Based on network pharmacology-molecular docking and experimental exploration, the preventive and therapeutic effects of dapagliflozin on gouty arthritis in rats were investigated

Based on network pharmacology-molecular docking and experimental exploration, the preventive and therapeutic effects of dapagliflozin on gouty arthritis in rats were investigated | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Based on network pharmacology-molecular docking and experimental exploration, the preventive and therapeutic effects of dapagliflozin on gouty arthritis in rats were investigated Tao Ye, Jingfang Du, Pian Li, Na Shen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5723942/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Exploring the preventive and therapeutic effects of dapagliflozin (DAPA) on gouty arthritis (GA) in rats, and revealing its potential mechanism of action. Methods Potential targets of DAPA were identified from DrugBank, Swiss Target Prediction, CTD, and PharmMapper databases. Targets associated with gouty arthritis (GA) were retrieved from Gene Cards, DisGeNET, and NCBI databases. By taking the intersection of these two sets, common targets of DAPA and GA were determined. These common targets were then subjected to Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Use the CB-DOCK2 online molecular docking platform to dock DAPA with the core target and perform visual analysis. Thirty-two SPF-grade male SD rats were randomly divided into four groups, with eight rats in each: a blank control group, a model group, a 20 mg/kg DAPA group, and a 40 mg/kg DAPA group. Rats received daily gavage administration of the corresponding medication for eight consecutive days. On the fifth day, monosodium urate (MSU) crystal suspension was injected into the left ankle joint to establish an acute GA model. Samples were collected one hour after the final gavage. The swelling of the ankle joints was recorded at various time points. Hematoxylin and eosin (HE) staining was used to observe pathological changes in the synovial tissue of the ankle joints. Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the levels of inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the peripheral blood of the rats. Western blotting was performed to detect the expression levels of signaling pathway proteins in the synovial tissue of the ankle joints. Results Based on network pharmacology analysis and molecular docking, it was found that targets were significantly enriched in the nucleotide binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway, and the binding energies between the related core targets and DAPA were all <-7.0 kcal/mol. In animal experiments, regarding ankle joint swelling: compared with the model group, the 20 mg/kg DAPA group showed a significant reduction in ankle joint swelling at 72 hours post-modeling (p<0.05), and the 40 mg/kg DAPA group exhibited significant reductions in ankle joint swelling at both 48 and 72 hours post-modeling (p<0.01). For ankle joint HE staining: compared with the model group, DAPA-treated groups showed varying degrees of attenuation in pathological damage, including inflammatory cell infiltration, synovial tissue proliferation, and vascular proliferation in the ankle joints. Peripheral blood ELISA results: the levels of IL-1β and TNF-α in DAPA-treated groups were significantly lower than those in the model group (p<0.05). As for the protein expression levels of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) and cysteinyl aspartate-specific proteinase-1 (Caspase-1) in ankle joint synovium: compared with the model group, the expression of NLRP3 and Caspase-1 proteins was significantly reduced in DAPA-treated groups (p<0.05). Conclusion DAPA may alleviate the inflammatory response in acute GA in rats by inhibiting the NLRP3/Caspase-1 pathway. Health sciences/Rheumatology/Rheumatic diseases/Acute inflammatory arthritis Health sciences/Rheumatology/Rheumatic diseases/Metabolic bone disease Gout Gouty arthritis Dapagliflozin Rat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Gout is an acute articular inflammation triggered by the deposition of monosodium urate (MSU) crystals in joints, cartilage, bursae, tendons, or soft tissues, resulting from disorders in purine metabolism and uric acid excretion [ 1 ] . This condition has a long history, dating back to ancient Egypt in 2640 BC, and was later described by Hippocrates, the father of Western medicine, as a "disease that prevents walking" [ 2 ] . With the continuous improvement of socioeconomic status and quality of life, the prevalence of gout has been on the rise annually across different countries and regions. According to the 2019 Global Burden of Diseases (GBD) statistics, there are approximately 16.2 million cases of gout in China, with age-standardized prevalence rates (ASPR) of 12.3‰ and 3.9‰ for males and females, respectively. The disease is predominantly concentrated in coastal areas and Taiwan, posing a heavy burden on the healthcare system [ 3 ] . Dapagliflozin (DAPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is an anti-diabetic drug that lowers blood glucose levels by inhibiting the reabsorption of glucose in the proximal tubules [ 4 ] . Current research indicates that DAPA not only regulates blood glucose but also exhibits anti-inflammatory and antioxidant properties [ 5 ] , as well as the ability to reduce serum uric acid levels [ 6 ] . Reports suggest that, in patients with type 2 diabetes, the risk of gout attacks is significantly reduced in those treated with SGLT2 inhibitors compared to those receiving dipeptidyl peptidase-4 inhibitors, particularly in patients receiving DAPA [ 7 ] . This finding has been further confirmed in subsequent meta-analyses [ 8 ] . Although these studies provide preliminary evidence for the potential application of DAPA in gout treatment, its efficacy and mechanism of action in gouty arthritis (GA) remain to be elucidated. Therefore, this study employs a network pharmacology-molecular docking approach and establishes an MSU crystal-induced rat model of GA to evaluate the preventive and therapeutic effects of DAPA on GA in rats, and to explore its potential mechanisms of action. The aim is to provide new theoretical bases and therapeutic strategies for the clinical treatment of gout. Methods and Materials 1.1 Network Pharmacology and Molecular Docking 1.1.1 Collection of DAPA Target Genes Using "Dapagliflozin" as the search term, the primary targets of DAPA were obtained from Drugbank ( https://go.drugbank.com/ ), Swiss Target Prediction ( https://www.swisstargetpredictionch/ ), CTD ( https://ctdbase.org/ ), and PharmMapper ( http://www.lilab-ecust.cn/pharmmapper/ ) databases. Subsequently, these targets were standardized and corrected using the Uniprot database ( https://www.uniprot.org/ ), and then integrated and deduplicated. 1.1.2 Collection of GA targets Using "Gouty arthritis" and "Acute gouty arthritis" as search terms, disease targets were retrieved from Gene Cards ( https://www.genecards.org/ ), DisGeNET ( https://www.disgenet.org/ ), and NCBI ( https://www.ncbi.nlm.nih.gov/ ) databases, respectively. Subsequently, these targets were standardized and corrected using the Uniprot database ( https://www.uniprot.org/ ), and then integrated and deduplicated. 1.1.3 Construction of the interaction network diagram between DAPA and GA proteins and establishment of core targets Upload the targets obtained in items 1.1.1 and 1.1.2 to the online VENN diagram generation platform ( https://bioinfogp.cnb.csic.es/tools/venny/ ) to obtain the intersection of DAPA and GA. Then, upload the intersection targets as the common action targets of DAPA and GA to the STRING platform ( https://cn.string-db.org/ ), select the species as Homo sapiens, and hide the nodes disconnected from the network to obtain the Protein-Protein Interaction (PPI) network. Finally, import the network in tsv format into Cytoscape3.10.1 software for optimization analysis, and filter by degree to determine the core targets. 1.1.4 GO and KEGG pathway enrichment analysis Import the intersection target of DAPA and GA into DAVID( https://david.ncifcrf.gov/home.jsp )Perform Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the database. The output results are screened in descending order of gene count values to identify the top 10 biological processes (BP), cellular components (CC), molecular functions (MF), and KEGG pathways, and then analyzed in the field of microbiology༈ https://www.bioinformatics.com.cn/༉Create an enrichment analysis chart. 1.1.5 Component Target Molecular Docking From PubChem database( https://pubchem.ncbi.nlm.nih.gov/)Obtai n the 3D structure of DAPA (Dapagliflozin; Compound CID: 9887712). Further through the PDB database༈ https://www.rcsb.org/ ༉Obtain the structure of the core target protein. Finally, upload the structures of DAPA and related core targets to the online molecular docking platform CB-DOCK2༈ https://cadd.labshare.cn/cb-dock2/php/index.php ༉Perform molecular docking to evaluate the binding activity of compounds with targets. In molecular docking, the absolute value of Vina score is directly proportional to the stability of the binding, with a score<-5.0 kcal/mol indicating good binding between the compound and the target, and a score<-7.0 kcal/mol indicating strong binding activity between the two. 1.2 Animal Experiment Verification 1.2.1 Experimental Animals Thirty-two healthy male SPF-grade SD rats, aged 6–8 weeks, with a body weight of 160 ~ 180g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. [SCXK (Beijing) 2024-0001]. All experimental rats were housed in individually ventilated cages at the Animal Experiment Center of Hebei Engineering University Affiliated Hospital [SYXK (Ji) 2023-001], where the environmental temperature was maintained at 20 ~ 25°C and humidity at 55 ~ 65%. During the experiment, the rats were subjected to a 12-hour light/dark cycle and had free access to food and water. This experiment was approved by the Experimental Animal Ethics Committee of Hebei Engineering University Affiliated Hospital (IACUC-Hebeu-2023-0029).The experiment strictly followed the ARRIVE guidelines. 