Signaling pathways in uric acid homeostasis and gout: From pathogenesis to therapeutic interventions.

Uric acid is a product of purine degradation, and uric acid may have multiple physiologic roles, including the beneficial effects as an antioxidant and neuroprotector, maintenance of blood pressure during low salt ingestion, and modulation of immunity. However, overproduction of metabolic uric acid, and/or imbalance of renal uric acid secretion and reabsorption, and/or underexcretion of extrarenal uric acid, e.g. gut, will contribute to hyperuricemia, which is a common metabolic disease. Long-lasting hyperuricemia can induce the formation and deposition of monosodium urate (MSU) crystals within the joints and periarticular structures. MSU crystals further induce an acute, intensely painful, and sterile inflammation conditions named as gout by NLRP3 inflammasome-mediated cleavage of pro-IL-1ß to bioactive IL-1ß. Moreover, hyperuricemia and gout are associated with multiple cardiovascular and renal disorders, e.g., hypertension, myocardial infarction, stroke, obesity, hyperlipidemia, type 2 diabetes mellitus and chronic kidney disease. Although great efforts have been made by scientists of modern medicine, however, modern therapeutic strategies with a single target are difficult to exert long-term positive effects, and even some of these agents have severe adverse effects. The Chinese have used the ancient classic prescriptions of traditional Chinese medicine (TCM) to treat metabolic diseases, including gout, by multiple targets, for more than 2200 years. In this review, we discuss the current understanding of urate homeostasis, the pathogenesis of hyperuricemia and gout, and both modern medicine and TCM strategies for this commonly metabolic disorder. We hope these will provide the good references for treating hyperuricemia and gout.

Tailored hydrogel composite membrane for the regulated crystallization of monosodium urate monohydrate within coffee's metabolites system.

Herein, a facile bionic research platform with fabricated hydrogel composite membrane (HCM) is constructed to uncover the effects of the main components of coffee's metabolites on MSUM crystallization. Tailored and biosafety polyethylene glycol diacrylate/N-isopropyl acrylamide (PEGDA/NIPAM) HCM allows the proper mass transfer of coffee's metabolites and can well simulate the process of coffee's metabolites acting in the joint system. With the validations of this platform, it is shown that chlorogenic acid (CGA) can hinder the MSUM crystals formation from 45 h (control group) to 122 h (2 mM CGA), which is the most likely reason that reduces the risk of gout after long-term coffee consumption. Molecular dynamics simulation further indicates that the high interaction energy (Eint) between CGA and MSUM crystal surface and the high electronegativity of CGA both contribute to the restraint of MSUM crystal formation. In conclusion, the fabricated HCM, as the core functional materials of the research platform, presents the understanding of the interaction between coffee consumption and gout control.

Anaerobic purinolytic enzymes enable dietary purine clearance by engineered gut bacteria.

Uric acid, the end product of purine degradation, causes hyperuricemia and gout, afflicting hundreds of millions of people. The debilitating effects of gout are exacerbated by dietary purine intake, and thus a potential therapeutic strategy is to enhance purine degradation in the gut microbiome. Aerobic purine degradation involves oxidative dearomatization of uric acid catalyzed by the O2-dependent uricase. The enzymes involved in purine degradation in strictly anaerobic bacteria remain unknown. Here we report the identification and characterization of these enzymes, which include four hydrolases belonging to different enzyme families, and a prenyl-flavin mononucleotide-dependent decarboxylase. Introduction of the first two hydrolases to Escherichia coli Nissle 1917 enabled its anaerobic growth on xanthine as the sole nitrogen source. Oral supplementation of these engineered probiotics ameliorated hyperuricemia in a Drosophila melanogaster model, including the formation of renal uric acid stones and a shortened lifespan, providing a route toward the development of purinolytic probiotics.

Mechanisms of theaflavins against gout and strategies for improving the bioavailability.

