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1.
Life Sci ; 240: 117138, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31809715

RESUMO

Pyroptosis is a form of cell death mediated by gasdermin D (GSDMD); it is characterised by NLRP3 inflammasome activation, caspase activation, cell membrane pore formation, and the release of interleukin-1ß and interleukin-18. NLRP3 inflammasome activation plays a central role in pyroptosis. Recent research has suggested that NLRP3 inflammasome activation may be involved in the occurrence and development of diabetes mellitus and its associated complications. This finding provided the impetus for us to clarify the significance of pyroptosis in diabetes. In this review, we summarise the current understanding of the molecular mechanisms involved in pyroptosis, as well as recent advances in the role of NLRP3 inflammasome activation and pyroptosis in the development of diabetes and diabetic complications.


Assuntos
Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/genética , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/genética , Inflamassomos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/genética , Animais , Complicações do Diabetes/patologia , Diabetes Mellitus/patologia , Humanos , Inflamassomos/efeitos dos fármacos , Piroptose/efeitos dos fármacos
2.
Life Sci ; 241: 117165, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31838136

RESUMO

AIMS: Previous work has reported the closely correlation between inflammation and carcinogenesis, while the role of NALP3, the key component of inflammasome activation in NSCLC remains elusive. This study was to unravel the mechanism of NALP3 on modulating NSCLC cancer cell growth. METHODS: IHC and immuno-blot were performed to analyze expression of NALP3 and indicated molecules. CCK-8 and xenograft nude mice assay were used to evaluate cell growth in vitro and in vivo. Bioenergetics assay was performed to measure OXPHOS and aerobic glycolysis. siRNA and shRNA were constructed to knockdown endogenous NALP3 and DNMT1. Co-immunoprecipitation was applied to confirm the interaction between NALP3 and DMAP1. BioProfile FLEX analyzer and Lactate Reagent Kit were used to measure relative level glucose uptake and lactate production. KEY FINDINGS: We reported NALP3 were up-regulated in NSCLC tumor tissues. NALP3 depletion suppressed cancer cell growth in vitro and in vivo. Moreover, data showed depletion of NALP3 promoted cell bioenergetics switch from aerobic glycolysis to OXPHOS. Additionally, we found NALP3 interacted with DMAP1 and alteration of NALP3 increased DNMT1 level. Subsequently, we clarified depletion of DNMT1 significantly suppressed NSCLC cell growth and orchestrated cellular metabolism which was similar to the effects of NALP3 knockdown. Finally, our data showed high NALP3 was associated with poor outcomes, and correlated with TNM stage and differentiation. SIGNIFICANCE: Current study elucidated NALP3 could promote metabolic reprogramming to regulate NSCLC cell growth and suggested that NALP3 may be considered as a novel biomarker and therapeutic target for NSCLC patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/secundário , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Glicólise , Neoplasias Pulmonares/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Estudos de Coortes , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Ecotoxicol Environ Saf ; 188: 109905, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31706245

