Lipoxin A4 reduces inflammation caused by uric acid crystals by lowering Nrf2 and raising TXNRD2 levels

A total of 66 male gout patients from Lanzhou University Second Hospital from January 2020 to December 2020 were enrolled in this study. Patients had no acute inflammatory diseases except gouty arthritis and no history of infection, cancer, or other autoimmune diseases. Patients with acute gout (AG) and intercritical gout (IG) were selected based on the American College of Rheumatology (ACR) classification criteria; IG patients were those with complete remission of acute gout with normal erythrocyte sedimentation rate or C-reactive protein. Moreover, age-matched male individuals admitted for regular physical examinations at Lanzhou University Second Hospital were selected as healthy controls (HCs).

This study was approved by the Ethics Committee of the Lanzhou University Second Hospital, and all patients provided informed consent.

Human monocytic leukemia (THP-1) cells were grown in Roswell Park Memorial Institute (RPMI)-1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PYG0016; Boster Bio, Wuhan, China). Cells were cultured in a humidified atmosphere containing 5% CO₂/95% air at 37°C. THP-1 cells were treated with 100 ng/ml phorbol myristate acetate (PMA; S1819, Beyotime Biotechnology, Shanghai, China) for 24 h to differentiate into macrophages.

Bone-marrow-derived macrophages (BMDMs) were obtained via the differentiation of bone marrow progenitors from the tibia and femur of C57BL/6J mice in Dulbecco’s modified Eagle’s medium (DMEM) with L929 supernatant (20%), 10% FBS, and 1% penicillin/streptomycin for 7 days. The culture medium was refreshed every other day. The BMDM purity was assessed by flow cytometry using CD11b and F4/80 antibodies.

BMDMs and THP-1 macrophages were primed with 300 ng/ml lipopolysaccharide (LPS) (S1732; Beyotime Biotechnology, Shanghai, China) for 3 h and then pretreated with different concentrations of LXA4 (GC18552; GlpBio, Montclair, CA, USA) for 1 h, and subsequently, 100 μg/ml MSU crystal was added to stimulate for 24 h. MSU crystals were prepared as previously described (10).

For inhibitor experiments, THP-1 macrophages were primed with 300 ng/ml LPS for 3 h and then pretreated with NAC (S0077; Beyotime Biotechnology, Shanghai, China), MitoTEMPO (HY-112879; MCE, NJ, USA), and DPI (HY-100965; MCE, NJ, USA) for 1 h, followed by 100 μg/ml MSU crystal stimulation for 24 h. Moreover, THP-1 macrophages primed with LPS were pretreated with 150 nM LXA4 and tBHQ (HY-100489; MCE, NJ, USA) or auranofin (HY-B1123; MCE, NJ, USA) for 1 h, followed by 100 μg/ml MSU stimulation for 24 h.

Cytokine levels in serum or conditioned media were detected using an ELISA kit following the manufacturer’s instructions, including LXA4, tumor necrosis factor alpha (TNF-α) (EMC102a; NeoBioScience, Shenzhen, China) and IL-1β (EMC001b; NeoBioScience, Shenzhen, China).

Proteins for Western blotting were prepared as previously described (25) or performed according to the manufacturer’s instructions from Membrane and Cytosol Protein Extraction Kit (P0033; Beyotime Biotechnology, Shanghai, China) and Nuclear and Cytoplasmic Protein Extraction Kit (P0028; Beyotime Biotechnology, Shanghai, China). Proteins were first separated, transferred, and blocked, after which membranes were incubated with antibodies (see Supplementary Materials).

BMDMs or THP-1 macrophages were treated as described above. Total cells were lysed in cold radioimmunoprecipitation assay (RIPA) buffer (P0013B; Beyotime Biotechnology, Shanghai, China) containing 0.5%Triton X-100, 50 mM Tris–HCl, and 1 mM dithiothreitol for 30 min. The lysates were centrifuged at 330×g for 10 min at 4°C, and then, the pellets were collected and washed twice with ice-cold phosphate buffered saline (PBS). The pellets were then resuspended in 500 μl cold PBS containing 4 mM disuccinimidyl suberate (DSS) (S1885; Sigma, St. Louis, MO, USA) and incubated with rotation at room temperature for 30 min. The samples were centrifuged at 1,000×g for 10 min to obtain cross-linked pellets, after which Western blotting was performed to analyze ASC oligomerization.