1.2.2 Main Drugs and Reagents DAPA (No: HJ20170119) was purchased from AstraZeneca Pharmaceuticals; Sodium Urate (No: U2875) was obtained from Sigma; Interleukin-1β (IL-1β) kit (No: E-NW0419Ra) and Tumor Necrosis Factor-α (TNF-α) kit (No: E-NW0635Ra) were acquired from Inova Technologies; Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (No: P16858) was sourced from Servicebio Technology; NOD-like Receptor Thermal Protein Domain Associated Protein 3 (NLRP3) (No: ab263899) was purchased from Abcam; and Cysteinyl Aspartate Specific Proteinase-1 (Caspase-1) (No: DY1200) was obtained from Abways Biotechnology. 1.2.3 Animal Grouping, Modeling, and Drug Administration After one week of adaptive feeding, the 32 SD rats were randomly divided into 4 groups (n = 8): a control group, a model group, a 20mg/kg DAPA group, and a 40mg/kg DAPA group. Each rat was given the corresponding drug at 9:00 AM daily. The control and model groups received an equal volume of 0.5% sodium carboxymethyl cellulose gavage, once daily for 8 consecutive days. On the fifth day of the experiment, one hour after drug administration, the rats were anesthetized with isoflurane gas. Following the method described by Coderre et al. [ 9 ] , 100µl of 25mg/ml MSU crystal suspension was injected into the left ankle joint of the rats to establish the AGA rat model. Rats in the control group received an equal volume of saline injection. After injection, a cotton swab was used to press the site for 10 ~ 30 seconds to prevent fluid leakage and successful injection was confirmed by the swelling of the contralateral ankle joint. 1.2.4 Ankle Joint Swelling The ankle joint circumference of each rat was measured using a vernier caliper at 0h before modeling and at 6h, 12h, 24h, 48h, and 72h after modeling to assess ankle joint swelling. Ankle joint swelling = (ankle joint circumference at each time point after modeling - ankle joint circumference at 0h before modeling). 1.2.5 Peripheral Blood Inflammatory Factors On the eighth day, one hour after gavage, the rats were anesthetized by inhalation of 2% isoflurane. Approximately 4 milliliters of blood were collected from the abdominal aorta using a blood collection needle and placed in a coagulation-promoting tube. After standing for 4 hours, the blood was centrifuged (3000 rpm, 10 minutes), and the supernatant was collected. Using a random number table, serum samples from 5 rats in each group were selected. Following the instructions of the ELISA kit strictly, the levels of TNF-α and IL-1β in the serum were determined. 1.2.6 Pathology of the Ankle Joint After blood collection, the rats were euthanized by cervical dislocation. Centered on the left ankle joint, the bones were cut approximately 0.5cm above and below the joint, and the skin was removed. The specimens were fixed in 4% paraformaldehyde and then decalcified in decalcifying solution until a needle could easily penetrate the ankle joint. After decalcification, the specimens underwent routine dehydration, embedding, sectioning, and staining. Finally, the pathological changes of the synovial tissue in the left ankle joint were observed under a microscope. 1.2.7 Related Proteins in Ankle Joint Synovial Tissue Using a random number table, ankle joint synovial tissues from 3 rats in each group were randomly selected. After lysis and centrifugation, the supernatant was collected, and the protein concentration was determined using the BCA method. The samples were then subjected to denaturation, electrophoresis, membrane transfer, blocking, and incubation (with GAPDH, NLRP3, and Caspase-1 antibodies). Protein bands were visualized using the ECL method. With GAPDH serving as an internal reference, the grayscale values of the bands were analyzed using ImageJ software. 1.2.8 Statistical Analysis Method Graphs were plotted and analyzed using GraphPad Prism 8.0 software. Data were expressed as mean ± standard deviation ( 𝑥̄ ± s ). Comparisons between groups were conducted using one-way ANOVA. For homogeneous variances, the Least Significant Difference (LSD) method was used for multiple comparisons between groups, while for heterogeneous variances, Dunnett's T3 method was employed. A P-value < 0.05 was considered statistically significant. Results 2.1 Network Pharmacology and Molecular Docking 2.1.1 Targets of DAPA A total of 1, 59, 103, and 287 DAPA targets were retrieved from the DrugBank, CTD, Swiss Target Prediction, and PharmMapper databases, respectively. After standardization and removal of duplicates using the UniProt database, a final set of 410 targets was obtained. 2.1.2 Related Targets of GA We obtained 232, 209, and 27 GA-related targets from the GeneCards, DisGeNET, and NCBI databases, respectively. Following standardization and deduplication using the UniProt database, a total of 404 unique targets were identified. 2.1.3 Potential Targets of DAPA for GA Treatment and Construction of the PPI Network Diagram The targets obtained in sections 2.1.1 and 2.1.2 were uploaded to an online VENN diagram generator, resulting in the identification of 49 common targets for DAPA and GA. These 49 targets were then imported into the STRING database to construct a Protein-Protein Interaction (PPI) network diagram, which was subsequently optimized using Cytoscape 3.10.1 software. In the network analysis, the average node degree was 15.91304, with 23 nodes having a degree higher than the average and thus being identified as core targets. (Fig. 1) 2.1.4 GO and KEGG Pathway Enrichment Analysis Enrichment analysis was conducted on the 49 common targets. The results showed that, in terms of Biological Process (BP), the targets were mainly involved in transcription regulation, inflammatory response, signal transduction, and immune response. For Cellular Component (CC), they were primarily located in the cytoplasm, nucleus, plasma membrane, and mitochondria. As for Molecular Function (MF), the targets were predominantly associated with protein binding, ATP binding, enzyme binding, and DNA binding. The KEGG pathway enrichment analysis revealed involvement in various pathways, including lipid metabolism disorders and atherosclerosis, cancer-related signaling pathways, cellular metabolic pathways, COVID-19 virus-host interactions, MAPK signaling pathway, AGE-RAGE signaling pathway in diabetic complications, C-type lectin receptor signaling pathway, Yersinia infection, and NOD-like receptor (NLR) signaling pathway. (Fig. 2) 2.1.5 Molecular Docking Based on the KEGG pathway enrichment analysis, we selected the NOD-like receptor signaling pathway as the focus for further investigation. From the 23 core targets, we chose four targets closely related to the NOD-like receptor signaling pathway, namely IL-1β, TNF, Caspase-1, and NLRP3, for molecular docking. The results indicated that the binding energies for all four targets were less than − 7.0 kcal/mol, suggesting that DAPA may exert its effects on GA by influencing the NLRP3 pathway within the NOD-like receptor signaling pathway. (Fig. 3 and Table 1 ) Table 1 Molecular docking results Target pdb ID Vina score(kcal/mol) Center (x, y, z) Docking size (x, y, z) NLRP3 7alv -9.4 17,31,142 23,23,23 TNF 2e7a -9.5 -2, -7,3 23,23,23 IL-1β 1itb -8.3 43,14,19 23,23,23 Caspase-1 3e4c -8.2 2, -9,3 35,35,23 2.2 In Vivo Experimental Validation 2.2.1 Ankle Swelling in Rats Across Groups Compared with the blank control group, the swelling degree of the model group at each time point significantly increased, with a significant statistical difference (p < 0.01), and reached its peak at 24 hours after modeling, indicating successful model construction; Compared with the model group, the swelling degree of rats in the 20mg/kg DAPA group significantly decreased at 72 hours after modeling (p < 0.05), and the swelling degree of rats in the 40mg/kg DAPA group significantly decreased at both 48 hours and 72 hours after modeling (p < 0.01).(Figure 4 and Table 2 ) Table 2 Swelling degree of ankle joint at different time points in each group of rats (n = 8) ( 𝑥̄ ± s , n = 8) Time Control Model 20mg/kg DAPA 40mg/kg DAPA 6h 3.25 ± 0.76 ** 5.51 ± 0.53 ## 5.72 ± 0.46 5.49 ± 0.56 12h 2.96 ± 0.74 ** 6.36 ± 0.38 ## 6.23 ± 0.53 5.84 ± 0.84 24h 2.03 ± 0.54 ** 6.78 ± 0.32 ## 6.41 ± 0.33 6.39 ± 0.73 48h 0.99 ± 0.42 ** 5.81 ± 0.31 ## 5.27 ± 0.82 4.91 ± 0.56 ** 72h 0.49 ± 0.18 ** 5.18 ± 0.37 ## 4.22 ± 0.63 * 3.82 ± 0.87 ** Note: Compared with the model, * p < 0.05, ** p < 0.01; Compared with the blank control, # p < 0.05, ## p < 0.01. 