RESUMO
Objective: This study aimed to evaluate the role of absent in melanoma 2 (AIM2) inflammasome-mediated pyroptosis in the pathogenesis of acute gouty arthritis (AGA) and asymptomatic hyperuricemia(AHU). Methods: A cohort of 30 AGA patients, 30 AHU individuals, and 30 healthy controls (HC) was assembled. Demographic and biochemical data, along with blood samples, were collected. Serum double-stranded DNA (dsDNA) levels were quantified using a fluorescent assay. Transcriptomic and proteomic analysis of AIM2, Caspase-1, GSDMD, IL-1ß, and IL-18 in peripheral blood mononuclear cells was performed using qRT-PCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) was employed to measure serum IL-1ß and IL-18. Spearman correlation analysis was utilized to assess relationships between variables. Results: Both AGA and AHU groups demonstrated elevated metabolic indicators and serum levels of dsDNA, IL-1ß, and IL-18 compared to the HC group. AGA patients exhibited higher inflammatory markers than the AHU group. In the AGA group, there was a significant increase in the mRNA and protein levels of AIM2, Caspase-1, GSDMD, IL-1ß, and IL-18 (P<0.05 to P<0.001). The AHU group showed higher AIM2, Caspase-1, GSDMD, and IL-18 mRNA levels than the HC group (P<0.001 to P<0.01), with a non-significant increase in AIM2, GSDMD, and IL-1ß proteins (P>0.05). In contrast, Caspase-1 and IL-18 proteins were significantly higher in the AHU group (P<0.05). Notable correlations were observed between AIM2 protein expression and levels of Caspase-1 and GSDMD in both AGA and AHU groups. In the AGA group, AIM2 protein correlated with IL-1ß, but not in the AHU group. The AIM2 protein in the AHU group was positively associated with IL-18, with no such correlation in the AGA group. Conclusion: AIM2 inflammasome may play a role in the inflammatory processes of AGA and AHU and that its activation may be related to the pyroptosis pathway.
Assuntos
Artrite Gotosa , Proteínas de Ligação a DNA , Hiperuricemia , Inflamassomos , Piroptose , Humanos , Masculino , Inflamassomos/metabolismo , Artrite Gotosa/imunologia , Artrite Gotosa/sangue , Artrite Gotosa/metabolismo , Pessoa de Meia-Idade , Hiperuricemia/sangue , Hiperuricemia/imunologia , Feminino , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Adulto , Interleucina-18/sangue , Idoso , Estudos de Casos e Controles , Biomarcadores/sangue , Caspase 1/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Trifolium alexandrinum L. (TA), has traditionally been used in folk medicine for its anti-inflammatory properties against hyperuricemia and gout. However, the specific mechanisms of action of TA have not been thoroughly studied. AIM OF THE WORK: This study aimed to evaluate the protective effects of irradiated (TR25) and non-irradiated (TR0) Trifolium alexandrinum L. aqueous extract (TAAE), along with two isolated compounds, caffeine (CAF) and saponin (SAP), in a rat model of acute gouty arthritis (GA). MATERIALS AND METHODS: The GA model was established by injecting a monosodium urate (MSU) suspension into the knee joint. Synovial tissue pathology was assessed, and levels of TNF-α, IL-6, IL-1ß, NF-κB, mTOR, AKT1, PI3K, NLRP3, and ASC were measured by ELISA. mRNA expression of ERK1, JNK, and p-38 MAPK was detected using qRT-PCR, and Caspase-1 protein expression was assessed by immunohistochemical analysis. Knee swelling, uric acid levels, liver and kidney function, and oxidative stress markers were also evaluated. RESULTS: TAAE analysis identified 170 compounds, with 73 successfully identified using LC-HR-MS/MS, including caffeine citrate and theasapogenol B glycoside as the main constituents. The studied materials demonstrated significant protective effects against GA. TR25 administration significantly mitigated knee joint circumference compared to other treatments. It demonstrated potential in alleviating hyperuricemia, renal and hepatic impairments induced by MSU crystals. TR25 also alleviated oxidative stress and reduced levels of IL1ß, IL-6, TNF-α, and NF-κB. Weak Caspase-1 immune-positive staining was observed in the TR25 group. TR25 decreased NLRP3 and ASC expression, reducing inflammatory cytokine levels in GA. It effectively inhibited the PI3K, AKT, and mTOR signaling pathways, promoting autophagy. Additionally, TR25 suppressed ERK1, JNK, and p-38 MAPK gene expression in synovial tissue. These effects were attributed to various components in TAAE, such as flavonoids, phenolic acids, tannins, alkaloids, and triterpenes. CONCLUSION: Importantly, irradiation (25 KGy) enhanced the antioxidant effects and phtchemical contents of TAAE. Additionally, TR0, TR25, CAF, and SAP exhibited promising protective effects against GA, suggesting their therapeutic potential for managing this condition. These effects were likely mediated through modulation of the NLRP3/ASC/Caspase-1 and ERK/JNK/p-38 MAPK signaling pathways, as well as regulation of the PI3K/AKT/mTOR pathway. Further research is warranted to fully elucidate the underlying mechanisms and optimize their clinical applications.