BACKGROUND: Gout is a crystal related arthropathy caused by monosodium urate deposition. At present, the identification of appropriate treatments and new drugs to reduce serum uric acid levels and gout risk is a major research area. PURPOSE: Theaflavins are naturally occurring compounds characterized by a benzodiazepine skeleton. The significant benefits of theaflavins have been well documented. A large number of studies have been carried out and excellent anti-gout results have been achieved in recent years. STUDY DESIGN: A comprehensive analysis of the mechanism of the anti-gout effect of theaflavins is presented through a literature review and network pharmacology prediction, and strategies for increasing the bioavailability of theaflavins are summarized. METHODS: In this review, the active components and pharmacological mechanisms of theaflavins in the treatment of gout were summarized, and the relationship between theaflavins and gout, the relevant components, and the potential mechanisms of anti-gout action were clarified by reviewing the literature on the anti-gout effects of theaflavins and network pharmacology. RESULTS: Theaflavins exert anti-gout effects by down regulating the gene and protein expression of glucose transporter 9 (GLUT9) and uric acid transporter 1 (URAT1), while upregulating the mRNA expression levels of organic anion transporter 1 (OAT1), organic cation transporter N1 (OCTN1), organic cation transporters 1/2 (Oct1/2), and organic anion transporter 2 (OAT2). Network pharmacology prediction indicate that theaflavins can regulate the AGE-RAGE and cancer signaling pathways through ATP-binding cassette subfamily B member 1 (ABCB1), recombinant mitogen activated protein kinase 14 (MAPK14), telomerase reverse tranase (TERT), signal transducer and activator of transcription 1 (STAT1), matrix metalloproteinase 2 (MMP2), B-cell lymphoma-2 (BCL2), and matrix metalloproteinase 14 (MMP14) targets for anti-gout effects. CONCLUSION: This review presents the mechanisms of anti-gout action of theaflavins and strategies for improving the bioavailability of theaflavins, as well as providing research strategies for anti-gout treatment measures and the development of novel anti-gout drugs.

Ouratea spectabilis and its Biflavanone Ouratein D Exert Potent Anti-inflammatory Activity in MSU Crystal-induced Gout in Mice.

Gouty arthritis (GA) is an inflammatory arthritis triggered by the deposition of monosodium urate monohydrate (MSU) crystals, causing pain, inflammation, and joint damage. Several drugs are currently employed to manage acute flares of GA, but they either have limited effectiveness or induce severe adverse reactions. Ouratea spectabilis is traditionally used in Brazil to treat gastric ulcers and rheumatism. The ethanolic extract of O. spectabilis stems (OSpC) and four biflavanones (ouratein A - D) isolated thereof were evaluated in a murine model of GA induced by the injection of MSU crystals. The underlying mechanism of action of ouratein D was investigated in vitro in cell cultures by measurement of IL-1ß levels by ELISA and Western blot analysis. The administration of OSpC (10, 30 or 100 mg/Kg, p. o.) reduced the migration of total inflammatory cells, monocytes, and neutrophils and diminished the levels of IL-1ß and CXCL1 in the synovial tissue. Among the tested compounds, only ouratein D (1 mg/Kg) reduced the migration of the inflammatory cells and it was shown to be active up to 0.01 mg/Kg (equivalent to 0.34 nM/Kg, p. o.). Treatment of pre-stimulated THP-1 cells (differentiated into macrophages) or BMDMs with ouratein D reduced the release of IL-1ß in both macrophage lines. This biflavanone reduced the activation of caspase-1 (showed by the increase in the cleaved form) in supernatants of cultured BMDMs, evidencing its action in modulating the inflammasome pathway. The obtained results demonstrate the anti-gout properties of O. spectabilis and point out ouratein D as the bioactive component of the assayed extract.

Anti-inflammatory potential of stevia residue extract against uric acid-associated renal injury in mice.

