Network Pharmacology-Based Study of Active Ingredients and Mechanisms of Compound Xuanju for Rheumatoid Arthritis

1Medical Experimental Center, The Third Affiliated Hospital of Guizhou Medical University, Duyun, Guizhou, China 2College of Biology and Engineering, Guizhou Medical University, Guiyang, Guizhou, China Abstract Objective: Compound Xuanju has good effects in treating rheumatoid arthritis (RA), but its composition is complex, and its active ingredients and mechanism have not been fully defined. In this study, the active ingredients and mechanism of compound Xuanju for the treatment of RA were explored through network pharmacological methods. Methods: TCMSP and TCMID, Pubmed, CNKI, Wanfang, and VIP databases were used to screen, select active pharmaceutical ingredients and targets; Drugbank disease target screening database, GeneCards database, Therapeutic The Target Database (TTD) database and DisGeNET database were used to collect RA targets, and OmicShare was used to screen compound Xuanju and RA for common targets and construct a Venn diagram. A protein target database String was used to construct a common target interaction network. OmicShare mapping software builds a ”drug-active ingredient-target” network and analyzes their associations. DAVID online software performs gene annotation (GO) and KEGG pathway enrichment analysis on key targets. Results: A total of 73 effective ingredients of compound Xuanju were obtained, and corresponding to 229 targets; 2337 targets for RA. 155 key targets for potential active ingredients of compound Xuanju predicted therapeutic effect of RA, the key targets map 55 active ingredients of compound Xuanju capsules. These targets mainly involve signaling pathways such as Toll-like receptor signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, and TNF signaling pathway acting on RA. Conclusion: Compound Xuanju may via its potential 55 active ingredients act on 155 targets to treat RA through Toll-like receptor signaling pathway, PI3K-Akt signaling pathway, NF-κB signaling pathway, HIF1 signaling pathway and TNF signaling pathway. This study lays the theoretical basis for the widespread application of compound Xuanju in clinical practice.


INTRODUCTION
R heumatoid arthritis (RA) is a chronic autoimmune disease with pathological features characterized by excessive hyperplasia of synovial tissue, joint swelling, vascular formation, and cartilage erosion (1). The high disability rate, poor prognosis, recurrent and long treatment cycle which brings a heavy economic burden on patients' families and society. The incidence of RA worldwide amounts to 0.18-1.07%, and it is increasing year by year (2). Depending on the U.S. administrative health insurance claims database, the prevalence of RA from 2004 to 2014 was from 0.41% to 0.54% (3). The prevalence of Chinese patients is about 0.4%. For patients who have not been diagnosed and treated promptly, the disability rate is 50% after 2 years, and 70% of patients after 3 years have varying degrees of disability, and their quality of life is severely affected (4). Current research shows that the pathogenesis of RA is related to multiple mechanisms such as infection, genetics, immune system disorders, and metabolic abnormalities (5,6), but it has not yet been conclusively determined, and effective preventive and therapeutic drugs are continue to be lacking.
Western medicines currently available include nonsteroidal anti-inflammatory drugs, glucocorticoids, anti-rheumatic drugs (DMARD) to relieve disease, and biological agents. However, its potential side effects may affect patient compliance with treatment (7). In addition, pathophysiological and genetic differences between patients also limit the therapeutic effectiveness of such drugs in RA treatment. Therefore, as an adjuvant treatment for patients with chronic diseases, complementary therapies have become more and more common (8). The most prevalent of these complementary therapies is traditional Chinese medicine (TCM). In China, a nationwide study of the incidence of TCM use in RA and non-RA populations has demonstrated that RA patients have a higher TCM use tendency compared to the general population, with a range of 30% (9).
Studies have shown (10) that compound Xuanju have significant anti-inflammatory and immuneregulating effects. Compound Xuanjuare mainly composed of Chinese herbal ingredients such as Xuanju (Polyrhachis vicine Roger), Epimrdii Herba, Cnidii Fructus, and Lycii Fructus. Xuanju is salty, sour, and mild in nature, which can make stronger, nourish blood, strengthen pain, and relieve pain and Quyu Tongluo. Lycii Fructus is sweet and flat, achieves strong bones, and nourishes liver and kidney; Epimrdii Herba can remove wind and dehumidification, kidney and aphrodisiac. Cnidii Fructus tastes bitter, mild, has warm kidney and impotence Cold effect. The compatibility of these drugs can achieve the effects of removing wind and dispel cold and warming kidney, and it can treat the RA from the two aspects of righting and eliminating evil. Although some studies have shown that compound Xuanjucan effectively treat RA (11,12), there is still an urgent need to further clarify and describe its underlying material basis and mechanism of action. Therefore, this study uses network pharmacology to conduct a comprehensive network analysis of the effects of compound Xuanju for RA targets, and to construct a RA target network with hub genes, in order to explain the key active ingredients of compound Xuanju and their mechanism of action. The workflow is shown in Figure 1.  drugs and experimental drugs that have undergone FDA approval procedures (13). The TTD database provides information on known therapeutic protein and nucleic acid targets, targeted disease conditions, pathway information, and target drug/ligand counterparts (14) . DisGeNET (http://www.disgenet.org /) is used to retrieve the molecular basis of specific human diseases, analysis of disease gene characteristics, generation of hypothesis for drug treatment effects and adverse drug reactions, verification of predicted disease genes and evaluation of text mining the performance of the method etc (15) . The chemical composition and target data obtained from the three databases are combined, duplicate and invalid targets are deleted, and the full names of the selected targets are entered into DrugBank to obtain the gene abbreviation and Uniprot ID.