Assuntos
Artrite Gotosa , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Extratos Vegetais , Animais , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Caspase 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ácido ÚricoRESUMO
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.
Assuntos
Artrite Gotosa , Autofagia , Fosfatase 1 de Especificidade Dupla , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ácido Úrico , Humanos , Autofagia/efeitos dos fármacos , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Artrite Gotosa/genética , Artrite Gotosa/metabolismo , Artrite Gotosa/imunologia , Artrite Gotosa/induzido quimicamente , Ácido Úrico/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Inflamassomos/metabolismo , Inflamassomos/imunologia , Células THP-1 , Masculino , Monócitos/metabolismo , Monócitos/imunologia , Monócitos/efeitos dos fármacos , Estudos de Casos e Controles , Feminino , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: In gout, monosodium urate crystals are taken up by macrophages, triggering the activation of the NLRP3 inflammasome and the maturation of IL-1ß. This study aimed to investigate the role of integrin CD11b in inflammasome activation in macrophages stimulated by MSU. METHODS: BMDM from WT and CD11b KO mice were stimulated in vitro with MSU crystals. Cellular supernatants were collected to assess the expression of the inflammatory cytokines by enzyme-linked immunosorbent assay and western blot methods. The role of integrin CD11b in MSU-induced gouty arthritis in vivo was investigated by intra-articular injection of MSU crystals. Real-time extracellular acidification rate and oxygen consumption rate of BMDMs were measured by Seahorse Extracellular Flux Analyzer. RESULTS: We demonstrate that CD11b-deficient mice developed exacerbated gouty arthritis with increased recruitment of leukocytes in the joint and higher IL-1ß levels in the sera. In macrophages, genetic deletion of CD11b induced a shift of macrophage metabolism from oxidative phosphorylation to glycolysis, thus decreasing the overall generation of intracellular ATP. Upon MSU stimulation, CD11b-deficient macrophages showed an exacerbated secretion of IL-1ß. Treating wild-type macrophages with a CD11b agonist, LA1, inhibited MSU-induced release of IL-1ß in vitro and attenuated the severity of experimental gouty arthritis. Importantly, LA1, was also effective in human cells as it inhibited MSU-induced release of IL-1ß by peripheral blood mononuclear cells from healthy donors. CONCLUSION: Our data identified the CD11b integrin as a principal cell membrane receptor that modulates NLRP3 inflammasome activation by MSU crystal in macrophages, which could be a potential therapeutic target to treat gouty arthritis in human patients.
Assuntos
Artrite Gotosa , Antígeno CD11b , Inflamassomos , Macrófagos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ácido Úrico , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos/metabolismo , Antígeno CD11b/metabolismo , Inflamassomos/metabolismo , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Camundongos , MasculinoRESUMO
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.