2.2.2 Histopathological Changes in Ankle Synovial Tissue of Rats In the blank control group, the ankle joint structures of the rats were clear, with neatly arranged synovial cells and loose connective tissue. Mild infiltration of inflammatory cells was occasionally observed, but there was no synovial cell proliferation or neovascularization. Compared to the blank control group, the model group exhibited significant pathological changes, including extensive neovascularization, widespread infiltration of inflammatory cells, and synovial tissue hyperplasia. In contrast to the model group, both the 20 mg/kg and 40 mg/kg DAPA groups showed varying degrees of reduction in ankle inflammatory cell infiltration, vascular proliferation, and synovial tissue pathological damage. (Figure 5 ) 2.2.3 Levels of Inflammatory Cytokines in Peripheral Serum of Rats The results indicated that, compared to the blank control group, the levels of IL-1β and TNF-α in the peripheral serum of rats in the model group were significantly increased (p < 0.01). In comparison to the model group, the levels of IL-1β and TNF-α in the peripheral serum of rats treated with 20 mg/ml and 40 mg/ml DAPA were significantly decreased (p < 0.05, p < 0.01). (Figure 6 ) 2.2.4 Expression Levels of Caspase-1 and NLRP3 Proteins in Ankle Synovial Tissue of Rats The results demonstrated that, compared to the blank control group, the expression of Caspase-1 and NLRP3 in the ankle synovial tissue of rats in the model group was significantly upregulated (p < 0.01). In contrast to the model group, the expression of both Caspase-1 and NLRP3 in the synovial tissue of rats treated with 20 mg/ml DAPA was significantly decreased (p < 0.05, p < 0.01). Similarly, rats treated with 40 mg/ml DAPA showed significant reductions in the expression of Caspase-1 and NLRP3 in their synovial tissue (p < 0.01). (Figure 7 and Table 3 ) Table 3 Expression of Caspace-1 and NLRP3 proteins in ankle synovial tissue of rats in each group (n = 3) ( 𝑥̄ ± s , n = 3) Group Caspace-1/GAPDH NLRP3/GAPDH Control 0.39 ± 0.16 ** 0.07 ± 0.04 ** Model 1.23 ± 0.10 ## 1.17 ± 0.09 ## 20mg/kg DAPA 0.81 ± 0.18 *# 0.65 ± 0.09 ** ## 40mg/kg DAPA 0.61 ± 0.14 ** 0.44 ± 0.08 ** ## Note: Compared with the model, * p < 0.05, ** p < 0.01; Compared with the blank control, # p < 0.05, ## p < 0.01. Discuss Gout, a metabolic inflammatory disease triggered by abnormal urate metabolism, is pathologically underpinned by elevated serum uric acid levels that surpass the solubility threshold in the blood, leading to the deposition of monosodium urate (MSU) crystals in tissues or joints. These deposited MSU crystals are recognized as endogenous danger signals—damage-associated molecular patterns (DAMPs)—by pattern recognition receptors (PRRs) in the innate immune system, including Toll-like receptors and NOD-like receptors [ 10 ] . Notably, the NLRP3 inflammasome within the NLR family plays a pivotal role [ 11 ] . Activated by MSU crystals, the NLRP3 inflammasome cleaves pro-IL-1β and pro-IL-18 via Caspase-1, converting them into mature IL-1β and IL-18, which are then released extracellularly to further promote the production of inflammatory cytokines and chemokines, such as IL-6, IL-8, and TNF-α [ 12 ] . The synergistic effects of these cytokines enhance the recruitment and activation of inflammatory cells and activate nociceptors, directly exacerbating joint pain and inflammation [ 13 ] . Without timely intervention, MSU crystals continue to accumulate in the body. Even during gout remission, MSU crystals can persistently trigger systemic inflammatory responses by activating inflammatory pathways, enhancing interactions between inflammatory cells, and elevating arachidonic acid metabolic activity [ 14 ] . This persistent inflammatory state ultimately leads to severe complications such as joint deformity and gouty nephropathy [ 15 ] . Therefore, effective intervention and management of gout patients (GA) are crucial. DAPA, a member of the SGLT2 inhibitor family, selectively inhibits the high-capacity glucose transporter SGLT2 in the renal proximal tubules, reducing glucose reabsorption in the kidneys and thereby lowering glucose levels independently of insulin [ 4 ] . However, as research on this class of drugs has deepened, it has been discovered that, apart from lowering blood glucose, they also possess anti-inflammatory, antioxidant properties, promote weight loss, regulate blood pressure, and improve cardiovascular and chronic kidney disease outcomes [ 16 ] . Recently, a large meta-analysis of its urate-lowering efficacy showed that DAPA significantly reduced serum uric acid levels in a dose-dependent manner (5–50 mg, P = 0.014) compared to controls [ 17 ] . Regarding gout attacks, multiple clinical trials and meta-analyses have indicated that DAPA significantly reduces the risk of gout in patients with type 2 diabetes or heart failure and decreases the need for antigout medications in gout patients [ 8 , 18 ] . This phenomenon is primarily attributed to its urate-lowering effect [ 19 ] . Nevertheless, a recent meta-analysis by Mainak Banerjee et al. found no statistically significant correlation between the urate-lowering efficacy of SGLT2 inhibitors and the reduction in gout attack risk (P > 0.3) [ 20 ] . Combined with DAPA's vast potential in anti-inflammatory, antioxidant, and immune metabolism fields—such as attenuating lipopolysaccharide-induced acute lung injury in rats by regulating the AMP-activated protein kinase/nuclear factor kappa B pathway and inhibiting NLRP3 activation [ 21 ] ;enhancing colonic autophagy and inhibiting apoptosis to improve colitis in rats through the nuclear factor E2-related factor 2/heme oxygenase-1 pathway [ 22 ] ༛and delaying osteoarthritis progression by reducing endoplasmic reticulum stress in chondrocytes [ 23 ] . Based on this, the role of DAPA in anti-gout may involve multifaceted physiological mechanisms. Network pharmacology, as an interdisciplinary combining systems biology, computer science and pharmacology, studies the interaction between drugs and targets by analyzing biomolecular networks [ 24 ] . In this study, we used network pharmacology to intersect the targets of DAPA and GA disease-related targets, and obtained 49 common targets of both. Among them, there are 23 core targets, including IL1β、TNF、TGF-β、Caspase-1、RELA、NLRP3、PTGS2、SIRT1, etc. At. Meanwhile, further combining the results of go and KEGG pathway enrichment analysis, nlrp3/caspase-1 signaling pathway in NOD like receptor signaling pathway was selected to verify the potential mechanism of DAPA in anti-GA effect. On this basis, the core targets closely related to the nlrp3/caspase-1 signaling pathway, IL-1 β, TNF, caspase-1 and NLRP3, were selected for molecular docking. The results showed that the binding energies of the above four targets with DAPA were significantly lower than − 7.0 kcal / mol, suggesting that DAPA may play an anti-GA role by regulating the nlrp3/caspase-1 signaling pathway. Subsequently, to further validate this pathway, we established a rat model of GA and administered DAPA for treatment. The results indicated that, compared with the model group, the DAPA-treated group exhibited improved ankle joint swelling and synovial tissue hyperplasia, reduced inflammatory cell infiltration, and decreased levels of inflammatory cytokines IL-1β and TNF-α in the peripheral blood. Additionally, by quantifying the protein expression levels of NLRP3 and Caspase-1 in the ankle joint synovial tissue, we found that DAPA treatment significantly downregulated the expression of both NLRP3 and Caspase-1 compared to the model group. These findings suggest that DAPA may alleviate the symptoms of acute gouty arthritis by inhibiting the activation of the NLRP3/Caspase-1 pathway, thereby reducing the inflammatory response. Conclusions To sum up, our study shows that DAPA has a preventive and therapeutic effect on MSU crystal induced GA in rat models, which may be achieved by reducing joint tissue damage, reducing the level of inflammatory cytokines in the blood and inhibiting the NLRP3/caspase-1 signal pathway. However, this study only selected four key targets closely related to NLRP3/caspase-1 signaling pathway for preliminary verification, and did not carry out in vitro experiments for further confirmation. At the same time, in view of the significant potential of DAPA in anti-inflammatory, antioxidant and immune metabolism, its multiple mechanisms of action in the treatment of acute GA still need to be further explored in future research. Abbreviations DAPA dapagliflozin GA gouty arthritis MSU monosodium urate GBD Global Burden of Diseases SGLT2 sodium-glucose cotransporter 2 GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes BP Biological Process CC Cellular Component MF Molecular Function IL-1β interleukin-1β TNF-α tumor necrosis factor-α GAPDH glyceraldehyde-3-phosphate dehydrogenase NLRP3 NOD-like receptor thermal protein domain associated protein 3 Caspase-1 cysteinyl aspartate specific proteinase-1 PRRs Pattern Recognition Receptors DAMPs Damage-associated molecular patterns NLR NOD-like receptor Declarations Acknowledgements None. Funding This work was supported by the observational study on the involvement of PKM2 in the regulation of malignant cell function in diffuse large B-cell lymphoma [No.21377797D]. Authors and Affiliations School of Clinical Medicine, Hebei University of Engineering, Handan, Hebei, 056002, China Ye Tao and Li Pian Affiliated Hospital of Hebei University of Engineering，Handan, Hebei, 056002, China Du Jingfang and Shen Na Contributions Tao Ye was responsible for the experimental operation, data statistics and paper writing; Du Jingfang was responsible for the methodology design; Li Pian was responsible for the experimental data collection; and Na Shen provided program planning, project funding, and guidance for writing and revising the paper. Corresponding author Na Shen Ethics declarations The animal experiments involved in this study have been reviewed and approved by the Laboratory Animal Ethical and Welfare Committee of Affiliated Hospital of Hebei Engineering University (Approval No.: IACUC-Hebeu-2023-0029). The experimental process was strictly carried out in accordance with the relevant laws, regulations, and rules on experimental animals in China. 