Dual Mechanisms of Naru Sanwei Pills in Gout: NLRP3 Inflammasome Inhibition and Uric Acid Regulation

Introduction

Gout, one type of arthritis that causes inflammation of the joints due to excessive uric acid, affects 1–4% of the global population with an incidence of 0.1–0.3%, with higher prevalence in developed nations compared to developing ones.^1,2 This condition, characterized by recurrent episodes of severe pain, swelling, warmth, and redness in the joints, is often associated with several other diseases, including hypertension, hyperlipidemia, gouty nephropathy, diabetes, and obesity.³ Due to a number of factors, including obesity and metabolic syndrome, the prevalence of gouty diseases has been increasing in recent 30 years, and the incidence of gouty patients has also been on the rise,⁴ creating a significant public health concern. Recurrent gout attacks, the complexity of treatment, and the increasing economic burden on patients have made gout a significant global health concern.

Gouty arthritis (GA) is considered an autoinflammatory disease (AID), gout develops when the concentration of uric acid in the blood is too high, uric acid precipitates in the form of monosodium urate (MSU) crystals that accumulate in the joints, triggering inflammation.⁵ MSU crystals activate the innate immune system, particularly macrophages, triggering a robust inflammatory response in localized joints and peri-articular tissues.^6,7 The NLRP3 inflammasome is a multiprotein complex consisting of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD (ASC), which plays a pivotal role in the regulation of inflammatory processes. Functioning as a pattern recognition receptor for invading pathogens, NLRP3 facilitates the cleavage of pro-IL-1β and pro-IL-18 into their mature, active forms, thereby initiating a robust inflammatory response.^8,9 Studies have shown that NLRP3 inflammasome activation by MSU crystals leads to the release of cytokines IL-1β and IL-18, ultimately contributing to cell death. Furthermore, IL-1β sensitizes or directly activates nociceptors in the peripheral sensory nervous system, causing severe pain.⁶ Therefore, proinflammatory cytokines, particularly IL-1β, play a pivotal role in gout.¹⁰

Hyperuricemia, defined by increased concentrations of uric acid in the bloodstream, constitutes a critical factor in the development and advancement of gout. Hyperuricemia also poses an independent risk for various complications, including kidney damage, joint deformities, periodontitis and metabolic syndrome.^11–15

Therefore, managing both hyperuricemia and the inflammatory response is crucial for effective treatment of gout. However, current clinical drugs often produce serious adverse reactions. For example, febuxostat and colchicine, commonly used drugs for hyperuricemia and gout, have been shown to cause side effects.¹⁶ Moreover, current studies have primarily focused on either anti-inflammatory effects or uric acid-lowering strategies, and there remains a lack of comprehensive investigations targeting both aspects simultaneously. Therefore, therapies with dual regulatory effects on inflammation and uric acid metabolism are of significant clinical interest. Plant medicines, which often possess uric acid-lowering and anti-inflammatory properties, offer potential alternatives. Therefore, finding a safe and efficient drug candidate for the treatment of gout is essential.

Naru Sanwei Pills (Naru-3) is a traditional herbal formulation commonly used in Mongolian clinical medicine. It is composed of three herbs: Piper longum L (biba.), Terminalia chebula Retz (hezi.) and Aconitum kus-nezoffi Rchb (caowu). Naru-3 is known for its effects of dispelling wind, relieving pain, and dispersing cold.¹⁷ It is used for various conditions, including rheumatism, joint pain, cold pain in the back and legs, toothache, and diphtheria.¹⁸ Commonly used clinically in the treatment of gouty arthritis.¹⁹ Naru-3 has been found to be significantly effective in the treatment of intervertebral disc degeneration (IDD).²⁰ In addition, Naru-3 showed significant anti-inflammatory effects in traumatic spinal cord injury (TSCI) and initiation of neuroinflammation of neuropathic pain.^21,22 Naru-3 is also commonly used to alleviate the symptoms of rheumatoid arthritis and works by inhibiting joint inflammation, synovial hyperplasia and neovascularisation.²³ However, anti-gout mechanism of Naru-3 is not clear. We hypothesized that Naru-3 exerts its anti-gout effects through a dual mechanism, simultaneously suppressing NLRP3 inflammasome-mediated inflammation and correcting hyperuricemia. Therefore, the present study seeks to investigate the potential effects of Naru-3 on uric acid reduction and anti-gout activity utilizing a PO-induced hyperuricemia mouse model as well as a MSU-induced acute gouty arthritis mouse model.

Materials and Methods

Reagents and Drugs

Naru-3 (Inner Mongolia, China); Uric acid sodium salt (Sigma-Aldrich); PO (aladdin); Allopurinol (Hefei Jiulian Pharmaceutical); Colchicine (Xishuangbanna Banna Pharmaceutical Co); Trizol reagent (Invitrogen); Myristic Acid, Luteolin, Azelaic Acid, Quercetin, Formononetin and Matairesinol (Yuanye); Kits for XOD, ADA, UA, Cre, and BUN (Nanjing Jiancheng); BCA protein concentration assay kit (Beyotime); HPLC grade methanol and acetonitrile (Merck); ELISA kits for TNF-α and IL-1β (R&D). Anti-NLRP3 (CST, #15101), Anti-Caspase-1 (CST, #24232), Anti-ASC (CST, #67824), and Anti-IL-1β (CST, #31202); Anti-GLUT9 (Proteintech, 26486) and Anti-URAT1 (Proteintech, 82964); Anti-GAPDH (ABclonal, A19056), Anti-β-actin (ABclonal, AC038) and Anti-OAT3 (ABclonal, A14575).

