DUSP1 Mitigates MSU-Induced Immune Response in Gouty Arthritis Reinforcing Autophagy.

BACKGROUND: Persistent hyperuricemia can lead to the generation and deposition of monosodium urate (MSU) crystals. This can trigger gouty arthritis (GA), which in turn induces inflammation. Activation of the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome plays a critical role in the onset and progression of GA. Autophagy may have a dual effect on GA with regard to the NLRP3 inflammasome. Therefore, the present study aimed to gain a deeper comprehension of the interaction between autophagy and NLRP3 inflammasome activation is imperative for developing more efficacious treatments for GA. METHODS: Peripheral blood monocytes (PBMCs) were first isolated from GA patients and healthy controls and underwent bulk RNA sequencing analysis. Overexpression and knockdown of dual specificity phosphatase 1 (DUSP1) was performed in THP-1 monocytes to investigate its role in the immune response and mitochondrial damage. The luciferase assay and Western blot analysis were used to study the interaction between autophagy and NLRP3 inflammasome activation. RESULTS: Bulk RNA sequencing analysis showed significant upregulation of DUSP1 expression in PBMCs from GA patients compared to healthy controls. This result was subsequently verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). DUSP1 expression in human THP-1 monocytes was also shown to increase after MSU treatment. Downregulation of DUSP1 expression increased the secretion of inflammatory cytokines after MSU treatment, whereas the overexpression of DUSP1 decreased the secretion levels. Lipopolysaccharides (LPS) combined with adenosine-triphosphate (ATP) led to mitochondrial damage, which was rescued by overexpressing DUSP1. DUSP1 overexpression further increased the level of autophagy following MSU treatment, whereas downregulation of DUSP1 decreased autophagy. Treatment with the autophagy inhibitor 3-Methyladenine (3-MA) restored inflammatory cytokine secretion levels in the DUSP1 overexpression group. MSU caused pronounced pathological ankle swelling in vivo. However, DUSP1 overexpression significantly mitigated this phenotype, accompanied by significant downregulation of inflammatory cytokine secretion levels in the joint tissues. CONCLUSIONS: This study revealed a novel function and mechanism for DUSP1 in promoting autophagy to mitigate the MSU-induced immune response in GA. This finding suggests potential diagnostic biomarkers and anti-inflammatory targets for more effective GA therapy.

Sirt1 inhibits macrophage polarization and inflammation in gouty arthritis by inhibiting the MAPK/NF-κB/AP-1 pathway and activating the Nrf2/HO-1 pathway.

OBJECTIVE AND DESIGN: To elucidate Sirt1's role in gouty arthritis inflammation and its potential mechanisms. MATERIAL: Constructed murine models of gouty arthritis and conducted THP-1 cell experiments. TREATMENT: 1 mg of MSU crystals injected into mice ankle joints for a 72-h intervention. After a 3-h pre-treatment with Sirt1-specific inhibitor (EX527) and agonist (SRT2104), inflammation was induced for 21 h using lipopolysaccharide (LPS) plus MSU crystals. METHODS: We assessed gouty arthritis severity through joint inflammation index, swelling, and hematoxylin and eosin (H&E) staining, and measured CD68 mononuclear macrophages and Sirt1 expression in synovial tissue via immunohistochemistry. ELISA, NO assay, RT-qPCR, Flow cytometry, and Western blot were utilized to examine macrophage inflammatory factors, polarization, reactive oxygen species(ROS), MAPK/NF-κB/AP-1 and Nrf2/HO-1 pathways proteins. RESULTS: Significant joint swelling, synovial tissue edema, and inflammatory cell infiltration were observed. CD68 mononuclear macrophages and Sirt1 expression were elevated in synovium. Sirt1 activation decreased inflammatory factors, M1 polarization, and ROS generation. Sirt1 activation reduced p38/JNK phosphorylation, thereby inhibiting downstream NF-κB p65/AP-1 and enhancing Nrf2/HO-1, thus suppressing inflammation. CONCLUSIONS: Sirt1 alleviates M1 macrophage polarization and inflammation in gouty arthritis by inhibiting the MAPK/NF-κB/AP-1 pathway and activating the Nrf2/HO-1 pathway. Thus, activating Sirt1 may provide a new therapeutic target for gouty arthritis.