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Gout: a disease involved with complicated immunoinflammatory responses: a narrative review [J]. Clin. Rheumatol. 39 (10), 2849–2859 (2020). GU, H. & YU, H. MSU crystal deposition contributes to inflammation and immune responses in gout remission [J]. Cell. Rep. 42 (10), 113139 (2023). TAO, H. et al. A review on gout: Looking back and looking ahead [J]. Int. Immunopharmacol. 117 , 109977 (2023). COWIE M R, F. I. S. H. E. R. M. SGLT2 inhibitors: mechanisms of cardiovascular benefit beyond glycaemic control [J]. Nat. reviews Cardiol. 17 (12), 761–772 (2020). ZHAO, Y. et al. Effects of sodium-glucose co-transporter 2 (SGLT2) inhibitors on serum uric acid level: A meta-analysis of randomized controlled trials [J]. Diabetes Obes. Metab. 20 (2), 458–462 (2018). WANG, A. et al. Newer Glucose-Lowering Drugs and Risk of Gout: A Network Meta-Analysis of Randomized Outcomes Trials [J]. Clin. Ther. 46 (11), 851–854 (2024). SOMAGUTTA M K R, LUVSANNYAM, E. et al. Sodium glucose co-transport 2 inhibitors for gout treatment [J]. (Craiova, Romania), 10(3): e152. (2022). BANERJEE, M. et al. Serum uric acid lowering and effects of sodium-glucose cotransporter-2 inhibitors on gout: A meta-analysis and meta-regression of randomized controlled trials [J]. Diabetes Obes. Metab. 25 (9), 2697–2703 (2023). ABD EL-FATTAH E E, S. A. B. E. R. S. et al. The dynamic interplay between AMPK/NFκB signaling and NLRP3 is a new therapeutic target in inflammation: Emerging role of dapagliflozin in overcoming lipopolysaccharide-mediated lung injury [J] 147112628 (Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 2022). ARAB H H, AL-SHORBAGY M Y, SAAD, M. A. Activation of autophagy and suppression of apoptosis by dapagliflozin attenuates experimental inflammatory bowel disease in rats: Targeting AMPK/mTOR, HMGB1/RAGE and Nrf2/HO-1 pathways [J]. Chemico-Biol. Interact. 335 , 109368 (2021). LIU, Z. et al. Dapagliflozin suppress endoplasmic reticulum stress mediated apoptosis of chondrocytes by activating Sirt1 [J]. Chemico-Biol. Interact. 384 , 110724 (2023). NOOR, F. et al. Machine learning for synergistic network pharmacology: a comprehensive overview [J]. Brief. Bioinform. , 24 (3). (2023). Additional Declarations No competing interests reported. Supplementary Files ARRIVEguidelinesAuthorChecklist.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5723942","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":398902260,"identity":"45295f2f-5f9b-4b18-b4ff-584d1296b3d1","order_by":0,"name":"Tao Ye","email":"","orcid":"","institution":"Hebei University of Engineering","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Ye","suffix":""},{"id":398902261,"identity":"9d4a3437-f19c-4dab-b6cc-5503239e18d4","order_by":1,"name":"Jingfang Du","email":"","orcid":"","institution":"Affiliated Hospital of Hebei University of Engineering","correspondingAuthor":false,"prefix":"","firstName":"Jingfang","middleName":"","lastName":"Du","suffix":""},{"id":398902262,"identity":"6a89caee-7d6c-45f7-9a77-586c33eb3bd7","order_by":2,"name":"Pian Li","email":"","orcid":"","institution":"Hebei University of Engineering","correspondingAuthor":false,"prefix":"","firstName":"Pian","middleName":"","lastName":"Li","suffix":""},{"id":398902263,"identity":"c5a196c9-7490-4066-9386-950404c7af6d","order_by":3,"name":"Na Shen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYDACCQgpx8/efIyBsQHMMyBGi4WxZM+xNJK0VCRuuJFjRpwW+dnNxx4X1EgkzmzI+fbg4w6bxAb25m0SDDV3cGoxuHMs3XjGMQnjfoaz2w1nnklLbOA5VibBcOwZbi0SOWbSPGwSsjMbe7dJ87YdTmwAikgwNhzG7bAZ+d+kef5JMG44zPNM+m/b/8QG+Tf4tTDcyGEDGi6huOEYD5s0Y9sBoC08+LUY3Egzk+btkwAGMpuZZO+ZZOM2nrRii4Rj+ByW/Eya51udHL/842cSP3fYyfazH95440MNHodhADYQkUCChlEwCkbBKBgFmAAAND1UX47mFPMAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of Hebei University of Engineering","correspondingAuthor":true,"prefix":"","firstName":"Na","middleName":"","lastName":"Shen","suffix":""}],"badges":[],"createdAt":"2024-12-28 01:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5723942/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5723942/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73416626,"identity":"28d2bcc1-72a4-444b-85ce-b122bbdcb858","added_by":"auto","created_at":"2025-01-09 17:18:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7020446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVENN and PPI network diagrams of DAPA and GA intersection targets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: Figure A shows the VENN plot of the intersection targets of DAPA and GA; Figure B: PPI network diagram of DAPA and GA intersection targets\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/fa2175d496ed3b1adaffb519.jpeg"},{"id":73416446,"identity":"e61be2f6-153d-46ef-b743-113777e3897b","added_by":"auto","created_at":"2025-01-09 17:10:22","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2852091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnrichment analysis of GO and KEGG pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: Figure A shows GO functional enrichment analysis; Figure B shows KEGG pathway enrichment analysis\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/7a5651b40818f47ffc9b0b0d.jpeg"},{"id":73416447,"identity":"a47a8d57-2e64-4cbf-a6a4-09c8fedd618b","added_by":"auto","created_at":"2025-01-09 17:10:22","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4834556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking visualization results.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/175227bc86e0bc2260314d45.jpeg"},{"id":73416625,"identity":"339fb2ab-6783-40f6-8f37-f7fa14190ace","added_by":"auto","created_at":"2025-01-09 17:18:23","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8297864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnkle joint morphology of rats in each group 72 hours after injection of MSU crystals\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/84397448b33f9fdcc5ac9ada.jpeg"},{"id":73416629,"identity":"43c6e8e8-8755-489b-a42c-c533c163baff","added_by":"auto","created_at":"2025-01-09 17:18:23","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12166583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathological changes of synovial membrane in ankle joint of rats in each group\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/cac4ff09b15271cfd4933de4.jpeg"},{"id":73416452,"identity":"36550c43-ce7a-49e4-a33f-8ea7146cb8f5","added_by":"auto","created_at":"2025-01-09 17:10:23","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2785636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLevels of IL-1 β and TNF - α in peripheral serum of rats in each group (n=5)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/43afff668c0ef93dac2796ac.jpeg"},{"id":73416627,"identity":"8569d21c-5808-492c-9cb0-85c792666ed5","added_by":"auto","created_at":"2025-01-09 17:18:23","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1667947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrophoresis of NLRP3 and Caspase-1 protein expression in rat ankle joint tissue.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/02f631494d8198a59310dbf5.jpeg"},{"id":91613645,"identity":"1d122106-b3cf-4c59-bdba-1c2cbee05388","added_by":"auto","created_at":"2025-09-18 10:18:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":38949901,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/695c4208-00c3-406d-b52b-8fa0038c5441.pdf"},{"id":73416449,"identity":"ecbf87bc-b59e-4bdc-9aac-e59b232d9ccf","added_by":"auto","created_at":"2025-01-09 17:10:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":209590,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEguidelinesAuthorChecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5723942/v1/9d981f813b2671736f7cac38.