Highly specific characterization and discrimination of monosodium urate crystals in gouty arthritis based on aggregation-induced emission luminogens

Existing technologies used to detect monosodium urate(MSU)crystals for gout diagnosis are not ideal due to their low sensitivity and complexity of operation.The purpose of this study was to explore whether aggregation-induced emission luminogens(AIEgens)can be used for highly specific imaging of MSU crystals to assist in the diagnosis of gout.First,we developed a series of luminogens(i.e.,tetraphenyl ethylene(TPE)-NH_(2),TPE-2NH_(2),TPE-4NH_(2),TPE-COOH,TPE-2COOH,TPE-4COOH,and TPE-Ketoalkyne),each of which was then evenly mixed with MSU crystals.Next,optimal fluorescence imaging of each of the luminogens was characterized by a confocal laser scanning microscope(CLSM).This approach was used for imaging standard samples of MSU,hydroxyapatite(HAP)crystals,and mixed samples with 1:1 mass ratio of MSU/HAP.We also imaged samples from mouse models of acute gouty arthritis,HAP deposition disease,and comorbidities of interest.Subsequently,CLSM imaging results were compared with those of compensated polarized light microscopy,and we assessed the biosafety of TPE-Ketoalkyne in the RAW264.7 cell line.Finally,CLSM time series and three-dimensional imaging were performed on MSU crystal samples from human gouty synovial fluid and tophi.As a promising candidate for MSU crystal labeling,TPE-Ketoalkyne was found to detect MSU crystals accurately and rapidly in standard samples,animal samples,and human samples,and could precisely distinguish gout from HAP deposition disease.This work demonstrates that TPE-Ketoalkyne is suitable for highly specific and timely imaging of MSU crystals in gouty arthritis and may facilitate future research on MSU crystal-related diseases.

Therapeutic Mechanism of Santeng Dingtong Recipe (三藤定痛方) on Monosodium Urate Crystal-Induced Rabbit Arthritis

Objective: To study the therapeutic mechanism of Santeng Dingtong recipe (STDT) on monosodium urate crystal (MSU) induced rabbit arthritis Methods: Forty-two rabbits were randomly divided into six groups, 7 in each group. Group 1 received 0.9% saline 2. 5 ml/kg per day by gastrogavage (ig) for 10 days; Group 2, 3 and 4 received STDT 0.125 g/kg, 1.0 g/kg and 8.0 g/kg per day respectively by ig for 10 days; Group 5 received colchicine 4. 5 mg/kg per day by ig for 4 days; and Group 6 was untreated. MSU crystals 10 mg /500ul containing polymyxin B 10 u/ml was injected into the knee joints of Group 1-5 to make rabbit arthritis models. Leukocytes in synovial lavage fluids was then counted and differentiated; pathological injury of synovial membranes was observed under HE staining; interleukin-1 beta (IL-1B), tumor necrosis factor alpha (TNFa) and leukotriene B4 (LTB4) content in synovial lavage fluids were determined by ELISA. Results: MSU caused a rapid leukocyte infiltration and increased production of IL-1B, TNFa and LTB4 2 hrs after intra-articular injection. STDT inhibited neutrophil infiltration in synovial fluids dose-dependently, protected the synovial membrane against pathological injury and reduced the production of IL-1B, TNFa and LTB4; while colchicine did not decrease the level of TNFa, but significantly inhibited neutrophil infiltration in synovial fluid and reduced the production of IL-11B and LTB4. Conclusion: STDT exerts an anti-inflammatory effect in rabbit model of acute MSU arthritis, its mechanism being probably due to the decrease of XL-1B, TNFa and LTB4 synthesis.