Mechanosensitive TRPV4 is required for crystal-induced inflammation.

Crystal structures activate innate immune cells, especially macrophages and initiate inflammatory responses. We aimed to understand the role of the mechanosensitive TRPV4 channel in crystal-induced inflammation. Real-time RT-PCR, RNAscope in situ hybridisation, and Trpv4eGFP mice were used to examine TRPV4 expression and whole-cell patch-clamp recording and live-cell Ca2+ imaging were used to study TRPV4 function in mouse synovial macrophages and human peripheral blood mononuclear cells (PBMCs). Both genetic deletion and pharmacological inhibition approaches were used to investigate the role of TRPV4 in NLRP3 inflammasome activation induced by diverse crystals in vitro and in mouse models of crystal-induced pain and inflammation in vivo. TRPV4 was functionally expressed by synovial macrophages and human PBMCs and TRPV4 expression was upregulated by stimulation with monosodium urate (MSU) crystals and in human PBMCs from patients with acute gout flares. MSU crystal-induced gouty arthritis were significantly reduced by either genetic ablation or pharmacological inhibition of TRPV4 function. Mechanistically, TRPV4 mediated the activation of NLRP3 inflammasome by diverse crystalline materials but not non-crystalline NLRP3 inflammasome activators, driving the production of inflammatory cytokine interleukin-1ß which elicited TRPV4-dependent inflammatory responses in vivo. Moreover, chemical ablation of the TRPV1-expressing nociceptors significantly attenuated the MSU crystal-induced gouty arthritis. In conclusion, TRPV4 is a common mediator of inflammatory responses induced by diverse crystals through NLRP3 inflammasome activation in macrophages. TRPV4-expressing resident macrophages are critically involved in MSU crystal-induced gouty arthritis. A neuroimmune interaction between the TRPV1-expressing nociceptors and the TRPV4-expressing synovial macrophages contributes to the generation of acute gout flares.

Tetrahydropalmatine attenuates MSU crystal-induced gouty arthritis by inhibiting ROS-mediated NLRP3 inflammasome activation.

Activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome plays a crucial role in the inflammatory responses of monosodium urate (MSU) crystal-induced gouty arthritis. Therefore, the molecular basis of NLRP3 inflammasome is very valuable in developing potential therapeutic drugs for gout. Tetrahydropalmatine (THP), the main active component of the traditional Chinese medicinal herb Corydalis yanhusuo, has shown prominent anti-inflammatory and analgesic activities, but to date, these effects have not been investigated exhaustively on gout. This study indicated that THP attenuated pain and swelling in an MSU-induced acute gout model by decreasing pro-inflammatory cytokine production and inflammatory cell infiltration. THP exerted its actions by suppressing NLRP3 inflammasome activation and subsequent formation of caspase-1. Furthermore, results showed that THP alleviated MSU-induced reactive oxygen species (ROS) generation, upstream of NLRP3 inflammasome activation, by an increase in the activities of antioxidant enzymes both in vitro and in vivo. In conclusion, our study suggests that THP suppressed ROS-mediated NLRP3 inflammasome activation in MSU-induced inflammatory responses, which highlights its therapeutic potential in gouty arthritis.

The inhibitor effect of RKIP on inflammasome activation and inflammasome-dependent diseases.