Assuntos
Artrite Gotosa , Heme Oxigenase-1 , Macrófagos , Fator 2 Relacionado a NF-E2 , NF-kappa B , Sirtuína 1 , Fator de Transcrição AP-1 , Animais , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Artrite Gotosa/imunologia , Sirtuína 1/metabolismo , Sirtuína 1/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Humanos , Masculino , NF-kappa B/metabolismo , Heme Oxigenase-1/metabolismo , Camundongos , Fator de Transcrição AP-1/metabolismo , Células THP-1 , Camundongos Endogâmicos C57BL , Inflamação , Transdução de Sinais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Carbazóis , Proteínas de MembranaRESUMO
BACKGROUND: Gouty arthritis (GA) is characterized by monosodium urate (MSU) crystal accumulation that instigates NLRP3-mediated pyroptosis; however, the underlying regulatory mechanisms have yet to be fully elucidated. The present research endeavors to elucidate the regulatory mechanisms underpinning this MSU-induced pyroptotic cascade in GA. METHODS: J774 cells were exposed to lipopolysaccharide and MSU crystals to establish in vitro GA models, whereas C57BL/6 J male mice received MSU crystal injections to mimic in vivo GA conditions. Gene and protein expression levels were evaluated using real-time quantitative PCR, Western blotting, and immunohistochemical assays. Inflammatory markers were quantified via enzyme-linked immunosorbent assays. Pyroptosis was evaluated using immunofluorescence staining for caspase-1 and flow cytometry with caspase-1/propidium iodide staining. The interaction between MDM2 and PPARγ was analyzed through co-immunoprecipitation assays, whereas the interaction between BRD4 and the MDM2 promoter was examined using chromatin immunoprecipitation and dual-luciferase reporter assays. Mouse joint tissues were histopathologically evaluated using hematoxylin and eosin staining. RESULTS: In GA, PPARγ was downregulated, whereas its overexpression mitigated NLRP3 inflammasome activation and pyroptosis. MDM2, which was upregulated in GA, destabilized PPARγ through the ubiquitin-proteasome degradation pathway, whereas its silencing attenuated NLRP3 activation by elevating PPARγ levels. Concurrently, BRD4 was elevated in GA and exacerbated NLRP3 activation and pyroptosis by transcriptionally upregulating MDM2, thereby promoting PPARγ degradation. In vivo experiments showed that BRD4 silencing ameliorated GA through this MDM2-PPARγ-pyroptosis axis. CONCLUSION: BRD4 promotes inflammation and pyroptosis in GA through MDM2-mediated PPARγ degradation, underscoring the therapeutic potential of targeting this pathway in GA management.
Assuntos
Artrite Gotosa , PPAR gama , Proteínas Proto-Oncogênicas c-mdm2 , Piroptose , Fatores de Transcrição , Animais , Masculino , Camundongos , Artrite Gotosa/metabolismo , Artrite Gotosa/genética , Artrite Gotosa/patologia , Artrite Gotosa/induzido quimicamente , Proteínas que Contêm Bromodomínio , Linhagem Celular , Modelos Animais de Doenças , Inflamassomos/metabolismo , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas Nucleares , PPAR gama/metabolismo , PPAR gama/genética , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ácido Úrico/metabolismoRESUMO
BACKGROUND: Gout is caused by monosodium urate (MSU) crystals deposition to trigger immune response. A recent study suggested that inhibition of Class I Histone deacetylases (HDACs) can significantly reduce MSU crystals-induced inflammation. However, which one of HDACs members in response to MSU crystals was still unknown. Here, we investigated the roles of HDAC3 in MSU crystals-induced gouty inflammation. METHODS: Macrophage specific HDAC3 knockout (KO) mice were used to investigate inflammatory profiles of gout in mouse models in vivo, including ankle arthritis, foot pad arthritis and subcutaneous air pouch model. In the in vitro experiments, bone marrow-derived macrophages (BMDMs) from mice were treated with MSU crystals to assess cytokines, potential target gene and protein. RESULTS: Deficiency of HDAC3 in macrophage not only reduced MSU-induced foot pad and ankle joint swelling but also decreased neutrophils trafficking and IL-1ß release in air pouch models. In addition, the levels of inflammatory genes related to TLR2/4/NF-κB/IL-6/STAT3 signaling pathway were significantly decreased in BMDMs from HDAC3 KO mice after MSU treatment. Moreover, RGFP966, selective inhibitor of HDAC3, inhibited IL-6 and TNF-α production in BMDMs treated with MSU crystals. Besides, HDAC3 deficiency shifted gene expression from pro-inflammatory macrophage (M1) to anti-inflammatory macrophage (M2) in BMDMs after MSU challenge. CONCLUSIONS: Deficiency of HDAC3 in macrophage alleviates MSU crystals-induced gouty inflammation through inhibition of TLR2/4 driven IL-6/STAT3 signaling pathway, suggesting that HDAC3 could contribute to a potential therapeutic target of gout.