Abnormal uric acid level result in the development of hyperuricemia and hallmark of various diseases, including renal injury, gout, cardiovascular disorders, and non-alcoholic fatty liver. This study was designed to explore the anti-inflammatory potential of stevia residue extract (STR) against hyperuricemia-associated renal injury in mice. The results revealed that STR at dosages of 150 and 300 mg/kg bw and allopurinol markedly modulated serum uric acid, blood urea nitrogen, and creatinine in hyperuricemic mice. Serum and renal cytokine levels (IL-18, IL-6, IL-1Β, and TNF-α) were also restored by STR treatments. Furthermore, mRNA and immunohistochemistry (IHC) analysis revealed that STR ameliorates UA (uric acid)-associated renal inflammation, fibrosis, and EMT (epithelial-mesenchymal transition) via MMPS (matrix metalloproteinases), inhibiting NF-κB/NLRP3 activation by the AMPK/SIRT1 pathway and modulating the JAK2-STAT3 and Nrf2 signaling pathways. In summary, the present study provided experimental evidence that STR is an ideal candidate for the treatment of hyperuricemia-mediated renal inflammation. PRACTICAL APPLICATIONS: The higher uric acid results in hyperuricemia and gout. The available options for the treatment of hyperuricemia and gout are the use of allopurinol, and colchicine drugs, etc. These drugs possess several undesirable side effect. The polyphenolic compounds are abundantly present in plants, for example, stevia residue extract (STR) exert a positive effect on human health. From this study results, we can recommend that polyphenolic compounds enrich STR could be applied to develop treatment options for the treatment of hyperuricemia and gout.

Protective effects of di-caffeoylquinic acids from Artemisia selengensis Turcz leaves against monosodium urate-induced inflammation via the modulation of NLRP3 inflammasome and Nrf2 signaling pathway in THP-1 macrophages.

Artemisia selengensis Turcz (AST) as a common vegetable is rich in di-caffeoylquinic acids (di-CQAs) and has been reported to possess multiple health benefits. However, whether di-CQAs from AST leaf extracts (ASTE) could alleviate gout inflammation is still unknown. Herein, this study explored the inhibitory mechanism of ASTE on gout inflammation in THP-1 macrophages. Results suggested that ASTE suppressed the secretion and mRNA levels of inflammatory cytokines including interleukin-18, interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Pretreatment with ASTE inhibited lipopolysaccharide-induced of IκBα degradation, p65 phosphorylation and up-regulation of Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome proteins. Moreover, ASTE inhibited monosodium urate-induced the up-regulation of active caspase-1 and interleukin-1ß, promoted nuclear factor E2-related factor2 (Nrf2) to translocate into the nucleus, reducing the generation of MSU-induced reactive oxygen species. These results suggested that ASTE alleviated gout inflammation via inhibiting NLRP3 inflammasome activation and activating Nrf2 signaling pathway. PRACTICAL APPLICATIONS: Artemisia selengensis Turcz (AST) as a common vegetable in China belongs to genus Artemisia, which are rich in di-caffeoylquinic acids. This study aimed to investigate the effect of ASTE on alleviating gout inflammation and whether NLRP3 inflammasome and Nrf2 signaling pathways are involved in the protection of ASTE against gout inflammation. Our findings are significant for developing di-CQAs from AST by-products as an effective functional food for preventing gout.

Taxifolin blocks monosodium urate crystal-induced gouty inflammation by regulating phagocytosis and autophagy.

Gout is a chronic disease caused by monosodium urate (MSU) crystal deposition in the joints and surrounding tissues. We examined the effects of Taxifolin, a natural flavonoid mainly existing in vegetables and fruits, on MSU-induced gout. Pretreatment with Taxifolin significantly reduced IL-1ß, Caspase-1 and HMGB1 levels, upregulation of autophagy-related protein, LC3, as well as improved phagocytosis of macrophages. This study indicated that Taxifolin-attenuated inflammatory response in MSU-induced acute gout model by decreasing pro-inflammatory cytokine production and promoting the autophagy and phagocytic capacity of macrophages. Dietary supplementation with Taxifolin induces the autophagy and attenuated inflammatory response, which in consequence modulates acute gout. A preventive strategy combining dietary interventions with Taxifolin may offer a potential therapeutic alternative to pharmacological treatment to reduce inflammatory response to gout.