Animal Preparation

Male C57BL/6 mice, weighing between 18 and 22 grams, were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Certificate No. SCXK 24 2020-0001), located in Benxi, China. NLRP3^−/− and Caspase-1^−/− mice on a C57BL/6 genetic background were purchased from Shanghai Nanmo Biotechnology Co., Ltd. (Certificate NO. SYXK 2017-0012, Shanghai, China). Age- and sex-matched wild-type C57BL/6 mice were used as controls in the experiments to ensure a comparable genetic background. To adapt to the unfamiliar environment, the mice were housed in an specific pathogen free (SPF) environment with a temperature of 24–25 °C, a humidity of 60–70%, a 12 h light-dark light cycle every day, and a normal diet for one week. All animal experiments are conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of South-Central Minzu University. The animal study was reviewed and approved by Animal Ethics Committee of Renmin Hospital of Wuhan University (approval number: WDRY2021-KS013).

RNA Sequencing and Transcriptomic Analysis

Sample Preparation, Sequencing, and Data Processing

Total RNA was extracted from the ankle joint synovial tissues using Trizol reagent and assessed for integrity and purity using an Agilent 2100 Bioanalyzer. High-quality RNA samples were used to construct sequencing libraries, which were subsequently sequenced on an Illumina platform to generate paired-end reads. Raw data were filtered to remove adapters and low-quality sequences. The resulting clean reads were aligned to the Mus musculus reference genome using HISAT2, and gene expression levels were quantified using featureCounts and normalized as Fragments Per Kilobase of transcript per Million mapped reads (FPKM).

Differential Expression and Functional Enrichment Analysis

Statistical analyses were performed in R 4.5.1. The global similarity of gene expression profiles among groups was visualized using a Pearson correlation heatmap. Differential expression analysis was conducted using the DESeq2 package, with an adjusted P-value < 0.05 and |log2FoldChange|≥1serving as the thresholds for identifying differentially expressed genes (DEGs). To elucidate the underlying molecular mechanisms, KEGG pathway enrichment analysis was performed using the clusterProfiler package. Furthermore, Gene Set Enrichment Analysis (GSEA) was utilized to evaluate the activation or suppression of specific signaling pathways, based on the Normalized Enrichment Score (NES) and false discovery rate (FDR).

Characterization of Naru-3 Constituents Using UPLC-Q/TOF-MS Analysis

This study employed ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) to identify and characterize the chemical constituents of Naru-3. Chromatographic separation was achieved using an Acquity HSS T3 column (2.1 mm × 100 mm, 1.8 μm) with a binary mobile phase gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The flow rate was maintained at 0.4 mL/min, and the column temperature was set to 40 °C. Mass spectrometric detection was carried out using an electrospray ionization (ESI) source in both positive and negative ion modes.

Network Pharmacology

The files of the compound structures were first downloaded from the PubChem database, and the downloaded files were imported into the Swiss ADME website, and the compounds were screened for their gastrointestinal absorption “High” and three or more “Yes” in the five principles of drug-like substances. Swiss Target Prediction can be used as a tool to get Naru-3 compound targets. Then, we searched “Gout” in GeneCards database to obtain the gout target. The Venny platform was used to plot the intersection of the two targets in a Wayne diagram to obtain the potential targets of Naru-3 for the treatment of gout. The obtained drug-disease potential targets were imported into Cytoscape software to construct the drug-component-target-disease interaction diagram. We also searched the STRING database for potential targets of Naru-3 for gout, predicted the gene-target relationship, and then PPI network diagrams were obtained using Cytoscape software. GO and KEGG enrichment of the above obtained core targets were performed using Enrichr and OmicShare web platforms and the resultant maps were obtained. The potential pathways and biological processes of Naru-3 were analyzed in conjunction with the resultant maps.

Molecular Docking Studies

To perform molecular docking simulations, the three-dimensional structures of the identified compounds were obtained from the PubChem database. The crystal structures of the target proteins, all of human origin, were downloaded from the Protein Data Bank (PDB, https://www.rcsb.org/). The carrier proteins were dehydrogenated, hydrogenated and molecularly docked with small molecule compounds using SYBYL 2.1.1 software, and the larger the Total Score of the docking result indicates the higher binding capacity of the protein to the compound, the final visualization was done by PyMOL software.

Preparation of Naru-3

Naru-3 is supplied by Inner Mongolia Meng Pharmaceutical Co. We ground the pill into powder and dissolved it with 0.5% CMC-Na. The dose of 140 mg/kg was determined based on the conversion of the body surface area between humans and mice. In addition, the group’s prior research demonstrated that when administered at a dose of 280 mg/kg for seven days, the mice exhibited smooth and glossy fur, normal behavioral patterns, unobstructed breathing, a regular diet and no abnormal lesions in their internal organs, and no toxic reactions were detected (unpublished data).