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Based on network pharmacology-molecular docking and experimental exploration, the preventive and therapeutic effects of dapagliflozin on gouty arthritis in rats were investigated","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGout is an acute articular inflammation triggered by the deposition of monosodium urate (MSU) crystals in joints, cartilage, bursae, tendons, or soft tissues, resulting from disorders in purine metabolism and uric acid excretion\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. This condition has a long history, dating back to ancient Egypt in 2640 BC, and was later described by Hippocrates, the father of Western medicine, as a \"disease that prevents walking\"\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. With the continuous improvement of socioeconomic status and quality of life, the prevalence of gout has been on the rise annually across different countries and regions. According to the 2019 Global Burden of Diseases (GBD) statistics, there are approximately 16.2\u0026nbsp;million cases of gout in China, with age-standardized prevalence rates (ASPR) of 12.3\u0026permil; and 3.9\u0026permil; for males and females, respectively. The disease is predominantly concentrated in coastal areas and Taiwan, posing a heavy burden on the healthcare system\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDapagliflozin (DAPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is an anti-diabetic drug that lowers blood glucose levels by inhibiting the reabsorption of glucose in the proximal tubules\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Current research indicates that DAPA not only regulates blood glucose but also exhibits anti-inflammatory and antioxidant properties\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, as well as the ability to reduce serum uric acid levels\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Reports suggest that, in patients with type 2 diabetes, the risk of gout attacks is significantly reduced in those treated with SGLT2 inhibitors compared to those receiving dipeptidyl peptidase-4 inhibitors, particularly in patients receiving DAPA\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. This finding has been further confirmed in subsequent meta-analyses\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Although these studies provide preliminary evidence for the potential application of DAPA in gout treatment, its efficacy and mechanism of action in gouty arthritis (GA) remain to be elucidated. Therefore, this study employs a network pharmacology-molecular docking approach and establishes an MSU crystal-induced rat model of GA to evaluate the preventive and therapeutic effects of DAPA on GA in rats, and to explore its potential mechanisms of action. The aim is to provide new theoretical bases and therapeutic strategies for the clinical treatment of gout.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Network Pharmacology and Molecular Docking\u003c/h2\u003e \u003cp\u003e1.1.1 Collection of DAPA Target Genes\u003c/p\u003e \u003cp\u003eUsing \"Dapagliflozin\" as the search term, the primary targets of DAPA were obtained from Drugbank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Swiss Target Prediction (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.swisstargetpredictionch/\u003c/span\u003e\u003cspan address=\"https://www.swisstargetpredictionch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), CTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ctdbase.org/\u003c/span\u003e\u003cspan address=\"https://ctdbase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and PharmMapper (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.lilab-ecust.cn/pharmmapper/\u003c/span\u003e\u003cspan address=\"http://www.lilab-ecust.cn/pharmmapper/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases. Subsequently, these targets were standardized and corrected using the Uniprot database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and then integrated and deduplicated.\u003c/p\u003e \u003cp\u003e1.1.2 Collection of GA targets\u003c/p\u003e \u003cp\u003eUsing \"Gouty arthritis\" and \"Acute gouty arthritis\" as search terms, disease targets were retrieved from Gene Cards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), DisGeNET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.disgenet.org/\u003c/span\u003e\u003cspan address=\"https://www.disgenet.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases, respectively. Subsequently, these targets were standardized and corrected using the Uniprot database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and then integrated and deduplicated.\u003c/p\u003e \u003cp\u003e1.1.3 Construction of the interaction network diagram between DAPA and GA proteins and establishment of core targets\u003c/p\u003e \u003cp\u003eUpload the targets obtained in items 1.1.1 and 1.1.2 to the online VENN diagram generation platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to obtain the intersection of DAPA and GA. Then, upload the intersection targets as the common action targets of DAPA and GA to the STRING platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), select the species as Homo sapiens, and hide the nodes disconnected from the network to obtain the Protein-Protein Interaction (PPI) network. Finally, import the network in tsv format into Cytoscape3.10.1 software for optimization analysis, and filter by degree to determine the core targets.\u003c/p\u003e \u003cp\u003e1.1.4 GO and KEGG pathway enrichment analysis\u003c/p\u003e \u003cp\u003eImport the intersection target of DAPA and GA into DAVID( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/home.jsp\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/home.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e )Perform Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the database. The output results are screened in descending order of gene count values to identify the top 10 biological processes (BP), cellular components (CC), molecular functions (MF), and KEGG pathways, and then analyzed in the field of microbiology༈ \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/༉Create\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/༉Create\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e an enrichment analysis chart.\u003c/p\u003e \u003cp\u003e1.1.5 Component Target Molecular Docking\u003c/p\u003e \u003cp\u003eFrom PubChem database( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/)Obtai\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/)Obtai\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003en the 3D structure of DAPA (Dapagliflozin; Compound CID: 9887712). Further through the PDB database༈ \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ༉Obtain the structure of the core target protein. Finally, upload the structures of DAPA and related core targets to the online molecular docking platform CB-DOCK2༈ \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cadd.labshare.cn/cb-dock2/php/index.php\u003c/span\u003e\u003cspan address=\"https://cadd.labshare.cn/cb-dock2/php/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ༉Perform molecular docking to evaluate the binding activity of compounds with targets. In molecular docking, the absolute value of Vina score is directly proportional to the stability of the binding, with a score\u0026lt;-5.0 kcal/mol indicating good binding between the compound and the target, and a score\u0026lt;-7.0 kcal/mol indicating strong binding activity between the two.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1.2 Animal Experiment Verification\u003c/h3\u003e\n\u003cp\u003e1.2.1 Experimental Animals\u003c/p\u003e \u003cp\u003eThirty-two healthy male SPF-grade SD rats, aged 6\u0026ndash;8 weeks, with a body weight of 160\u0026thinsp;~\u0026thinsp;180g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. [SCXK (Beijing) 2024-0001]. All experimental rats were housed in individually ventilated cages at the Animal Experiment Center of Hebei Engineering University Affiliated Hospital [SYXK (Ji) 2023-001], where the environmental temperature was maintained at 20\u0026thinsp;~\u0026thinsp;25\u0026deg;C and humidity at 55\u0026thinsp;~\u0026thinsp;65%. During the experiment, the rats were subjected to a 12-hour light/dark cycle and had free access to food and water. This experiment was approved by the Experimental Animal Ethics Committee of Hebei Engineering University Affiliated Hospital (IACUC-Hebeu-2023-0029).The experiment strictly followed the ARRIVE guidelines.\u003c/p\u003e \u003cp\u003e1.2.2 Main Drugs and Reagents\u003c/p\u003e \u003cp\u003eDAPA (No: HJ20170119) was purchased from AstraZeneca Pharmaceuticals; Sodium Urate (No: U2875) was obtained from Sigma; Interleukin-1β (IL-1β) kit (No: E-NW0419Ra) and Tumor Necrosis Factor-α (TNF-α) kit (No: E-NW0635Ra) were acquired from Inova Technologies; Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (No: P16858) was sourced from Servicebio Technology; NOD-like Receptor Thermal Protein Domain Associated Protein 3 (NLRP3) (No: ab263899) was purchased from Abcam; and Cysteinyl Aspartate Specific Proteinase-1 (Caspase-1) (No: DY1200) was obtained from Abways Biotechnology.\u003c/p\u003e \u003cp\u003e1.2.3 Animal Grouping, Modeling, and Drug Administration\u003c/p\u003e \u003cp\u003eAfter one week of adaptive feeding, the 32 SD rats were randomly divided into 4 groups (n\u0026thinsp;=\u0026thinsp;8): a control group, a model group, a 20mg/kg DAPA group, and a 40mg/kg DAPA group. Each rat was given the corresponding drug at 9:00 AM daily. The control and model groups received an equal volume of 0.5% sodium carboxymethyl cellulose gavage, once daily for 8 consecutive days. On the fifth day of the experiment, one hour after drug administration, the rats were anesthetized with isoflurane gas. Following the method described by Coderre et al.\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, 100\u0026micro;l of 25mg/ml MSU crystal suspension was injected into the left ankle joint of the rats to establish the AGA rat model. Rats in the control group received an equal volume of saline injection. After injection, a cotton swab was used to press the site for 10\u0026thinsp;~\u0026thinsp;30 seconds to prevent fluid leakage and successful injection was confirmed by the swelling of the contralateral ankle joint.