Method for analyzing action mechanism of wind-dispelling five-herb decoction for treating gout based on network pharmacology and molecular docking and application

Abstract

The invention provides a method for analyzing the action mechanism of wind-dispelling five-herb decoction for treating gout based on network pharmacology and molecular docking and application, and relates to the technical field of traditional Chinese medicine information analysis, the method comprises the following steps: acquiring a common target spot of a drug target spot of the wind-dispelling five-herb decoction and a gout disease target spot; the wind-dispelling five-herb decoction comprises lysimachia christinae hance, oldenlandia diffusa, lalang grass rhizome, plantain herb Constructing a protein interaction network based on the common target; screening core targets in the protein interaction network, and identifying core active components of the Chinese herbal medicine prescription; performing pathway enrichment analysis and function enrichment analysis on the common target spot to determine a drug action mechanism; and verifying the interaction strength of the core active component and the core target spot through molecular docking. By directly calling a plurality of biological databases and integrating molecular docking verification, the analysis process is simplified, and the mechanism that the wind-dispelling five-herb decoction acts on key targets and signal pathways through core active components to treat gout is accurately disclosed.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics analysis technology in traditional Chinese medicine, and in particular to a method and application for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking. Background Technology

[0002] The statements in this section are merely background information relating to this disclosure and do not necessarily constitute prior art.

[0003] Gout is a chronic metabolic disease caused by the deposition of sodium urate crystals due to long-term hyperuricemia. Clinical manifestations include joint redness, swelling, heat, pain, and functional impairment; in severe cases, it can lead to kidney damage and cardiovascular disease. In recent years, with changes in dietary structure, the incidence of gout has continued to rise and is showing a trend towards affecting younger people. Currently, Western medicine treatment mainly uses uric acid-lowering drugs such as allopurinol, but these have side effects such as liver and kidney damage. Traditional Chinese medicine, on the other hand, has unique advantages in regulating metabolism and reducing inflammation, especially compound preparations which demonstrate significant efficacy through multi-target synergistic effects.

[0004] Network pharmacology and molecular docking technology are important tools for revealing the mechanisms of action of multiple components, targets, and pathways in traditional Chinese medicine. However, existing research methods based on network pharmacology usually require researchers to master programming skills such as R for data processing and to perform complex conversions between databases with different gene nomenclature standards. These methods are difficult to learn, cumbersome to operate, and prone to errors, thus increasing the research threshold and time costs.

[0005] Therefore, there is an urgent need to develop a simpler, more intuitive, and more accurate analytical method to reveal the specific molecular mechanism of the Five Herbs Decoction for Dispelling Wind in treating gout, so as to provide a scientific basis for its clinical application and development. Summary of the Invention

[0006] To address at least one of the technical problems mentioned above, this invention proposes a method and application for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking. This method does not require complex programming, but simplifies the analysis process by directly calling multiple biological databases and integrating molecular docking verification, and accurately reveals the mechanism by which Qufeng Wucao Decoction treats gout by acting on key targets and signaling pathways through its core active ingredients.

[0007] The first aspect of this invention provides a method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking, comprising: The common target of the drug target of Qufeng Wucao Decoction and the target of gout disease were obtained. The common target was determined by the intersection analysis of the Chinese medicine database and the disease database. Qufeng Wucao Decoction includes Lysimachia christinae, Hedyotis diffusa, Imperata cylindrica, Plantago asiatica, and corn silk. A protein-protein interaction network is constructed based on the common target, and the network is implemented using a protein-protein interaction database. Screening the core targets in the protein-protein interaction network and identifying the core active ingredients of the wind-dispelling five-herb decoction; Pathway enrichment analysis and functional enrichment analysis were performed on the common targets to determine the drug action mechanism; The interaction strength between the core active ingredient and the core target was verified by molecular docking.

[0008] Furthermore, the traditional Chinese medicine database is the TCMSP database, and the screening criteria for effective ingredients are oral bioavailability greater than or equal to a preset threshold and drug-likeness greater than or equal to a preset threshold; the disease database includes the GeneCards database and the OMIM database, and disease targets are retrieved using "Gout" as the keyword; the intersection analysis is implemented using Venn diagram tools.

[0009] Furthermore, the protein interaction database is the STRING database, with specific parameters including: species set to "Homo sapiens", interaction score greater than a preset threshold, and removal of free target sites.