Grouping and Details of Animals

Air Pouch Inflammation Model

Based on previous studies, a model of air pouch inflammation was prepared.²⁴ The mice were anesthetized, the back hair above 2 cm from the tail of the mice was shaved with a shaver, and the remaining hair was cleaned by applying depilatory cream to expose the back skin. After disinfection with 75% alcohol, 5 mL of sterile air treated with a 0.22 μm filter was injected near the depilation position at the base of the tail to form an air pouch. On the 4th day, check whether the back air pouch is complete, and then add 3 mL of sterile air. On the 6th day, there was no air leakage, inflammation, etc.

On day 6, all mice were divided into 9 groups (n=6/group): (1) Control group (a, 0.5% CMC-Na); (2) MSU group (b, 3 mg/mL MSU); (3) Colchicine (c, 0.8 mg/kg) group; (4) Myristic Acid (d, 10 mg/kg) group; (5) Luteolin (e, 10 mg/kg) group; (6) Azelaic Acid (f, 10 mg/kg) group; (7) Quercetin (g, 10 mg/kg) group; (8) Formononetin (h, 10 mg/kg) group; (9) Matairesinol (i, 10 mg/kg) group. The colchicine group was given intragastric administration, The remaining groups were injected with 1 mL of the corresponding drug in the air pouch. After 1 h of administration, except the blank group, which was injected with 1 mL of sterile PBS solution into the air pouch, the other groups were injected with 1 mL of MSU to induce synovial inflammation.

Acute Gouty Arthritis Model

The model based on previous research protocol.²⁵ All mice were divided into 6 groups (n=7/group): (1) Control group (0.5% CMC-Na); (2) MSU group; (3) Colchicine group (0.8 mg/kg) group; (4) Naru-3 low dose group (70 mg/kg); (5) Naru-3 medium dose (140 mg/kg) group; (6) Naru-3 high dose (280 mg/kg) group. Colchicine and Naru-3 were freshly suspended in 0.5% CMC-Na. The Colchicine group and Naru-3 groups (70, 140, and 280 mg/kg) were given corresponding doses of drugs intragastric administration, respectively. The control group and MSU group were administered an equal volume of 0.5% CMC-Na by gavage. Three days after administration, except for the control group, all other groups were injected into the ankle joint with 20 μL/1 mg MSU crystal suspension to induce an acute gout model in mice. The experiment was carried out for 5 days.

Hyperuricemic Nephropathy Mouse Model

The hyperuricemic nephropathy model was established following the methodology described in previous research.²⁶ Specifically, hyperuricemic nephropathy was induced in mice via intraperitoneal administration of potassium oxonate (PO) at a dose of 250 mg/kg. The experimental subjects were divided into six groups (n = 8 per group): (1) Control group (0.5% CMC-Na); (2) PO group; (3) allopurinol (ALL) group receiving 5 mg/kg; (4) Naru-3 group receiving 70 mg/kg; (5) Naru-3 group receiving 140 mg/kg; and (6) Naru-3 group receiving 280 mg/kg. Hyperuricemia was induced by intraperitoneal injection of PO at 300 mg/kg one hour prior to drug administration. Both allopurinol and Naru-3 powders were suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) solution and administered orally via gavage. One hour following PO injection, the ALL group received allopurinol at 5 mg/kg, while the treatment groups were administered Naru-3 at doses of 70, 140, or 280 mg/kg. All treatments were administered once daily for a duration of 28 consecutive days.

Pretreatment of Samples

Within 48 hours of treatment in the acute gouty arthritis model, various indicators were detected. The mice were euthanized by CO₂ inhalation, the skin tissue of the hindlimb on the modeling side was peeled off, the tissue at 0.5 cm above and below the ankle joint was cut, 4 joints were randomly selected in each group, the joint cavity was washed by 500 μL of pre-cooled PBS from a syringe, and the lavage fluid was collected for ELISA detection. Joint tissue was taken for Western blot analysis. The remaining ankle joints were stripped of excess tissue and then fixed in 4% paraformaldehyde for pathological examination.

The animals were euthanized by CO₂ inhalation after 6 h of treatment in the air pouch inflammation model. Pre-cooled PBS (4 mL) was injected into the air pouch, and the mice were gently shaken repeatedly to collect the lavage fluid, which was then analyzed using ELISA kits. The excised air pouch tissue was fixed in 4% paraformaldehyde for pathological examination.

After 28 days of modeling and treatment, mice were placed in metabolic cages to collect urine, and the urine was centrifuged to remove impurities. Blood was collected via orbital venous plexus sampling, after which the mice were euthanized by CO₂ inhalation. Kidneys were fixed in 4% paraformaldehyde for pathological examination. Blood samples were centrifuged for 10 minutes, and the supernatant was collected as serum. All urine, serum, and tissue samples were properly stored for further analysis.

Analysis of Urine and Serum

Levels of UA, Cre, BUN were measured using Nanjing Jiancheng kits according to instructions. FEUA according to a previously reported formula,²⁷ namely: FEUA = (Uur×Scr)/(Sur×Ucr)× 100%. Uur (urine uric acid); Scr (serum creatinine); Sur (serum uric acid); Ucr (urine creatinine).