\u003c/p\u003e \u003cp\u003e1.2.4 Ankle Joint Swelling\u003c/p\u003e \u003cp\u003eThe ankle joint circumference of each rat was measured using a vernier caliper at 0h before modeling and at 6h, 12h, 24h, 48h, and 72h after modeling to assess ankle joint swelling. Ankle joint swelling = (ankle joint circumference at each time point after modeling - ankle joint circumference at 0h before modeling).\u003c/p\u003e \u003cp\u003e1.2.5 Peripheral Blood Inflammatory Factors\u003c/p\u003e \u003cp\u003eOn the eighth day, one hour after gavage, the rats were anesthetized by inhalation of 2% isoflurane. Approximately 4 milliliters of blood were collected from the abdominal aorta using a blood collection needle and placed in a coagulation-promoting tube. After standing for 4 hours, the blood was centrifuged (3000 rpm, 10 minutes), and the supernatant was collected. Using a random number table, serum samples from 5 rats in each group were selected. Following the instructions of the ELISA kit strictly, the levels of TNF-α and IL-1β in the serum were determined.\u003c/p\u003e \u003cp\u003e1.2.6 Pathology of the Ankle Joint\u003c/p\u003e \u003cp\u003eAfter blood collection, the rats were euthanized by cervical dislocation. Centered on the left ankle joint, the bones were cut approximately 0.5cm above and below the joint, and the skin was removed. The specimens were fixed in 4% paraformaldehyde and then decalcified in decalcifying solution until a needle could easily penetrate the ankle joint. After decalcification, the specimens underwent routine dehydration, embedding, sectioning, and staining. Finally, the pathological changes of the synovial tissue in the left ankle joint were observed under a microscope.\u003c/p\u003e \u003cp\u003e1.2.7 Related Proteins in Ankle Joint Synovial Tissue\u003c/p\u003e \u003cp\u003eUsing a random number table, ankle joint synovial tissues from 3 rats in each group were randomly selected. After lysis and centrifugation, the supernatant was collected, and the protein concentration was determined using the BCA method. The samples were then subjected to denaturation, electrophoresis, membrane transfer, blocking, and incubation (with GAPDH, NLRP3, and Caspase-1 antibodies). Protein bands were visualized using the ECL method. With GAPDH serving as an internal reference, the grayscale values of the bands were analyzed using ImageJ software.\u003c/p\u003e \u003cp\u003e1.2.8 Statistical Analysis Method\u003c/p\u003e \u003cp\u003eGraphs were plotted and analyzed using GraphPad Prism 8.0 software. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u003cem\u003e\u0026#119909;̄ \u0026plusmn; s\u003c/em\u003e). Comparisons between groups were conducted using one-way ANOVA. For homogeneous variances, the Least Significant Difference (LSD) method was used for multiple comparisons between groups, while for heterogeneous variances, Dunnett's T3 method was employed. A P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Network Pharmacology and Molecular Docking\u003c/h2\u003e\n \u003cp\u003e2.1.1 Targets of DAPA\u003c/p\u003e\n \u003cp\u003eA total of 1, 59, 103, and 287 DAPA targets were retrieved from the DrugBank, CTD, Swiss Target Prediction, and PharmMapper databases, respectively. After standardization and removal of duplicates using the UniProt database, a final set of 410 targets was obtained.\u003c/p\u003e\n \u003cp\u003e2.1.2 Related Targets of GA\u003c/p\u003e\n \u003cp\u003eWe obtained 232, 209, and 27 GA-related targets from the GeneCards, DisGeNET, and NCBI databases, respectively. Following standardization and deduplication using the UniProt database, a total of 404 unique targets were identified.\u003c/p\u003e\n \u003cp\u003e2.1.3 Potential Targets of DAPA for GA Treatment and Construction of the PPI Network Diagram\u003c/p\u003e\n \u003cp\u003eThe targets obtained in sections 2.1.1 and 2.1.2 were uploaded to an online VENN diagram generator, resulting in the identification of 49 common targets for DAPA and GA. These 49 targets were then imported into the STRING database to construct a Protein-Protein Interaction (PPI) network diagram, which was subsequently optimized using Cytoscape 3.10.1 software. In the network analysis, the average node degree was 15.91304, with 23 nodes having a degree higher than the average and thus being identified as core targets. (Fig. 1)\u003c/p\u003e\n \u003cp\u003e2.1.4 GO and KEGG Pathway Enrichment Analysis\u003c/p\u003e\n \u003cp\u003eEnrichment analysis was conducted on the 49 common targets. The results showed that, in terms of Biological Process (BP), the targets were mainly involved in transcription regulation, inflammatory response, signal transduction, and immune response. For Cellular Component (CC), they were primarily located in the cytoplasm, nucleus, plasma membrane, and mitochondria. As for Molecular Function (MF), the targets were predominantly associated with protein binding, ATP binding, enzyme binding, and DNA binding. The KEGG pathway enrichment analysis revealed involvement in various pathways, including lipid metabolism disorders and atherosclerosis, cancer-related signaling pathways, cellular metabolic pathways, COVID-19 virus-host interactions, MAPK signaling pathway, AGE-RAGE signaling pathway in diabetic complications, C-type lectin receptor signaling pathway, Yersinia infection, and NOD-like receptor (NLR) signaling pathway. (Fig. 2)\u003c/p\u003e\n \u003cp\u003e2.1.5 Molecular Docking\u003c/p\u003e\n \u003cp\u003eBased on the KEGG pathway enrichment analysis, we selected the NOD-like receptor signaling pathway as the focus for further investigation. From the 23 core targets, we chose four targets closely related to the NOD-like receptor signaling pathway, namely IL-1\u0026beta;, TNF, Caspase-1, and NLRP3, for molecular docking. The results indicated that the binding energies for all four targets were less than \u0026minus;\u0026thinsp;7.0 kcal/mol, suggesting that DAPA may exert its effects on GA by influencing the NLRP3 pathway within the NOD-like receptor signaling pathway. (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMolecular docking results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTarget\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epdb ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVina score(kcal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCenter (x, y, z)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDocking size (x, y, z)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNLRP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7alv\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17,31,142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23,23,23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2e7a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-9.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2, -7,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23,23,23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIL-1\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1itb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43,14,19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23,23,23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaspase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3e4c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2, -9,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35,35,23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e2.2 In Vivo Experimental Validation\u003c/h3\u003e\n\u003cp\u003e2.2.1 Ankle Swelling in Rats Across Groups\u003c/p\u003e\n\u003cp\u003eCompared with the blank control group, the swelling degree of the model group at each time point significantly increased, with a significant statistical difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and reached its peak at 24 hours after modeling, indicating successful model construction; Compared with the model group, the swelling degree of rats in the 20mg/kg DAPA group significantly decreased at 72 hours after modeling (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the swelling degree of rats in the 40mg/kg DAPA group significantly decreased at both 48 hours and 72 hours after modeling (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).(Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eSwelling degree of ankle joint at different time points in each group of rats (n\u0026thinsp;=\u0026thinsp;8)\u003c/strong\u003e (\u003cem\u003e𝑥̄ \u0026plusmn; s\u003c/em\u003e, n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20mg/kg DAPA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e40mg/kg DAPA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eNote: Compared with the model, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Compared with the blank control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e2.2.2 Histopathological Changes in Ankle Synovial Tissue of Rats\u003c/p\u003e\n\u003cp\u003eIn the blank control group, the ankle joint structures of the rats were clear, with neatly arranged synovial cells and loose connective tissue. Mild infiltration of inflammatory cells was occasionally observed, but there was no synovial cell proliferation or neovascularization. Compared to the blank control group, the model group exhibited significant pathological changes, including extensive neovascularization, widespread infiltration of inflammatory cells, and synovial tissue hyperplasia. In contrast to the model group, both the 20 mg/kg and 40 mg/kg DAPA groups showed varying degrees of reduction in ankle inflammatory cell infiltration, vascular proliferation, and synovial tissue pathological damage. (Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.2.3 