THP-1 macrophages were fixed in 4% paraformaldehyde and then permeabilized with ice-cold methanol. Samples were then incubated with an anti-ASC antibody (1:100) overnight at 4°C and Alexa Fluor 488 goat-anti-mouse IgG (1:100) for 1 h at room temperature. Finally, samples were then stained with 4′,6-diamidino-2-phenylindole (DAPI) (D8200; Solarbio Science, Beijing, China). ASC-speck formation was imaged using confocal laser scanning microscopy (Olympus, Tokyo, Japan).

BMDMs and THP-1 macrophages treated as described above were lysed, and the lysis was collected. The samples were incubated with the antibody against ASC (1:100) and Nek7 (1:100) overnight at 4°C to obtain antibody–target protein complexes, and the complexes were gathered with Protein G agarose beads for 2 h at 4°C. Next, sediment was resuspended in 1× sodium dodecyl sulfate (SDS) loading buffer and analyzed by Western blotting.

THP-1 macrophages were seeded in 24-well plates and treated as described above. Cells were stained with propidium iodide (8 μg/ml) and Hoechst dye (8 μg/ml) (C0003; Beyotime Biotechnology, Shanghai, China) and imaged using a fluorescent microscope (Olympus, Tokyo, Japan). PI-positive cells suggested cell death.

THP-1 macrophages were seeded in 12-well plates and treated as described above. Then, 120 μl conditioned supernatant was transferred to 96-well plates and mixed with 60 μl LDH Cytotoxicity Assay Kit reagent (C0016; Beyotime Biotechnology, Shanghai, China) and incubated for 30 min at 37°C. The absorbance was determined at 490 nm using a microplate reader. The cellular LDH release was calculated following the manufacturer’s instructions.

Intracellular chloride ions were detected by MQAE (S1082; Beyotime Biotechnology, Shanghai, China), a fluorescence probe for chloride ions. A higher fluorescence intensity indicated a lower level of chloride ions and vice versa. Intracellular potassium ions were detected by EPG-2 AM (MX4513; Maokang Biotechnology, Shanghai, China), a fluorescence indicator for potassium ions.

THP-1 macrophages treated as described above were stained following manufacturer’s instructions. The fluorescence images of the cell-loaded probe were captured using confocal laser scanning microscopy, and the Image software was applied to evaluate fluorescence intensity.

THP-1 macrophages were seeded in 12-well plates and treated as described above. Next, cells were incubated with 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (S0033S; Beyotime Biotechnology, Shanghai, China), a fluorescent probe, for 30 min at 37°C. Fluorescence images of cell-loaded probe were captured using a fluorescent microscope, and fluorescence intensity was measured by a microplate reader.

For the flow cytometry assay, THP-1 macrophages were treated and loaded with DCFH-DA. The cells were collected and suspended in ice-cold PBS, and the flow cytometry and FlowJo software were performed to analyze the level of ROS.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was determined using lucigenin-enhanced chemiluminescence as previously described (26). THP-1 macrophages were lysed. Samples (30 μl) were then incubated with 300 μl reaction buffer containing 5 mM K₂HP0₄, 1 mM EGTA, 150 mM sucrose, 5 μM Lucigenin (GC33485; GlpBio, Montclair, CA, USA), 100 μM NADPH (GC34058; GlpBio, Montclair, CA, USA), and 200 μl mixtures were transferred to 96-cell plates. Luminescence [arbitrary light units (ALUs)] was measured using a microplate reader.

Mitochondrial ROS was evaluated by MitoSOX (M36005↗; Invitrogen, Carlsbad, CA, USA), a specific indicator for mitochondrial ROS. Mito-Track Green (C1048; Beyotime Biotechnology, Shanghai, China) stained mitochondria in live cells and was used for mitochondrial localization. THP-1 macrophages treated as described above were incubated with 2 μM MitoSOX and 50 nM Mito-Track Green for 30 min at 37°C and then analyzed by a confocal laser scanning microscopy.

Mitochondrial membrane potential (ΔΨm) was measured by a JC-1 probe (M8650; Solarbio Science, Beijing, China) according to the manufacturer’s instructions. JC-1 located on mitochondria under normal conditions is shown in red, while JC-1 appears in green under cell depolarization. THP-1 macrophages treated as described above were stained by JC-1 and subsequently imaged by confocal laser scanning microscopy. The Image software was applied to evaluate fluorescence intensity, and the ratio of red/green indicated the degree of depolarization.