Assay for the Activity of XOD and ADA

The liver tissue was weighed, 9 times the volume of cold 0.9% NaCl solution was added to each 100 mg, and mechanical homogenization was performed to make into a 10% homogenate, centrifuged for 10 min at 4 °C, and the upper layer was collected as sample. XOD activity and ADA activity of sample were assayed according to the instructions.

ELISA Assay

Centrifuge the lavage solution and take the upper layer to remove impurities. Levels of IL-1β and TNF-α were measured using R&D ELISA kits according to the kit instructions.

Histological Analysis

All tissues, including air pouch, joint, and kidney samples, were fixed in 4% paraformaldehyde for a duration exceeding 24 hours. Subsequently, paraffin-embedded sections were prepared and stained with H&E following standard protocols. Notably, joint tissues underwent decalcification using EDTA prior to paraffin embedding. The processed sections were then individually examined and imaged using a light microscope.

To quantitatively assess tissue damage, histopathological scoring was performed by two independent pathologists blinded to the experimental groups. For kidney sections, tubular injury was evaluated on a scale of 0 to 4 based on the percentage of affected area (tubular dilation, epithelial atrophy, and cast formation): 0, normal; 1, <25%; 2, 25%–50%; 3, 51%–75%; and 4, >75%. For joint tissues, the severity of arthritis was scored from 0 to 4 considering synovial hyperplasia, inflammatory cell infiltration, and cartilage erosion: 0, normal; 1, slight; 2, moderate; 3, marked; and 4, severe. For air pouch samples, inflammation was quantified by scoring the thickness of the lining layer and the degree of leukocyte infiltration on a scale of 0 to 4. Five random fields were selected per section for analysis, and the average score was calculated for each sample.²⁸

Western Blot Analysis

Western blot analysis was conducted following the protocol established by Hornung et al²⁹ RIPA lysate (containing protease inhibitors) was added to the homogenization tube at a volume of 1:10, and grinding beads were added, and cryogenically ground. The protein solution was obtained by centrifugation and assayed using the BCA kit. The total protein was then mixed with 1/4 of the loading buffer and boiled in a metal bath for 10 minutes. The prepared protein solution was separated using a suitable SDS-PAGE gel, blocked with 5% nonfat milk blocking solution and transferred to PVDF membranes. Incubate the membrane with the appropriate primary antibody overnight followed by a secondary anti-IgG antibody for 90 minutes. Detect proteins using a bioradiation chemiluminescence imaging system. Analyze protein bands by Image J.

Gene Expression Analysis

Total RNA was isolated from kidney tissues using Trizol reagent following standard protocols. RNA samples were reverse transcribed into cDNA using the PrimeScript RT kit. Quantitative real-time PCR (qRT-PCR) was performed on a CFX system (Bio-Rad) using SYBR Green Master Mix Kit to quantify the mRNA levels of GLUT9, URAT1, and OAT3. Relative mRNA expression levels were calculated using the 2^−ΔΔCt method, and normalized to GAPDH as an internal control. The primer sequences used for qRT-PCR are listed in Table 1.

Table 1 Primer Sequences Used RT-PCR Gene Expression Studies

Statistical Analysis

All data were statistically analyzed using GraphPad Prism software. Data are presented as mean ± standard deviation. During data quantification and statistical analysis, investigators were blinded to group allocation to minimize potential bias. Multiple comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. P-value less than 0.05 was considered statistically significant.

Results

Naru-3 Relieves MSU-Induced Acute Gouty Arthritis

To investigate the anti-gout efficacy of Naru-3, we induced acute gouty arthritis in mice by injecting MSU into their ankle joints. The results showed that Naru-3 treatment effectively reduced paw swelling compared to MSU-treated mice (Figure 1A). Clinical studies have established that MSU-induced gouty arthritis elicits a strong pain response. It was observed that MSU injection resulted in severe weight-bearing imbalance, while Naru-3 mitigated this effect (Figure 1B). Gouty arthritis, induced by MSU in the ankle joint, also leads to a local temperature increase. The temperature change was assessed, and it was found that Naru-3 significantly inhibited MSU-induced temperature elevation (Figure 1C). Furthermore, the response of gouty arthritis mice to mechanical pain stimulation and applying plantar thermal stimulation was also evaluated. Naru-3 exhibited a significant analgesic effect, reducing the paw withdrawal and licking responses (Figure 1D and E). Histological analysis revealed that MSU-induced gouty arthritis was characterized by significant joint swelling, severe tissue damage, extensive inflammatory cell infiltration, and synovial hyperplasia. Naru-3 treatment significantly inhibited inflammatory cell influx and alleviated MSU-induced inflammatory symptoms, and significantly reduced its pathological score (Figure 1F and G). Those results show that Naru-3 effectively inhibited the release of IL-1β and TNF-α (Figure 1H and I).