[0010] Furthermore, Cytoscape 3.8.2 software and the MCODE and cytoHubba plugins were used to calculate the connectivity of common target nodes in order to screen core targets.

[0011] Furthermore, enrichment analysis was performed using the DAVID database to screen for the N pathways most closely related to gout, and KEGG enrichment bubble charts and GO functional enrichment three-model charts were generated using the MicroBioinformatics platform.

[0012] Furthermore, the protein crystal structure of the core target is obtained from the PDB database, with priority given to conformations with a resolution less than or equal to a preset threshold and containing complete active sites; the 3D structure of the core active ingredient is obtained from the PubChem database.

[0013] Furthermore, the molecular docking tools include AutoDockTools and AutoDockVina, and the docking algorithm adopts the Lamarkian genetic algorithm.

[0014] Furthermore, the core active ingredients of the wind-dispelling five-herb decoction include quercetin, luteolin, and kaempferol, which regulate the core targets TP53, IL6, and TNF by inhibiting the IL-17 / TNF / NF-kappa B signaling pathway.

[0015] Furthermore, the quercetin is derived from Lysimachia christinae and Hedyotis diffusa; the luteolin is derived from Plantago asiatica and corn silk; and the kaempferol is derived from Lysimachia christinae.

[0016] A second aspect of the present invention provides an application of the method described in the first aspect of the present invention in analyzing the mechanism of action of the Five Herbs Decoction for Dispelling Wind in the treatment of gout.

[0017] Compared with existing technologies, the present invention provides a method and application for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking, which has the following beneficial effects: (1) This invention provides a simplified and efficient analysis process. By directly calling professional databases such as TCMSP, GeneCards, OMIM, STRING, DAVID, PDB, and PubChem, and using tools such as DrawVennDiagram, Cytoscape (including MCODE and cytoHubba plugins), MicroBioinformatics Platform, and AutoDockTools / Vina for analysis, it avoids the complex R language programming and gene identifier conversion operations between databases in traditional network pharmacology methods, significantly reducing the technical threshold and learning cost, and improving the analysis efficiency and accuracy.

[0018] (2) This invention precisely reveals the mechanism of action of the Five-Herb Decoction for Dispelling Wind. By integrating network pharmacology prediction and molecular docking verification, it systematically elucidates for the first time that the Five-Herb Decoction for Dispelling Wind (including Lysimachia christinae, Hedyotis diffusa, Imperata cylindrica, Plantago asiatica, and Zea mays) acts on key targets (such as TP53, IL6, TNF, AKT1, JUN, IL1B, and HSP90AA1) through its core active ingredients (quercetin, luteolin, kaempferol, etc.), regulating the IL-17 signaling pathway, TNF signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, and NOD-like receptor signaling pathway. This allows it to synergistically treat gout by inhibiting uric acid production, promoting uric acid excretion, suppressing inflammatory responses, and regulating cell apoptosis and proliferation. Molecular docking results further confirm the good binding activity between the core active ingredients and the core targets.

[0019] (3) This invention provides a new approach to the modernization of research on traditional Chinese medicine compound prescriptions. The method provided by this invention combines bioinformatics technology with traditional Chinese medicine pharmacological analysis, providing a research paradigm that can be promoted for elucidating the mechanism of action of traditional Chinese medicine compound prescriptions in treating complex diseases, scientifically explaining the molecular mechanism of traditional Chinese medicine compound prescriptions, and assisting in clinical application and new drug development. Attached Figure Description

[0020] The accompanying drawings, which form part of this disclosure, are used to provide a further understanding of this disclosure. The illustrative embodiments of this disclosure and their descriptions are used to explain this disclosure and do not constitute an undue limitation of this disclosure.