Mitochondrial Ca²⁺ was assessed with Rhod-2M (40776ES50; Yeasen, Shanghai, China) following manufacturers’ instructions. In brief, THP-1 macrophages treated as described above were incubated with 2 μM Rhod-2M and 50 nM Mito-Track Green for 30 min at 37°C. Then, cells were imaged using confocal laser scanning microscopy.

THP-1 macrophages were seeded at slides and treated as described above. Next, cells were fixed with 4% paraformaldehyde for 20 min and blocked with 1% bovine serum albumin (BSA) containing 0.3% Triton X-100 for 1 h, after which they were incubated with anti-Nrf2 antibody (1:100) overnight at 4°C and subsequently stained with Alexa Fluor 488 goat-anti-mouse IgG for 1 h and sealed with DAPI. The specific fluorescence was imaged using confocal laser scanning microscopy.

Total RNA was extracted from THP-1 macrophages with TRIzol Reagent (15596026; Thermo Fish, Waltham, MA, US), and RNA was reverse transcribed into cDNA with a Reverse Transcriptase kit (2690S; TakaRa, Tokyo, Japan). Real-time quantitative PCR (RT-qPCR) was performed using a real-time system with SYBR Green Master Mix (3735S; TakaRa, Tokyo, Japan). The β-actin gene was employed as an endogenous control. The relative gene expression levels were analyzed by the 2^−ΔΔCT method. The primer sequences (Sangon Biotech, Shanghai, China) are shown in Supplementary Table S1.

The interaction between Nrf2 and Klf9 promoter and Klf9 and TXNRD2 promoter was assessed using the ChIP-IT (P2078; Beyotime Biotechnology, Shanghai, China), and qPCR analysis was performed following the manufacturer’s protocol from the kit. The following antibodies were used: control IgG and antibodies specific to Nrf2 and Klf9. The primer sequences (Sangon Biotech, Shanghai, China) were as follows: Klf9 promoter, forward 5’-CGCTAGAGTTACGAAACAGGG-3’; reverse 5’-GAAAGGCCATCCGTTCATGC-3’; TXNRD2 promoter, forward 5′-AACCCTCCCTTCCCAGTTTTG-3′, reverse 5′-AAAAAGCTGGCTCCATGCTG-3′; and GAPDH promoter, forward 5′-TACTAGCGGTTTTACGGGCG-3′, reverse 5′- TCGAACAGGAGCAGAGAGCGA-3′.

THP-1 macrophages were seeded at six-cell plates or slides and then transfected with TXNRD2 siRNA and negative control siRNA (GenePharma, Shanghai, China) using Lipofectamine 2000 (12566014; Invitrogen, Carlsbad, CA, USA) according to manufacturers’ instructions. The specific TXNRD2 siRNA sequence is shown in. Then, the cells were primed with 300 ng/ml LPS for 3 h, pretreated with 150 nM LXA4 for 1 h, and subsequently stimulated with 100 μg/ml MSU for 24 h. 1

Male adult Sprague–Dawley rats (6 weeks, 180–250 g) were purchased from SPF Biotechnology Company (Beijing, China). All the animals were housed in an environment with a temperature of 22 ± 1°C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. The euthanasia was conducted using mild carbon dioxide (30%) asphyxiation. All animal studies were done in compliance with the regulations and guidelines of Lanzhou University Second Hospital Institutional Animal Care and conducted according to the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the Institutional Animal Care and Use Committee (IACUC) guidelines.

The model of gouty arthritis was induced as previously described (10). Rats were then divided into three groups: control group, model group, and LXA4 group. The LXA4 group received 100 μl LXA4 into the ankle joint (10 μg/kg); the same volume of sterile PBS was injected into a rat of the control and model groups. After 1 h, the model and LXA4 groups were injected with 100 μl MSU crystals suspension (500 μg/ml) into the ankle joint, while no treatment was given to the control group. The ankle perimeter of rats was assessed with a string at 12, 24, 48, and 72 h after MSU crystals injection, following which rats were sacrificed. The serum and synovium tissue were collected for further study.

The synovium tissue samples were fixed in the 4% paraformaldehyde solution, embedded in paraffin, and sliced into paraffin sections. The sections were stained with H&E as previously described (10). Meanwhile, the synovitis score was estimated as previously described (27, 28).