Levels of Inflammatory Cytokines in Peripheral Serum of Rats\u003c/p\u003e\n\u003cp\u003eThe results indicated that, compared to the blank control group, the levels of IL-1\u0026beta; and TNF-\u0026alpha; in the peripheral serum of rats in the model group were significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In comparison to the model group, the levels of IL-1\u0026beta; and TNF-\u0026alpha; in the peripheral serum of rats treated with 20 mg/ml and 40 mg/ml DAPA were significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). (Figure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003e2.2.4 Expression Levels of Caspase-1 and NLRP3 Proteins in Ankle Synovial Tissue of Rats\u003c/p\u003e\n\u003cp\u003eThe results demonstrated that, compared to the blank control group, the expression of Caspase-1 and NLRP3 in the ankle synovial tissue of rats in the model group was significantly upregulated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast to the model group, the expression of both Caspase-1 and NLRP3 in the synovial tissue of rats treated with 20 mg/ml DAPA was significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, rats treated with 40 mg/ml DAPA showed significant reductions in the expression of Caspase-1 and NLRP3 in their synovial tissue (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). (Figure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eExpression of Caspace-1 and NLRP3 proteins in ankle synovial tissue of rats in each group (n\u0026thinsp;=\u0026thinsp;3)\u003c/strong\u003e (\u003cem\u003e𝑥̄ \u0026plusmn; s\u003c/em\u003e, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCaspace-1/GAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNLRP3/GAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20mg/kg DAPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003e*#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003e** ##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40mg/kg DAPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003e** ##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003eNote: Compared with the model, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Compared with the blank control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discuss","content":"\u003cp\u003eGout, a metabolic inflammatory disease triggered by abnormal urate metabolism, is pathologically underpinned by elevated serum uric acid levels that surpass the solubility threshold in the blood, leading to the deposition of monosodium urate (MSU) crystals in tissues or joints. These deposited MSU crystals are recognized as endogenous danger signals—damage-associated molecular patterns (DAMPs)—by pattern recognition receptors (PRRs) in the innate immune system, including Toll-like receptors and NOD-like receptors\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Notably, the NLRP3 inflammasome within the NLR family plays a pivotal role\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Activated by MSU crystals, the NLRP3 inflammasome cleaves pro-IL-1β and pro-IL-18 via Caspase-1, converting them into mature IL-1β and IL-18, which are then released extracellularly to further promote the production of inflammatory cytokines and chemokines, such as IL-6, IL-8, and TNF-α\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The synergistic effects of these cytokines enhance the recruitment and activation of inflammatory cells and activate nociceptors, directly exacerbating joint pain and inflammation\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Without timely intervention, MSU crystals continue to accumulate in the body. Even during gout remission, MSU crystals can persistently trigger systemic inflammatory responses by activating inflammatory pathways, enhancing interactions between inflammatory cells, and elevating arachidonic acid metabolic activity\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. This persistent inflammatory state ultimately leads to severe complications such as joint deformity and gouty nephropathy\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Therefore, effective intervention and management of gout patients (GA) are crucial.\u003c/p\u003e\u003cp\u003eDAPA, a member of the SGLT2 inhibitor family, selectively inhibits the high-capacity glucose transporter SGLT2 in the renal proximal tubules, reducing glucose reabsorption in the kidneys and thereby lowering glucose levels independently of insulin\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, as research on this class of drugs has deepened, it has been discovered that, apart from lowering blood glucose, they also possess anti-inflammatory, antioxidant properties, promote weight loss, regulate blood pressure, and improve cardiovascular and chronic kidney disease outcomes\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Recently, a large meta-analysis of its urate-lowering efficacy showed that DAPA significantly reduced serum uric acid levels in a dose-dependent manner (5–50 mg, P = 0.014) compared to controls\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Regarding gout attacks, multiple clinical trials and meta-analyses have indicated that DAPA significantly reduces the risk of gout in patients with type 2 diabetes or heart failure and decreases the need for antigout medications in gout patients\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. This phenomenon is primarily attributed to its urate-lowering effect\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, a recent meta-analysis by Mainak Banerjee et al. found no statistically significant correlation between the urate-lowering efficacy of SGLT2 inhibitors and the reduction in gout attack risk (P \u0026gt; 0.3) \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Combined with DAPA's vast potential in anti-inflammatory, antioxidant, and immune metabolism fields—such as attenuating lipopolysaccharide-induced acute lung injury in rats by regulating the AMP-activated protein kinase/nuclear factor kappa B pathway and inhibiting NLRP3 activation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e;enhancing colonic autophagy and inhibiting apoptosis to improve colitis in rats through the nuclear factor E2-related factor 2/heme oxygenase-1 pathway\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e༛and delaying osteoarthritis progression by reducing endoplasmic reticulum stress in chondrocytes\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Based on this, the role of DAPA in anti-gout may involve multifaceted physiological mechanisms.\u003c/p\u003e\u003cp\u003eNetwork pharmacology, as an interdisciplinary combining systems biology, computer science and pharmacology, studies the interaction between drugs and targets by analyzing biomolecular networks\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In this study, we used network pharmacology to intersect the targets of DAPA and GA disease-related targets, and obtained 49 common targets of both. Among them, there are 23 core targets, including IL1β、TNF、TGF-β、Caspase-1、RELA、NLRP3、PTGS2、SIRT1, etc. At. Meanwhile, further combining the results of go and KEGG pathway enrichment analysis, nlrp3/caspase-1 signaling pathway in NOD like receptor signaling pathway was selected to verify the potential mechanism of DAPA in anti-GA effect. On this basis, the core targets closely related to the nlrp3/caspase-1 signaling pathway, IL-1 β, TNF, caspase-1 and NLRP3, were selected for molecular docking. The results showed that the binding energies of the above four targets with DAPA were significantly lower than − 7.0 kcal / mol, suggesting that DAPA may play an anti-GA role by regulating the nlrp3/caspase-1 signaling pathway.\u003c/p\u003e\u003cp\u003eSubsequently, to further validate this pathway, we established a rat model of GA and administered DAPA for treatment. The results indicated that, compared with the model group, the DAPA-treated group exhibited improved ankle joint swelling and synovial tissue hyperplasia, reduced inflammatory cell infiltration, and decreased levels of inflammatory cytokines IL-1β and TNF-α in the peripheral blood. Additionally, by quantifying the protein expression levels of NLRP3 and Caspase-1 in the ankle joint synovial tissue, we found that DAPA treatment significantly downregulated the expression of both NLRP3 and Caspase-1 compared to the model group. These findings suggest that DAPA may alleviate the symptoms of acute gouty arthritis by inhibiting the activation of the NLRP3/Caspase-1 pathway, thereby reducing the inflammatory response.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo sum up, our study shows that DAPA has a preventive and therapeutic effect on MSU crystal induced GA in rat models, which may be achieved by reducing joint tissue damage, reducing the level of inflammatory cytokines in the blood and inhibiting the NLRP3/caspase-1 signal pathway. However, this study only selected four key targets closely related to NLRP3/caspase-1 signaling pathway for preliminary verification, and did not carry out in vitro experiments for further confirmation. At the same time, in view of the significant potential of DAPA in anti-inflammatory, antioxidant and immune metabolism, its multiple mechanisms of action in the treatment of acute GA still need to be further explored in future research.