Mechanism of ShuiJingDan in Treating Acute Gouty Arthritis Flares Based on Network Pharmacology and Molecular Docking

Introduction

Acute gouty arthritis (GA) flares are mainly caused by the reaction of macrophages and neutrophils with monosodium urate (MSU),¹ and are mainly characterized by redness, swelling, heat, and severe pain in the joints. GA is the most common form of arthritis around the world,² and a large number of people suffer from gout-related pain every day. Colchicine, non-steroidal anti-inflammatory drugs (NSAIDs), or glucocorticoids are currently used to treat acute flares of GA,³ but these drugs often cause adverse reactions such as gastrointestinal bleeding, immunosuppression, and liver injury. There is thus an urgent need to find an efficient, safe, and convenient treatment method. ShuiJingDan (SJD) is an internal formulation of the Department of Chinese Medicine of Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital. It consists of Rheum palmatum L., Carthamus tinctorius L., Natrii sulfas, Alum, which has been clinically used for nearly 60 years, but the use of three of them (Rheum palmatum L., Carthamus tinctorius L., Natrii sulfas) has been shown to be more effective in the treatment of gouty arthritis after a lot of clinical practice, and so this study focuses on exploring the anti-inflammatory mechanisms of these three herbs in gouty arthritis. According to previous clinical observations, SJD has a good effect on acute inflammatory diseases, such as acute GA flares, cellulitis,⁴ acute hematoma,⁵ infusion exudation⁶ and chemotherapy-induced phlebitis,⁷ acute parotitis, and acute hemorrhoids.⁸ The treatment of acute GA flares can quickly relieve the symptoms of joint redness, swelling, heat, and pain; shorten the course of disease; reduce the required dosages of NSAIDs and glucocorticoids; and delay disease progression.^9,10 As a natural drug, SJD has the advantages of high curative effects, low cost, and good safety,⁹ but there are few studies on its mechanism of action. It was previously found that SJD can reduce interleukin-1β (IL-1β)- and TNF-α-induced inflammatory factors in the synovial fluid of MSU-induced GA rats,¹¹ but as a compound formulation, SJD has multiple components and multiple targets. Its anti-inflammatory mechanism is still unclear.

With the rapid development of bioinformatics, network pharmacology analysis, which emphasizes the concept of “network-targeting, multi-component therapy”, is very consistent with the overall perspective of Chinese medicine (CM),^12,13 and has been proven to be a powerful tool.^13–15 Since network pharmacology provides a systematic understanding of drug action and disease complexity, pharmacological models are beneficial for dissecting the impact of CM on specific diseases.¹³ The application of molecular docking has led to the discovery of many key pharmacodynamic components, revealed the pharmacodynamic basis of complex systems, and improved the efficiency and pertinence of the CM screening process.^16,17 However, computation cannot completely replace experiments, and therefore in many cases it must be combined with other methods to validate the results experimentally for the rational design of polypharmacology.¹⁸

In the present study, we combine network pharmacology, molecular docking simulations, and experimental validation to elucidate the mechanism of action of SJD in the treatment of acute GA flares. Our results indicate that SJD is a new drug candidate for GA treatment.

Material and Methods

Reagents and Materials

SJD Preparation and Topical Application

SJD was provided by the Department of Traditional Chinese Medicine of Sichuan Provincial People’s Hospital. It was made by crushing Rheum palmatumnn L., Carthamus tinctorius L., and Natrii sulfas, and mixing them at a 5:8:1 (w/w/w) ratio. After dissolving the medicine in pure water at a ratio of 1:5 (w/v), the solution was boiled for 1 min, filtered, and absorbed into cotton with a thickness of 1.0–1.5 cm. Next, the liquid was squeezed out and the cotton was applied wet onto the affected joint, covered with polyethylene film, fixed with a bandage, and changed once a day.¹¹

Animals

Sprague-Dawley rats were purchased from Chengdu Dashuo Biological Co., Ltd. (Chengdu, Sichuan Province, China) and bred under SPF conditions, with 12-hour day-night cycle light and free access to food and water. The experiment was approved by the Ethics Committee of Chengdu Sport University (2021-43). All procedures were in accordance with the guidelines issued by the China Animal Care Committee.