The data are presented as the mean ± SEM or median with an interquartile range. Statistical analysis was performed using one-way analysis of variance (ANOVA) and subsequent least significant difference (LSD) analysis or signed-rank test or Spearman correlation analysis. p-value < 0.05 was considered to be statistically significant.

We detected the levels of LXA4 and inflammatory markers in serum. The levels of IL-1β and TNF-α in patients with AG increased compared with patients with IG and HC (Figures 1A, B). We also found that LXA4 was significantly increased in patients with acute gout (Figure 1C), and the ratio of IL-1β to LXA4 was significantly increased (Figures 1D, E). Moreover, LXA4 showed a significant negative correlation with IL-1β and TNF-α in these patients (Figure 1F, G). These results suggest that LXA4 is insufficient to fight inflammatory response in patients with acute gout.

To explore the intervention potential of LXA4 on NLRP3 inflammasome triggered by MSU crystals in macrophages, we stimulated lipopolysaccharide (LPS)-primed BMDMs and PMA-differentiated THP-1 macrophages with MSU crystals to activate NLRP3 inflammasome, as previously described (29). As shown in Figures 2A, B, LXA4 significantly reduced the concentration of IL-1β in macrophages in a dose-dependent manner. The NLRP3 inflammasome activation enhances cleaved caspase-1 manufactured from pro-caspase-1 and matured IL-1β cleaved from pro-IL-1β, and both are released out of the cell. Immunoblotting results further showed elevated cleaved caspase-1 (p20) and matured IL-1β (p17) in the culture supernatants upon MSU crystals stimulation. This process was reversed by LXA4 (Figures 2C, D). Yet, LXA4 did not inhibit the precursors of IL-1β and caspase-1 protein expression (Figures 2C, D). These findings indicate that LXA4 may interfere with the activation signal rather than the priming signal of NLRP3 inflammasome.

Besides promoting IL-1β secretion, the activation of NLRP3 inflammasome can cause inflammatory death to cells, especially NLRP3-mediated pyroptosis (7, 10, 11, 30, 31). Immunoblotting results showed that the MSU crystal enhances the GSDMD-N in the cell supernatant of macrophages, the execution protein for pyroptosis (32), while LXA4 attenuates the elevated GSDMD-N (Figures 2E, F).

Next, we adopted propidium iodide (PI) uptake (red fluorescence) and cellular LDH release to evaluate pyroptosis directly. LXA4 prevented the PI staining (Figures 2G, H) and decreased the cellular LDH release of differentiated THP-1 macrophages (Figure 2I). Taken together, we concluded that LXA4 attenuates the MSU-crystal-induced NLRP3 inflammasome activation in macrophages.

Next, we sought to investigate the mechanisms through which LXA4 attenuates the NLRP3 inflammasome activation induced by MSU crystals. We observed that LXA4 barely affects NLRP3 and ASC expression consistent with pro-caspase-1 in macrophages (Figures 3A, B), suggesting again that LXA4 may affect the assembly but not the expression of NLRP3 inflammasome. The formation of perinuclear structures called ASC speck and oligomerization of ASC are indispensable for assembling and activating the NLRP3 inflammasome (31, 33). Our results found that LXA4 significantly reduced the formation of ASC specks induced by MSU crystal in differentiated THP-1 macrophages (Figures 3C, D). Furthermore, we used DSS to cross-link ASC and investigated ASC oligomerization by immunoblotting and found a similar result that LXA4 markedly repressed ASC oligomerization in macrophages (Figures 3E, F).

The premise of NLRP3 inflammasome activation is that the components are completely assembled (34). Herein, we employed co-immunoprecipitation to analyze the interaction between ASC with NLRP3 and pro-caspase-1. ASC-NLRP3 interaction triggered by MSU crystal was markedly reduced by LXA4 in macrophages (Figures 3G, H); yet, LXA4 had no effect on the ASC-pro-caspase-1 interaction (Figures 3G, H). It has been reported that the interaction between NLRP3 and NIMA-related kinase 7 (NEK7) enhances inflammasome activation (11, 35). However, in our study, LXA4 did not affect the expression of NEK7 (Supplementary Figures S1A, C) or NEK7–NLRP3 interaction (Supplementary Figures S1B, D). These data suggest that LXA4 antagonizes the assembly of the NLRP3 inflammasome by impeding the specks formation and oligomerization of ASC and the ASC-NLRP3 interaction.