Aberrant inflammasome activation contributes to the pathogenesis of various human diseases, including atherosclerosis, gout, and metabolic disorders. Elucidation of the underlying mechanism involved in the negative regulation of the inflammasome is important for developing new therapeutic targets for these diseases. Here, we showed that Raf kinase inhibitor protein (RKIP) negatively regulates the activation of the NLRP1, NLRP3, and NLRC4 inflammasomes. RKIP deficiency enhanced caspase-1 activation and IL-1ß secretion via NLRP1, NLRP3, and NLRC4 inflammasome activation in primary macrophages. The overexpression of RKIP in THP-1 cells inhibited NLRP1, NLRP3, and NLRC4 inflammasome activation. RKIP-deficient mice showed increased sensitivity to Alum-induced peritonitis and Salmonella typhimurium-induced inflammation, indicating that RKIP inhibits NLRP3 and NLRC4 inflammasome activation in vivo. Mechanistically, RKIP directly binds to apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and competes with NLRP1, NLRP3, or NLRC4 to interact with ASC, thus interrupting inflammasome assembly and activation. The depletion of RKIP aggravated inflammasome-related diseases such as monosodium urate (MSU)-induced gouty arthritis and high-fat diet (HFD)-induced metabolic disorders. Furthermore, the expression of RKIP was substantially downregulated in patients with gouty arthritis or type 2 diabetes (T2D) compared to healthy controls. Collectively, our findings suggest that RKIP negatively regulates NLRP1, NLRP3, and NLRC4 inflammasome activation and is a potential therapeutic target for the treatment of inflammasome-related diseases.

Silencing of Gasdermin D by siRNA-Loaded PEI-Chol Lipopolymers Potently Relieves Acute Gouty Arthritis through Inhibiting Pyroptosis.

Gasdermin D (GSDMD) plays a causal role in NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis eruption, which has been regarded as a potential therapeutic target for pyroptosis-related diseases including acute gouty arthritis. In the present study, the synthesized PEI-Chol (cholesterol grafted polyethylenimine) was assembled with GSDMD small interfering RNA (siRNA) to form PEI-Chol/siGSDMD polyplexes, which provided high transfection efficiency for siRNA-mediated GSDMD knockdown. Then we evaluated the effect of GSDMD siRNA-loaded PEI-Chol on inflammatory cascades in bone-marrow-derived macrophages (BMDMs) and acute gouty arthritis animal models under MSU exposure. When accompanied by pyroptosis blockade and decreased release of interleukin-1 beta (IL-1ß), NLRP3 inflammasome activation was also suppressed by GSDMD knockdown in vivo and in vitro. Moreover, in MSU-induced acute gouty arthritis mice, blocking GSDMD with siRNA significantly improved ankle swelling and inflammatory infiltration observed in histopathological analysis. Furthermore, investigation using a mouse air pouch model verified the effect of siGSDMD-loaded PEI-Chol on pyroptosis of recruited macrophages and related signaling pathways in response to MSU. These novel findings exhibited that GSDMD knockdown relieved acute gouty arthritis through inhibiting pyroptosis, providing a possible therapeutic approach for MSU-induced acute gouty arthritis molecular therapy using PEI-Chol as a nucleic acid delivery carrier.

The Extraction Solvent Influences the Anti-Inflammatory Effects of Jakyakgamcho-Tang in Lipopolysaccharide-Stimulated Macrophages and Mice with Gouty Arthritis.

Jakyakgamcho-Tang (JGT) is a traditional medicine used to treat muscular tension, spasm, and pain. Several studies have reported its clinical use as an anti-inflammatory and in gynaecological treatment. This study aimed to compare the anti-inflammatory effects of JGT according to extraction solvent, water (JGTW) and 30% EtOH (JGTE) on lipopolysaccharide (LPS)-stimulated macrophages and in mice with monosodium urate (MSU)-induced gouty arthritis. We evaluated the production of inflammatory mediators and cytokines and the expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. We also examined oedema, pain, and inflammation in MSU-induced mice by measuring affected hind paw swelling, weight-bearing, pro-inflammatory cytokines levels, and myeloperoxidase (MPO) activity. In LPS-stimulated RAW264.7 cells, JGTW and JGTE significantly decreased prostaglandin (PG) E2(PGE2) production via suppressing COX-2 expression and cytokines interleukin-1ß and interleukin-6. Only JGTE reduced the production of NO and cytokines and the mRNA levels of iNOS and cytokines. In MSU-induced mice, JGTE and JGTW efficiently decreased paw swelling and attenuated joint pain. JGTE (200 and 300 mg/kg) effectively suppressed inflammation by downregulating pro-inflammatory cytokines (tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) and MPO activity, which were only slightly reduced by JGTW. Our data demonstrate the anti-inflammatory activity of JGT in macrophage and gouty arthritis animal models and show that JGTE is more effective than JGTW at lower concentrations.