Construction and analysis of the active ingredienttarget network
OmicShare were used to construct and visualize the collected traditional Chinese medicine ingredients and their target information. The nodes represent the active ingredient and the target, and the edges represent the interaction between them, and the connectivity analysis is performed. The more connected knots are, the more important the compound or target is. The key active components and central genes of compound Xuanju against RA were determined by analyzing the strength of network connectivity.

Analysis of key target functions annotation and pathway enrichment
Through the DAVID database (https://david.ncifcrf. gov/), P <0.05 was used as the screening condition to obtain key target proteins for gene ontology (GO) functional enrichment analysis and Kyoto Gene and Genome Encyclopedia (KEGG) pathway enrichment analysis. Three modules including biological process, cellular component and molecular function were selected for mapping. Use GraphPad Prism 5.0 to visualize the top 20 entries of biological processes, cell composition, and molecular function entries in GO function enrichment analysis as a visual bar chart. Use the OmicShare to present the channel enrichment results in the form of a bubble chart for KEGG pathway analysis on key targets.  Figure 2A) showed that the potential active ingredients of compound Xuanju intersect with RA. The module predicts a total of 155 targets Table 2. A protein-protein interaction (PPI) network model was constructed with 155 common targets in the String database ( Figure 2B). These 155 common targets may be the key targets for the active ingredients of compound Xuanju to treat RA.

Analysis of key active ingredients and targets of compound Xuanju against RA
In order to further understand the mechanism of action of key genes, we identified 155 overlapping genes based on the predicted results and reversely searched for compounds that interacted with them. Finally, 55 compounds were identified from Xuanju, Epimrdii Herba, Cnidii Fructus, and Lycii Fructus. The compounds are shown in Table 3 . And visualize the traditional Chinese medicine-chemical composition-target network Figure 3. The results showed that the three components with the strongest connectivity were: Quercetin (96), Kaempferol (64), and Luteolin (46); the four targets with the strongest connectivity were: PGR (30), PTGS2 (28), PTGS1 (19) and DDP4 (17). It is suggested that compound Xuanju mainly treat targets such as PGR, PTGS2, PTGS1 and DDP through active ingredients such as quercetin, kaempferol and luteolin to treat RA.