Assuntos
Acrilamidas , Gota , Histona Desacetilases , Macrófagos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenilenodiaminas , Ácido Úrico , Animais , Ácido Úrico/toxicidade , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/deficiência , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Gota/metabolismo , Gota/patologia , Camundongos , Inflamação/metabolismo , Inflamação/induzido quimicamente , Masculino , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Artrite Gotosa/patologia , Modelos Animais de Doenças , Transdução de Sinais/efeitos dos fármacosRESUMO
Citronella and Nutmeg are two common spices used for seasoning and medicinal purposes, both of which have significant economic value. This study aimed to investigate whether Citronella essential oil and Nutmeg essential oil (NEO) can ameliorate monosodium urate (MSU)-induced gouty arthritis in rats and the potential mechanisms. The results showed that CEO and NEO reduced swelling and redness at joint sites, inhibited neutrophil infiltration, and limited proinflammatory mediator secretion in mice with MSU-induced gouty arthritis. Based on the results of network pharmacology, molecular docking, and western blotting, CEO and NEO may exert anti-gouty arthritis effects by reducing the expression of reactive oxygen species and oxidative stress and downregulating the phosphorylation of the PI3K/AKT/mTOR signaling pathway, thereby inhibiting the production of the NLRP3 inflammasome and inhibiting the production of inflammatory cytokines. Therefore, these two essential oils show potential for use as adjuvant treatments for gouty arthritis in specific aromatherapy products or food seasonings.
Assuntos
Artrite Gotosa , Proteína 3 que Contém Domínio de Pirina da Família NLR , Óleos Voláteis , Estresse Oxidativo , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Serina-Treonina Quinases TOR , Óleos Voláteis/farmacologia , Óleos Voláteis/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Ratos , Masculino , Myristica/química , Ácido Úrico/metabolismo , Simulação de Acoplamento Molecular , Ratos Sprague-DawleyRESUMO
OBJECTIVES: Cellular NAD+ declines in inflammatory states associated with increased activity of the leukocyte-expressed NADase CD38. In this study, we tested the potential role of therapeutically targeting CD38 and NAD+ in gout. METHODS: We studied cultured mouse wild type and CD38 knockout (KO) murine bone marrow derived macrophages (BMDMs) stimulated by monosodium urate (MSU) crystals and used the air pouch gouty inflammation model. RESULTS: MSU crystals induced CD38 in BMDMs in vitro, associated with NAD+ depletion, and IL-1ß and CXCL1 release, effects reversed by pharmacologic CD38 inhibitors (apigenin, 78c). Mouse air pouch inflammatory responses to MSU crystals were blunted by CD38 KO and apigenin. Pharmacologic CD38 inhibition suppressed MSU crystal-induced NLRP3 inflammasome activation and increased anti-inflammatory SIRT3-SOD2 activity in macrophages. BMDM RNA-seq analysis of differentially expressed genes (DEGs) revealed CD38 to control multiple MSU crystal-modulated inflammation pathways. Top DEGs included the circadian rhythm modulator GRP176, and the metalloreductase STEAP4 that mediates iron homeostasis, and promotes oxidative stress and NF-κB activation when it is overexpressed. CONCLUSIONS: CD38 and NAD+ depletion are druggable targets controlling the MSU crystal- induced inflammation program. Targeting CD38 and NAD+ are potentially novel selective molecular approaches to limit gouty arthritis.
Assuntos
ADP-Ribosil Ciclase 1 , Inflamação , Macrófagos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD , Ácido Úrico , Animais , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Inflamação/tratamento farmacológico , Camundongos , NAD/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Células Cultivadas , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Artrite Gotosa/genética , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacosRESUMO
Gouty arthritis (GA) is an inflammatory disease caused by the deposition of monosodium urate (MSU) crystals into joints. Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid extracted from the root of Stephania tetrandra and can exert an anti-inflammatory function in different diseases. Nevertheless, the specific function of TET in GA remains unclear. We established the GA mouse model by MSU injection into joints of mice. Paw volume and gait score were detected for measuring the degree of joint swelling and the situation of joint dysfunction. Western blot were utilized to test the alterations of M1-related factors (IL-6, IL-1ß, TNF-α, IL-12, and iNOS) and M2-related factors (Mgl1, Mgl2, Pgc1-ß, Arg-1, and IL-10). The activity of NF-κB p65 in tissues was determined. The interaction of NF-κB p65 and Lcp1 was measured by ChIP and luciferase reporter assay. Lcp1 KO mice were utilized to detect the effect of Lcp1 depletion on GA process. TET treatment markedly suppressed MSU-induced joint swelling, joint dysfunction, and joint injury in GA mice. TET can also reduce inflammatory reactions in MUS-induced mice. Furthermore, we proved that TET facilitated M2 macrophage polarization and inhibited M1 macrophage polarization in GA mice. In addition, TET was found to inhibit NF-κB activity and NF-κB-mediated Lcp1 expression. Lcp1 knockdown can improve joint injury and promote M2 macrophage polarization in GA mice, while this effect was further enhanced by TET. TET alleviates inflammation and facilitates macrophage M2 polarization in GA by NF-κB-mediated Lcp1.
