Analysis method for application of wolfberry in preparation of food and medicine for rheumatoid arthritis

Abstract

This invention discloses an analytical method for the application of wolfberry in the preparation of foods and drugs for rheumatoid arthritis. First, 36 active ingredients and 201 drug targets of wolfberry were obtained through a traditional Chinese medicine systems pharmacology database and analysis platform. Then, 536 targets for rheumatoid arthritis were predicted using Gene Cards and TTD databases, and 55 intersection targets were obtained through Venn diagram analysis. An intersection target network diagram and a "drug-ingredient-target-disease" network diagram were constructed. Topological analysis screened out four core targets: IL1B, JUN, IL6, and TNF, and five core ingredients. Molecular docking results showed that quercetin, genistein, β-sitosterol, stigmasterol, and lycopene strongly bound to the four core targets. This invention provides a theoretical reference for the development of drugs for the treatment of rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to the field of network pharmacology analysis technology of traditional Chinese medicine, specifically to an analytical method for the application of wolfberry in the preparation of foods and medicines for rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease, primarily characterized by synovitis, and its incidence is increasing annually. Common clinical manifestations include chronic symmetrical joint pain and swelling. If left untreated, it can lead to joint deformities and loss of function, making it a leading cause of disability. This not only severely reduces patients' quality of life but also imposes a heavy economic burden. The occurrence of RA may be related to environmental, genetic, inflammatory, immune, and gut microbiota factors. Treatment for RA often involves nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids and glucocorticoids, antirheumatic drugs, and biologics. Some of these medications are expensive, and frequent use can cause adverse reactions such as gastrointestinal ulcers, osteoporosis, muscle atrophy, hypertension, atherosclerosis, and diabetes. Therefore, there is an urgent need to find more economical and safer traditional Chinese medicine treatments for RA.

[0003] Goji berries are dried, ripe fruits, sweet in taste and neutral in nature, entering the liver and kidney meridians. They nourish the liver and kidneys, benefit essence and improve eyesight. They are often used to nourish yin and qi, and strengthen the body's resistance. Goji berries are a food and medicine homology substance, containing a large amount of goji polysaccharides, betaine, free amino acids, vitamins, and carotene, etc., and have good pharmacological effects such as protecting the liver, lowering blood lipids, lowering blood sugar, regulating the body's immunity, and anti-oxidation. Rheumatoid arthritis can lead to the erosion and destruction of cartilage and bone tissue. The "Compendium of Materia Medica" records that "goji berries specifically enter the kidneys, and also the liver, strengthening tendons and bones, and replenishing essence and yang." However, the material basis, target, and mechanism of action of goji berries in treating rheumatoid arthritis are still unclear and require further investigation and research.

[0004] Network pharmacology is a comprehensive discipline that combines pharmacology, bioinformatics, and computer network technology. It can predict the components of traditional Chinese medicine and their targets, create biological maps at the molecular level, and assist in analyzing the process of drug treatment of diseases in a component-target-pathway manner. Molecular docking is a computer-based analysis technique that simulates the binding process between small molecules and proteins, predicting the binding modes and affinity of drugs with proteins, nucleic acids, etc. Summary of the Invention

[0005] The purpose of this invention is to provide an analytical method for the application of wolfberry in the preparation of foods and medicines for rheumatoid arthritis. This method systematically elucidates the mechanism of wolfberry's synergistic treatment of rheumatoid arthritis through "multi-component-multi-target-multi-pathway" mechanisms, providing a theoretical reference for the development of drugs for the treatment of rheumatoid arthritis.

[0006] An analytical method for the use of wolfberry in the preparation of foods and medicines for rheumatoid arthritis includes the following steps:

[0007] (1) Obtain the target sites of the components of wolfberry and convert the obtained target site names into standard gene names; (2) To identify the target of action for rheumatoid arthritis; (3) Take the intersection of the target points obtained in step (1) and the target points obtained in step (2) to obtain the potential target points of wolfberry for treating rheumatoid arthritis; (4) Import the intersection target of wolfberry and rheumatoid arthritis into the STRING database, create a protein-protein interaction network diagram, export the analysis results in TSV format, perform network topology analysis using Cytoscape software, and screen core targets. (5) Construct a visual network diagram of "Traditional Chinese Medicine-Components-Targets-Diseases" using Cytoscape software; (6) The intersection targets of wolfberry and rheumatoid arthritis were analyzed by GO (functional) enrichment and KEGG (pathway) enrichment using the microbioinformatics platform and bubble charts were generated. (7) Perform molecular docking of “core component-core target”.

[0008] As a preferred embodiment, step (1) includes using the TCM Systems Pharmacology Database and Analysis Platform TCMSP database to search and screen the active ingredients of wolfberry and the corresponding targets of the ingredients, and using the Uniprot database to convert the "Target name" of the active ingredients into a standard gene name.