[0021] Figure 1This is a flowchart of the method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking, provided by the present invention. Figure 2 This is a progressive dependency relationship in the technology chain provided by the present invention; Figure 3 This invention provides a Venn diagram of potential targets for the Five Herbs Decoction drug to intervene in gout. Figure 4 This is a network diagram of the common target gene PPI of the Five Herbs Decoction for gout provided by the present invention; Figure 5 This is a concentric circle relationship diagram of the Five Herbs Decoction and the core target of gout provided by the present invention; Figure 6 This is a diagram showing the relationship between the top ten Degree values ​​and core targets for gout intervention provided by this invention. Figure 7 This is a diagram showing the relationship between the Five Herbs Decoction, its active ingredients, and the drug targets of gout genes provided by this invention. Figure 8 This is a bubble chart of KEGG pathway enrichment analysis provided by the present invention; Figure 9 This is a diagram of the three-model GO functional enrichment analysis provided by the present invention; Figure 10 This is a schematic diagram of the TP53-quercetin molecular docking results provided by the present invention; Figure 11 This is a schematic diagram of the IL6-quercetin molecular docking results provided by the present invention; Figure 12 This is a schematic diagram of the TNF-quercetin molecular docking results provided by the present invention; Figure 13 This is a schematic diagram of the TP53-luteolin molecular docking results provided by the present invention; Figure 14 This is a schematic diagram of the IL6-luteolin molecular docking results provided by the present invention; Figure 15 This is a schematic diagram of the TNF-luteolin molecular docking results provided by the present invention; Figure 16 This is a schematic diagram of the TP53-kaempferol molecular docking results provided by the present invention; Figure 17 This is a schematic diagram of the IL6-kaempferol molecular docking results provided by the present invention; Figure 18 This is a schematic diagram of the TNF-kaempferol molecular docking results provided by the present invention. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. Furthermore, it should be understood that the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0024] Where there is no conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0025] All data acquisition in this embodiment is carried out in accordance with laws and regulations and with user consent, and the data is used legally.

[0026] Example 1 Please refer to the instruction manual appendix. Figure 1 This invention provides a method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking, comprising: obtaining the common target of the drug target of Qufeng Wucao Decoction and the target of gout disease, wherein the common target is determined by the intersection analysis of a traditional Chinese medicine database and a disease database; Qufeng Wucao Decoction includes Lysimachia christinae, Hedyotis diffusa, Imperata cylindrica, Plantago asiatica, and corn silk; A protein-protein interaction network is constructed based on the common target, and the network is implemented using a protein-protein interaction database. Screening the core targets in the protein-protein interaction network and identifying the core active ingredients of the wind-dispelling five-herb decoction; Pathway enrichment analysis and functional enrichment analysis were performed on the common targets to determine the drug action mechanism; The interaction strength between the core active ingredient and the core target was verified by molecular docking.

[0027] Specifically, the traditional Chinese medicine database is the TCMSP database, and the screening criteria for effective ingredients are oral availability (OB) greater than or equal to a preset threshold (in one specific embodiment, OB ≥ 30%) and drug-likeness (DL) greater than or equal to a preset threshold (in one specific embodiment, DL ≥ 0.18%). The disease database includes the GeneCards database and the OMIM database, and disease targets are retrieved using "Gout" as the keyword. The intersection analysis is performed using Venn diagram tools.

[0028] The TCMSP database is a dedicated database for the systems pharmacology of traditional Chinese medicine. Thresholds of OB ≥ 30% and DL ≥ 0.18% are used to screen out ineffective components (such as high-molecular-weight polysaccharides), ensuring that active ingredients can be absorbed by the body and act on their targets. GeneCards & OMIM, through complementary validation using two disease databases (GeneCards integrates multi-source targets, while OMIM focuses on genetically related targets), avoid missing key target genes (such as URAT1 / SLC2A9) from a single database. The Venn diagram tool visually presents the intersection of drug and disease targets, pinpointing the specific target group of Qufeng Wucao Tang (a traditional Chinese medicine formula).

[0029] Specifically, the protein interaction database is the STRING database, and the specific parameters include: the species is set to "Homo sapiens", the interaction score is greater than a preset threshold (in a specific embodiment, the interaction score > 0.9), and free target sites are removed.

[0030] The STRING database is an authoritative protein interaction database. It filters out low-reliability interactions (such as predicted interactions that have not been validated in the literature) based on a confidence level >0.9. It removes free targets, eliminating isolated targets without interaction relationships (such as certain zymogens), and focuses on core functional modules.

[0031] Specifically, Cytoscape 3.8.2 software and the MCODE and cytoHubba plugins were used to calculate the connectivity of common target nodes in order to screen core targets.

[0032] CytoHubba identifies pivotal targets by sorting them by degree value (e.g., IL6 with a degree of 32 indicates it is located at the center of the signaling network). MCODE clusters closely interacting target groups (e.g., inflammatory clusters formed by IL1B / TNF / JUN). It avoids subjective selection bias by quantifying target importance based on topology.