Equipment and Reagents

MSU was purchased from Sigma-Aldrich (St. Louis, MO, USA, batch number U2875). The electronic thermometer and pain threshold meter were provided by the Institute of Chengdu Sports University. IL-1β and cyclooxygenase 2 (COX-2) ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, Hubei Province, China, batch number BNDGW4WZ4F, D89SKIVT8A) and the IL-8 ELISA kit was purchased from Jingmei Biotechnology Co., Ltd. (Yancheng, Jiangsu Province, China, batch number 202107). RNA TRIzol reagent was purchased from Hefei Bomei Biotechnology Co., Ltd. (Hefei, Anhui Province, China, batch number BM1144); isopropanol was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. (Shanghai, China, batch number B422BA0020); and the PrimeScript RT reagent Kit and TB Green TM Premix Ex Taq^TM II (Tli RNaseH Plus) were purchased from Baori Doctor Biotechnology CoLtd. (Beijing, China, batch numbers RR047A and RR820A).

Network Pharmacology Analysis and Molecular Docking of SJD

Acquisition and Screening of Active Ingredients and Targets for SJD

Candidate chemical components and corresponding protein targets were searched for in the Traditional Chinese Medicine Systems Pharmacology Analysis Platform (TCMSP) database using the Chinese pinyin of palmatumnn L. and Carthamus tinctorius L. as query criteria. With reference to Zhu et al’s method of screening effective components of external drugs by using network pharmacology,¹⁹ components with drug-like properties (DL) > 0.18 were considered as possibly having medicinal properties for the human body. The components and corresponding targets obtained from the TCMSP database were screened for activity and pharmacological action on the basis of DL > 0.18. Natrii sulfas (Na₂SO₄·10H₂O) was searched in the Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-TCM) database. All drug targets were combined and sorted, duplicate drug targets were eliminated, and a “medicinal materials–chemical composition–drug” target network was established.

Retrieval and Acquisition of Disease Target

The disease target genes related to gout were obtained using the PharmGKB, OMIM, GeneCards, and TTD databases using “gout” as the keyword and limiting the search conditions to “gene” and “Homo sapiens”. All target genes were combined and duplicate target genes were eliminated.

Venn Analysis of Potential Targets for the Treatment of GA

The total drug targets and total disease targets were imported into the online mapping platform BioVenn, and the intersection of drug targets and disease targets was obtained, which are the potential targets of SJD for the treatment of GA.

Establishment of Protein–Protein Interaction Networks

The intersection targets obtained by Venn analysis were imported into the STRING database. The species was defined as human, the information on protein–protein interactions (PPIs) was obtained, and the information obtained was imported into Cytoscape to generate a PPI network. The topology of the network was analyzed using the Cytoscape 3.8.0 plug-in cytoNCA, including the degree of centrality (DC), medium centrality (BC), eigenvector centrality (EC), and closeness centrality (CC).

GO Functional Enrichment and KEGG Pathway Enrichment Analyses

The enrichment analysis included functional annotation and signal pathway analyses, taking the P-value of enrichment results as reference value. The data downloaded from the online database of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), cellular component (CC), biological process (BP), and molecular function (MF) were used in this study. Using default parameters for GO and KEGG analyses, the functional categories and signal pathways were screened. The first 20 functional annotations and signal pathways were selected for analysis. The results are displayed in the form of a bar chart and a bubble chart, respectively. The enrichment data of drug targets in the first 20 signal pathways were analyzed.

Preparation of Data of Small-Molecule Ligands and Protein Receptors

Ligand files for small molecules were extracted using PubChem. Three-dimensional conversion of small-molecule ligand files was performed using Chemoffice software. Protein receptor files for the target genes were obtained by searching the BDP website, and then the water molecules and small-molecule ligands for the protein receptor were removed using Pymol software.

Active Pocket Detection and Molecular Docking

VINA software was used to determine the active pocket (where the target is a protein or enzyme and the active site is the domain [active pocket] in which the protein can bind to small-molecule ligands). In addition, VINA software was used to determine the location of small-molecule ligands and protein receptors and to predict the best binding sites of effective CM components and drug core targets. The ligand file and the protein receptor file were introduced into VINA for molecular docking.

Animal Experiments

Experimental Animals and Grouping

In total, 32 adult male rats were randomly divided into 4 groups: the control group (C), model group (M), natural group (N), and SJD treatment group (SJ).