RESUMO

Cooking oil fumes-derived PM2.5 (COFs-derived PM2.5) is the main source of indoor pollution. Exposure to COFs-derived PM2.5 can cause oxidative stress and affect angiogenesis. Here we investigated the roles of vitamin D3 (VD3) in protecting tubule formation injury induced by COFs-derived PM2.5, and the roles of ROS/NLRP3/VEGF signaling pathway in the effects. Human umbilical vein endothelial cells (HUVECs) were exposed to 0 (1‰ DMSO), 1000 nmol/l VD3, 100 µg/ml PM2.5, and 1000 nmol/l VD3 + 100 µg/ml PM2.5, respectively. Cell viability and tube formation, as well as protein and mRNA levels were measured. The results showed that exposure of COFs-derived PM2.5 dose-and time-dependently reduced the viability of HUVECs, increased the levels of mitochondrial and intracellular ROS, and changed the mitochondrial membrane potential level. While co-incubation with VD3 rescued these adverse effects. Both Western blot and real-time PCR (RT-PCR) showed that the expressions of NLRP3, caspase-1, Interleukin (IL)-1ß, and IL-18 in COFs-derived PM2.5 exposure group increased significantly, which could be effectively decreased by co-incubation with VD3. COFs-derived PM2.5 exposure could also reduce the expression of VEGF, while co-incubating HUVECs with VD3 evidently up-regulated the protein level of VEGF in HUVECs. In addition, COFs-derived PM2.5 could also inhibit the tube formation of HUVECs in vitro, which could be effectively rescued by the co-incubation of VD3. Our study proved that COFs-derived PM2.5 could damage the tubule formation of HUVECs in vitro, which could be effectively rescue by co-incubation with VD3, in which processes the ROS/NLRP3/VEGF signaling pathway played a crucial role. It provides a new theoretical basis for further study on the toxicity of PM2.5 to umbilical cord blood vessels.


Assuntos
Colecalciferol/farmacologia , Culinária , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Material Particulado/toxicidade , Veias Umbilicais/citologia , Poluição do Ar em Ambientes Fechados/efeitos adversos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/crescimento & desenvolvimento , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Gastroenterology ; 158(1): 253-269.e14, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31593700

RESUMO

BACKGROUND & AIMS: Pancreatitis starts with primarily sterile local inflammation that induces systemic inflammatory response syndrome, followed by compensatory anti-inflammatory response syndrome (CARS). We investigated the mechanisms of these processes in mice and human serum. METHODS: We induced severe acute pancreatitis by partial duct ligation with caerulein stimulation or intraperitoneal injection of l-arginine in mice with deletion of interleukin (IL)12B, NLRP3, or IL18 and in mice given MCC950, a small molecule inhibitor of the NLRP3-inflammasome. Pancreata were collected from mice and analyzed by histology, and cytokine levels were measured in serum samples. We measured activation of adaptive immune responses in mice with pancreatitis by flow cytometry analysis of T cells (CD25 and CD69) isolated from the spleen. Differentiation of T-helper (Th1) cells, Th2 cells, and T-regulatory cells was determined by nuclear staining for TBET, GATA3, and FOXP3. We performed transcriptome analysis of mouse lymph nodes and bone marrow-derived macrophages after incubation with acini. We measured levels of cytokines in serum samples from patients with mild and severe acute pancreatitis. RESULTS: Activation of the adaptive immune response in mice was initiated by macrophage-derived, caspase 1-processed cytokines and required activation of NLRP3 (confirmed in serum samples from patients with pancreatitis). Spleen cells from mice with pancreatitis had increases in Th2 cells but not in Th1 cells. Bone marrow-derived macrophages secreted IL1B and IL18, but not IL12, after co-incubation with pancreatic acini. T-cell activation and severity of acute pancreatitis did not differ significantly between IL12B-deficient and control mice. In contrast, NLRP3- or IL18-deficient mice had reduced activation of T cells and no increase in Th2 cell-mediated responses compared with control mice. The systemic type 2 immune response was mediated by macrophage-derived cytokines of the IL1 family. Specifically, IL18 induced a Th2 cell-mediated response in the absence of IL12. MCC950 significantly reduced neutrophil infiltration, T-cell activation, and disease severity in mice. CONCLUSIONS: In mice with severe pancreatitis, we found systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome developed in parallel. Infiltrating macrophages promote inflammation and simultaneously induce a Th2 cell-mediated response via IL18. Inhibition of NLRP3 reduces systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome and might be used to treat patients with severe pancreatitis.