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MENG ET AL.

Analysis of key target functions annotation and pathway enrichment
To further understand the function and enrichment of key targets, GO and KEGG analyses were performed. The results showed that the highest scores in the biological process, cellular component, and molecular function of GO functional enrichment analysis were positive regulation of transcription from RNA polymerase II promoter (GO: 0045944), extracellular space (GO: 000561), and protein homodimerization activity (GO : 0042803). KEGG pathway enrichment analysis showed that compound Xuanju mainly passed Toll-like receptor signaling pathway (ssc04620), PI3K-Akt signaling pathway (ssc04151), NF-κB signaling pathway (ssc04064), HIF-1 signaling pathway (ssc04066), TNF Signaling pathways (ssc04668) and other signaling pathways act on RA. Figure 4

DISCUSSION
The ingredient of traditional Chinese medicine is a complex chemical system, and the biological body is also a complex system. The interaction between the chemical substance entity of the traditional Chinese medicine product and the biological activity of the body has become an important entry point for modern Chinese medicine research. Network pharmacology researches the pharmacology of relating drugs through big data analysis. It takes herbs, chemical components, targets, diseases and other perspectives as the starting point to achieve a comprehensive and systematic comprehensive network analysis of traditional Chinese medicine research. It has the unique advantage and great potential to predicts and identifies active ingredients of traditional Chinese medicine, groups and targets, clarify the mechanism of action, reveal the formulas, discover new indications and new active compounds. Moreover, network pharmacology emphasizing on "one target, one  drug" to "network target, multiple groups paradigm shift. This gives us a better understanding of the mechanism of action of drugs, then it is not difficult to understand that network pharmacology is widely used in drug research (18) In the treatment of RA,compound Xuanju have received more attention due to its obvious effectiveness and fewer side effects. Therefore, in this study, we utilized network pharmacology to study the potential targets of compound Xuanju for the treatment of RA. Construct and analyze target networks, perform enrichment analysis on hub genes, and uncover their potential mechanism.
In this study, we first excavated the targets of RA, the active ingredients and targets of compound Xuanju capsules. By mapping and matching the targets, we obtained the key targets of compound Xuanju for RA; Point the corresponding active ingredients; construct the protein-protein interaction network of key targets, and then perform GO analysis and KEGG analysis to clarify the functional annotation and enrichment of key targets.
The results of this study show that the four most active ingredients are: quercetin in Lycii Fructus and Epimrdii Herba, kaempferol in Epimrdii Herba, luteolin in Lycii Fructus and Epimrdii Herba, βsitosterol in Xuanju and Cnidii Fructus. This component group may play a therapeutic role in RA through highly connected targets such as PGR, PTGS2, PTGS1, and DDP4. Among them, quercetin has the effects of anti-oxidation and scavenging of free radicals, and also has anti-inflammatory, immunomodulatory, anti-cancer, antibacterial, antiviral, hypoglycemic and antihypertensive effects, and cardiovascular protection (19,20) Jayanti et al (21) reported that quercetin gel preparation can effectively treat rheumatoid arthritis. Kaempferol has anti-inflammatory, antibacterial, anti-cancer and other physiological activities (22,23). β-sitosterol (beta-sitosterol) inhibit tumor cell proliferation and inhibit blood angiogenesis (24). PTGS1 is thought to play a key role in the pathophysiology of inflammation, arthritic disease, and cancer (25) Clinical studies have shown that as a specific inhibitor of PTGS, low-dose aspirin can effectively exert antipyretic, analgesic and anti-inflammatory effects (26). Importantly, PTGS1 inhibitors or silencing of PTGS1 ex-pression through negative regulation greatly reduces the inflammatory response caused by LPS (27). Wang et al (28) showed that knockout of PTGS1 promotes osteogenic differentiation of ASCs in vitro and in vivo by targeting p65 cytoplasm/nuclear translocation, and that during bone remodeling, PTGS1 silencing may provide regulation of the inflammatory microenvironment a potential means. Wei et al (29) reported that PTGS2 plays an important role in inflammation and fibroblast-like synoviocytes in RA. MiR-101-3p reduced joint swelling and arthritis index in RA rats by down-regulating PTGS2. The negative expression of miR-101-3p and PTGS2 was found in the synovial tissues of RA patients and RA rats, overexpressed MiR-101-3p and silenced PTGS2. RA-FLS was observed to have reduced cell proliferation, migration and invasion, promote cells apoptosis.