[0009] As a preferred embodiment, step (2) includes searching for relevant targets of rheumatoid arthritis using the Gene Cards and TTD online databases with "rheumatoid arthritis" as the keyword.

[0010] As a preferred embodiment, step (3) includes drawing a Venn diagram of drug targets and rheumatoid arthritis disease targets through a microbioinformatics platform, and taking the intersection of the two targets as the potential target for the treatment of rheumatoid arthritis by wolfberry.

[0011] As a preferred implementation, step (4) includes importing the intersection target points into the STRING database, selecting "Homo sapiens" as the Organizations, constructing a protein-protein interaction network diagram, saving the obtained analysis results in TSV format, importing them into Cytoscape software for network topology analysis, and filtering core target points by degree value.

[0012] As a preferred embodiment, step (7) includes energy minimization treatment of the obtained effective active ingredient structure, and the obtained protein structure needs to be dehydrated, deligated, hydrogenated, and then molecularly docked.

[0013] Compared with the prior art, the present invention has the following advantages: This invention is the first to employ network pharmacology and molecular docking technology to analyze a method for treating rheumatoid arthritis using wolfberry (Lycium barbarum). It reveals for the first time the core active ingredients, core targets, and mechanisms of action of wolfberry in treating rheumatoid arthritis. Molecular docking technology verifies that the core active ingredients of wolfberry have good binding activity with the core targets. This suggests that wolfberry may treat rheumatoid arthritis through multiple components, multiple targets, and multiple pathways. The obtained active ingredients can be directly used to develop drugs, health products, or functional foods for treating rheumatoid arthritis, providing a theoretical reference for the treatment of rheumatoid arthritis. Attached Figure Description

[0014] Figure 1 Venn diagram of the target of the active ingredient of wolfberry in this invention - rheumatoid arthritis; Figure 2 This is a PPI network diagram illustrating the protein-protein interactions of the active ingredients of wolfberry in the treatment of rheumatoid arthritis according to the present invention. Figure 3 This diagram illustrates the target points of the active ingredients from wolfberry in the treatment of rheumatoid arthritis according to the present invention. Figure 4 This is the network diagram of "Traditional Chinese Medicine-Components-Targets-Diseases" of this invention; Figure 5 This is a bar chart showing the results of the GO functional enrichment analysis (BP-CC-MF) of this invention; Figure 6 Bubble diagram for KEGG signal pathway enrichment analysis in this invention; Figure 7 The heatmap shows the docking and binding energy between the active ingredients of wolfberry and the core target molecules. Detailed Implementation

[0015] To better understand the technical solutions of the embodiments of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0016] It should be understood that the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0017] Example This embodiment provides an analytical method for the application of wolfberry in the preparation of foods and medicines for rheumatoid arthritis, such as... Figure 1-7 As shown, it includes the following steps: 1. Screening of active ingredients and collection of targets from wolfberry: Using the TCM Systems Pharmacology Database and Analysis Platform (TCMSP) (http: / / tcmspw.com / tcmsp.php), active ingredients were retrieved using "goji berries" as the keyword. The selection criteria included oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18. This yielded 36 ingredients and their corresponding target names that met the criteria.

[0018] 2. Convert target names to standard gene names: After combining the target names corresponding to the 36 components and removing duplicates, the names were converted into standard gene names using the UniProt database (https: / / www.uniprot.org / ). The "Reviewed" option was selected, and "Human" was chosen for "Popular organisms".

[0019] 3. Collection of targets related to rheumatoid arthritis (RA): Using "rheumatoidarthritis" as the keyword, we searched for relevant targets of rheumatoid arthritis in the online databases Gene Cards (https: / / www.genecards.org) and TTD (https: / / ttd.idrblab.cn / ). The screening criteria in Gene Cards was "Score > 10". After combining the targets collected from the two databases and removing duplicates, we obtained 536 targets of rheumatoid arthritis.

[0020] 4. Draw a Venn diagram of active ingredients from wolfberry and their targets in rheumatoid arthritis: The target of wolfberry and the target of rheumatoid arthritis were compared using "VennDiagrams - free online drawing tool (jvenn)" (https: / / www.bioinformatics.com.cn / static / others / jvenn / example.html). By constructing a Venn diagram, the intersection points obtained from the two methods represent the potential targets for wolfberry in treating rheumatoid arthritis. A total of 55 intersection points were identified. (See...) Figure 1 .