[0033] Specifically, enrichment analysis was performed using the DAVID database to screen the N pathways with the highest correlation to gout (in one specific embodiment, N is 10), and KEGG enrichment bubble charts and GO functional enrichment three-model charts were generated using the MicroBioinformatics platform.

[0034] The DAVID database integrates over 200 biological databases, including KEGG / GO, to address the fragmentation problem of pathway annotation. The microbioinformatics platform uses bubble charts for intuitive visualization of key pathways.

[0035] Specifically, the protein crystal structure of the core target is obtained from the PDB database, with priority given to conformations with a resolution less than or equal to a preset threshold and containing complete active sites; the 3D structure of the core active ingredient is obtained from the PubChem database.

[0036] PDB database: Resolution ≤ 2Å ensures accurate protein activity pocket structure (e.g., TNF crystal 2AZ5 resolution 1.8Å). PubChem: Provides 3D conformations of small molecules (e.g., protonated conformation of quercetin CID5280343), avoiding errors from manual modeling.

[0037] Specifically, the molecular docking tools include AutoDockTools and AutoDockVina, and the docking algorithm uses the Lamarkian genetic algorithm.

[0038] Specifically, the core active ingredients of the wind-dispelling five-herb decoction include quercetin, luteolin, and kaempferol, which regulate the core targets TP53, IL6, and TNF by inhibiting the IL-17 / TNF / NF-kappa B signaling pathway.

[0039] Specifically, the quercetin is derived from Lysimachia christinae and Hedyotis diffusa; the luteolin is derived from Plantago asiatica and corn silk; and the kaempferol is derived from Lysimachia christinae.

[0040] like Figure 2 As shown, this method, through a direct database pipeline (non-programmed interaction), achieves for the first time a complete chain mechanism analysis of compound components, gout targets, inflammatory pathways, and three-dimensional binding. Individually separating any step can trigger cascading errors. For example, missing intersection targets can lead to the inclusion of non-gout-related targets (such as the hypertension target ACE) in the PPI network, resulting in deviation from the core mechanism; skipping core target screening will prevent focusing on the top 10% of pivotal targets (such as TP53), leading to generalization of molecular docking targets; if molecular docking is omitted, pathway prediction remains at the hypothesis level, lacking empirical evidence of binding energy (such as quercetin-TNF = -7.9 kcal / mol), etc. Only through the complete analysis pipeline provided by this invention can a multi-layered network relationship diagram of "multi-component, multi-target, and multi-pathway" be constructed (e.g., Figure 5 , Figure 7As shown in the diagram, this invention visually reveals how multiple Chinese medicinal herbs and their components work synergistically to inhibit uric acid production, promote uric acid excretion, and reduce inflammation by acting on different targets and pathways. Conventional pharmacological experimental methods are time-consuming and labor-intensive, making it difficult to efficiently and comprehensively describe the action spectrum provided by this invention.

[0041] In one specific embodiment, the following steps are performed: S1: Acquisition of target genes for each drug in the Five Herbs Decoction formula The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https: / / tcmspw.com / tcmsp.php) was used to obtain the effective drug targets. TCMSP, a key database in the field of TCM research, covers a large amount of chemical components and target information of TCM, providing a rich data source for exploring the potential active ingredients and targets of the Wucao Tang formula. It is important to note that drug targets need to be screened for major active ingredients in the TCMSP database: OB (oral bioavailability) was set to ≥30%, and DL (drug-likeness) was set to ≥0.18%. After screening, all TCM components in the database were sequentially retrieved to obtain all their targets. Subsequently, all targets were integrated into the human protein index from the globally renowned protein database website (uniprot, https: / / www.uniprot.org). During this process, the VLOOKUP function was applied for data efficiency indexing to accurately retrieve the genes corresponding to the targets, and then each retrieved gene was processed.

[0042] S2: Acquisition of target genes for gout First, you need to know the specific English name of the disease: gout. Then, use this as a keyword to search for disease genes in two databases: GeneCard (https: / / www.genecards.org), a comprehensive human genome database, and OMIM (https: / / omim.org), an online database of human genes and genetic diseases. This will allow you to obtain comprehensive target genes for gout.