In GO analysis, the biological process mainly involves positive regulation of transcription from RNA polymerase II promoter, adenylate cyclaseactivating adrenergic receptor signaling pathway, and lipopolysaccharide-mediated signaling pathway; Molecular function mainly involves protein homodimerization activity, G-protein coupled acetylcholine receptor activity, and steroid hormone receptor activity. The KEGG analysis mainly involved TNF signaling pathway, HIF-1 signaling pathway, T cell receptor signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, NF-κB signaling pathway and p53 signaling pathway. The NF-κB signaling pathway plays an important role in the inflammatory process of rheumatoid arthritis. Its transcription factor NF-κB is involved in regulating the expression of multiple cytokines and cell adhesion factors during the inflammation process (30). Activation can lead to T cell activation and thereby mediate the inflammatory response. It can also induce abnormal proliferation of RA-FLS and stimulate the proliferation and activation of osteoclasts. As a result, joint deformities and bone erosion can be caused, which can aggravate the condition of RA. In addition, after activation of NF-κB, cytokines and acute response proteins are produced to activate the immune response and increase the expression of adhesion molecules. These immune molecules exacerbate the inflammation level of RA (31) Toll-NETWORK PHARMACOLOGY-BASED STUDY OF ACTIVE INGREDIENTS AND MECHANISMS OF COMPOUND XUANJU FOR RHEUMATOID ARTHRITIS like receptors (TLRs) in the toll-like receptor signaling pathway are type I transmembrane protein receptors, which are widely distributed on the surface of immune cells and epithelial cells. TLRs/NF-κB in signal transduction pathways that mediate immunity signaling pathways play an extremely important role in immune regulation (32,33) TNF-α in the TNF signaling pathway can induce the expression of multiple inflammatory cytokines (such as IL-6'IL-8) and matrix metal protein (MMP) in synovial cells, which is a persistent and prolonged progression of rheumatoid arthritis key factors (34,35). Study found that P53 is mutated in synovial tissue of RA patients. Mutated P53 can cause cells to over express IL-6. It can be seen that abnormal proliferation T cell differentiation, and various inflammations of RA synovial tissue is regulated by the P53 signal pathway Factor production (36).
In summary, we utilized network pharmacological methods to discover 73 ingredients and 229 potential targets in compound Xuanju, and 2337 targets related to RA disease, of which compound Xuanju-RA common targets 155 points, and through the GO biological process and KEGG signal pathway enrichment analysis of these targets, it is predicted that compound Xuanju may regulate the TNF signal pathway, toll-like receptors signal pathway, NF-κB signal pathway, P53 signal pathway to act on PTGS2, PTGS1, DDP4, and PGR targets. Thereby inhibiting inflammatory response, regulating immune function and regulating apoptosis to treat RA. But it's worth noting that network pharmacology develops rapidly. Our research present certain limitations. First of all, this study is based on a network pharmacological approach to study the potential targets of compound Xuanju for RA, but the specific mechanism of action remains to be confirmed by cell and animal experiments. Second, due to the difficulty of obtaining data, the database may not include all known or unknown active ingredients, targets, and protein-protein interactions. However, it is undeniable that the results of this study can provide ideas and reference for experimental research, and provide a theoretical basis for the wide application of compound Xuanju in clinical practice.

Conflicts of Interests :
The authors have no conflicts of interests.