Efficacy of Xixiancao (Herba Siegesbeckiae Orientalis) on interactions between nuclear factor kappa-B and inflammatory cytokines in inflammatory reactions of rat synovial cells induced by sodium urate.

OBJECTIVE: To investigate the interaction between nuclear factor kappa-B (NF-κB) and inflammatory cytokines in synovial cell inflammatory responses induced by sodium urate, and to evaluate the efficacy of Xixiancao (Herba Siegesbeckiae Orientalis) on these interactions. METHODS: The interactions between NF-κB and inflammatory cytokines/mediators in synovial cells in acute gouty arthritis were investigated. We observed the expressions of NF-κB, interleukin (IL)-1ß, IL-8, and tumor necrosis factor alpha (TNF-α) in synovial cells at different timepoints in an in vitro model of synovial cell inflammatory responses induced by sodium urate and in an in vivo model of gouty arthritis. Changes in the expressions of NF-κB, IL-1ß, IL-8, and TNF- in synovial cells of all experimental groups were compared and observed after treatment with different doses of Xixiancao (Herba Siegesbeckiae Orientalis) and colchicine. The interactions between NF-κB and IL-1ß, IL-8, and TNF-α were analyzed. Pathological changes in synovial tissues were observed in rats with acute gouty arthritis. RESULTS: Compared with the blank group, the expression levels of NF-κB, IL-1ß, IL-8, and TNF-α were increased significantly at different timepoints in the in vitro model of synovial cell inflammatory responses induced by sodium urate, and in the in vivo model of gouty arthritis. Compared with the model group, the expressions of NF-κB, IL-1ß, IL-8, and TNF-α in synovial cells induced by sodium urate were decreased in the different Xixiancao (Herba Siegesbeckiae Orientalis) dose groups and the colchicine group. The effect was more obvious in the high dose Xixiancao (Herba Siegesbeckiae Orientalis) group. The expression of NF-κB in synovial cells was positively correlated with the expressions of IL-1ß, IL-8, and TNF-. Histopathological examination of synovial tissues in the high dose Xixiancao (Herba Siegesbeckiae Orientalis) group and Colchicine group showed that the characteristics of acute gouty arthritis were reduced, and there was a trend towards a positive correlation between NF-κB and inflammatory cytokine expressions. CONCLUSION: The activation of NF-κB is associated with the activation of IL-1ß, IL-8, and TNF-α during the pathogenesis of acute gouty arthritis, leading to the continuation and enhancement of the inflammatory response. Expressions of IL-1ß, IL-8, and TNF-α in synoviocytes during acute gouty arthritis effectively inhibit local inflammation.

Cyr61 Promotes Inflammation of a Gouty Arthritis Model in Rats.

BACKGROUND: Cyr61 is considered a novel proinflammatory factor. Gouty arthritis (GA) is a self-limited inflammatory reaction caused by monosodium urate (MSU) crystals. In this study, we assessed the role of Cyr61 in the inflammatory process of GA. METHODS: We investigated the expression of Cyr61 in MSU-induced rat gout models and MSU-stimulated rat fibroblast-like synovial (FLS) cells. After inhibiting the expression of Cyr61, levels of IL-1ß, TNF-α, and IL-6 were detected by ELISA, qPCR, western blot, and immunohistochemical methods. We probed the downstream NF-κB signaling pathway using the NF-κB inhibitor PDTC, and levels of NF-κB and p-NF-κB were detected by western blot and qPCR. RESULTS: Our results demonstrate that Cyr61 plays a potent role in the formation of local inflammation in vitro and in vivo. Cyr61 was highly expressed in synovial tissues of gout models, and the expression of Cyr61 protein was also significantly increased in MSU-stimulated FLS cells. Cyr61 promoted MSU-induced acute inflammation via the NF-κB signaling pathway. CONCLUSIONS: Our study has revealed that Cyr61 is an important regulatory factor for the initiation of inflammation in GA. The high expression of Cyr61 protein can induce synovial cells to produce many inflammatory cytokines, such as IL-1ß, TNF-α, and IL-6, partly in an NF-κB-dependent manner. Thus, inhibition of Cyr61 could be a new target and strategy for the prevention and treatment of GA.

CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout.

Gout is a chronic arthritis caused by the deposition of poorly soluble monosodium urate monohydrate (MSU) crystals in peripheral joints. Resident macrophages initiate inflammation in response to MSU mediated by NF-κB nuclear translocation and NLRP3 inflammasome activation. We investigated the role of CD44, a transmembrane receptor, in mediating MSU phagocytosis by macrophages. We used an antibody that sheds the extracellular domain (ECD) of CD44 to study the role of the receptor and its associated protein phosphatase 2A (PP2A) in macrophage activation. We also studied the significance of CD44 in mediating MSU inflammation in-vivo. Cd44-/- BMDMs showed reduced MSU phagocytosis, LDH release, IL-1ß expression and production compared to Cd44+/+ BMDMs. Elevated CD44 staining was detected intracellularly and CD44 colocalized with α-tubulin as a result of MSU exposure and ECD-shedding reduced MSU phagocytosis in murine and human macrophages. Anti-CD44 antibody treatment reduced NF-κB p65 subunit nuclear levels, IL-1ß expression, pro-IL-1ß and IL-8 production in MSU stimulated THP-1 macrophages (p < 0.01). The effect of the antibody was mediated by an enhancement in PP2A activity. CD44 ECD-shedding reduced the conversion of procaspase-1 to active caspase-1, caspase-1 activity and resultant generation of mature IL-1ß in macrophages. Neutrophil and monocyte influx and upregulated production of IL-1ß was evident in wildtype mice. MSU failed to trigger neutrophil and monocyte recruitment in Cd44-/- mice and lower IL-1ß levels were detected in peritoneal lavages from Cd44-/- mice (p < 0.01). Anti-CD44 antibody treatment reduced neutrophil and monocyte recruitment and resulted in reduced lavage IL-1ß levels in the same model. CD44 plays a biologically significant role in mediating phagocytosis of MSU and downstream inflammation and is a novel target in gout treatment.

Direct Binding to NLRP3 Pyrin Domain as a Novel Strategy to Prevent NLRP3-Driven Inflammation and Gouty Arthritis.

OBJECTIVE: The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases. METHODS: A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1ß (IL-1ß) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout. RESULTS: Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of ß-carotene to the pyrin domain (PYD) of NLRP3 (KD = 3.41 × 10-6 ). Molecular modeling and mutation assays revealed the interaction mode between ß-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of ß-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, ß-carotene reduced IL-1ß secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout. CONCLUSION: Our results present ß-carotene as a selective and direct inhibitor of NLRP3, and the binding of ß-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis.

Urate-induced immune programming: Consequences for gouty arthritis and hyperuricemia.

Trained immunity is a process in which innate immune cells undergo functional reprogramming in response to pathogens or damage-associated molecules leading to an enhanced non-specific immune response to subsequent stimulation. While this capacity to respond more strongly to stimuli is beneficial for host defense, in some circumstances it can lead to maladaptive programming and chronic inflammation. Gout is characterized by persistent low-grade inflammation and is associated with an increased number of comorbidities. Hyperuricemia is the main risk factor for gout and is linked to the development of comorbidities. Several experimental studies have shown that urate can mechanistically alter the inflammatory capacity of myeloid cells, while observational studies have indicated an association of hyperuricemia to a wide spectrum of common adult inflammatory diseases. In this review, we argue that hyperuricemia is a main culprit in the development of the long-term systemic inflammation seen in gout. We revisit existing evidence for urate-induced transcriptional and epigenetic reprogramming that could lead to an altered functional state of circulating monocytes consisting in enhanced responsiveness and maladaptive immune responses. By discussing specific functional adaptations of monocytes and macrophages induced by soluble urate or monosodium urate crystals and their contribution to inflammation in vitro and in vivo, we further enforce that urate is a metabolite that can induce innate immune memory and we discuss future research and possible new therapeutic approaches for gout and its comorbidities.