Monosodium urate crystals trigger Nrf2- and heme oxygenase-1-dependent inflammation in THP-1 cells

Gouty arthritis is an inflammatory disease that is caused by an accumulation of monosodium urate （MSU） crystals in the joints. MSU is capable of activating the nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 （NLRP3） inflammasome, leading to interleukin-lβ （IL-lβ） secretion. Reactive oxygen species （ROS） are major mediators of the NLRP3/IL-lβ interaction. Although nuclear factor E2-related factor 2 （Nrf2） is recognized as a transcription factor that is involved in the response to oxidative stress, the effect of MSU on Nrf2 and on Nrf2-mediated antioxidant enzymes remains unclear. The treatment of THP-1 monocytes using phorbol 12-myristate 13-acetate （PMA） was shown to initiate inflammatory responses. Here, we showed that THP-1 cells, following treatment with MSU crystals, significantly increased IL-1β release, NLRP3 inflammasome activation and ROS production. MSU also promoted the nuclear translocation of Nrf2 and activated lysosomal destabilization. Moreover, the levels of heme oxygenase-1 （HO-1） in gene and protein expressions were upregulated by MSU. MSU-induced IL-lβ secretion and NLRP3 inflammasome activation were inhibited by the knockdown of Nrf2 and via the HO-1 inhibitor zinc （11） protoporphyrin IX （ZnPP）. In addition, HO-1 inhibition increased the level of superoxide anion production and the consumption of glutathione. These findings suggest that Nrf2 and HO-1 mediate redox homeostasis and interact with pro-inflammatory factors in MSU-challenged THP-1 cells, thereby providing new insight into how MSU-induced gouty inflammation is mediated by specific mechanisms that are involved in the Nrf2/Ho-1 antioxidant signaling pathway.

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 mR NA 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, SaO S-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-1β and TNFα) were recorded. MEMH inhibited NFκB signaling pathway and COX-2 protein expression in chondrocytes. MSU-induced mR NA 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-1β 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.

Suppression of monosodium urate crystal-induced inflammation by inhibiting TGF-β-activated kinase 1-dependent signaling:role of the ubiquitin proteasome system

Monosodium urate(MSU)crystals activate inflammatory pathways that overlap with interleukin-1β(IL-1β)signaling.However,the post-translational mechanisms involved and the role of signaling proteins in this activation are unknown.In the present study,we investigated the intracellular signaling mechanisms involved in MSU-induced activation of THP-1 macrophages and human nondiseased synovial fibroblasts(NLSFs)and the in vivo efficacy of an inhibitor of tumor grovArth factor-β(TGF-β)-activated kinase 1(TAK1),5Z-7-oxozeaenoI,in MSU-induced paw inflammation in C57BL76 mice.THP-1 macrophage activation with MSU crystals(25-200 ng/ml)resulted in the rapid and sustained phosphorylation of interleukin-1 receptor-activated kinase 1(IRAK1 Thr^(209))and TAK1(Thr^(184/187))and their association with the E3 ubiquitin ligase TRAF6.At the cellular level,MSU inhibited the deubiquitinases A20 and UCHL2 and increased 20s proteasomal activity,leading to a global decrease in K^(63)-linked ubiquitination and increase in K^(48)-linked ubiquitination in THP-1 macrophages.While MSU did not stimulate cytokine produaion in NLSFs,it significantly amplified IL-1β-induced IL-6,IL-8,and ENA-78/CXCL5 production.Docking studies and MD simulations followed by TAK1 in vitro kinase assays revealed that uric acid molecules are capable of arresting TAK1 in an active-state conformation,resulting in sustained TAK1 kinase activation.Importantly,MSU-induced proinflammatory cytokine production was completely inhibited by 5Z-7-oxozeaenol but not IRAK1/4 or TRAF6 inhibitors.Administration of 5Z-7-oxozeaenol(5 or 15 mg/kg;orally)significantly inhibited MSU-induced paw inflammation in C57BL/6 mice.Our study identifies a novel post-translational mechanism of TAK1 activation by MSU and suggests the therapeutic potential of TAK1 in regulating MSU-induced Inflammation.