Modeling of Acute GA Flares

After 1 week of adaptive feeding, rats in groups M, N, and SJ were injected with 0.5 mL of 25 mg/mL MSU solution in the right knee joint cavity, and rats in group C were injected with the same dose of normal saline. The contralateral bulge of the joint puncture point was set as the injection success criterion, and the acute GA model was induced by the above method. Swelling of the ankle joint, increased skin temperature, and restricted joint movement were clearly observed in groups M, N, and SJ after 12 h, which proved that the model was established successfully.²⁰

Experimental Intervention Protocol

After successful modeling, the SJ group was treated with topical application of SJD, while the C and N groups were treated with topical application of pure water in the same way, once per day for 6 h, for a total of 3 days.

Sample Collection and Preservation

After anesthesia with sodium pentobarbital (ip, 70 mg/kg body weight) 24 hours after molding in group M and 72 hours after intervention in groups C, N, and SJ, 0.5 mL of saline was injected into the right ankle joint and the joint was moved for 1 minute, the ankle skin was incised, the synovial fluid was aspirated with an empty needle, and the synovial fluid was centrifuged at 3000 rpm for 5 minutes and stored at −80°C.

Joint Swelling Degree, Right Ankle Skin Temperature, and Paw Withdrawal Threshold Measurement

The circumference of the same part of the right ankle joint was measured before and after modeling and before sampling using a non-elastic soft tape. This was repeated three times and the resulting values were averaged. The joint swelling degree was calculated as follows: joint swelling degree = (post-modeling joint circumference − pre-modeling joint circumference)/(pre-modeling joint circumference). The local skin temperature of the right hind ankle joint was measured by an electronic thermometer. The right hind limb ankle joint was stimulated bottom-up with von Frey filament as follows: The rat was placed on a grid platform surrounded by a transparent box, and a 15-gauge filament was used to stimulate the rat ankle from below. The paw was retracted due to pain. At this time, the maximum value generated by the force sensor under the filament was taken as the paw retraction threshold. This was repeated five times and the average value was taken.

H&E Staining of Ankle Synovial Membrane

The synovial tissues of each group were fixed in 10% paraformaldehyde solution, and the synovial tissues were decalcified, paraffin-embedded, sectioned, de-paraffinized, hydrated, and stained with H&E. The histopathology of the synovial tissues was observed under a light microscope.

Detection of Synovial Fluid Cytokines by ELISA

The standard operation procedures according to the instructions of the ELISA kits were followed.

Detection of IL1B, CXCL8, and PTGS2 mRNA in Synovial Tissue by Real-Time Quantitative PCR

The entire gene sequence was searched from the National Center for Biotechnology Information (NCBI) database, and gene-specific primers were designed and screened using Premier primer design software. All primers were designed and synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. and purified by ULTRAPAGE. The primer sequences are shown in Table 1. To measure mRNA levels, 50 mg of synovial tissue was taken and total RNA was extracted using TRIzol. The extracted total RNA was reverse transcribed into cDNA following the kit’s instructions, and the cDNA was used for real-time quantitative PCR.

Table 1 Primer Sequence Table

Statistical Analysis

Data have been presented as mean values ± standard deviation. One-way analysis of variance (ANOVA) with Tukey multiple comparison test is used for data analysis. P < 0.05 was considered statistically significant.

Results

Screening of Active Ingredients

The database of CM was searched, using the screening condition DL > 0.18. A total of 27 active ingredients of Rheum palmatumnnL., Carthamus tinctoriusL., and Natrii sulfas, were selected. Among them, rhein, aloe-emodin, quercetin, luteolin, beta-carotene, etc., have a wide range of pharmacological effects, such as anti-inflammatory and antioxidant effects. The targets in the database of active ingredients of CM were retrieved, the target gene names were obtained from the UniProt database, invalid and duplicate targets were deleted, and 221 targets of active ingredients were obtained. A total of 1591 GA gene targets were obtained by searching and integrating the Gene Card, OMIM, PharmGKB, and TTD databases (Figure 1A) and 79 drug–disease common target genes were obtained (Figure 1B). Components without corresponding targets and duplicate targets were excluded, and the resulting 23 drug components (Table 2) and 79 disease-related targets were mapped. Among them, DH represents Rheum palmatum L., HH represents Carthamus tinctorius L., HD represents common ingredients of Carthamus tinctorius L. and Rheum palmatum L., and MX represents Natrii sulfas. The data were imported into Cytoscape to construct the component target network (Figure 1C), where one or more active components can correspond to a target and multiple targets can correspond to the same active component, indicating that the group has a multi-component and multi-target profile for the treatment of GA.