Figure 1 The effects of Naru-3 treatment on gouty arthritis in mice. (A) Paw swelling, (B) Two-paw gravity distribution, (C) Changes in paw temperature, (D) Paw withdrawal threshold for mechanical and (E) Thermal stimulation were detected at different time points. (F) Ankle photograph taken 24 h after intra-articular injection of MSU crystals, with H&E staining of the joint and (G) scoring. Scale bars, 500 μm and 200 μm. (H) IL-1β and (I) TNF-α levels of the lavage fluid of joint cavity determined by ELISA. Data are presented as mean ± SD (n = 7). Statistical analysis was performed using one-way analysis of variance (ANOVA). For figure (A–E) comparisons were performed at each time point: #Indicates comparison with the Control group at the same time point; *Indicates comparison with the MSU group at the same time point. ###p < 0.001, compared with Control group; *p< 0.05, **p< 0.01, ***p < 0.001, compared with MSU group.

Study on the Anti-Inflammatory Active Targets and Active Components of Naru-3

To further elucidate the anti-inflammatory mechanisms of Naru 3, transcriptomic analysis of the ankle joint synovium was performed. The sample correlation heatmap revealed distinct gene expression profiles between the Model and Control groups, whereas the Naru 3-treated group exhibited a transcriptional signature closer to that of the Control group (Figure 2A). Compared with the Control group, the Model group showed 1876 upregulated and 2112 downregulated differentially expressed genes (DEGs). Conversely, Naru 3 treatment resulted in the upregulation of 1730 genes and the downregulation of 1513 genes compared to the Model group (Figure 2B and C). KEGG pathway enrichment analysis highlighted significant activation of the NOD-like receptor and NF-kappa B signaling pathway in the Model group. Notably, Naru 3 treatment effectively reversed the aberrant activation of these inflammatory pathways (Figure 2D and E). Gene Set Enrichment Analysis (GSEA) The plots further confirmed that the model group was significantly enriched with NOD-like receptor and NF-kappa B signaling pathway (Figure 2F and G), and Naru 3 reversed this result (Figure 2H and I).

Figure 2 Transcriptomic analysis of synovial tissues in gout model mice treated with Naru 3. (A) Sample correlation heatmap showing the clustering of gene expression profiles among Control, Model, and Naru 3 groups (n=3 per group). The color scale indicates the Pearson correlation coefficient. (B and C) Volcano plots of differentially expressed genes (DEGs) in (B) Model vs. Control and (C) Naru 3 vs. Model comparisons. Red: upregulated; Blue: downregulated. (D and E) KEGG pathway enrichment analysis of (D) genes upregulated in the Model group and (E) genes downregulated by Naru 3 treatment. The size of the dots represents the number of genes, and the color gradient represents the significance level. (F–I) Gene Set Enrichment Analysis (GSEA) plots, (F and G) Enrichment of the NOD-like receptor and NF-κB signaling pathways in the Model group compared to Control, (H and I) Suppression of the NOD-like receptor and NF-κB signaling pathways in the Naru 3 group compared to Model. Abbreviation: NES, normalized enrichment score.

Study on the Anti-Inflammatory Active Targets and Active Components of Naru-3

UPLC-Q/TOF-MS analysis was performed to characterize the chemical composition of Naru-3, and total ion chromatograms were obtained in both positive and negative ion modes (Figure 3A). After removing duplicate entries, a total of 159 compounds with composite scores greater than 80 were identified (Supplementary Table 1 and Supplementary Appendix 1). Subsequent screening based on gastrointestinal absorption properties yielded 93 candidate compounds. These compounds were subjected to target prediction analysis, resulting in 245 potential targets (Supplementary Table 2). In parallel, 1913 gout-related targets were retrieved from the GeneCards database (Supplementary Table 3). Intersection analysis identified 91 common targets, which were considered potential therapeutic targets of Naru-3 (Supplementary Figure 1A, B and Supplementary Table 4). A protein–protein interaction (PPI) network was constructed, leading to the identification of 17 core active compounds (Supplementary Figure 1C).

Figure 3 Proposed Anti-Gout Mechanism of Naru-3. (A) Total ion flow diagrams in negative (column 1, black) and positive (column 2, red) ion modes. (B) GO biological process enrichment analysis. (C) KEGG pathway enrichment analysis. (D) Molecular docking results of Formononetin, Luteolin, and Quercetin with NLRP3 protein. (E) Molecular docking results of Myristic Acid, Azelaic Acid and Matairesinol with Caspase-1 protein. (F) Molecular docking results of Myristic Acid, Azelaic Acid and Matairesinol with ASC protein. (G) Molecular docking results of Luteolin, Azelaic Acid and Matairesinol with IL-1β protein. (H) Hematoxylin and eosin (H&E) staining of skin tissue (scale bar = 200 μm). (I and J) Enzyme-linked immunosorbent assay (ELISA) for TNF-α (I) and IL-1β (J) levels in air pouch washings. Data are presented as mean ± standard deviation (n = 6). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. Symbols indicate comparisons between the indicated groups. ###p < 0.001, compared with Control group; *p< 0.01, **p< 0.01, ***p < 0.001, compared with MSU group.

GO and KEGG enrichment analyses revealed significant enrichment in inflammatory pathways, including TNF signaling, NF-kappa B signaling, and NOD-like receptor signaling pathways (Figure 3B and C).

