functional modules, intuitively. The mechanism of action of "Bi xie and Tu fuling" in intervening gouty arthritis. Among them, the protein network constructed by STRING 11.0 was first imported into Cytoscape software to obtain a primary protein interaction diagram: the network diagram contains 58, 359 relationship lines, 46 edges are expected, and the PPI concentration is p＜1.0e-16, The network action relationship is complex and cannot directly reflect the action relationship of core targets. Through further cluster analysis, two sub-protein networks are obtained, one with IL1β, MAPK1, and IL6 as the core, including 15 target proteins and 65 action lines. The score is 9.286; the other is VEGFA, IL10, PTGS2, CXCL8, including 16 target proteins, 60 lines of action, with a score of 8.0. By decomposing the complex interaction diagram, the target interaction is simple and intuitive. Therefore, it is predicted that IL1β,VEGFA, MAPK1, IL10, and PTGS2 in "Bi xie and Tu fuling" are the core proteins for the treatment of gouty arthritis.(Figure 3)

Figure 3 Network diagram of PPI network construction and target protein cluster analysis

3.5 Biological pathway enrichment analysis

The results of GO function enrichment analysis showed that 1095 GO entries were identified in biological processes, mainly including biological stimulus, biological regulation, and neurotransmitter metabolism; cell components contained 55 GO entries, mainly including Cytoplasm, intracellular organelle part, extracellular region; molecular functions mainly include 97 GO entries, mainly by protein binding, ion binding, signal receptors Signalling receptor binding. The enrichment information was screened based on the number of gene enrichment, and it was found that the effects of the drug "Bi xie and Tu fuling" on the possible response to toxic substances, extracellular stimuli, protein-polypeptide responses, inorganic responses, vascular development, and cell differentiation Regulation of other pathways to play its role in anti-gouty arthritis. KEGG pathway analysis was performed through the DIVID database, and the common target genes obtained in 2.2 were uploaded to the database, and pathway annotation was performed to clarify the role relationship. Define P＜0.05 as the significant enrichment pathway, integrate and enrich the pathway with a higher Count value, and construct a bar chart for the first 20 pathways. The KEGG pathway enrichment analysis found that a total of 152 pathways were related to the treatment of gouty arthritis with“Bi xie and Tu fuling” drugs, and some signal pathways (P＜9.802×10-7). The "Bi xie and Tu fuling" drug pair may act on the IL-4 signal pathway, IL-10 signal pathway, IL-13 signal pathway, IL-17 signal pathway, PID REG GR pathway, PID ATF2 channel, and HIF-1 signal pathway The TNF signal pathway and other pathways affect gouty arthritis. Therefore, it is speculated that the kidney "Bi xie and Tu fuling" medicine is mainly used for the treatment of gouty arthritis by anti-inflammatory immune mechanism.(Figure 4、5)

Figure 4 Heat map of the "Bi xie and Tu fuling" medicine for intervening gouty arthritis

Figure 5 Analysis of the enrichment analysis of "Bi xie and Tu fuling" medicine on intervention of gouty arthritis

Biological pathway heat map: English letters on the right, the bottom number represents the significance of enrichment (-log10 (P value), the darker the color, the greater the value, the more significant the enrichment), and the right Y-axis represents Enriched GO, KEGG Terms.

Enrichment analysis chart: the relationship diagram of the enrichment pathway information network. The different colored dots in Figure A represent the GO and KEGG pathway information, and the depth of the dots in Figure B represents the Rich factor, which is the degree of enrichment The darker the color, the more significant the enrichment.