Polysaccharide extract from Isatidis Radix inhibits multiple inflammasomes activation and alleviate gouty arthritis.

The polysaccharide extract from Isatidis Radix exhibits potent antiinflammatory and antiviral activities, but the mechanism of Isatidis Radix polysaccharide (IRP) remains obscure. Herein, we reported that IRP blocked the activation of nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, leading to the inhibiting of caspase-1 cleavage and IL-1ß secretion. Mechanistically, IRP did not inhibit NLRP3 inflammasome through suppressing mitochondrial reactive oxygen species (mtROS) production. However, IRP can significantly suppress the oligomerization of apoptosis-associated speck-like protein (ASC) and subsequently block the formation of inflammasome. Next, we evaluate the role of IRP in monosodium urate (MSU)-induced gout in vivo which is a NLRP3-associated disease. We also observed that oral administration of IRP can reduce the increased ankle thickness and the secretion of IL-1ß, IL-18, IL-6, TNF-α and MPO of the mouse ankle joints caused by MSU crystals. Furthermore, flow cytometry analysis highlighted a significant modulation of T helper 17 cells (Th17)/regulatory T cells (Treg) following IRP treatment in MSU induced gout. Overall, our findings suggest that IRP has comprehensive and potent antiinflammatory effects and provide a reasonable therapeutic strategy in preventing inflammasome-associated diseases, such as inflammatory gouty arthritis.

Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

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.

Network Pharmacology Study on Morus alba L. Leaves: Pivotal Functions of Bioactives on RAS Signaling Pathway and Its Associated Target Proteins against Gout.

M. alba L. is a valuable nutraceutical plant rich in potential bioactive compounds with promising anti-gouty arthritis. Here, we have explored bioactives, signaling pathways, and key proteins underlying the anti-gout activity of M. alba L. leaves for the first-time utilizing network pharmacology. Bioactives in M. alba L. leaves were detected through GC-MS (Gas Chromatography-Mass Spectrum) analysis and filtered by Lipinski's rule. Target proteins connected to the filtered compounds and gout were selected from public databases. The overlapping target proteins between bioactives-interacted target proteins and gout-targeted proteins were identified using a Venn diagram. Bioactives-Proteins interactive networking for gout was analyzed to identify potential ligand-target and visualized the rich factor on the R package via the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on STRING. Finally, a molecular docking test (MDT) between bioactives and target proteins was analyzed via AutoDock Vina. Gene Set Enrichment Analysis (GSEA) demonstrated that mechanisms of M. alba L. leaves against gout were connected to 17 signaling pathways on 26 compounds. AKT1 (AKT Serine/Threonine Kinase 1), γ-Tocopherol, and RAS signaling pathway were selected as a hub target, a key bioactive, and a hub signaling pathway, respectively. Furthermore, three main compounds (γ-Tocopherol, 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene) tyramine, and Lanosterol acetate) and three key target proteins-AKT1, PRKCA, and PLA2G2A associated with the RAS signaling pathway were noted for their highest affinity on MDT. The identified three key bioactives in M. alba L. leaves might contribute to recovering gouty condition by inactivating the RAS signaling pathway.

Quercetin decreases the antinociceptive effect of diclofenac in an arthritic gout-pain model in rats.

