The analysis of the application and mechanism of Traditional Chinese Medicine in osteoarthritis based on Data Mining and Network Pharmacology

: Background: Using Data Mining to retrieve the core drug of osteoarthritis in clinic , predicting the drug molecular action target through the Network Pharmacology, combining with the related targets of osteoarthritis to identify the key nodes of the interaction, exploring the pharmacological mechanism of Traditional Chinese Medicine against osteoarthritis and other possible mechanisms of actions. Methods: Pubmed, CNKI, VIP, CBM and WanFang Database was used to retrieve the commonly used therapeutic formulations for osteoarthritis patients in clinical, and screen out the core drugs through the Ancient and Modern Medical Case Cloud Platform and software Gephi, filtered out the core drug molecules and targets combined with TCMSP database and the targets of osteoarthritis in Genecard, OMIM database, impoting those datas into R project and Cytoscape to construct the intersection model of Drug molecule-osteoarthritis, carrying out PPI network and GO and KEGG enrichment analysis with String database. Vina molecular docking was implemented to draw molecular docking diagram, and the results were analyzed after comprehensive analysis. Results: The core drug pairs were identified as "Eucommiae Cortex - Achyranthis Bidentatae Radix" through correlation analysis, complex network analysis basing on the coefficient. "Eucommiae Cortex Achyranthis Bidentatae Radix" can intervene cell behaviors through multiple pathways and regulate cell metabolism, cytokine synthesis, oxidative , cellular immunity as a consequence of topology analysis in String Database. Conclusions: "Eucommia bark - achyranthes" drug molecules can be combined with the target to produce hydrogen bond, hydrophobic function and Pi-Pi directly or indirectly affecting the corresponding targets, to participate in the regulation of osteogenesis and osteoclast proliferation, protect the extracellular matrix, inhibition of cell apoptosis and anti-inflammatory for resistance to osteoarthritis, also, providing the basis for interpretation of its action mechanism. phenylpropanoids, iridoids, polysaccharides, etc. Flavonoids are the intersection of two Traditional Chinese Medicines and one of the main components of the medicine. This study found 156 predictive targets and 35 active drug compounds, suggesting that the drug components have a synergistic effect and play an important role in the course of osteoarthritis ， 4 drug compounds and 8 targets were selected and Estrogen Receptor (ESR1), Prostaglandin Endoperoxidase Synthase 2 (PTGS2), Androgen Receptor (AR), Heat Shock Protein (HSP90AA1), Calmodulin (CALM1), and Prostaglandin Endoperoxidase Synthase 1 (PTGS1) are both the target of drug active ingredients and the disease target of osteoarthritis according to the Degree (≥10) and topology results. GO annotation result shows, The biological process (BP) of the positive regulation of protein kinase B may be related to the effect of drugs on osteoarthritis. Protein kinase B is a serine/threonine protein kinase that plays a key role in the P13k-Akt pathway. It has the functions of promoting cell growth, proliferation, inhibiting cell apoptosis, and promoting angiogenesis. The molecular function (MF) level shows that drug molecules can play a role through calcium ions, adenosine triphosphate enzyme, cytokines, etc. Cell level (CC) shows that this mechanism is closely related to molecular chaperone complexes, melanosomes, lysosomes, clathrin, etc. suggesting that the mechanism of drug action may be related to autophagy ， Combining the results of BP, CC, and MF, it can be concluded that the drug molecule directly or indirectly exerts the biological behavior of anti-osteoarthritis by binding to different targets. key molecules pass acid

development of osteoarthritis, and the normal performance of its function may be related to exosomes [5]. This research is to use data mining technology to collect prescriptions commonly used in the treatment of osteoarthritis, analyze the composition of the drugs, and screen out the core drugs to interpret the mechanism of action and association between drugs and diseases through network pharmacology and molecular docking Methodss, and predict other possible mechanisms of action.

Literature sources
Search CNKI, VIP, CBM, Pubmed, WanFang Database with "osteoarthritis", "degenerative arthritis", "Bizheng", "Gubi syndrome", "Chinese medicine" as the subject terms, starting from January 2010 to June 2020. A total of 3,319 duplicate documents were excluded, and a total of 207 clinical observations, case analyses, medical record summaries, master and doctoral dissertations on the treatment of osteoarthritis with Traditional Chinese Medicine were screened out. The data involved were extracted by two independent personnel.