Phosphoinositide-3 kinase gamma regulates caspase-1 activation and leukocyte recruitment in acute murine gout.

This study investigates the participation of PI3Kγ in the development of joint inflammation and dysfunction in an experimental model of acute gout in mice. Acute gout was induced by injection of monosodium urate (MSU) crystals into the tibiofemoral joint of mice. The involvement of PI3Kγ was evaluated using a selective inhibitor and mice deficient for PI3Kγ (PI3Kγ-/- ) or with loss of kinase activity. Neutrophils recovered from the inflamed joint were quantified and stained for phosphorylated Akt (pAkt) and production of reactive oxygen species (ROS). The adherence of leukocytes to the joint microvasculature was assessed by intravital microscopy and cleaved caspase-1 by Western blot. Injection of MSU crystals induced massive accumulation of neutrophils expressing phosphorylated Akt. In the absence of PI3Kγ, there was reduction of pAkt expression, chemokine production, and neutrophil recruitment. Genetic or pharmacological inhibition of PI3Kγ reduced the adherence of leukocytes to the joint microvasculature, even in joints with established inflammation. Neutrophils from PI3Kγ-/- mice produced less ROS than wild-type neutrophils. There was decreased joint damage and dysfunction in the absence of PI3Kγ. In addition, in the absence of PI3Kγ activity, there was reduction of cleaved caspase-1 and IL-1ß production in synovial tissue after injection of MSU crystals and leukotriene B4 . Our studies suggest that PI3Kγ is crucial for MSU crystal-induced acute joint inflammation. It is necessary for regulating caspase-1 activation and for mediating neutrophil migration and activation. Drugs that impair PI3Kγ function may be useful to control acute gout inflammation.

Novel findings from determination of common expressed plasma exosomal microRNAs in patients with psoriatic arthritis, psoriasis vulgaris, rheumatoid arthritis, and gouty arthritis.

Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy. Localized tumors can be treated successfully with surgical excision, but the presence of micrometastases, recurrence, and advanced disease are associated with high mortality rates, despite the use of chemotherapy with etoposide, doxorubicin, cisplatin (EDP), and mitotane. During the past decade, the characterization of ACC using genomic profiling and next-generation sequencing (NGS) has resulted in the proposed new targeted therapies for patients with advanced ACC. In 2018, the European Society of Endocrinology in collaboration with the European Network for the Study of Adrenal Tumors (ENSAT) published clinical practice guidelines for the management of ACC. However, the authors of these new guidelines concluded that the evidence to support clinical management recommendations remains weak, as there remains a requirement for large-scale controlled clinical trials to support new targeted therapies. This review discusses the recent developments in the diagnosis, staging, and management of ACC, and the molecular changes that may be the basis for future personalized or targeted therapy, if supported by data from clinical trials.

Mechanistic insight into the attenuation of gouty inflammation by Taiwanese green propolis via inhibition of the NLRP3 inflammasome.