We next determined whether LXA4 can hamper the upstream event dependent on NLRP3 inflammasome activation. Previous studies have confirmed that chloride efflux, potassium efflux, and ROS production are important upstream events for activation of NLRP3 inflammasome (15, 16, 36, 37). The MQAE is a fluorescence probe for intracellular chloride ions; the higher intensity of MQAE fluorescence shows a lower level of intracellular chloride ions and vice versa. The MQAE data revealed that the MSU crystal triggered a marked decrease in chloride efflux; nevertheless, LXA4 did not improve this decrease (Figures 4A, B). Consistently, EPG-2M data (a fluorescence probe for intracellular potassium) showed that LXA4 had no effect on the potassium efflux induced by MSU crystal (Figures 4C, D).

Furthermore, we used DCFH (a ROS fluorescence probe) to detect ROS levels. The results from fluorescence microscopy, microplate reader, and flow cytometry analysis demonstrated that MSU crystal resulted in a significant increase in ROS; yet, this process was reversed by LXA4 (Figures 4E–G), suggesting that LXA4 suppressed the MSU-crystal-triggered ROS generation. We speculate that LXA4 attenuates NLRP3 inflammasome activation induced by MSU crystal, probably through blocking ROS production.

We further studied the effect of LXA4 on the source of ROS. To inquire about the source of ROS in our experimental model, we pretreated cells with inhibitors of ROS, including NAC (a cellular ROS scavenger), MitoTEMPO (a mitochondrial ROS scavenger), and DPI (a specific inhibitor for NADPH oxidase). As shown in Figures 5A, B, all inhibitors could depress the release of cleaved caspase-1 and matured IL-1β, indicating that ROS from activated NADPH oxidase and damaged mitochondria are the mediator for the NLRP3 inflammasome activation induced by MSU crystal, which is consistent with other studies (13, 38, 39). Accordingly, we further examined whether LXA4 could prevent NADPH oxidase activation and mitochondria dysfunction. We found that the MSU-crystal-triggered NADPH oxidase activation was markedly reduced by LXA4 (Figure 5C).

p47phox and p22phox are the essential cytoplasmic and membrane subunit of NADPH oxidase (40). We found that pretreatment with LXA4 hardly affects the p47phox but decreases the p22phox protein level (Figure 5D). Moreover, MSU crystal decreased p47phox in cytosol lysates and increased p47phox in membrane lysates, but LXA4 overturned the changes (Figure 5E), indicating that LXA4 hampers the transfer of p47phox from the cytoplasm to the membrane.

Meanwhile, we tested the mitochondrial ROS generation by mitochondrial ROS-specific indicator MitoSOX staining and found that MSU-crystal-triggered ROS augmentation in mitochondria was reduced by LXA4 (Figures 5F, G). The depolarization of membrane potential and grave Ca²⁺ overload are the inducements and signs of serious mitochondrial damage (41, 42). Using the specific indicator, we found that LXA4 relieved the depolarization of the membrane potential and Ca²⁺ overload in mitochondria at least partly (Figures 5H–K). We propose that LXA4 blunts MSU-crystal-induced ROS generation due to the suppression of NADPH oxidase activation and improvement of mitochondrial dysfunction.

Nrf2 is a classical and pivotal regulator with an antioxidant role (43–45). LXA4 can arouse Nrf2 activation to mediate antioxidant and anti-inflammatory activity (17–19). Additionally, MSU evoking IL-1β/NLRP3 inflammasome is dependent of Nrf2 activation (11, 20). Consequently, we wondered whether LXA4 could regulate Nrf2 signaling. Consistent with previous reports (11, 20), our results revealed that the MSU crystal increased Nrf2 in differentiated THP-1 macrophages; however, LXA4 mitigated the protein level of Nrf2 (Figure 6A). The immunofluorescence analysis further showed that LXA4 relieved the nuclear translocation of Nrf2 triggered by the MSU crystal (Figure 6B). Simultaneously, immunoblotting results revealed that LXA4 rectified the MSU-crystal-caused increase in Nrf2 in nuclear lysates (Figure 6C), suggesting that LXA4 prevents the nuclear movement of Nrf2. Interestingly, the antioxidants encoded by Nrf2, including HO-1, SOD, and GPx and their mRNA and protein expression were strongly depressed by LXA4 (Figures 6D–G). These results indicate that LXA4 inhibits the activation of Nrf2.