4. Discussion

Traditional Chinese medicine believes that gouty arthritis belongs to the categories of "heat paralysis of paralysis" and "calendar". The name of the disease was first published in Zhu Danxi's "Gezhiyulun·Gout Theory": "Goutes, ... have a high rate of heat from blood , Has been boiling, and then involved in cold water, or standing wetland, or fan wind to get cold, or lying on the ground when the wind, cold and external beat, dirty and astringent so painful, night pain. " The pathogenesis of this disease lies in rheumatism and stasis, obstruction of the meridian joints, liver and spleen and kidney organs turbidity and metabolism abnormalities, which belongs to the evidence of this deficiency. Turbidity, blood stasis, coldness and dampness are common, and the frequency of dampness, heat and phlegm are the highest[8,9]. Modern pathophysiology research finds that gouty arthritis is divided into two types: primary and secondary gouty arthritis, which are both phagocytosed by autoimmune cells in the body's immune system, inflammatory mediators such as NLRP3 inflammatory bodies and interleukins The negative regulation is related[10]. There are three main types of clinical medications: currently there are three main types of drugs:①inhibit uric acid production, such as allopurinol, febuxostat, etc.;②promote uric acid excretion, such as benzbromarone, propanesulfonic acid, etc.;Pain relief, such as: non-steroidal anti-inflammatory drugs, colchicine and so on. The effect of comprehensive treatment with western medicine is remarkable, but gouty arthritis is a recurrent disease. Taking the drug for a long time can easily cause complications such as chronic renal insufficiency, liver injury, bone marrow suppression, hepatocyte necrosis and neurological toxicity. Clinical application must be cautious and should Regular review of blood routine, liver and kidney function[11].

Traditional Chinese medicine adheres to the principle of dialectical treatment, exorcism and righteousness, and the principle of treating disease should be based on the principle. Comprehensive application of traditional Chinese medicine to treat gouty arthritis has played a significant effect. "Treatment of Syndrome" records "gout, one of the symptoms of pain and palsy ... because of wind, cold, dampness and depression in the beginning, the heat will cause pain in the long run, and it will be more dramatic at night". Lin Peiqin formulated Bi xie and Tu fuling Soup, taking the essential medicine for treating limb constriction, Tu fuling for the treatment of dampness, turbidity, and spleen and stomach, get rid of rheumatism, joints, and spleen and stomach. It's bones and bones. "Compendium of Materia Medica" records: Bi xie, Foot Yangming, Jueyin meridian also. Juyin's main tendon belongs to wind, Yangming's main flesh is wet, and the power of yang is longer than rheumatism, so it can cure rheumatism who is weak, stubborn, turbid, and malignant sores. It is good at removing dampness and turbidity. Turbid clarification patent. " Modern pharmacological research believes that Poria cocos can reduce inflammation and analgesic, and promote uric acid excretion. It is used in a variety of compounds for treating gouty arthritis, such as Qingre Lishi Fang and Lishi Tongluo The prescriptions such as Fang and Tongfengkang Ⅱ decoction all contain the medicine pair of“Bi xie and Tu fuling”[12,13,14], which shows that the medicine of“Bi xie and Tu fuling”is of great significance in the clinical treatment of HF.The main chemical of Bi xie is a variety of steroidal saponins including steroids, diarylheptanes, lignans, diosgenin, and other ingredients, which have the effects of lowering uric acid, antiinflammatory and analgesic, and anti-osteoporosis[15, 16]. The main chemistry of Tu fuling is that it contains saponins such as astilbin,β-sitosterol, tannins, flavones, resins, etc. It has antitumor, antibacterial, anti-atherosclerosis, anti-inflammatory, analgesic, diuretic and detoxifying effects[17,18].In this study, TCMSP screened 17 biologically active components, and 180 possible targets were found. Intersect with the known 600 targets related to gouty arthritis and finally obtain 58 common targets, 1399 Possible interactions. The key targets of“Bi xie and Tu fuling”include IL1β, VEGFA, MAPK1, IL10, PTGS2, etc., and there are complex interactions with multiple chemical components. Among them, IL1β has been proven to be one of the first substances that need to be activated during the onset of gout inflammation. That is, MSU first activates NLRP3 to stimulate the release of caspase-1. Finally, the inactive pro-IL-1β is collectively called IL-1β, which causes inflammation reaction[19]; IL-10 is an anti-inflammatory cytokine that can inhibit the release of IL-8 proinflammatory mediators and prevent the activation of IL-1β[20]. VEGFA, or vascular endothelial growth factor, belongs to the pro-angiogenic factor, and has significance for angiogenesis, angiogenesis, and maintenance of vascular stability[21]. MAPK1 is one of the indicators of inflammation evaluation, and PTGS2 is mainly involved in the occurrence of inflammation and mitosis, and can judge whether the drug has anti-inflammatory effect[22,23]. The synovial fluid of patients with gouty arthritis contains high levels of the anti-inflammatory factor IL-10 and soluble TNF receptors. It has been preliminary proved that the core target of "Bi xie and Tu fuling" is related to the treatment of gouty arthritis. Further research in cell biology.