[0021] 5. Construction of a protein-protein interaction network diagram for the treatment of rheumatoid arthritis by active ingredients of wolfberry: Fifty-five intersecting target sites were imported into the STRING database (https: / / cn.string-db.org / ) and analyzed using the "Multiple Proteins" setting. The species "Organisms" were selected as "Homo sapiens," resulting in a PPI network diagram. The settings were configured to "minimum required interaction score = 0.900," and disconnected nodes in the network were hidden. All other parameters were left at their default values. This yielded the PPI network diagram of target protein interactions. (See...) Figure 2 Export the analysis results as TSV format.

[0022] 6. Construction of a concentric circle network diagram of the intersection targets of active ingredients from wolfberry and rheumatoid arthritis: Import the TSV format results exported from the STRING (https: / / cn.string-db.org / ) database into Cytoscape for network topology analysis. (See...) Figure 3 In the diagram, node size and color represent the degree value of the target; larger nodes with darker colors indicate a higher degree value. Targets are sorted according to their degree values, with higher values ​​likely indicating core targets. Using the Centiscape 2.2 plugin in Cytoscape, the top ten targets by degree centrality (the most direct measure of node centrality in network analysis; a higher degree indicates greater node centrality and importance in the network) were selected: Tumor Necrosis Factor (TNF), JUN AP-1 transcription factor subunit (JUN), Interleukin-6 (IL6), Interleukin-1β (IL1B), Cell Tumor Antigen p53 (TP53), Interleukin-8 (CXCL8), Serine / Threonine Protein Kinase 1 (AKT1), Chemokinin Ligand 2 (CCL2), Interferon-γ (IFNG), and Transcription Factor p65 (RELA), as shown in Table 1.

[0023] Table 1. Core targets of wolfberry in the treatment of rheumatoid arthritis (top ten targets)

[0024] 7. Constructing a network diagram of traditional Chinese medicine, its components, targets, and diseases: A Network file was created using wolfberry, its active ingredients, the target of wolfberry in treating rheumatoid arthritis, and rheumatoid arthritis (RA). A Type file was also created using wolfberry, its target of rheumatoid arthritis, its active ingredients, wolfberry, and rheumatoid arthritis (RA). Both the Network and Type files were imported into Cytoscape software for topology analysis, and a network diagram of traditional Chinese medicine, its components, its target, and the disease was drawn. Figure 4 The network involves one traditional Chinese medicine, 13 active ingredients, 55 targets, one disease, and 161 edges, indicating that wolfberry (goji berry) treats rheumatoid arthritis through the synergistic interaction of multiple components and targets. Analysis revealed that the top five active ingredients of wolfberry are quercetin, genistein, β-sitosterol, stigmasterol, and lycopene; these compounds are likely the main active ingredients in wolfberry for treating rheumatoid arthritis.

[0025] Table 2. Effective active ingredients of wolfberry

[0026] 8. Enrichment analysis of GO and KEGG: Data on drug-disease intersection targets were transformed and organized using the MicroBioInformatics - Online Bioinformatics Analysis and Visualization Cloud Platform (https: / / www.bioinformatics.com.cn / ), and GO gene function and KEGG pathway enrichment analysis were performed. The analysis yielded 47 GO function enrichment entries and 20 KEGG pathway enrichment entries.

[0027] GO functional enrichment involved 20 biological processes (BP), among which the top ten were response to lipopolysaccharide, inflammatory response, positive regulation of cell migration, leukocyte differentiation, regulation of cell-cell adhesion, negative regulation of cell population proliferation, response to tumor necrosis factor, regulation of apoptotic signaling pathway, response to growth factor, and response to decreased oxygen levels. There are nine cellular components (CCs), mainly enriched in the receptor complex, secretory granule lumen, endoplasmic reticulum lumen, extracellular matrix, external side of plasma membrane, transcription regulator complex, membrane raft, nuclear matrix, and lamellipodium.There are 18 molecular functions (MFs), with the top ten being cytokine receptor binding, transcription factor binding, protein homodimerization activity, ubiquitin-like protein ligase binding, serine-type endopeptidase activity, sulfur compound binding, kinase regulator activity, protein kinase activity, integrin binding, and phosphatase binding. For the GO functional analysis, the top ten items in each group were used to create a bar chart to visualize the results, see [link to GO functional analysis]. Figure 5 .

[0028] KEGG pathway enrichment analysis yielded 20 pathways, see [link / reference] Figure 6The bubble chart indicates that the mechanism by which wolfberry treats rheumatoid arthritis mainly involves lipid and atherosclerosis, cancer pathways, fluid shear stress and atherosclerosis, leishmaniasis, rheumatoid arthritis, endocrine resistance, transcriptional misregulation in cancer, the HIF-1 signaling pathway, bladder cancer, effecoxysis, diabetic cardiomyopathy, viral carcinogenesis, allograft rejection, integrin signaling, oxytocin signaling, p53 signaling, and serotonergic synapses. Synapse, natural killer cell-mediated cytotoxicity, cell adhesion molecule (CAM) interaction, and chemical carcinogenesis-DNA adducts.