NLRP3 inflammasome has a crucial role in mediating acute symptoms of gout, including MSU deposition and immune response in vivo, we performed molecular docking of the identified 17 active chemical compound with NLRP3 (PDB:6NPY), caspase-1 (PDB:8WRA), ASC (PDB:8DJT), and IL-1β (PDB:4GAF) proteins using SYBYL software (Supplementary Table 5). The experimental results showed that Formononetin, Luteolin, and Quercetin had a strong affinity for NLRP3 proteins (Figure 3D); Myristic Acid, Azelaic Acid, and Matairesinol demonstrated a strong affinity for Caspase-1 protein (Figure 3E); Myristic Acid, Azelaic Acid, and Matairesinol also showed a strong affinity for ASC protein (Figure 3F); and Luteolin, Azelaic Acid, and Matairesinol exhibited a strong affinity for IL-1β protein (Figure 3G). Through an extensive literature review of these six compounds, it was uncovered that they possess varying efficacies in alleviating inflammation. Subsequently, the air pouch experiment was employed to mimic the joint cavity, aiming to explore the anti-inflammatory impacts of these six compounds within a gout model. MSU stimulation led to a substantial accumulation of inflammatory cells in the air pouch. Notably, all six compounds remarkably diminished the accumulation of inflammatory cells, as well as pathological scores (Figure 3H and Supplementary Figure 2). By scrutinizing the levels of inflammatory factors in the air pouch lavage fluid, it was evident that all six compounds manifested different degrees of anti-inflammatory potential (Figure 3I and J). This implies that the anti-gout effect of Naru-3 is attained by modulating the NLRP3 signaling pathway.

Naru-3 Attenuates Gouty Arthritis by Inhibiting NLRP3 Inflammasome

Western blot analysis revealed that MSU significantly up-regulated NLRP3 pathway-related proteins. Conversely, Naru-3 significantly reduced the over-expression of these proteins (Figure 4A and B). NLRP3-/- and caspase-1-/- mice were further used to validate the effect of Naru-3 on gout. The results showed that Naru-3 significantly reduced the increase in IL-1β and TNF-α levels after MSU treatment. Importantly, in NLRP3 and caspase-1 deficient mice, the significant inhibition of IL-1β and TNF-α by the deficiency itself was not further enhanced by Naru-3 treatment (Figure 4C and D). Similarly, paw swelling, which was suppressed in MSU-induced NLRP3 and caspase-1 deficient mice, was not further improved by the combination of Naru-3 (Figure 4E and F). Taken together, these findings strongly suggest that Naru-3 prevents gout by inhibiting the NLRP3 inflammasome.

Figure 4 Naru-3 exerts anti-gout effects by inhibiting NLRP3 inflammasome. (A) Expression of NLRP3 pathway-related proteins and (B) Quantitative analysis (n = 3). (C) IL-1β and (D) TNF-α levels of the lavage fluid of joint cavity determined by ELISA. (E) Paw swelling was measured in Caspase-1 knockout mice. (F) Paw swelling was measured in NLRP3 knockout mice. The results are expressed as mean ± SD (n = 7), Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. In (B) #Compared with the Control group; *Compared with MSU group. (C–F) *Compared with WT group. ###p < 0.001, **p< 0.01, ***p < 0.001. Abbreviation: ns, no significance.

Naru-3 Alleviates Hyperuricemic Nephropathy in Mice

A hyperuricemic nephropathy model was established using potassium oxonate (PO). Serum uric acid levels were significantly elevated in the PO group, confirming successful model establishment (Figure 5A). Naru-3 treatment significantly reduced serum uric acid levels. In addition, serum creatinine (Scr) and blood urea nitrogen (BUN) levels were markedly increased in the PO group, indicating renal dysfunction, whereas these parameters were significantly improved following Naru-3 and allopurinol treatment (Figure 5B and C). Histopathological analysis further confirmed that Naru-3 alleviated renal tissue damage and reduced pathological scores (Figure 5D and Supplementary Figure 3). Urinary uric acid and creatinine (Cre) levels were significantly decreased in the PO group, whereas Naru-3 treatment restored these levels (Figure 5E and F). Furthermore, fractional excretion of uric acid (FEUA) was reduced in the PO group but significantly increased following Naru-3 administration (Figure 5G). These discoveries indicate that Naru-3 has positive effects on hyperuricemic nephropathy.

Figure 5 Effects of Naru-3 on UA, Cre, BUN and renal histopathology in PO-induced hyperuricemic nephropathy mice. (A) Serum uric acid. (B) Serum creatinine. (C) Blood urea nitrogen. (D) Gross appearance of kidneys photographed and representative H&E staining. Scale bars, 200 μm. (E) Urine uric acid. (F) Urine creatinine. (G) FEUA. The results are expressed as mean ± SD (n =8). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. ###p < 0.001, ####p < 0.0001, compared with Control group; *p< 0.05, **p< 0.01, ***p < 0.001, ****p < 0.0001, compared with PO group.

Naru-3 Alleviates Hyperuricemic Nephropathy by Reducing UA Production and Increasing UA Excretion

To investigate the underlying mechanisms, the activities of key enzymes involved in uric acid metabolism were assessed. Liver XOD, serum XOD, and serum ADA activities were significantly increased in the PO group (Figure 6A–C). Both allopurinol and Naru-3 significantly inhibited these enzyme activities. The expression of renal urate transporters was further evaluated. In the PO group, GLUT9 and URAT1 expression levels were significantly upregulated, whereas OAT3 expression was downregulated. Naru-3 treatment reversed these changes at both protein and mRNA levels (Figure 6D–J). These findings suggest that Naru-3 reduces uric acid production while promoting uric acid excretion, thereby exerting renoprotective effects.