Table 2 Effective Active Ingredients of Drugs

Figure 1 (A) The distribution of 1591 disease targets from the GeneCards, OMIM, PharmGKB, and TTD databases. (B) Venn diagram of potential targets of SJD for GA treatment. (C) The core drug group–target–disease Venn diagram, where green shows the target genes, blue shows the Carthamus tinctorius L. active ingredients, beige shows the active ingredients of Rheum palmatum L., purple shows the active ingredients of mirabilite, and dusky red shows the common active ingredients of Carthamus tinctorius L. and Rheum palmatum L..

PPI Network and GO and KEGG Enrichment Pathway Analysis

The common targets of SJD for GA treatment were imported into STRING (https://string-db.org/) to construct PPI networks. We obtained the GA-related PPI network. We observed 79 targets that could interact with proteins and 1124 edges representing PPIs, with a mean node degree of 28.5, a mean local clustering coefficient of 0.683, an expected number of edges of 308, and a PPI-enriched P-value of < 1.0e−16 (Figure 2A). The results of MF, BP, and CC analysis of 170 core targets showed that the key active components of SJD were highly enriched in 102 GO items, and target proteins were enriched in 158 KEGG pathways. The top 30 GOs were selected to analyze the results with a threshold of P < 0.05 to obtain the main pathways of action in the core drug group for the treatment of gout, including the NF-κB, MAPK, Toll-like receptor (TLR), PI3K–Akt, IL-17, TNF, and HIF-1 signaling pathways (Figure 2B and C).

Figure 2 PPI network of disease targets and GO and KEGG enrichment pathway analysis. (A) PPI network of disease targets. (B) Functional enrichment analysis of GO, the potential targets of SJD for gout treatment. (C) 30 KEGG pathways that were highly correlated with SJD treatment for gout.

PPI Core Gene Screening

The TSV format data from 32 PPIs were imported into Cytoscape and the PPIs based on DC, BC, CC, EC, NC, and lac parameters were analyzed using cytoNCA. The initial sieve threshold was Degree > 12, Magi Eigenvector > 0.073067062, Lac > 6, Magnetic Closeness > 37.74521856, Magical Closeness > 0.483660131, and Network > 6.75. There were 37 nodes and 163 edges. Then, 37 targets were further screened. Secondary screening thresholds were degree > 27, pr eigenvectors > 0.188253894, prelac > 11.71428571, recall mediator > 168.9082139, pr closeness > 0.587301587, and gradient network > 18.27076629. The second screen yielded 10 nodes with 29 edges, including JUN, PTGS2, VEGFA, MAPK1, IL1B, CXCL8, EGF, TP53, and MAPK8, suggesting that this could be a potentially important target for the treatment of gout (Figure 3).

Figure 3 Screening of core targets for SJD in the treatment of gout.

Figure 4 Molecular docking structure of SJD core small-molecule components and key therapeutic targets of gout. (A) IL1B-Quercetin; (B) CXCL8-Quercetin; (C) PTGS2-Quercetin; (D) PTGS2-Baicalein; (E) PTGS2-Kaempfero; (F) PTGS2-Quercetagetin; (G) PTGS2-(-)-catechin; (H) PTGS2-Rhein.

Molecular Docking

The core targets of IL1B,²¹ CXCL8,^22,23 and PTGS2²⁴ with the highest correlation with acute gout inflammation were analyzed and extracted, and the core small-molecule ligands (quercetin, emodin, rhein, etc.) were extracted from the PubChem and PDB databases. The corresponding small-molecule ligands were optimized by Chemo3D software to determine the optimal binding site between the visually effective compound SJD and the gout core target, as well as the molecular docking of core molecular ligands and protein receptors (Figure 4). Yellow in the figure shows hydrogen bond, and molecular binding energy is attached to Table 3.

Table 3 The Binding Energy of Each Molecule to the Protein Exhibit

Figure 5 Changes in ankle skin temperature, joint swelling degree, and paw withdrawal threshold. (A) The appearance of the ankle joint. GA rats have obvious redness and swelling, and SJD treatment can significantly improve the degree of swelling. (B) The skin temperature of the ankle joint. (C) The degree of swelling of the ankle joint. (D) The von Frey paw withdrawal threshold of the ankle joint. *P < 0.05; **P < 0.01; ****P < 0.0001. Abbreviation: Ns, not significant.