[0029] 9. Molecular docking between "core components" and "core targets": Using the top four core targets (IL1B, JUN, IL6, and TNF) with the highest Degree values ​​in the core target network analysis diagram as receptors, and their corresponding components (quercetin, genistein, β-sitosterol, stigmasterol, and lycopene) as ligands, molecular docking was performed. Generally, a binding energy less than -5.0 kcal / mol indicates good and strong binding activity between the ligand and receptor. The docking results heatmap shows that the binding free energy of wolfberry with each protein is less than -5 kcal / mol, indicating that wolfberry has strong binding activity with the predicted targets. Figure 7As shown, the value in each small square represents the binding energy between the active ingredient and the core target molecule. The lower the binding energy, the closer it is to blue; the higher the binding energy, the closer it is to red. Specifically, quercetin has a binding energy of -9.1 kcal / mol with JUN and -8.8 kcal / mol with TNF; glycitein has a binding energy of -8.2 kcal / mol with IL1B; and stigmasterol has a binding energy of -8.4 kcal / mol with TNF, -8.2 kcal / mol with JUN, and -8.3 kcal / mol with IL1B.

[0030] This invention focuses on utilizing network pharmacology and molecular docking technology to analyze the mechanism of action of Lycium barbarum in treating rheumatoid arthritis (RA), predicting and identifying relevant target genes and mechanisms of action. Based on a network pharmacology platform, 13 active components in Lycium barbarum for treating RA were screened. Among them, quercetin, genistein, β-sitosterol, stigmasterol, and lycopene are potential core active components for treating RA. Molecular docking results indicate that these components may be key components in the treatment of RA by Lycium barbarum. GO functional enrichment analysis shows that the active components of Lycium barbarum—potential targets for RA—are mainly involved in a series of biological processes such as lipopolysaccharide response, inflammatory response, positive regulation of cell migration, and leukocyte differentiation. KEGG pathway enrichment analysis shows that the mechanism of Lycium barbarum in treating RA is closely related to lipids and atherosclerosis, cancer pathways, hypoxia-inducible factor-1 signaling pathway, p53 signaling pathway, and leishmaniasis. In summary, wolfberry can treat rheumatoid arthritis through the interaction of multiple components, multiple targets, and multiple pathways, which can provide new ideas for the treatment of rheumatoid arthritis.

[0031] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. An analytical method for the application of wolfberry in the preparation of foods and medicines for rheumatoid arthritis, characterized in that, Includes the following steps: (1) Obtain the target sites of the components of wolfberry and convert the obtained target site names into standard gene names; (2) To identify the target of action for rheumatoid arthritis; (3) Take the intersection of the target points obtained in step (1) and the target points obtained in step (2) to obtain the potential target points of wolfberry for treating rheumatoid arthritis; (4) Import the intersection target of wolfberry and rheumatoid arthritis into the STRING database, create a protein-protein interaction network diagram, export the analysis results in TSV format, perform network topology analysis using Cytoscape software, and screen core targets. (5) Construct a visual network diagram of "Traditional Chinese Medicine-Components-Targets-Diseases" using Cytoscape software; (6) The intersection targets of wolfberry and rheumatoid arthritis were analyzed by GO (functional) enrichment and KEGG (pathway) enrichment using the microbioinformatics platform and bubble charts were generated. (7) Perform molecular docking of "core component-core target".

2. The method according to claim 1, characterized in that, Step (1) includes using the TCM Systems Pharmacology Database and Analysis Platform TCMSP database to search and screen the active ingredients of wolfberry and their corresponding targets, and using the Uniprot database to convert the "Target name" of the active ingredients into a standard gene name.

3. The method according to claim 1, characterized in that, Step (2) involves searching for relevant targets for rheumatoid arthritis using the keywords "rheumatoid arthritis" through the Gene Cards and TTD online databases.

4. The method according to claim 1, characterized in that, Step (3) involves drawing a Venn diagram of drug targets and rheumatoid arthritis disease targets using the MicroBioinformatics platform, and taking the intersection of the two targets as the potential target for the treatment of rheumatoid arthritis by wolfberry.

5. The method according to claim 1, characterized in that, Step (4) includes importing the intersection targets into the STRING database, selecting "Homo sapiens" as the Organizations, constructing a protein-protein interaction network diagram, saving the obtained analysis results in TSV format, importing them into Cytoscape software for network topology analysis, and filtering core targets by degree value.

6. The method according to claim 1, characterized in that, Step (7) involves minimizing the energy of the obtained effective active ingredient structure. The obtained protein structure needs to be dehydrated, deligated, and hydrogenated before molecular docking.

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