Figure 6 Effects of Naru-3 treatment on XOD, ADA and protein of uric acid transporter. (A) Liver XOD. (B) Serum XOD. (C) Serum ADA. (D) Expression of GLUT9, URAT1 and OAT3 proteins was detected by WB (n =3). Quantification of (E) GLUT9, (F) OAT3 and (G) URAT1 expression by Image software. Expression levels of (H) GLUT9 mRNA, (I) URAT1 mRNA and (J) OAT3 mRNA. The results are expressed as mean ± SD (n =8). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. ##p < 0.01, ###p < 0.001, compared with Control group; *p< 0.05, **p< 0.01, ***p < 0.001, compared with PO group.

Discussion

MSU crystals within the joints stimulate the activation of the NLRP3 inflammasome, leading to the cleavage of pro-caspase-1. The activated caspase-1 subsequently initiates a pro-inflammatory form of pyroptosis and facilitates the maturation of pro-IL-1β into its active form, IL-1β. As a potent pro-inflammatory cytokine, IL-1β promotes the recruitment of innate immune cells to the joint tissue, thereby contributing to joint inflammation, pain, and swelling.³⁰ We successfully established a gouty arthritis model by injecting MSU crystals into the ankle joint cavity. We used colchicine, a drug commonly used in clinical treatment of acute gout attacks, as a positive control. Consistent with previous reports, we found that colchicine (0.8 mg/kg) significantly reversed the toe swelling rate in mice with gouty arthritis, Naru-3 also effectively alleviated the ankle swelling rate in mice. Joint pathological features further confirmed these findings, as Naru-3 slowed the infiltration of inflammatory cells and reduced synovial hyperplasia. Gout attacks can cause severe pain. Our study revealed that Naru-3 alleviated hindlimb gravitational imbalance, increased the intensity of mechanical stimulation, and delayed paw withdrawal from thermal pain stimulation. These results suggest that Naru-3 effectively alleviates MSU-induced inflammation and pain.

Transcriptomic analysis provided system-level support for the anti-inflammatory effects of Naru-3. MSU stimulation induced a pronounced shift toward a pro-inflammatory transcriptional profile in synovial tissue, whereas Naru-3 treatment largely restored gene expression patterns toward the control state. Functional enrichment analysis consistently highlighted innate immune and inflammatory signaling pathways, particularly those related to NOD-like receptor and NF-κB signaling, which are closely linked to NLRP3 inflammasome activation. It is noteworthy that several infection-related pathways were also enriched in the transcriptomic data. This likely reflects the shared molecular machinery between pathogen-induced responses and MSU-induced sterile inflammation, such as the recruitment of common inflammatory mediators and the activation of conserved signaling hubs like NF-κB and MAPKs.^31–33 These transcriptomic trends were highly consistent with the observed phenotypic and molecular findings, supporting the notion that Naru-3 suppresses gouty inflammation through coordinated regulation of NLRP3-related inflammatory cascades.

Given the multi-component nature of Naru-3 and the system-level regulation revealed by transcriptomics, we next sought to identify its bioactive constituents and molecular targets. The chemical composition of Naru-3 was experimentally characterized by UPLC-Q/TOF-MS, providing a solid basis for subsequent network pharmacology analysis. Target enrichment analysis based on the identified compounds revealed significant involvement in inflammatory and innate immune pathways, particularly the NOD-like receptor signaling pathway, in close agreement with the transcriptomic results.

Molecular docking further indicated that several representative compounds, including Myristic Acid, Luteolin, Azelaic Acid, Quercetin, Formononetin, and Matairesinol, exhibited favorable binding affinity toward NLRP3 inflammasome–associated proteins. These compounds have been widely reported to exert anti-inflammatory effects in multiple inflammatory diseases,^34,35 such as osteoarthritis,^36,37 ulcerative colitis,^38–40 psoriasis,⁴¹ atherosclerosis,⁴² and non-alcoholic fatty liver disease.⁴³ While these compounds have been reported to exhibit anti-inflammatory effects in various diseases, the present study is the first to demonstrate their collective potential to inhibit the NLRP3 inflammasome in the context of MSU-induced gout, highlighting their contribution to the therapeutic effects of Naru-3. Consistently, functional validation using an MSU-induced air pouch model demonstrated that all six compounds significantly reduced inflammatory cell infiltration and suppressed IL-1β and TNF-α release. Together, these findings suggest that Naru-3 exerts its anti-gout effects through the coordinated action of multiple bioactive constituents targeting the NLRP3 inflammasome–associated inflammatory network.