Assuntos
Furanos/administração & dosagem , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Pancreatite/imunologia , Sulfonamidas/administração & dosagem , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Células Acinares , Imunidade Adaptativa , Animais , Arginina/toxicidade , Células Cultivadas , Ceruletídeo/toxicidade , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Injeções Intraperitoneais , Interleucina-18/imunologia , Interleucina-18/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Pâncreas/citologia , Pâncreas/imunologia , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Cultura Primária de Células , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Células Th2/imunologia , Células Th2/metabolismo
5.
Gene ; 725: 144163, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31639433

RESUMO

BACKGROUND: Previous studies have established that coronary artery disease is associated with excess inflammation. These studies have shown an elevation of both pro and anti-inflammatory cytokines in sufferers of coronary artery disease. There is increasing interest in the role played by the inflammasome Nod Like Receptor family pyrin domain containing 3 (NLRP3) in the aetiology of coronary artery disease. Increased severity of coronary artery disease correlates with higher levels of expression of NLRP3. Does NLRP3 polymorphisms play a role in the aetiology of coronary artery disease? METHOD: In a cohort of Vietnam War (n-299) veterans who have been previously exposed to trauma, NLRP3 polymorphisms were analysed for association with coronary calcium scores using analyses of variance. Independent t-test was used to analyse genotypes. In samples with a small representation of minor homozygotes, genotypes were combined and analysed using independent t-test. If any of the genotype analysis suggested the potential for a dominant or a recessive model the model was further explored. Hardy-Weinberg Equilibrium was calculated using Hardy-Weinberg equilibrium calculator including analysis for ascertainment bias. RESULTS: The NLRP3 polymorphism, rs10159239 was significantly associated (p = 0.001) with a higher raised coronary calcium score. The Single Nucleotide Polymorphism rs10159239 was examined by logistic regression with known risk factors for Coronary artery disease and remained significant (0.035). This is the first time rs10159239 A-allele has been associated with raised coronary calcium score. CONCLUSIONS: This is the first time rs10159239 A-allele has been associated with raised coronary calcium score. Further research is needed to replicate our results in larger well-characterised cohorts.


Assuntos
Doença da Artéria Coronariana/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Idoso , Idoso de 80 Anos ou mais , Alelos , Proteínas de Transporte/genética , Estudos de Coortes , Doença da Artéria Coronariana/metabolismo , Citocinas/genética , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genótipo , Humanos , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Veteranos , Guerra do Vietnã
6.
Sheng Li Xue Bao ; 71(6): 846-854, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31879740

RESUMO

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 µmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1ß (IL-1ß) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1ß and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1ß and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.


Assuntos
Regulação da Expressão Gênica , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Albumina Sérica , Caspase 1 , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/efeitos dos fármacos , Albumina Sérica/farmacologia
7.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570558

RESUMO

Schistosomiasis is a parasitic helminth disease that can cause severe inflammatory pathology, leading to organ damage, in humans. During a schistosomal infection, the eggs are trapped in the host liver, and products derived from eggs induce a polarized Th2 cell response, resulting in granuloma formation and eventually fibrosis. Previous studies indicated that the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is involved in schistosomiasis-associated liver fibrosis and that taurine could ameliorate hepatic granulomas and fibrosis caused by Schistosoma japonicum infection. Nevertheless, the precise role and molecular mechanism of the NLRP3 inflammasome and the protective effects of taurine in S. japonicum infection have not been extensively studied. In this study, we investigated the role of the NLRP3 inflammasome and the hepatoprotective mechanism of taurine in schistosoma-induced liver injury in mice. NLRP3 deficiency ameliorated S. japonicum-infection-induced hepatosplenomegaly, liver dysfunction, and hepatic granulomas and fibrosis; it also reduced NLRP3-dependent liver pyroptosis. Furthermore, taurine suppressed hepatic thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation in mice with S. japonicum infections, thereby inhibiting the activation of downstream inflammatory mediators such as interleukin-1ß and subsequent pyroptosis. Our results suggest that the TXNIP/NLRP3 inflammasome pathway and mediating pyroptosis are involved in S. japonicum-induced liver injury and may be a potential therapeutic target for schistosomiasis treatment. In addition, taurine may be useful to alleviate or to prevent the occurrence of schistosomiasis-associated liver fibrosis.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Schistosoma japonicum/imunologia , Esquistossomose Japônica/imunologia , Taurina/farmacologia , Tiorredoxinas/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Fígado/lesões , Fígado/parasitologia , Cirrose Hepática/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/imunologia , Esquistossomose Japônica/parasitologia , Transdução de Sinais/imunologia
8.
Oral Dis ; 25(8): 2030-2039, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31529565