Dysregulation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including gouty arthritis. Activation of the NLRP3 inflammasome requires priming and activation signals: the priming signal controls the expression of NLRP3 and interleukin (IL)-1ß precursor (proIL-1ß), while the activation signal leads to the assembly of the NLRP3 inflammasome and to caspase-1 activation. Here, we reported the effects of the alcoholic extract of Taiwanese green propolis (TGP) on the NLRP3 inflammasome in vitro and in vivo. TGP inhibited proIL-1ß expression by reducing nuclear factor kappa B activation and reactive oxygen species (ROS) production in lipopolysaccharide-activated macrophages. Additionally, TGP also suppressed the activation signal by reducing mitochondrial damage, ROS production, lysosomal rupture, c-Jun N-terminal kinases 1/2 phosphorylation and apoptosis-associated speck-like protein oligomerization. Furthermore, we found that TGP inhibited the NLRP3 inflammasome partially via autophagy induction. In the in vivo mouse model of uric acid crystal-induced peritonitis, TGP attenuated the peritoneal recruitment of neutrophils, and the levels of IL-1ß, active caspase-1, IL-6 and monocyte chemoattractant protein-1 in lavage fluids. As a proof of principle, in this study, we purified a known compound, propolin G, from TGP and identified this compound as a potential inhibitor of the NLRP3 inflammasome. Our results indicated that TGP might be useful for ameliorating gouty inflammation via inhibition of the NLRP3 inflammasome.

Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.

Oridonin (Ori) is the major active ingredient of the traditional Chinese medicinal herb Rabdosia rubescens and has anti-inflammatory activity, but the target of Ori remains unknown. NLRP3 is a central component of NLRP3 inflammasome and has been involved in a wide variety of chronic inflammation-driven human diseases. Here, we show that Ori is a specific and covalent inhibitor for NLRP3 inflammasome. Ori forms a covalent bond with the cysteine 279 of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7, thereby inhibiting NLRP3 inflammasome assembly and activation. Importantly, Ori has both preventive or therapeutic effects on mouse models of peritonitis, gouty arthritis and type 2 diabetes, via inhibition of NLRP3 activation. Our results thus identify NLRP3 as the direct target of Ori for mediating Ori's anti-inflammatory activity. Ori could serve as a lead for developing new therapeutics against NLRP3-driven diseases.

MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis.

BACKGROUND: Gout is considered one of the most painful acute conditions caused by deposition of monosodium urate (MSU) crystals within joints. Recent studies have shown that interleukin (IL)-1ß is a key inflammatory mediator in acute gouty arthritis (GA), and its level is regulated by microRNAs (miRNAs). However, the molecular mechanisms of the regulation remain unclear. METHODS: A miRNA microarray was used to analyze the miRNA expression profiles in peripheral white blood cells (WBCs) of patients with GA. THP-1 cells were transfected with miRNA mimics, stimulated by MSU crystals, and then subjected to quantitative real-time polymerase chain reaction or Western blot analysis. Levels of IL-1ß, IL-8, and tumor necrosis factor (TNF)-α in culture supernatants of THP-1 cells were measured by enzyme-linked immunosorbent assay. A luciferase reporter assay was conducted to confirm the interaction of miRNA and IL-1ß 3'-untranslated regions (UTRs). RESULTS: Combining bioinformatics and miRNA expression profiles, we found five miRNAs (hsa-miR-30c-1-3p, hsa-miR-488-3p, hsa-miR-550a-3p, hsa-miR-663a, and hsa-miR-920) that possibly target IL-1ß. Then, we demonstrated that miR-488 and miR-920 were significantly decreased in the WBCs of patients with GA and that MSU crystals could inhibit expression of miR-488 and miR-920. Upregulation of miR-488 and miR-920 could suppress MSU-induced IL-1ß protein expression in THP-1 cells, but no significant difference in IL-1ß messenger RNA levels was observed. Moreover, we found that miR-488 and miR-920 could directly target the 3'-UTR of IL-1ß. Overexpression of miR-488 and miR-920 could significantly inhibit the gene and protein expression of IL-8 and TNF-α in MSU-induced THP-1 cells. CONCLUSIONS: This study demonstrates the roles of miR-488 and miR-920 in regulating the production of proinflammatory cytokines in the pathogenesis of GA. These findings suggest that miR-488 and miR-920 could serve as potential therapeutic targets in the treatment of GA.

Anti-inflammatory effects of traditional mixed extract of medicinal herbs (MEMH) on monosodium urate crystal-induced gouty arthritis.