Moreover, we adopted tBHQ, an activator for Nrf2, to resume the diminished Nrf2 caused by LXA4. We found that the LXA4-induced depression in the release of cleaved caspase-1 and matured IL-1β was reversed by tBHQ (Figures 6H, I). These results indicate that LXA4 prevents MSU-crystal-triggered NLRP3 inflammasome activation via suppressing Nrf2.

As LXA4 inhibited Nrf2 and antioxidants encoded by Nrf2, we turned our attention to the Klf9/TXNRD2 axis; still, a signaling pathway mediated by Nrf2 was involved in oxidative stress (46). As shown in Figures 7A–C, MSU crystal increased Klf9 and decreased TXNRD2 (both protein and mRNA levels), while LXA4 reversed this process. To validate the regulation of LXA4 on the Nrf2/Klf9/TXNRD2 axis, we adopted tBHQ to resume the diminished Nrf2 caused by LXA4. We observed that tBHQ overthrows the inhibitory effect of LXA4 on Klf9 and the promotional effect of LXA4 on TXNRD2 (Figures 7D–F). These results suggest that LXA4 interdicts the Nrf2/Klf9/TXNRD2 signaling pathway.

Given that Nrf2 can occupy the promoter of Klf9 and Klf9 can occupy the promoter of TXNRD2 (46), we performed a ChIP assay to determine whether LXA4-induced repression of Klf9 and promotion of TXNRD2 are associated with a decrease in these occupancies. Results showed that a significant enrichment of Klf9- and TXNRD2-promoter-specific DNA was detected in the chromatin material precipitated with Nrf2- and Klf9-specific antibodies in THP-1 macrophages induced by the MSU crystal, respectively; however, LXA4 depressed both (Figures 7G, H), indicating that LXA4 disturbs the interaction between Nrf2 and the promoter of Klf9, and Klf9 and the promoter of TXNRD2. These findings further confirm the regulation of LXA4 on the Nrf2/Klf9/TXNRD2 pathway.

TXNRD2 is a mediator endowed with antioxidant ability and located on the Nrf2/Klf9/TXNRD2 axis terminal. Given that the above results suggested that LXA4 could elevate TXNRD2 expression by suppressing Nrf2, we further explored whether LXA4-induced attenuation of the MSU-crystal-triggered NLRP3 inflammasome activation is dependent on TXNRD2. To address this, we employed a TXNRD2 silence strategy and established TXNRD2-depleted cells by small interfering RNA in differentiated THP-1 macrophages (Supplementary Figures S2A, B). The results showed that siRNA-TXNRD2 could block the inhibitory impact of LXA4 on the IL-1β release, specks formation, and oligomerization of ASC, and the ASC-NLRP3 interaction (Figures 8A–E, Supplementary Figure S3). Similarly, auranofin, a specific inhibitor for TXNRD2, also neutralized LXA4-induced inhibition of the IL-1β release (Supplementary Figures S4A, B). Interestingly, we observed that siRNA-TXNRD2 also impaired the inhibitory influence of LXA4 on superior total ROS generation (Figures 8F, G). In addition, siRNA-TXNRD2 reversed the suppressive action of LXA4 on NADPH oxidase activation and mitochondria ROS production (Figures 8H–J). We conclude that LXA4 interferes with ROS generation to prevent NLRP3 inflammasome activation dependent on TXNRD2.

To investigate the therapeutic potential of LXA4 for gout in vivo, we established a gouty arthritis rat model. Our results showed that LXA4 markedly relieved acute joint swelling of gouty arthritis rats (Figures 9A, B). Histological analysis further showed that LXA4 inhibited the massive infiltration of inflammatory cells in joint tissues (Figures 9C, D). In addition, LXA4 could significantly depress serum IL-1β and TNF-α in gouty arthritis rats (Figures 9E, F). Interestingly, LXA4 also significantly inhibited the expression of NLRP3, cleaved caspase-1, and matured IL-1β in the joint of gouty arthritis rats (Figure 9G).

To validate the regulatory effect of LXA4 on the Nrf2/Klf9/TXNRD2 signaling pathway, we detected the key protein of this pathway in a rat model. As shown in Figure 9H, LXA4 also subverted the increase in Nrf2 and Klf9 and depression of TXNRD2 in gouty arthritis rats, which is consistent with in vitro data. Thus, these results approve that LXA4 may be a potential candidate in the treatment of gouty arthritis.