Quantification of uric acid in vasculature of patients with gout using dual-energy computed tomography

BACKGROUNDGout, caused by hyperuricemia and subsequent deposition of aggregatedmonosodium urate crystals (MSU) in the joints or extra-articular regions, is themost common inflammatory arthritis. There is increasing evidence that gout is anindependent risk factor for hypertension, cardiovascular disease progression andmortality.AIMTo evaluate if dual energy computed tomography (DECT) could identify MSUwithin vessel walls of gout patients, and if MSU deposits within the vasculaturediffered between patients with gout and controls. This study may help elucidatewhy individuals with gout have increased risk for cardiovascular disease.METHODS31 gout patients and 18 controls underwent DECT scans of the chest andabdomen. A material decomposition algorithm was used to distinguish regions ofMSU (coded green), and calcifications (coded purple) from soft tissue (uncoded). Volume of green regions was calculated using a semi-automated volumeassessment program. Between-group differences were analyzed using Mann-Whitney U exact test and nonparametric rank regression.RESULTSGout patients had significantly higher volume of MSU within the aorta comparedto controls [Median (Min-Max) of 43.9 (0-1113.5) vs 2.9 (0-219.4), P = 0.01].Number of deposits was higher in gout patients compared to controls [Median(Min-Max) of 20 (0-739) vs 1.5 (0-104), P = 0.008]. However, the difference wasinsignificant after adjustment for age, gender, history of cardiovascular diseaseand diabetes. Increased age was positively associated with total urate volume (rs =0.64;95% confidence interval: 0.43-0.78).CONCLUSIONThis pilot study showed that DECT can quantify vascular urate deposits withvariation across groups, with gout patients possibly having higher deposition.This relationship disappeared when adjusted for age, and there was a positiverelationship between age and MSU deposition. While this study does not provethat green coded regions are truly MSU deposition, it corroborates recent studiesthat show the presence of vascular deposition.

Case Report: Gouty Tophus of the Larynx

Gout is a metabolic disorder that is characterized by a deposition of monosodium urate crystals in the joint or soft tissue. Gout manifestations in head and neck region are rare, and larynx is an infrequently affected location. We recently managed a case of a gouty tophus over the larynx. A 65-year-old male with a long history of gout without medical control. He has suffered from increasing hoarseness for six months. Laryngoscopy revealed a mass lesion over the right vocal process region without vocal paralysis. Laryngo-micro-surgery biopsy was taken, and pathology confirmed laryngeal gouty tophus without evidence of malignancy.

Melatonin Alleviates Acute Gouty Inflammation In Vivo and In Vitro

The aim of this study was to identify the effects of melatonin on acute gouty inflammation and to investigate the underlying mechanisms.We found significantly lower serum melatonin levels in gout patients in the acute phase than in those in the remission phase or in normal individuals.The mRNA expression of melatonin receptor 2(MT2)was also lower in gout patients than in normal individuals.To verify the in-vivo role of melatonin,a gouty arthritis model was established by intraarticular injection of monosodium urate(MSU,1 mg)crystals into the paws of C57BL/6 mice.Joint inflammation in the mouse model was evaluated by measuring the thickness of the right paw/left paw,and the inflammation index was determined by examining infiltrating neutrophils with haematoxylin and eosin(H&E)staining.Melatonin was found to reduce both paw thickness and the inflammation index in the mouse model,and melatonin also reduced the mRNA levels of interleukin-1 beta(IL-1β),IL-6 and NLR family pyrin domain containing 3(NLRP3)inflammasome.To mimic gouty inflammation in vitro,mouse peritoneal macrophages were stimulated with lipopolysaccharides(LPS)plus MSU.Melatonin was revealed to reduce IL-1βsecretion by stimulated macrophages.The mRNA expression levels of IL-1βand IL-6 were also inhibited by melatonin.Western blot analysis showed that the expression of NLRP3,caspase-1 and pro-IL-1βwas also inhibited by melatonin.In conclusion,our study demonstrated that melatonin alleviated gouty inflammation in vivo and in vitro,and the underlying mechanism may involve inhibiting the assembly of the NLRP3 inflammasome.