Assuntos
Artrite Gotosa , Benzilisoquinolinas , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Benzilisoquinolinas/efeitos adversos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Macrófagos , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Ácido Úrico/efeitos adversos , Ácido Úrico/metabolismo , Animais , CamundongosRESUMO
The present study aims to explore the therapeutic effect of Stefin B on gouty arthritis (GA) and the polarization of macrophages in mice. Stefin B-overexpressed or knockdown M0 macrophages were constructed. The GA model was established in mice by injecting 25 mg/mL MSU, followed by a single injecting of Stefin B-overexpressing adenovirus vector (GA model + Stefin B OE) or an empty vector (GA model + Stefin B OE NC). Stefin B was found lowly expressed in M1 macrophages. CD206 was markedly upregulated and IL-10 release was signally increased in Stefin B-overexpressed macrophages. In gouty arthritis mice, marked redness and swelling were observed in the ankle joint. Dramatical infiltration of inflammatory cells was observed in the GA model and GA model + Stefin B OE NC groups, which was suppressed in the Stefin B OE group. Increased proportion of F4/80+CD86+ cells observed in GA mice was markedly repressed by Stefin B overexpression, accompanied by the declined level of Caspase-1 and IL-17. Collectively, Stefin B alleviated the GA in mice by inducing the M2 polarization of macrophages.
Assuntos
Artrite Gotosa , Macrófagos , Animais , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/patologia , Artrite Gotosa/metabolismo , Artrite Gotosa/induzido quimicamente , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Masculino , Lectinas Tipo C/metabolismo , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Lectinas de Ligação a Manose/metabolismo , Modelos Animais de Doenças , Receptor de Manose , Interleucina-10/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Sanmiao wan (SMW), a classical traditional Chinese medicine (TCM) formula, has been employed to treat gouty diseases in clinic as early as Yuan dynasty. It shows remarkably therapeutic effects in acute gouty arthritis (GA). However, the potential mechanisms of SMW are still not fully revealed. AIM OF THE STUDY: The objective of this project is to evaluate the pharmacological effects and possible mechanisms of SMW in a rat model of acute GA. MATERIALS AND METHODS: Monosodium urate (MSU) suspension was injected into the ankle joint of rats to establish acute GA model. The inflammation was evaluated by measuring the posterior ankle diameter. The pathological status of synovial tissue was assessed by hematoxylin eosin (HE), Masson, and picrosirius red staining. The level of IL-6 was measured using ELISA kit. The levels of blood urea nitrogen (BUN), creatinine (CR), UA (uric acid), and xanthine oxidase (XOD) in the serum were measured using standard diagnostic kits. The percentage of Th17 cells in blood samples was performed using flow cytometry. Moreover, RT-qPCR was performed to examine the mRNA level of RANK, RORγt, RANKL, and STAT3 in the synovial tissue. Furthermore, immunofluorescence was carried out to assess the expression of STAT3 in the synovial tissue. RESULTS: SMW effectively alleviated the inflammation and improved the pathological status of the ankle joint in rats with acute GA. It significantly suppressed the release of proinflammatory cytokine (IL-6). Meanwhile, the levels of UA, BUN, and CR were markedly reduced after SMW treatment. A remarkable reduction of XOD activity was observed in the study. Importantly, SMW treatment significantly reduced the frequency of Th17 cells, decreased the mRNA levels of RANK, RORγt, RANKL, and STAT3 in the synovial tissue. Furthermore, the suppression of STAT3 was also demonstrated using immunofluorescence in SMW-treated group. CONCLUSION: SMW showed significant anti-inflammatory and hypouricemic effects in a rat model of GA. It is an effective TCM formula for GA therapy.