[0043] S3: Acquisition of common target genes for gout-related diseases using Wucaotang (Five-Herb Decoction) medication. Using the gout disease and Wucaotang drug targets obtained by the methods described in S1 and S2, an intersection analysis was performed on the set of active drug target targets of Wucaotang and the set of gout disease target targets to identify common targets, i.e., potential targets of Wucaotang for treating gout. A Venn diagram was then drawn using the data intersection system DrawVennDiagram (https: / / bioinfogp.cnb.csic.es / tools / venny) to obtain a Venn diagram of potential targets of Wucaotang for intervening in gout. Figure 3), and obtain the intersection genes for subsequent protein-protein inter-peptide (PPI) network construction.

[0044] S4: Construction of a common protein-protein interaction (PPI) network for drug-disease interactions The common target genes of the Five Herbs Decoction and gout were imported into the STRING (https: / / cn.string-db.org) database to build a PPI network. The protein classification was set to "Homo sapiens", the interaction score was >0.9 (highest confidence), and other parameters of the PPI network were set to default. Free target sites were removed, and the PPI network diagram was constructed. Figure 4 Simultaneously, a PPI network relationship table was obtained. Through network visualization analysis, the multi-component, multi-target synergistic effect mode of the Wucao Tang formula in treating gout was preliminarily revealed.

[0045] S5: Acquisition of core target genes The obtained PPI network relationship table was imported into the graphing and analysis software Cytoscape 3.8.2 (https: / / cytoscape.org) to create a PPI network relationship diagram of the Wucaotang drug against potential targets for intervention in gout. The intersection gene network of "Wucaotang-target-gout" was constructed using Cytoscape software analysis, and a concentric circle relationship diagram of the core target was drawn based on the Degree value. Figure 5 This visually presents the complex relationships between targets. Simultaneously, MCODE is opened, and the top ten Degree values ​​of the gout targets in the Five Herbs Decoction are calculated using the CytoHubba plugin. Furthermore, a core target relationship diagram is created using the MCODE plugin. Figure 6 ), and core targets were selected.

[0046] S6: Acquisition of core active ingredients First, the target points corresponding to the components of the five herbs in the Five Herbs Decoction formula were compiled and made into an Excel spreadsheet. Then, the spreadsheet was imported into Cytoscape software. A "Five Herbs Decoction - Active Ingredients - Target Points" network was constructed using Cytoscape software to create a drug component-target relationship diagram. Figure 7 The PPI network above visually presents the complex relationship between the three. The drug (such as Lysimachia christinae, Hedyotis diffusa, etc.) node, the active ingredient node (such as quercetin, luteolin, etc.), and the target node (such as TP53, IL6, etc.) are connected to each other by edge lines. The thickness of the edge lines is related to the connection strength, reflecting the close relationship between the ingredient and the target, and between the target and the disease.

[0047] S7: KEGG pathway enrichment analysis and GO functional enrichment analysis to explore the specific mechanisms of drug action. Import the intersection targets of the above-obtained Wucao Decoction and gout disease into the DAVID database (https: / / david.ncifcrf.gov / ), query its Gene Ontology (GO) functional enrichment analysis pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis pathway, select ten pathways with the highest relevance to gout from them, and import the pathways into the KEGG enrichment dot bubble module of the bioinformatics platform (https: / / www.bioinformatics.com.cn). Select the color as the p-value -log10 transformation, and set the length and width range to 6 - 8 to generate the KEGG enrichment dot bubble diagram ( Figure 8 ), and at the same time import the common target genes of the Wucao Decoction - gout disease into the GO-KEGG enrichment analysis module (gokeggpathwayenrichment) of the bioinformatics platform to generate a three-model diagram of GO functional enrichment analysis ( Figure 9 ), providing clues for further exploration of the action sites.

[0048] S8: Molecular docking for further exploration of drug mechanisms Among the above-screened core targets and core active ingredients, select the top five for subsequent molecular docking to explore their drug action mechanisms. The specific steps are as follows: First, protein receptor structure preprocessing is required. Obtain the crystal structures of macromolecular target proteins from the PDB (https: / / www.rcsb.org) database (PDBIDs are TP53: 2OCJ, TNF: 2AZ5, IL6: 1ALU, AKT1: 4EKL, JUN: 1JUN). Conformations with a resolution ≤ 2.0 Å and containing complete active sites can be preferentially selected, and then use the obgui software to convert and save them in the pdb format.