OBJECTIVE: To analyse the antinociceptive interaction between quercetin (QUER) and diclofenac (DIC) in experimental arthritic gout-pain. METHODS: The antinociceptive effect of DIC and QUER alone and in combination were evaluated using an arthritic gout-pain model. Pain was induced through intra-articular administration of uric acid in the rats and the treatments were administered 2 h later. Additionally, the cyclooxygenase (COX) activity was determined in rats treated with DIC, QUER and their combination. KEY FINDINGS: DIC induced a maximal effect of 69.7 ± 2.7% with 3.1 mg/kg; whereas QUER only produced 17.6 ± 2.6% with the maximal dose (316 mg/kg). Ten of twelve DIC + QUER combinations showed a lesser antinociceptive effect than DIC alone did (P < 0.05). Moreover, DIC reduced total-COX (70.4 ± 1.3 versus 52.4 ± 1.8 and 77.4 ± 9.0 versus 56.1 ± 1.3, P < 0.05) and COX-2 (60.1 ± 1.0 versus 42.4 ± 1.8 and 58.1 ± 2.4 versus 48.7 ± 1.3, P < 0.05) activity after 1 and 3 h, respectively. Nevertheless, only the COX-2 activity induced by DIC was prevented in the presence of QUER (63.2 ± 3.0 versus 60.1 ± 1.0 and 56.6 ± 1.3 versus 58.1 ± 2.4 at 1 and 3 h, respectively). CONCLUSIONS: All these data demonstrated that the simultaneous administration of QUER + DIC produces an unfavorable interaction on the antinociceptive effect of DIC. Therefore, this combination might not be recommendable to relieve arthritic gout-pain.

Cleroda-4(18),13-dien-15,16-olide as novel xanthine oxidase inhibitors: An integrated in silico and in vitro study.

In the present study, in silico predictions and molecular docking were performed on five clerodane diterpenes (1-5) from Polyalthia longifolia seeds to evaluate their potential as xanthine oxidase (XO) inhibitors. The initial screening was conducted by target prediction using TargetNet web server application and only compounds 3 and 4 showed a potential interaction with XO. Compounds 3 and 4 were subsequently subjected to in silico analyses on XO protein structure (PDB: 1N5X) using Schrödinger Release 2020-3 followed by structural modeling & molecular simulation studies to confirm the initial prediction result and identify the binding mode of these compounds to the XO. Molecular docking results revealed that compounds 3 (-37.3 kcal/mol) and 4 (-32.0 kcal/mol) binds more stably to XO than the reference drug allopurinol (-27.0 kcal/mol). Interestingly, two residues Glu 802 and Thr 1010 were observed as the two main H-bond binding sites for both tested compounds and the allopurinol. The center scaffold of allopurinol was positioned by some π-π stacking with Phe 914 and Phe 1009, while that of compounds 3 and 4 were supported by many hydrophobic interactions mainly with Leu 648, Phe 649, Phe 1013, and Leu 1014. Additionally, the docking simulation predicted that the inhibitory effect of compounds 3 and 4 was mediated by creating H-bond with particularly Glu 802, which is a key amino acid for XO enzyme inhibition. Altogether, in vitro studies showed that compounds 3 and 4 had better inhibitory capacity against XO enzyme with IC50 values significantly (p < 0.001) lower than that of allopurinol. In short, the present study identified cleroda-4(18),13-dien-15,16-olide as novel potential XO inhibitors, which can be potentially used for the treatment of gout.

Plantaginis Semen polysaccharides ameliorate renal damage through regulating NLRP3 inflammasome in gouty nephropathy rats.

Gouty nephropathy (GN) is considered to be a prevalent renal disease and is an inflammatory event mainly induced by MSU crystals. Plantaginis Semen is a traditional Chinese herb that has been used in the treatment of gout, gouty arthritis and GN, but the mechanism and ingredients have been unclear. In this study, we explored and evaluated the preliminary structural characterizations of Plantaginis Semen polysaccharides (PSPs) and the activity of protecting against renal damage in GN rats. Three polysaccharide fractions, PSP-D, PSP-H and PSP-S, were sequentially extracted by different processes from the seed of Plantago asiatica L. The Fourier transform infrared spectral (FTIR) results showed that there were significant differences between PSP-S and the other two polysaccharides (PSP-D and PSP-H). PSP-D and PSP-H have pyrene monomers and linkages of ß-glycosides in their structures, and PSP-S has furanoside in the molecular structure. The scanning electron microscope (SEM) images of three polysaccharides showed that PSP-D has a smooth surface and a small curve, PSP-H is block-like and uneven in magnitude, whereas PSP-S is sea-tent-like and its surface is very distinct from the others. Main components and molar ratios are also different. Rats were randomly divided into six groups (n1/6 8 per group): the control group, model group, positive group, and three treatment groups (PSP-D, PSP-H and PSP-S). For all groups except the control group, rats were intragastrically administered the adenine suspension (50 mg kg-1 d-1) and fed with a high-yeast diet (15 g kg-1 d-1) for 28 days. On the 9th day, the control group and the model group were administered normal saline at the same time. Treatment groups were individually given corresponding drugs for 20 days. We found that PSPs could prevent renal damage, including decreasing the inflammatory response and regulating the (NOD)-like receptor protein 3 (NLRP3) protein in renal tissue. The underlying mechanism was related to NLRP3 inflammasome signal pathways, and it could take effect through the down-regulation of the protein expression levels of NLRP3, ASC and caspase-1 and inhibit the release of downstream inflammatory factors. PSPs are promising polysaccharides that could protect against renal injury through ameliorating renal inflammation in GN rats. Plantaginis Semen polysaccharides are potential functional food ingredients or pharmacological agents for treating GN in clinical practice.