Animal Experiment Results

Improvement of Acute GA Flare Symptoms by SJD

During the modeling process, one rat in the SJ group suffered lung contusion due to over-tight fixation, so it was excluded from the experiment. Significant swelling, increased local skin temperature, restricted joint movement, and a lower paw withdrawal threshold were observed in the ankle joints of rats in groups M, N, and SJ 12 h after molding, with no statistical difference between them (P > 0.05). Group C showed no significant changes in the ankle joint and was statistically different from groups M, N, and SJ (P < 0.0001). The rats in group N had a reduced joint circumference, lower local skin temperature, and higher paw withdrawal threshold than rats in group M, with more pronounced changes in group SJ compared to group M (Figure 5).

Effects of SJD on Acute GA Synovial Inflammation

The synovial joint tissue in group C was clear and intact; the lining layer consisted of a single or double layer of synovial cells, with no obvious inflammatory cell infiltration or fibrous tissue hyperplasia. Group M had mild to moderate infiltration of inflammatory cells in the sublayer, mainly lymphocytes with rounded, deeply stained nuclei, neutrophils with rod-shaped or lobulated nuclei, and plasma cells with nuclei on one side of the cell, and localized necrosis with necrotic cell nuclei solidified and disintegrated, cytoplasmic lysis, and more eosinophilic plasma material exuding from the necrotic area. Group N has varying degrees of inflammatory cell infiltration in the sublingual layer, mainly lymphocytes, neutrophils, and plasma cells, with proliferation of fibrous tissue and slight local hemorrhage. Inflammatory cell infiltration was observed in the sublingual layer of the SJ group, dominated by lymphocytes and neutrophils, with an increased number of fibroblasts with long oval or oval nuclei and slight local hemorrhage. The degree of pathological changes was significantly greater in group M compared to group C. Compared to group M, group N had relatively mild pathological changes and group SJ had the least pathological changes (Figure 6).

Figure 6 HE staining of ankle joint. Up (×100), down (×400), In the down (×400) of group N,lymphocytes (green↑), neutrophils (yellow↑), fibroblasts (blue↑), and bleeding (red↑).

Effects of SJD on Cytokine and IL1B, CXCL8, and PTGS2 mRNA Expression

MSU crystals can induce the expression of IL-1β, IL-8, and COX-2 in the synovial fluid of rats. Significant differences were observed between groups M and C. MSU crystals can increase the expression of IL1B, CXCL8, and PTGS2 mRNA in synovium. Compared with the non-medicated group N, the external application of SJD significantly reduced the levels of pro-inflammatory factors and IL1B, CXCL8, and PTGS2 mRNA in the synovial fluid of the GA rats (Figure 7).

Figure 7 Cytokine concentrations in synovial fluid and IL1B, CXCL8, and PTGS2 mRNA expression in synovium (nC = 8; nM = 8; nN = 8; nSJ = 7). (A) IL-1β concentration (ng/mL). (B) IL-8 concentration (ng/L). (C) COX-2 concentration (ng/L). (D) IL1B mRNA expression level. (E) CXCL8 mRNA expression level. (F) PTGS2 mRNA expression level. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Abbreviation: Ns, not significant.

Discussion

SJD is a widely used prescription in Sichuan Provincial People’s Hospital, which has good anti-inflammatory effects on acute GA flares. The SJD formula is refined, and the main ingredients are Rheum palmatum L., Carthamus tinctorius L., and Natrii sulfas. In the theory of CM, rhubarb is bitter and cold, and external use can have heat-clearing and detoxifying effects. It can be used to treat acute inflammation. Mirabilite is bitter and cold and can improve local swelling of acute GA flares. Safflower is pungent and warm in nature, which helps to unblock the blood vessels and disperse blood stasis, helping to improve local blood circulation due to acute inflammation.