Effect of Berberine on Activation of TLR4-NFκB Signaling Pathway and NLRP3 Inflammasome in Patients with Gout

Objective:To determine the effects of berberine(BBR) on the activation of toll-like receptor 4(TLR4),nuclear factor(NF) κB(NF-κB) signaling and NLRP3 inflammasome in patients with gout.Methods:Peripheral blood mononuclear cells(PBMCs) were obtained from 24 acute(AP) and 41 non-acute(NAP) phases of primary gout patients,respectively,as well as 30 healthy controls(HC).TLR4,NF-κB(p65),NLRP3,apoptosis-associated specklike protein containing a CARD(PYCARD),cysteinyl aspartate specific proteinase-1(CASP1),and interleukin-1 β(IL-1 β) mRNA expression levels in PBMCs were measured by quantitative reverse transcriptase polymerase chain reaction.The protein levels of TLR4,myeloid differentiation factor 88(MyD88),NF-κB(p50/65),inhibitor of kappa B kinase α/β(IKKα/β),NF-κB inhibitor α(IKB α),phospho-IKKα/β(p-IKKα/β),NLRP3,PYCARD,and CASP1were monitored by Western blotting.Serum IL-1 β protein level was measured using enzyme-linked immunosorbent assay(ELISA).In addition,PBMCs from HC and macrophages derived from a spontaneously immortalized monocyte-like cell line(THP-1) were stimulated using monosodium urate(MSU,100 μ g/mL),0.1% dimethyl sulfoxide,25 μmol/L BBR,and 10,25,and 50 μmol/L BBR+100 μg/mL MSU for different time periods.The protein levels of IL-1β and IL-18 in cell culture supernatants was measured by ELISA,and the protein expressions of TLR4,MyD88,NF-κB(p50/p65),IKK α/β,IκBα,p-IKKα/β,NLRP3, PYCARD,and CASP1 in macrophages were analyzed by Western blotting.Results:(1) TLR4,NF-κB(p65),PYCARD,CASP1,and IL-1β mRNA levels in PBMCs were significantly higher in the AP group than in the HC group(P<0.05).The NLRP3 mRNA expression levels in PBMCs were found to be significantly lower in the AP and NAP groups than in the HC group(P<0.05,P<0.01).(2) The protein levels of TLR4,IKKβ,MyD88,NF-κB,p-IKKα/β,PYCARD,and CASP1 in PBMCs were significantly higher,and those of IκBα,IKKα,and NLRP3 were found to be significantly lower in the AP group than in the HC group(P<0.05 or P<0.01).(3) The serum IL-1β protein levels were significantly higher in the AP and NAP groups than in the HC group(P<0.01).(4)The IL-1β protein level was significantly lower in the culture supernatants of the PBMCs stimulated with MSU for 3 and 6 h in the 25 and 50 μmoL/L BBR groups compared with that in the MSU group(P<0.01).(5) The protein levels of IL-1β and IL-18 were also significantly lower in the culture supernatants of macrophages stimulated with MSU for 3 and 6 h in BBR groups compared with those in the MSU group(P<0.01).(6) The protein levels of TLR4, MyD88,NF-κB(p50,p65,p105),IKKα/β,p-IκBα,p-IKKα/β,PYCARD,and CASP1 were significantly differed between the macrophages stimulated with MSU for 0.5 and 6 h in BBR groups compared with those in the MSU group(P<0.05 or P<0.01).Conclusions:Activation of TLR4-NF κB signaling and NLRP3 inflammasome by MSU crystals drives the progression of gout inflammation.BBR ameliorates gouty inflammation,which is mechanistically associated with its regulation of TLR4-NF-κB signaling and NLRP3inflammasome expression.