The Anti-Inflammatory and Uric Acid Lowering Effects of Si-Miao-San on Gout.

Background: Si-Miao-San (SMS) is a well-known traditional Chinese medicine. This study aims to evaluate the anti-inflammatory effects of SMS on gouty arthritis and its potential mechanism of action. Methods: The effects and mechanism of SMS were evaluated in monosodium urate (MSU)-treated mice or macrophages. The expression of cytokines and PI3K/Akt was analyzed using real-time PCR and Western blotting analyses. Macrophage polarization was assessed with immunofluorescence assays, real-time PCR, and Western blotting. Mass spectrometry was used to screen the active ingredients of SMS. Results: Pretreatment with SMS ameliorated MSU-induced acute gouty arthritis in mice with increased PI3K/Akt activation and M2 macrophage polarization in the joint tissues. In vitro, SMS treatment significantly inhibited MSU-triggered inflammatory response, increased p-Akt and Arg-1 expression in macrophages, and promoted M2 macrophage polarization. These effects of SMS were inhibited when PI3K/Akt activation was blocked by LY294002 in the macrophages. Moreover, SMS significantly reduced serum uric acid levels in the hyperuricemia mice. Using mass spectrometry, the plant hormones ecdysone and estrone were detected as the potentially effective ingredients of SMS. Conclusion: SMS ameliorated MSU-induced gouty arthritis and inhibited hyperuricemia. The anti-inflammatory mechanism of SMS may exert anti-inflammatory effects by promoting M2 polarization via PI3K/Akt signaling. Ecdysone and estrone might be the potentially effective ingredients of SMS. This research may provide evidence for the application of SMS in the treatment of gout.

RDP3, A Novel Antigout Peptide Derived from Water Extract of Rice.

Gout and hyperuricemia can seriously affect the quality of life; at present, however, existing medicines are unable to meet all clinical needs. In the current study, a novel peptide (i.e., rice-derived-peptide-3 (RDP3), AAAAMAGPK-NH2, 785.97 Da) in water extract obtained from shelled Oryza sativa fruits was identified. Testing revealed that RDP3 (minimum effective concentration 100 µg/kg) did not show both hemolytic and acute toxicity, and reduced uric acid levels in the serum of hyperuricemic mice by inhibiting xanthine oxidase activity and decreasing urate transporter 1 expression. RDP3 also alleviated renal injury in hyperuricemic mice by decreasing NLRP3 inflammasome expression. Furthermore, RDP3 alleviated formalin-induced paw pain and reduced monosodium urate crystal-induced paw swelling and inflammatory factors in mice. Thus, this newly identified peptide reduced uric acid levels and renal damage in hyperuricemic mice and showed anti-inflammatory and analgesic activities, indicating the potential of RDP3 as an antigout medicine candidate.

A mouse model of MSU-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1ß as relevant therapy for gout patients.