Korean oriental medicine prescription is widely used for the treatment of gouty diseases. In the present study, we investigated anti-inflammatory effects of modified Korean herbal formulation, mixed extract of medicinal herbs (MEMH), and its modulatory effects on inflammatory mediators associated with gouty arthritis. Both in vitro and in vivo studies were carried out to assess the anti-inflammatory efficacy of MEMH on monosodium urate (MSU) crystals-induced gouty inflammation. MSU crystals stimulated human chondrosarcoma cell line, SW1353, and human primary chondrocytes were treated with MEMH in vitro. The expression levels of pro-inflammatory mediators and metalloproteases were analyzed. The effect of MEMH on NFκB signaling pathway in SW1353 cells was examined. Effect of MEMH on the mRNA expression level of pro-inflammatory mediators and chemotactic factor from human monocytic cell line, THP-1, was also analyzed. The probable role of MEMH in the differentiation process of osteoblast like cells, SaOS-2, after MSU treatment was also observed. To investigate the effects of MEMH in vivo, MSU crystals-induced ankle arthritic model was established. Histopathological changes in affected joints and plasma levels of pro-inflammatory mediators (IL-1ß and TNFα) were recorded. MEMH inhibited NFκB signaling pathway and COX-2 protein expression in chondrocytes. MSU-induced mRNA expressions of pro-inflammatory mediators and chemotactic cytokines were suppressed by MEMH. In MSU crystals-induced ankle arthritic mouse model, administration of MEMH relieved inflammatory symptoms and decreased the plasma levels of IL-1ß and TNFα. The results indicated that MEMH can effectively inhibit the expression of inflammatory mediators in gouty arthritis, demonstrating its potential for treating gouty arthritis.

Molecular Pathophysiology of Gout.

Three contradictory clinical presentations of gout have puzzled clinicians and basic scientists for some time: first, the crescendo of sterile inflammation in acute gouty arthritis; second, its spontaneous resolution, despite monosodium urate (MSU) crystal persistence in the synovium; and third, immune anergy to MSU crystal masses observed in tophaceous or visceral gout. Here, we provide an update on the molecular pathophysiology of these gout manifestations, namely, how MSU crystals can trigger the auto-amplification loop of necroinflammation underlying the crescendo of acute gouty arthritis. We also discuss new findings, such as how aggregating neutrophil extracellular traps (NETs) might drive the resolution of arthritis and how these structures, together with granuloma formation, might support immune anergy, but yet promote tissue damage and remodeling during tophaceous gout.

Morin, a dietary bioflavonol suppresses monosodium urate crystal-induced inflammation in an animal model of acute gouty arthritis with reference to NLRP3 inflammasome, hypo-xanthine phospho-ribosyl transferase, and inflammatory mediators.

The anti-inflammatory effect of morin, a dietary bioflavanol was explored on monosodium urate (MSU) crystal-induced inflammation in rats, an experimental model for acute gouty arthritis. Morin treatment (30mg/kg b.wt) significantly attenuated the ankle swelling and the levels of lipid peroxidation, nitric oxide, serum pro-inflammatory cytokines (tumor necrosis factor (TNF) -α, interleukin (IL)-1ß, and IL-6), monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and articular elastase along with an increased anti-oxidant status (catalase (CAT) and superoxide dismutase (SOD)) in the joint homogenate of MSU crystal-induced rats. Histological assessment revealed that morin limited the diffusion of joint space, synovial hyperplasia, and inflammatory cell infiltrations. The mRNA expression of NLRP3 (nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3) inflammasome, caspase-1, pro-inflammatory cytokines, MCP-1, inflammatory enzymes (inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)), and nuclear factor-kappa B (NF-κB) p65 was found downregulated and HPRT (hypo-xanthine phospho-ribosyl transferase) mRNA expression was upregulated in morin treated MSU crystal-induced rats. In addition, morin treatment reduced the protein expression of NF-κB p65, p-NF-κB p65, iNOS, COX-2, and TNF-α. The results clearly demonstrated that morin exert a potent anti-inflammatory effect on MSU crystal-induced inflammation in rats.