Biological pathway enrichment analysis found that the IL-4 signaling pathway, IL-10 signaling pathway, IL-13 signaling pathway, IL-17 signaling pathway, PID REG GR pathway, PID ATF2 pathway, HIF-1 signaling pathway, and TNF signaling pathway are networks The key targets in the signal pathway are mainly enriched. The IL-4 signaling pathway, IL-10 signaling pathway, and IL-13 signaling pathway belong to the anti-inflammatory interleukin signaling pathway[24]. Among them, the IL-4 signaling pathway strongly inhibits the synthesis of inflammatory factors such as IL-1, IL-6, IL-8 and TNF-α, and plays an important role in suppressing and eliminating tissue inflammatory responses[25,26];IL-10 signaling pathway is mainly used to inhibit the synthesis of inflammatory factors, mainly produced by Th2, monocytes, macrophages, etc., by affecting the mRNA transcription and synthesis, blocking the release of proinflammatory cytokines [27]. The IL-13 signaling pathway works in concert with the IL-4 signaling pathway to participate in the anti-inflammatory response by reducing the production of IL-1β, IL-8, and NO, and promoting the release of the IL-1Rt2 receptor[28]. As an important pro-inflammatory cytokine, IL-17 is expressed by activated CD4+ T lymphocytes, and the expression level is positively correlated with the degree of cellular damage, and is related to the inflammatory response and immune response in vivo[29]. TNF is an important inflammatory mediator, produced by activated monocyte macrophages, which helps inflammatory cells to express adhesion molecules. HIF-1α is the most typical hypoxic response signal pathway in the body. In the case of hypoxia in the body, a series of biological reactions are initiated in time to increase the oxygen level. HIF-1α plays an important role in protecting cells. Increasing the content of HIF-1α can promote cell survival and angiogenesis. HIF-1α can induce the expression of PPARγ gene promoter and participate in energy metabolism[30,31]. Other pathways such as the PID REG GR pathway and PID ATF2 pathway may be related to the formation and excretion of uric acid. The specific mechanism has not been found in the current research, and it is necessary to focus on future research to clarify its role.

Traditional Chinese medicine treats diseases based on dialectical treatment. With the development of modern medicine, the treatment of gouty arthritis should be critically inherited, upheld and innovative. There are various compatibility of traditional Chinese medicine prescriptions. Clinical drugs should make full use of the advantages of the target and cooperate with the compatibility to make the drug, which often has the effect of 1+1＞ 2. The target of “Bi xie and Tu fuling” for the treatment of gouty arthritis is complex and the pathways are diverse. In this study, the anti-inflammatory and immune mechanism of the“Bi xie and Tu fuling”drug was initially identified with the help of network pharmacology. The mechanism of excretion and protection of liver and kidney function, no specific target pathway was obtained in this study. Subsequent research needs to strengthen the research on uric acid lowering, core targets for liver and kidney protection, and pathways, and further clarify the mechanism of action of “Bi xie and Tu fuling”on gouty arthritis. In short, the component-target-channel network constructed in this study provides a theoretical basis for the treatment of gouty arthritis, provides a direction for clinicians to use medicine, and provides an evidence-based basis for the modernization of traditional Chinese medicine.