Data extraction
Input the data within the literature into the EXCEL table and saved and imported Ancient and Modern Medical Record Cloud Platform (http://www.yiankb.com/). The association rule of analysis, drug frequency statistics, complex network construction and etc. were performed to analyze the drug compatibility law, and the pair of drug combination with confidence level> 0.9 and support level> 0.2 was selected as the core drug compatibility.

Network pharmacology
Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php)were using to search the chemical components and its targets of core Chinese medicine which was selected in the previous step with the filter condition OB≥30%. Then, got the gene targets of diseases through databases such as Genecards, GEO, OMIM, and used "Osteoarthritis, Arthritis, Degenerative" as search terms. After taking the intersection of the compound target contained in the drug and the disease gene target, imported Cytoscape 3.6.0 software to construct a "compound-target" network. Construction signaling pathway targets the core screened by STRING internet PPI protein interaction network, using R Project for KEGG GO annotation enrichment analysis and understanding of the core target.

Molecular docking
Searched the 3D structure diagram of the core target in the RSCB database and the 2D structure diagram of the compound through the PubChem database, using Chem 3D software to make it into a 3D structure diagram, and utilizing PyMOL, Autodock, Discovery Studio software to hydrogenate, dewatering, de-impurity and other preprocessing with the core target, whereafter, the Vina docking was performed, and the conformational map of the molecule with the lowest binding energy value was 3 docked with the 3D map of the protein. Ligplus was used to display the hydrogen bond distance and hydrophobicity of the drug molecule and the surrounding residues.

Drug frequency
According to the inclusion and exclusion criteria, a total of 3,292 articles were retrieved from various databases, and 184 articles were finally chosen and included in the study, including 94 prescriptions and 194 Traditional Chinese Medicines. The frequency statistics of the Chinese medicines contained in the prescriptions were performed, and 17 with a frequency of ≥45, which the top 5 were Achyranthes bidentata, Angelica, Licorice, Eucommia, Chuanxiong, (Table 1).  Fig 1).

Complex network analysis
Import data from correlation analysis into Gephi-0.9.2 for statistics, calculate topological characteristic parameters "Weighted degree", "Harmonic closeness centrality", "Eigen centrality", "Betweeness centrality", ect. The average node degree of the network is 2.429, and the graph density is 0.187. Achyranthis Bidentatae Radix, Angelicae Sinensis Radix, Chuanxiong Rhizoma, Gentiana Macrophylla Pall ranked top among them. (Table 3). platform. Using Genecard and OMIM database with "Arthritis, Degenerative" and "Osteoarthritis" as the subject terms, 5892 targets are retrieved in total, removing the duplicate targets, 3737 targets and chemical composition targets are intersected and 156 intersecting targets are obtained, (Fig 3), Build a complex network of "Achyranthis Bidentatae Radix-Eucommiae Cortex" compounds and disease targets in Cytoscape, (Fig 4), The Degree ≥ 10, Betweenness, and Closeness was used as the screening basis, and finally 14 compound components and 8 targets were obtained, Table 4. The top 5 compounds:

PPI network construction
Import the above targets into the STRING database, construct the PPI network diagram, and output the tsv table at the same time, and import the key nodes into the Cytoscape using the Cytohubba plug-in. The color more darker is, the more important its position in the network, (Fig 5-6 (Fig 7-9).