[0049] Use PyMOL to remove water molecules, cofactors, and irrelevant ligands, retain key metal ions (such as the Mg ion of AKT1), then use the spdbv reconstruction software to supplement missing residues, followed by hydrogen addition and charge assignment. Add polar hydrogen atoms in AutoDockTools, calculate the Kollman charge, and save it in the pdbqt format.

[0050] Subsequently, small molecule ligands were prepared. The 3D structures of the small molecule compounds (CID numbers quercetin 5280343, luteolin 5280445, and kaempferol 5280863) were downloaded from PubChem (https: / / pubchem.ncbi.nlm.nih.gov) and then converted and saved as pdb format using obgui software.

[0051] Protonation (pH 7.4) was performed using OpenBabelGUI software to generate a low-energy conformation. Gasteiger charges were assigned using AutoDockTools software, rotatable bonds were set, and the output was in pdbqt format.

[0052] Next, docking parameters were set, and a grid box was constructed centered on known active or allosteric sites (such as the DNA-binding domain of TP53 or the receptor-binding groove of TNF). The size of the grid box was ensured to cover the entire binding pocket. For the algorithm parameters, the Lamarkian genetic algorithm (LGA) was used, running 50 independent docking calculations. The population size was 150, and the maximum number of evaluations was 2.5 × 10⁻⁶. 6 The crossover rate was 0.8 and the mutation rate was 0.02.

[0053] Finally, molecular docking was performed using the AutoDockVina engine. Rigid docking (receptor fixed, ligand flexible) was executed, and each ligand-target combination was run three times independently. A molecular docking scoring table (Table 1) and docking results graph were generated. Figures 10 to 18 After molecular docking, the docking results need to be analyzed and verified. Cross-validation is used, and the output conformations are clustered according to the RMSD≤2.0Å standard. The representative conformation of the largest cluster is selected, as shown in the figure below. Figures 10-18 As shown. The binding energy was subsequently assessed, and the lowest binding energy and hydrogen-bonded hydrophobic interactions were recorded. The accuracy of the docking procedure was verified using a control positive ligand (RMSD < 1.5 Å). False positives were eliminated using a consistency scoring function (≥3 parameters required).

[0054] Table 1. Scoring of molecular docking between core target genes and core active ingredients.

[0055] In summary, the present invention provides a method for analyzing the mechanism of action of Chinese herbal medicine prescriptions in treating gout based on network pharmacology and molecular docking. The analysis found that the five-herb decoction plays an important role in treating gout by affecting seven core targets, namely TP53, IL6, TNF, AKT1, JUN, IL1B, and HSP90AA1. GO functional enrichment analysis suggests that the potential targets of the five-herb decoction-gout are mainly involved in a series of biological processes such as cell proliferation and apoptosis; KEGG pathway enrichment analysis suggests that the mechanism of action of the five-herb decoction on gout is closely related to signal pathways such as the IL-17 signaling pathway, TNF signaling pathway, NF-kappaB signaling pathway, Toll-like receptor signaling pathway, and NOD-like receptor signaling pathway. In summary, the results of the network pharmacology analysis and molecular docking research in the present invention provide an important basis for the further research on the treatment of gout diseases with Chinese herbal medicine prescriptions.

[0056] Example 2 Example 2 provides an application of the method as in Example 1 in analyzing the mechanism of action of the Qufeng Wucao Decoction in treating gout.

[0057] The Qufeng Wucao Decoction is composed of 30 g of Lysimachia christinae, 20 g of Hedyotis diffusa, 30 g of Imperata cylindrica, 15 g of Plantago asiatica, and 30 g of Cornus officinalis. The core active ingredient quercetin of this prescription mainly comes from the monarch drug Lysimachia christinae and the ministerial drug Hedyotis diffusa; luteolin mainly comes from the assistant drug Plantago asiatica and the monarch drug Cornus officinalis; kaempferol mainly comes from the monarch drug Lysimachia christinae. Through the application of this method, it has been successfully revealed that the Qufeng Wucao Decoction plays a role in reducing uric acid, anti-inflammation, regulating immunity and cell function, etc. through the coordination of multiple components (quercetin, luteolin, kaempferol, etc.), multiple targets (TP53, IL6, TNF, AKT1, etc.), and multiple pathways (IL-17, TNF, NF-kappa B and other signaling pathways), thereby treating the overall molecular mechanism of gout.