Naru-3 indisputably reduced IL-1β and TNF-α levels in MSU-induced gouty arthritis mice. Upon exposure to pathogen-associated molecular patterns, damage-associated molecular patterns, environmental stress, or other exogenous invaders, NLRP3 binds to the PYD in ASC. ASC then recruits the CARD after binding to the CARD domain in pro-caspase-1, forming the NLRP3 inflammatory complex. Pro-caspase-1 undergoes self-cleavage, resulting in the generation of activated caspase-1. Subsequently, activated caspase-1 activates the immature pro-inflammatory cytokines IL-1β and IL-18.⁴⁴ The findings of our investigation indicate that Naru-3 effectively suppresses the activation of the NLRP3 inflammasome, as demonstrated by a significant decrease in the protein expression levels of NLRP3, ASC, IL-1β, and caspase-1. Moreover, Naru-3 treatment did not result in a notable improvement in NLRP3^−/− and caspase-1^−/− mice. Our findings indicate that Naru-3 exerts an anti-gout effect by blocking the NLRP3/ASC/caspase-1 pathway, inhibiting the activation of NLRP3, and preventing the release of IL-1β.

Hyperuricemic, defined as elevated uric acid levels in the blood, is a primary contributor to the onset of gout. Moreover, clinical studies have demonstrated that long-term hyperuricemic is associated with an increased risk of various health complications, including cardiovascular disease,^11,12 chronic kidney disease,¹³ periodontitis,¹⁴ and metabolic syndrome.¹⁵ Therefore, controlling elevated uric acid levels is crucial for preventing and treating these associated diseases. Long-term hyperuricemic can lead to severe kidney damage. BUN and Scr levels are key indicators for evaluating renal function.⁴⁵ The results of our study demonstrated that after 21 days of Naru-3 treatment, there was a notable reduction in BUN, urine uric acid (Uur), and Scr levels, while urine creatinine (Ucr) and fractional excretion of uric acid (FEUA) levels exhibited a significant increase. These findings indicate that Naru-3 effectively reversed hyperuricemic-induced renal injury in mice, thereby demonstrating its beneficial kidney-protective properties. The results of renal pathology were also in alignment with these findings. In conclusion, our findings provide compelling evidence that Naru-3 plays a pivotal role in alleviating PO-induced renal impairment in hyperuricemic mice.

XOD is a pivotal enzyme in the synthesis of uric acid.⁴⁶ An increase in XOD activity may result in an excess of uric acid synthesis.⁴⁷ Therefore, inhibiting XOD activity plays a significant role in anti-hyperuricemic strategies. ADA is a crucial enzyme in purine metabolism, converting extracellular adenosine to inosine.⁴⁸ To gain further insight into the uric acid-lowering mechanism of Naru-3, we investigated the activity of XOD in both the liver and serum, as well as the activity of serum ADA. In accordance with prior findings, allopurinol, a prevalent XOD inhibitor in clinical settings, markedly suppressed XOD activity in both the liver and serum. It is noteworthy that Naru-3 demonstrated efficacy in inhibiting the activity of serum and liver XOD. Furthermore, Naru-3 demonstrated a notable inhibitory effect on ADA activity. This indicates that Naru-3 may suppress ADA activity, thereby reducing the production of xanthine and hypoxanthine in vivo. Moreover, it may inhibit XOD activity to prevent uric acid production and the formation of MSU crystals, thereby exerting a therapeutic effect on hyperuricemic. The excretion of uric acid by the kidneys is a complex process involving several stages, including glomerular filtration, tubular reabsorption, tubular secretion, and reabsorption. A reduction in uric acid excretion and an increase in uric acid reabsorption are both factors that contribute to the development of hyperuricemic. The serum uric acid concentration is regulated by urate transporters in the kidney and gut, particularly GLUT9, URAT1 and OAT3,⁴⁹ which are considered potential targets for the treatment of hyperuricemic. URAT1 and GLUT9 control renal uric acid re-absorption,⁵⁰ while OAT3, ABCC4, and ABCG2 are involved in uric acid secretion.⁵¹ The results demonstrated that Naru-3 down-regulated the protein and mRNA levels of URAT1 and GLUT9, while up-regulating the protein and mRNA levels of OAT3 in the kidneys of hyperuricemic mice. These findings indicate that treatment with Naru-3 resulted in a reduction in uric acid reabsorption by the kidneys and an increase in uric acid secretion and excretion, thereby exerting a nephroprotective effect in hyperuricemic mice.

This study also has some limitations, the main target of Naru-3 on NLRP3 signaling pathway is still unclear, in addition, Naru-3 contains many natural products of alkaloids, phenolic acids, terpenoids, and flavonoids, The specific mechanisms of those compounds with anti-gouty arthritis and anti-hyperuricemic could be more deeply explored to provide stronger support for the efficacy of Naru-3.

Conclusion

The objective of this study was to identify potential targets and active components of Naru-3 against gout using transcriptomic analysis, UPLC-Q/TOF-MS, network pharmacology, and molecular docking. In vivo experiments demonstrated that Naru-3 ameliorated gouty arthritis by reducing inflammatory responses and modulating the NLRP3 signaling pathway. In addition, Naru-3 improved hyperuricemia by decreasing uric acid production and promoting uric acid excretion.

Overall, these findings suggest that Naru-3 exerts dual therapeutic effects through coordinated regulation of inflammation and uric acid metabolism. These findings not only validate the traditional use of Naru-3 but also identify key bioactive components and mechanisms that may facilitate the development of novel multitarget therapeutic strategies for gout and hyperuricemia-related complications.