Assuntos
Artrite Gotosa , Ratos , Animais , Artrite Gotosa/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Interleucina-6 , Inflamação/tratamento farmacológico , Ácido Úrico , RNA MensageiroRESUMO
BACKGROUND: Gouty arthritis (GA) is a common form of inflammatory arthritis caused by intra-articular deposition of monosodium urate (MSU) crystals; however, there is a tremendous lack of safe and effective therapy in the clinic. OBJECTIVE: The goal of this work was to investigate a novel leflunomide analogue, N-(2,4- dihydroxyphenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTLOH-4e), for its potential to prevent/ treat gouty arthritis. METHODS: In this study, the anti-inflammatory activity of UTLOH-4e was evaluated by MSUinduced GA model in vivo and in vitro, and the molecular docking test was applied to estimate the affinity of UTLOH-4e/UTL-5g/b for MAPKs, NF-κB, and NLRP3. RESULTS: In vitro, UTLOH-4e (1~100 µM) treatment inhibited the inflammatory reaction with no obvious cytotoxicity in PMA-induced THP-1 macrophages exposed to MSU crystals for 24 h, involving the prominent decreased production and gene expression of IL-1ß, TNF-α, and IL-6. Western blot analyses demonstrated that UTLOH-4e (1~100 µM) significantly suppressed the activation of NLRP3 inflammasomes, NF-κB, and MAPK pathways. Furthermore, the data from the experiment on gouty rats induced by intra-articular injection of MSU crystal confirmed that UTLOH-4e markedly ameliorated rat paw swelling, articular synovium inflammation and reduced the concentration of IL-1ß and TNF-α in serum through down-regulating NLRP3 protein expression. CONCLUSION: These results manifested that UTLOH-4e ameliorates GA induced by MSU crystals, which contributes to the modulation of NF-κB/ NLRP3 signaling pathway, suggesting that UTLOH- 4e is a promising and potent drug candidate for the prevention and treatment of gouty arthritis.
Assuntos
Artrite Gotosa , Ratos , Animais , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Ácido Úrico/efeitos adversos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , NF-kappa B/metabolismo , Leflunomida/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismo , Simulação de Acoplamento Molecular , Inflamação/metabolismo , Inflamassomos/metabolismo , Transdução de SinaisRESUMO
Evodiamine (EVO) was tested on acute gouty arthritis rats to investigate its anti-inflammatory effect. Seventy-two male Sprague-Dawley (SD) rats were randomly assigned into the control, model, high, medium, and low dose of EVO groups and colchicine group. The ankle swelling degrees were measured at 2 h, 6 h, and 24 h following sodium urate injection into ankle joint. Histopathological examination was performed 24 h after injection. Reactive oxygen species (ROS) content in the ankle joint was detected using chemical fluorescence. Serum interleukin-1ß (IL-1ß), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α) content were determined by ELISA. Serum xanthine oxidase (XOD), superoxide dismutase (SOD), and malondialdehyde (MDA) were determined by spectrophotometry. The expressions of thioredoxin-interacting protein (TXNIP), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), pro-caspase-1, caspase-1, and apoptosis-related spot like protein (ASC) in synovium were detected by Western blot. Evodiamine alleviated the ankle swelling of the affected foot in gouty arthritis rats and reduced inflammatory cell infiltration in joint synovial tissue. Evodiamine also decreased the content of serum inflammatory factors including IL-1ß, IL-18, and TNF-α, and increased serum SOD activity, while it decreased serum XOD, MDA activity, and ROS level. Moreover, evodiamine downregulated the protein expression levels of TXNIP, NLRP3, pro-caspase-1, cleaved caspae-1, and ASC. The mechanism of EVO in treating gouty arthritis is associated with the inhibition of NLRP3 inflammasome by regulating the ROS/TXNIP/NLRP3 signaling pathway.