[0058] In a specific embodiment, the prescription ratio information of the Qufeng Wucao Decoction and the active ingredients of each drug related to gout are as follows: I. Qufeng Wucao Decoction: Monarch drugs - Lysimachia christinae, Cornus officinalis Ministerial drugs - Hedyotis diffusa, Imperata cylindrica Assistant drug - Plantago asiatica II. Ratio description 1. Lysimachia christinae 30 g Sweet, salty, slightly cold, attributing to the liver, gallbladder and kidney Promote uric acid excretion and inhibit the activity of xanthine oxidase 2. Hedyotis diffusa 20 g Bitter, sweet, cold, attributing to the stomach, large intestine and liver Anti-inflammatory and analgesic, reduce serum uric acid and creatinine levels 3. 30g of Imperata cylindrica root Sweet and cold in nature, it enters the lung, stomach, and bladder meridians. Alkalize urine and increase the solubility of uric acid. 4. 15g of plantain Sweet and cold in nature, it enters the liver, kidneys, lungs, and bladder. Diuretic and acid-excreting effects, regulating renal tubular uric acid transport proteins 5. 30g of corn silk Ganping, enters the bladder, liver and gallbladder meridians. Inhibits URAT1 uric acid reabsorption, lowers blood pressure and protects kidneys III. Compatibility Analysis 1. Principal herbs: Lysimachia christinae + corn silk Lysimachia christinae clears heat and promotes diuresis, dissolves stones and relieves urinary tract infections; a high dose of 30g can enhance uric acid excretion. Corn silk is mildly nourishing and diuretic, neutralizing the cold nature of the whole formula and preventing stomach upset.

[0059] Synergistically inhibits uric acid production and promotes excretion 2. Accompanying herbs: Oldenlandia diffusa + Imperata cylindrica root Hedyotis diffusa detoxifies and removes blood stasis, and is effective for joint redness, swelling, heat and pain (acute phase). Imperata cylindrica root cools the blood and stops bleeding, and prevents the formation of uric acid kidney stones.

[0060] Controlling inflammatory response and protecting kidney function 3. Adjuvant herb: Plantain It draws damp heat downwards, enhancing the diuretic effect. A dosage of 15g is recommended to avoid electrolyte imbalance.

[0061] The principal drug assists in strengthening the acid excretion pathway. IV. Dosage Adjustment and Subtraction Recommendations Adjust flexibly according to physical condition / symptoms 1. Spleen and stomach deficiency and cold Add 6g of dried ginger and 10g of stir-fried Atractylodes macrocephala. Neutralizes coldness and protects stomach qi. 2. Joints are red, swollen, and painful. Add 40g of Smilax glabra and 15g of Clematis chinensis. Enhances anti-inflammatory and analgesic effects, promotes uric acid dissolution 3. Uric acid stones Add 12g chicken gizzard and 10g sea gold sand (fried) Promote the expulsion of stones 4. Kidney deficiency and lower back pain Add 15g of Achyranthes bidentata and 10g of Eucommia ulmoides. Tonifies the kidneys and strengthens the lower back without promoting dampness and heat. 5. Obesity with phlegm and dampness Add 20g of raw hawthorn and 12g of Alisma plantago-aquatica. Regulate lipid metabolism and reduce uric acid production V. Contraindications 1. Pregnant women, patients with low blood pressure, and those with electrolyte imbalances should use with caution. 2. For those with Yang deficiency who are afraid of cold and have loose stools, warming herbs should be added to the prescription. VI. Preparation and Administration Methods and Course of Treatment 1. Decoction method: Soak the herbs in cold water for 40 minutes, then add 1500ml of water; Bring to a boil over high heat, then reduce to low heat and simmer for 30 minutes. Strain the liquid. Add 1000ml of water to the second decoction and simmer for another 20 minutes. Then combine the two decoctions.