Molecular docking of compound and target
Perform vina docking of the top 5 compounds in Degree with the 6 core targets, and calculate their binding energy, (Table 5), draw a heat map based on the binding energy, the higher the absolute value of affinity, the tighter the molecular binding (Fig 11).    shows that this mechanism is closely related to molecular chaperone complexes, melanosomes, lysosomes, clathrin, etc. suggesting that the mechanism of drug action may be related to autophagy， Combining the results of BP, CC, and MF, it can be concluded that the drug molecule directly or indirectly exerts the biological behavior of anti-osteoarthritis by binding to different targets. (GSH), PGH2 is formed into prostaglandin E2 (PGE2) [7,8]. A part of PGH2 diffuses directly into the cytoplasm through the endoplasmic reticulum membrane and is heterogeneous to form PGE2 by PTGES2 and PTGES3, while a part of PGH2 in the endoplasmic reticulum is heterogeneous to form thromboxane A2 (TXA2) by TBXAS1 [9,10]，Peng [11]detected that the levels of IL-18 and PGE2 in the joint fluid of patients with osteoarthritis were significantly higher than those of normal people, Hossein [12], Jun [13]reduce the synthesis of PGE2 and TXA2 by inhibiting and down-regulating the expression of PTGS2 gene to slow down the process of osteoarthritis. Estrogen diffuses freely into the cell and binds to the estrogen receptor (ER α/β) in the nucleus to regulate the proliferation and inhibition of downstream genes in the classic mechanism of estrogen [14]. In bone tissue, the content of ER β in nuclear receptors is higher than that of ER α. ER α regulates the growth of osteoblasts, while ER β is involved in bone formation and resorption. In the absence of ligands, ER contains HSP90 and P23. The complex with chaperone-related proteins releases ERs and HSP90 under the action of ATP hydrolase [15], HSP40 recaptures ERs through ligand binding and recaptures the chaperone complex recombination. The ligand can compete to bind to the HSP40 site to escape chaperone recombination and DNA binding [15]. LIU [16] found through in vitro experiments that the binding of estradiol to ER α inhibits the activation of ERK signaling pathway, promotes chondrocyte autophagy, inhibits apoptosis, and regulates chondrocyte proliferation. The classic mechanism of Renin secretion is involved in the regulation of blood pressure. With the discovery of RAAS components in bone tissue, the function of stimulating the formation of osteoclasts and inhibiting the activity of osteoblasts gradually appears [17].
The level of renin is positively correlated with the degree of osteoarthritis in the synovial tissue of osteoarthritis [18,19], involving in the formation of inflammation and the hypertrophy of chondrocytes [20]. Wang [21]found Among the five core drug molecules predicted in this study, the current research is as follows: quercetin [27,28] and kaempferol [29] can inhibit the increase of Matrix Metalloproteinases (MMPs) and PGE2 in joints induced by interleukin, HSP90 can interfere with vascular endothelial growth factor Flk-1 receptor and affect the angiogenesis of bone growth plate, quercetin [30] prevent the up-regulation of HSP70, but does not reduce the level of HSP90, wogonin [31] and baicalin [31,32] inhibit the production pathway of PGE2 induced by lipopolysaccharide, reduce the synthesis of PGE2, wogonin [28,33] has been proven to reduce the synthesis of PGE2 by activating the ROS/ERK/Nrf2 pathway of chondrocytes to exert anti-inflammatory and chondroprotective functions, β-carotene [34] inhibits lipopolysaccharide from stimulating inflammatory genes in macrophages by inhibiting the activation of redox group NF-kB, for the interaction mechanism between the drug and the target predicted in this study, further experiments are needed to verify.
The complexity of the components of Traditional Chinese Medicine has always been a limitation of the scientific research of Traditional Chinese Medicine. There is still a lack of effective relevant research reports on the treatment of osteoarthritis with Traditional Chinese Medicine. This study uses data mining, network pharmacology, and molecular docking technology to predict the key mechanism of disease target interaction, Showing the overall characteristics of "Achyranthis Bidentatae Radix-Eucommiae Cortex" in the treatment of osteoarthritis. The mechanism of action and safety evaluation of Network Pharmacology have a wide range of application value, and provide a theoretical basis and direction for the subsequent screening of pharmaceutical ingredients to establish a quantitative fingerprint to improve its quality evaluation.

Conclusion
"Eucommia bark -achyranthes" drug molecules can be combined with the target to produce hydrogen bond, hydrophobic function and Pi-Pi directly or indirectly affecting the corresponding targets, to participate in the regulation of osteogenesis and osteoclast proliferation, protect the extracellular matrix, inhibition of cell apoptosis and anti-inflammatory for resistance to osteoarthritis, also, providing the basis for interpretation of its action mechanism.