Assuntos
Artrite Gotosa , Proteína 3 que Contém Domínio de Pirina da Família NLR , Quinazolinas , Ratos , Masculino , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Interleucina-18/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Caspase 1/metabolismo , Ratos Sprague-Dawley , Superóxido Dismutase , Proteínas de Ciclo Celular/uso terapêuticoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: DaiTongXiao (DTX) is a traditional Chinese Dai folk formulation utilized for gouty arthritis treatment, with substantial evidence supporting its anti-inflammatory properties. The NLRP3 inflammasome disorder is tightly linked to the development of many inflammatory diseases. AIM OF THE STUDY: To elucidate the therapeutic efficacy of DTX in gouty arthritis and reveal its potential underlying mechanism. MATERIALS AND METHODS: The primary active constituents in DTX were determined through ultraviolet spectrophotometry and gas chromatography. Rats underwent induction with monosodium urate (MSU), followed by treatment of J774A.1 cells with adenosine triphosphate (ATP) activation and lipopolysaccharide (LPS) induction and the subsequent culture in Dulbecco's modified Eagle's medium. The degree of foot joint swelling in rats was assessed, and ankle joints were evaluated through H&E staining. Enzyme-linked immunosorbent assay was performed to measure the levels of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α in both serum and cells. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the relative mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 macrophages. The expression of NLRP3, ASC, Caspase-1, and NF-κB was examined by western blotting. RESULTS: DTX could alleviate MSU-induced joint swelling in rats, as evidenced by a reduction in joint inflammation. Moreover, DTX effectively enhanced the survival rate of J774A.1 cells following LPS induction and ATP activation. Furthermore, DTX significantly reduced IL-1ß, IL-6, IL-8, and TNF-α levels in both cell culture medium and rat serum. RT-PCR results revealed that DTX notably downregulated the mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 cells. Additionally, DTX downregulated NLRP3, ASC, NF-κB, and Caspase-1 expression in the joint tissue. CONCLUSIONS: DTX exerts a significant anti-gouty arthritis effect, with its mechanism being tightly linked to the NLRP3 inflammatory signaling pathway. This pathway may be modulated by inhibiting IL-1ß differentiation and maturation by downregulating NLRP3, ASC, Caspase-1, and NF-κB protein expression. This, in turn, leads to a reduction in the release of IL-6, IL-8, and TNF-α, ultimately impeding gouty arthritis progression.
Assuntos
Artrite Gotosa , Ratos , Animais , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6 , Lipopolissacarídeos , Interleucina-8 , Transdução de Sinais , Inflamassomos/metabolismo , Ácido Úrico , Caspase 1/metabolismo , Edema , Trifosfato de Adenosina , RNA MensageiroRESUMO
AIM: According to reports, long non-coding RNAs (lncRNAs) are involved in the regulation of many inflammatory diseases. Here, our main purpose was to ascertain the expression data of lncRNA SNHG14 in acute gouty arthritis (AGA) and to explore its possible mechanism in the regulation of AGA. METHOD: Reverse transcription quantitative polymerase chain reaction technology was supplied to detect the lncRNA SNHG14 expression. A receiver operating characteristics curve was drawn to estimate the accuracy of lncRNA SNHG14 in AGA diagnosis. An in vitro AGA cell model was constructed by inducing THP-1 cells with monosodium urate (MSU). The concentrations of inflammatory factors such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay. The luciferase reporter gene was used to verify the relationship between lncRNA SNHG14 and miR-223-3p. RESULTS: In clinical analysis, the levels of serum lncRNA SNHG14 in AGA patients were significantly higher than those in the control group. Abnormally elevated lncRNA SNHG14 has high sensitivity and specificity for AGA diagnosis. In in vitro cell experiments, silencing lncRNA SNHG14 inhibited the inflammatory response of THP-1 cells stimulated by MSU, and the luciferase reporter gene proved that lncRNA SNHG14 could bind to miR-223-3p. In addition, the level of miR-223-3p declined in AGA patients and the AGA cell model. Overexpression of miR-223-3p is helpful to alleviate an MSU-induced inflammatory response. CONCLUSION: In the AGA cell model, lncRNA SNHG14, as an miR-223-3p sponge, induces a cellular inflammatory response by controlling the level of miR-223-3p, so aggravating the disease progress of AGA.