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edapagliflozin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egouty arthritis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonosodium urate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGBD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlobal Burden of Diseases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSGLT2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium-glucose cotransporter 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBiological Process\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCellular Component\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolecular Function\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-1β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterleukin-1β\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor-α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLRP3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNOD-like receptor thermal protein domain associated protein 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCaspase-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecysteinyl aspartate specific proteinase-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePattern Recognition Receptors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAMPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDamage-associated molecular patterns\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNOD-like receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the observational study on the involvement of PKM2 in the regulation of malignant cell function in diffuse large B-cell lymphoma [No.21377797D].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchool of Clinical Medicine, Hebei University of Engineering, Handan, Hebei, 056002, China\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYe Tao and Li Pian\u003c/p\u003e\n\u003cp\u003eAffiliated Hospital of Hebei University of Engineering，Handan, Hebei, 056002, China\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDu Jingfang and Shen Na\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTao Ye was responsible for the experimental operation, data statistics and paper writing; Du Jingfang was responsible for the methodology design; Li Pian was responsible for the experimental data collection; and Na Shen provided program planning, project funding, and guidance for writing and revising the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNa Shen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments involved in this study have been reviewed and approved by the Laboratory Animal Ethical and Welfare Committee of Affiliated Hospital of Hebei Engineering University (Approval No.: IACUC-Hebeu-2023-0029). The experimental process was strictly carried out in accordance with the relevant laws, regulations, and rules on experimental animals in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNARANG R K. Pathophysiology of Gout [J]. \u003cem\u003eSemin Nephrol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e (6), 550\u0026ndash;563 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTANG S C W \u0026amp; Gout A Disease of Kings [J]. \u003cem\u003eContrib. Nephrol.\u003c/em\u003e \u003cb\u003e192\u003c/b\u003e, 77\u0026ndash;81 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHU, B. et al. 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(Craiova, Romania), 10(3): e152. (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBANERJEE, M. et al. Serum uric acid lowering and effects of sodium-glucose cotransporter-2 inhibitors on gout: A meta-analysis and meta-regression of randomized controlled trials [J]. \u003cem\u003eDiabetes Obes. Metab.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (9), 2697\u0026ndash;2703 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eABD EL-FATTAH E E, S. A. B. E. R. S. et al. \u003cem\u003eThe dynamic interplay between AMPK/NFκB signaling and NLRP3 is a new therapeutic target in inflammation: Emerging role of dapagliflozin in overcoming lipopolysaccharide-mediated lung injury [J]\u003c/em\u003e147112628 (Biomedicine \u0026amp; pharmacotherapy\u0026thinsp;=\u0026thinsp;Biomedecine \u0026amp; pharmacotherapie, 2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eARAB H H, AL-SHORBAGY M Y, SAAD, M. A. Activation of autophagy and suppression of apoptosis by dapagliflozin attenuates experimental inflammatory bowel disease in rats: Targeting AMPK/mTOR, HMGB1/RAGE and Nrf2/HO-1 pathways [J]. \u003cem\u003eChemico-Biol. Interact.\u003c/em\u003e \u003cb\u003e335\u003c/b\u003e, 109368 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLIU, Z. et al. Dapagliflozin suppress endoplasmic reticulum stress mediated apoptosis of chondrocytes by activating Sirt1 [J]. \u003cem\u003eChemico-Biol. Interact.\u003c/em\u003e \u003cb\u003e384\u003c/b\u003e, 110724 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNOOR, F. et al. Machine learning for synergistic network pharmacology: a comprehensive overview [J]. \u003cem\u003eBrief. Bioinform.\u003c/em\u003e, \u003cb\u003e24\u003c/b\u003e(3). (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gout, Gouty arthritis, Dapagliflozin, Rat","lastPublishedDoi":"10.21203/rs.3.rs-5723942/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5723942/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective \u003c/strong\u003eExploring the preventive and therapeutic effects of dapagliflozin (DAPA) on gouty arthritis (GA) in rats, and revealing its potential mechanism of action.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003ePotential targets of DAPA were identified from DrugBank, Swiss Target Prediction, CTD, and PharmMapper databases. Targets associated with gouty arthritis (GA) were retrieved from Gene Cards, DisGeNET, and NCBI databases. By taking the intersection of these two sets, common targets of DAPA and GA were determined. These common targets were then subjected to Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Use the CB-DOCK2 online molecular docking platform to dock DAPA with the core target and perform visual analysis. Thirty-two SPF-grade male SD rats were randomly divided into four groups, with eight rats in each: a blank control group, a model group, a 20 mg/kg DAPA group, and a 40 mg/kg DAPA group. Rats received daily gavage administration of the corresponding medication for eight consecutive days. On the fifth day, monosodium urate (MSU) crystal suspension was injected into the left ankle joint to establish an acute GA model. Samples were collected one hour after the final gavage. The swelling of the ankle joints was recorded at various time points. Hematoxylin and eosin (HE) staining was used to observe pathological changes in the synovial tissue of the ankle joints. Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the levels of inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the peripheral blood of the rats. Western blotting was performed to detect the expression levels of signaling pathway proteins in the synovial tissue of the ankle joints.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Based on network pharmacology analysis and molecular docking, it was found that targets were significantly enriched in the nucleotide binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway, and the binding energies between the related core targets and DAPA were all \u0026lt;-7.0 kcal/mol. In animal experiments, regarding ankle joint swelling: compared with the model group, the 20 mg/kg DAPA group showed a significant reduction in ankle joint swelling at 72 hours post-modeling (p\u0026lt;0.05), and the 40 mg/kg DAPA group exhibited significant reductions in ankle joint swelling at both 48 and 72 hours post-modeling (p\u0026lt;0.01). For ankle joint HE staining: compared with the model group, DAPA-treated groups showed varying degrees of attenuation in pathological damage, including inflammatory cell infiltration, synovial tissue proliferation, and vascular proliferation in the ankle joints. Peripheral blood ELISA results: the levels of IL-1β and TNF-α in DAPA-treated groups were significantly lower than those in the model group (p\u0026lt;0.05). As for the protein expression levels of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) and cysteinyl aspartate-specific proteinase-1 (Caspase-1) in ankle joint synovium: compared with the model group, the expression of NLRP3 and Caspase-1 proteins was significantly reduced in DAPA-treated groups (p\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eDAPA may alleviate the inflammatory response in acute GA in rats by inhibiting the NLRP3/Caspase-1 pathway.\u003c/p\u003e","manuscriptTitle":"Based on network pharmacology-molecular docking and experimental exploration, the preventive and therapeutic effects of dapagliflozin on gouty arthritis in rats were investigated","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 17:10:17","doi":"10.21203/rs.3.rs-5723942/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e397b2f7-d252-446c-90db-74204d90e81c","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42499819,"name":"Health sciences/Rheumatology/Rheumatic diseases/Acute inflammatory arthritis"},{"id":42499820,"name":"Health sciences/Rheumatology/Rheumatic diseases/Metabolic bone disease"}],"tags":[],"updatedAt":"2025-09-18T10:09:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-09 17:10:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5723942","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5723942","identity":"rs-5723942","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}