Rationale: The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1ß in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1ß secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Methods: Acute urate crystal inflammation was obtained by subcutaneous injections of MSU crystals in mice. Symptoms were followed by scoring, cytokine quantification by ELISA and western blot, gene expression by RT-qPCR and RNAseq; Magnetic Resonance Imaging was also used to assess inflammation. Results: We provide an extensive clinical, biological and molecular characterization of an acute uratic inflammation mouse model which accurately mimics human gout. We report the efficacy of topical imiquimod treatment and its impact on Interferon-dependent down modulation of Il-1ß gene expression in this experimental model. Conclusion: Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities for gout patients.

HSP60 Regulates Monosodium Urate Crystal-Induced Inflammation by Activating the TLR4-NF-κB-MyD88 Signaling Pathway and Disrupting Mitochondrial Function.

Acute gout is an inflammatory response induced by monosodium urate (MSU) crystals. HSP60 is a highly conserved stress protein that acts as a cellular "danger" signal for immune reactions. In this study, we aimed to investigate the role and molecular mechanism of HSP60 in gout. HSP60 expression was detected in peripheral blood mononuclear cells (PBMCs) and plasma of gout patients. The effect and molecular mechanism of HSP60 in gout were studied in MSU crystals treatment macrophages and C57BL/6 mice. JC-1 probe and MitoSOX Red were used to measure the mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS). HSP60 expression was significantly upregulated in the PBMCs and sera of patients with acute gout (AG) compared to those with intercritical gout (IG) or healthy controls (HCs). MSU crystals induced the expression and secretion of HSP60 in the macrophages. HSP60 knockdown or overexpression affects TLR4 and MyD88 expression, IκBα degradation, and the nuclear localization of NF-κB in MSU crystal-stimulated inflammation. Further, HSP60 facilitates MMP collapse and mtROS production and activates the NLRP3 inflammasome in MSU crystal-stimulated macrophages. In MSU crystal-induced arthritis mouse models pretreated with HSP60 vivo-morpholino, paw swelling, myeloperoxidase (MPO) activity, and inflammatory cell infiltration significantly decreased. Our study reveals that MSU crystal stimulates the expression of HSP60, which accelerates the TLR4-MyD88-NF-κB signaling pathway and exacerbates mitochondrial dysfunction.

Coptisine from Coptis chinensis blocks NLRP3 inflammasome activation by inhibiting caspase-1.

Inflammasome mediates the activation of caspase-1, which promotes the secretion of proinflammatory cytokines. In this work, we aimed to investigate whether natural compounds from a Traditional Chinese Medicine prescription called San-Huang-Xie-Xin-Tang exert its clinical efficacy by inhibiting inflammasome activation and the underlying mechanism. The inhibitory effects of compounds on caspase-1 were evaluated in recombinant expressed caspase-1 protein and macrophages. Molecular docking was conducted to examine the interaction between compounds and caspase-1. The effects of the compounds on pro-inflammatory cytokines were examined by enzyme-linked immunosorbent assay. The mechanism of the compounds on nucleotide oligomerization domain (NOD)-like receptor protein-3 (NLRP3) inflammasome activation was investigated in macrophages. The anti-inflammasome effects of compounds were examined in mice stimulated by lipopolysaccharide (LPS) and monosodium urate crystal (MSU). Coptisine was the most potent inhibitor of caspase-1 in the San-Huang-Xie-Xin-Tang prescription. Coptisine adopted a favorable conformation at the active site of caspase-1. Coptisine significantly attenuated mature interleukin (IL)-1ß secretion in RAW264.7 macrophages stimulated with LPS plus ATP, nigericin, or MSU, by blocking caspase-1 activation. Coptisine not only prevented NLRP3 inflammasome assembly by affecting the binding between pro-caspase-1 and apoptosis-associated speck-like protein containing a CARD, but also inhibited inflammasome priming by decreasing NLRP3 expression through inactivation of the nuclear factor-κB pathway. Moreover, coptisine prevented LPS-mediated IL-1ß production and MSU-mediated mice paw edema by blocking NLRP3 inflammasome activation in vivo. Coptisine blocks NLRP3 inflammasome activation by inhibiting caspase-1 and may be useful for treating NLRP3 inflammasome-involved gouty arthritis.