Assuntos
Artrite Gotosa , MicroRNAs , RNA Longo não Codificante , Humanos , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/genética , Artrite Gotosa/metabolismo , RNA Longo não Codificante/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Ácido Úrico , LuciferasesRESUMO
In the present study, a natural product database of compounds associated with herbs traditionally verified to treat gout/hyperuricemia/arthritis was constructed. 3D-shape and docking-based virtual screening was conducted. To identify potential xanthine oxidase (XOD) inhibitors in the database, eight compounds with commercial availability were identified as high 3D-shape similarity with febuxostat (1), a known XOD inhibitor. Docking was used to further predict the possible interactions between XOD and these compounds. Moracin C (2), moracin D (3), and isoformononetin (8) exhibited higher docking scores and binding energies than other compounds. In vitro, 2 inhibited XOD with an IC50 value of 0.25 ± 0.14 µM, which is similar to that of 1 (0.16 ± 0.08 µM). In a hyperuricemic mouse model, 5-20 mg/kg 2 exhibited satisfying urate-lowering and XOD inhibitory effects. Compound 2 also exhibited antiarthritis activities. In RAW264.7 cells, 2 at 1-10 µM inhibited the expression of IL-1ß and TNF-α induced by MSU. In an acute gouty arthritis model in SD rats, 5-20 mg/kg 2 significantly alleviated the toe swelling, inflammatory response, and dysfunction disorder caused by monosodium urate (MSU). Compound 2 inhibited serum IL-1ß and TNF-α cytokines as well as reduced the expression of the NLRP3/ASC/caspase-1 inflammasome in joints. In summary, 2 was an effective compound for the treatment of hyperuricemia/gouty arthritis.
Assuntos
Artrite Gotosa , Hiperuricemia , Camundongos , Ratos , Animais , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Fator de Necrose Tumoral alfa , Ratos Sprague-Dawley , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Ácido Úrico/efeitos adversos , Inibidores EnzimáticosRESUMO
Lycium ruthenicum Murr. (LR) has long been used as a unique nutritional and medicinal food to treat various diseases such as gouty arthritis. However, although recently the literature has focused on the protective roles of LR anthocyanins on gouty arthritis, there is no relevant research from a holistic perspective of lipid metabolism to study their anti-gout effects. In this study, a combined tissue lipidomics, network pharmacology, and molecular docking approach was performed to investigate the intervention mechanism of LR anthocyanins against a monosodium urate (MSU)-induced gout mouse model. 54 gout-related lipid markers were identified via lipidomic profiling of the mouse knee joint, including glycerophospholipids, sphingolipids, glycerolipids, and plasmalogens. Integrating with pathway analysis, network pharmacology, and molecular docking, the potential targets of LR anthocyanins for treating gouty arthritis were predicted, while pathways in cancer, prostate cancer, sphingolipid signaling, choline metabolism in cancer, arachidonic acid metabolism, and ovarian steroidogenesis were involved as shared critical pathways of lipidomic analysis and network pharmacology. Furthermore, the binding sites and patterns of 3 active components and 4 core targets with the lowest binding energies were explored. Western blotting was finally used to verify the expression levels of 4 core proteins: MMP2, MMP9, MAP2K1, and MAPK14. These results provide new insights into our understanding of gouty arthritis and the anti-gout mechanism of LR anthocyanins.
Assuntos
Artrite Gotosa , Gota , Lycium , Masculino , Camundongos , Animais , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Antocianinas/farmacologia , Antocianinas/química , Simulação de Acoplamento Molecular , Lycium/química , Lipidômica , Farmacologia em RedeRESUMO
Gout is a metabolic disease caused by the deposition of monosodium urate (MSU) crystals inside joints, which leads to inflammation and tissue damage. Increased concentration of serum urate is an essential step in the development of gout. Serum urate is regulated by urate transporters in the kidney and intestine, especially GLUT9 (SLC2A9), URAT1 (SLC22A12) and ABCG. Activation of NLRP3 inflammasome bodies and subsequent release of IL-1ß by monosodium urate crystals induce the crescendo of acute gouty arthritis, while neutrophil extracellular traps (NETs) are considered to drive the self-resolving of gout within a few days. If untreated, acute gout may eventually develop into chronic tophaceous gout characterized by tophi, chronic gouty synovitis, and structural joint damage, leading the crushing burden of treatment. Although the research on the pathological mechanism of gout has been gradually deepened in recent years, many clinical manifestations of gout are still unable to be fully elucidated. Here, we reviewed the molecular pathological mechanism behind various clinical manifestations of gout, with a view to making contributions to further understanding and treatment.
Assuntos
Artrite Gotosa , Armadilhas Extracelulares , Gota , Transportadores de Ânions Orgânicos , Humanos , Ácido Úrico , Gota/patologia , Artrite Gotosa/metabolismo , Inflamação/metabolismo , Armadilhas Extracelulares/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismoRESUMO
Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal-induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck-/-Fgr-/-Lyn-/- mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck-/-Fgr-/-Lyn-/- mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.