In this study, 27 active ingredients were screened. Among them, rhubarb acid, aloe rhodopsin, quercetin, lignan, and β-carotene are the main core compounds of SJD for GA treatment. The NLRP3 inflammasome has a prominent role in the pathogenesis of inflammatory arthritis, especially gout.²⁵ Rhein can inhibit the protease activity of caspase-1 and the pharmacological activity of IL-1, interfering with the NLRP3 complex.²⁶ β-Carotene can prevent the production of IL-1β and caspase-1, which may have potential as a future therapy for gout.²⁷ Xanthine oxidase (XO) catalyzes the production of uric acid from hypoxanthine and xanthine in human metabolism. Excessive production of uric acid can lead to hyperuricemia and eventually gout and other diseases. Aloe-emodin derivatives and luteolin can significantly inhibit XO.^28,29 In addition, luteolin can improve the inflammatory response of gout by inhibiting the TLR/MyD88/NF-κB pathway³⁰ and reducing the levels of IL-1β and TNF-α.³¹ Quercetin was found to reversibly inhibit XO activity by 50% and reduce uric acid production, and in in vitro studies, quercetin inhibited COX-I and II activity by 52.8% and 42.3%, respectively.³²

The results of this study show that the main potential targets of SJD for the treatment of gout are JUN, PTGS2, VEGFA, MAPK1, IL1B, CXCL8, EGF, TP53, and MAPK8. Among them, COX-2 is a key enzyme that induces the synthesis of prostaglandin E2 (PGE2), and PGE2 is an important factor involved in synovial and periarticular tissue inflammation and glomerular injury lesions.^32,33 In patients with gout, deposited MSU crystals may stimulate the generation of a systemic inflammatory response by upregulating PTGS2 (COX-2 gene) in peripheral mononuclear cells in advanced gout,³⁴ and selective COX-2 inhibitors are effective and important agents for the treatment of acute attacks of gout³⁵ to provide rapid relief of joint erythema and pain. Our experiments demonstrated that SJD can rapidly control inflammation by inhibiting PTGS2 gene expression, which seems to explain that SJD has a similar effect to COX-2 selective inhibitors. In gout, IL-1β production is mediated by MSU crystals triggering the inflammasome,¹ and sustained IL-1β secretion induces rapid recruitment of neutrophils to sites of crystal deposition, producing a more severe inflammatory response,³⁶ as well as inducing the production of matrix-degrading enzymes that break down cartilage and chondrocytes,³⁷ all of which are key factors in IL-1β-mediated inflammation. CXCL8 (IL-8) levels are increased in the synovial fluid and serum of gout patients independent of their disease activity, but higher CXCL8 levels are associated with cardiovascular disease in gout patients.³⁸ VEGFA was identified as the locus of hyperuricemia.³⁹ KEGG pathway enrichment analysis showed that the key pathways involved affected by SJD in gout treatment are the NF-κB, MAPK, and TLR signaling pathways. The NF-κB signaling pathway regulates the production and maturation of IL-1β, which is a key factor causing acute inflammation. The induction of IL-1β release by MSU requires the activation of NF-κB to stimulate the expression of pro-IL-1β.⁴⁰ TLRs are transmembrane proteins expressed by cells of the innate immune system; they recognize pathogen-associated molecular patterns and play important roles in immune and inflammatory responses to destroy invaders.⁴¹ MSU interacts with TLR2 and TLR4 of the TLR family, interacting with TLR2 on chondrocytes to induce nitric oxide production, thereby accelerating cartilage degeneration,⁴² and interacting with TLR2/TLR4 on macrophages, leading to IL-1β production to induce synovial inflammation.⁴³

It has been shown that SJD can quickly relieve the symptoms of acute GA flares, reduce key inflammatory factors, and inhibit inflammation during acute GA flares, and it may have a protective effect on the cardiovascular system of gout patients. SJD is an excellent option as a topical treatment during acute GA flares.

Conclusion

In this study, network pharmacology and molecular docking simulations combined with experimental verification were used to clarify the mechanism of SJD in the treatment of acute GA flares. The network pharmacology and molecular docking simulation results show that SJD mainly acts on target proteins such as IL-1β, IL-8, and COX-2 and regulates the NF-κB, TLR, IL-17, and MAPK signaling pathways in the treatment of acute GA. In addition, experiments in rats showed that SJD can significantly relieve the symptoms of GA, reduce the important pro-inflammatory factors IL-1β, IL-8, and COX-2 in the synovial fluid, and inhibit the expression of IL1B, CXCL8, and PTGS2 mRNA in the synovium membrane.