[0062] 2. Dosage: Take one dose daily, divided into two warm doses after meals (to reduce stomach irritation). Acute phase: Take for 7–14 days, then reduce the dose by half after uric acid levels return to normal. Chronic conditioning period: Take 3–4 doses per week for 1–3 months. VII. Supporting Basis from Modern Pharmacology 1. Lysimachia christinae: Contains flavonoid glycosides → inhibits XOD enzyme activity and reduces uric acid synthesis. 2. Corn silk: Saponins → downregulate URAT1 expression in renal tubules, reducing uric acid reabsorption. 3. Imperata cylindrica root: High potassium content → alkalizes urine, increasing uric acid solubility by more than 30%. VIII. Safety Tips 1. This formula is slightly cold in nature, and electrolytes (especially blood potassium) should be monitored during long-term use.

[0063] 2. During the course of treatment, drink ≥2000ml of water daily and avoid high-purine foods such as beer and animal organs.

[0064] 3. It is recommended to check blood uric acid every 2 weeks and adjust the dosage according to the value.

[0065] 4. This formula combines the three functions of "inhibiting production, promoting excretion, and protecting the kidneys" and can be flexibly combined and applied in clinical practice.

[0066] IX. Key Active Ingredients Related to Gout 1. Moneywort Related active ingredients: quercetin, kaempferol, isorhamnetin, and acetin. 2. Hedyotis diffusa Related active ingredients: Stigmasterol, quercetin, beta-sitosterol 3. Imperata cylindrica root (also known as white thatch grass) Related active ingredients: beta-sitosterol, stigmasterol 4. Plantain Related active ingredients: luteolin, baicalin, dinatin, stigmasterol 5. Corn silk Key active ingredients: Chryseriol, luteolin, and stigmasterol. While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking, characterized in that, include: The common target of the drug target of Qufeng Wucao Decoction and the target of gout disease were obtained. The common target was determined by the intersection analysis of the Chinese medicine database and the disease database. Qufeng Wucao Decoction includes Lysimachia christinae, Hedyotis diffusa, Imperata cylindrica, Plantago asiatica, and corn silk. A protein-protein interaction network is constructed based on the common target, and the network is implemented using a protein-protein interaction database. Screening the core targets in the protein-protein interaction network and identifying the core active ingredients of the wind-dispelling five-herb decoction; Pathway enrichment analysis and functional enrichment analysis were performed on the common targets to determine the drug action mechanism; The interaction strength between the core active ingredient and the core target was verified by molecular docking.

2. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 1, characterized in that, The traditional Chinese medicine database is the TCMSP database, and the screening criteria for effective ingredients are oral bioavailability greater than or equal to a preset threshold and drug-likeness greater than or equal to a preset threshold; the disease database includes the GeneCards database and the OMIM database, and disease targets are retrieved using "Gout" as the keyword; the intersection analysis is performed using Venn diagram tools.

3. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 1, characterized in that, The protein-protein interaction database is the STRING database, and the specific parameters include: the species is set to "Homo sapiens", the interaction score is greater than a preset threshold, and free target sites are removed.

4. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 1, characterized in that, Cytoscape 3.8.2 software and the MCODE and cytoHubba plugins were used to calculate the connectivity of common target nodes in order to screen core targets.

5. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 1, characterized in that, Enrichment analysis was performed using the DAVID database to screen for the N pathways most closely associated with gout, and KEGG enrichment bubble charts and GO functional enrichment tri-model charts were generated using the MicroBioinformatics platform.

6. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 1, characterized in that, The protein crystal structure of the core target was obtained from the PDB database, with priority given to conformations with a resolution less than or equal to a preset threshold and containing complete active sites; the 3D structure of the core active ingredient was obtained from the PubChem database.

7. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 1, characterized in that, The molecular docking tools include AutoDockTools and AutoDockVina, and the docking algorithm uses the Lamarkian genetic algorithm.

8. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 1, characterized in that, The core active ingredients of the Five Herbs Decoction for Dispelling Wind include quercetin, luteolin, and kaempferol, which regulate the core targets TP53, IL6, and TNF by inhibiting the IL-17 / TNF / NF-kappa B signaling pathway.

9. The method for analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout based on network pharmacology and molecular docking as described in claim 8, characterized in that, The quercetin is derived from Lysimachia christinae and Hedyotis diffusa; the luteolin is derived from Plantago asiatica and corn silk; and the kaempferol is derived from Lysimachia christinae.

10. The application of the method as described in any one of claims 1-9 in analyzing the mechanism of action of Qufeng Wucao Decoction in treating gout.

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