RESUMO

OBJECTIVE: The aim of this study is to detect pyroptosis in macrophages stimulated with Porphyromonas gingivalis and elucidate the mechanism by which P. gingivalis induces pyroptosis in macrophages. METHODS: The immortalized human monocyte cell line U937 was stimulated with P. gingivalis W83. Flow cytometry was carried out to detect pyroptosis in macrophages. The expression of miR-155 was detected by real-time PCR and inhibited using RNAi. Suppressor of cytokine signaling (SOCS) 1, cleaved GSDMD, caspase (CAS)-1, caspase-11, apoptosis-associated speck-like protein (ASC), and NOD-like receptor protein 3 (NLRP3) were detected by Western blotting, and IL-1ß and IL-18 were detected by ELISA. RESULTS: The rate of pyroptosis in macrophages and the expression of miR-155 increased upon stimulation with P. gingivalis and pyroptosis rate decreased when miR-155 was silenced. GSDMD-NT, CAS-11, CAS-1, ASC, NLRP3, IL-1ß, and IL-18 levels increased, but SOCS1 decreased in U937 cells after stimulated with P. gingivalis. These changes were weakened in P. gingivalis-stimulated U937 macrophages transfected with lentiviruses carrying miR-155 shRNA compared to those transfected with non-targeting control sequence. However, there was no significant difference in ASC expression between P. gingivalis-stimulated shCont and shMiR-155 cells. CONCLUSIONS: Porphyromonas gingivalis promotes pyroptosis in macrophages during early infection. miR-155 is involved in this process through regulating the NLRP3 inflammasome.


Assuntos
Inflamassomos , Macrófagos , MicroRNAs , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Porphyromonas gingivalis , Piroptose/genética , Humanos , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Neoplasias
9.
Nat Commun ; 10(1): 4375, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558756

RESUMO

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1ß. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1ß were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation.


Assuntos
Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Inflamassomos/genética , Inflamação/genética , Interleucina-1beta/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Metástase Neoplásica , Transdução de Sinais/genética , Microambiente Tumoral/genética
10.
In Vivo ; 33(5): 1493-1497, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471397

RESUMO

BACKGROUND/AIM: Neurological symptoms (neuro-Behçet's disease; NBD) occur in a fraction of Behçet's disease (BD) patients and often present with parenchymal brain lesions and clinical exacerbations. Our aim was to identify genes associated with attack and remission periods of NBD. MATERIALS AND METHODS: Microarray analysis was performed using peripheral blood mononuclear cell (PBMC) samples obtained during attack and remission periods of five NBD patients. Expression levels of the most significantly up-regulated genes were measured with real-time PCR using PBMC samples of 15 NBD patients and 20 healthy controls. RESULTS: During NBD attacks, the most remarkably up-regulated genes were defensin alpha 1B (DEFA1B) and NLR family, pyrin domain containing 3 (NLRP3). Real time PCR studies showed significantly increased DEFA1B and NLRP3 expression levels during attacks. CONCLUSION: Immunological factors showing the most significant increase in expression during NBD attacks were primarily associated with innate immunity functions. DEFA1B and NLRP3 can be used as biomarkers for estimation of disease activity in NBD.


Assuntos
Síndrome de Behçet/diagnóstico , Síndrome de Behçet/etiologia , Expressão Gênica , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fenótipo , alfa-Defensinas/genética , Biomarcadores , Progressão da Doença , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Imagem por Ressonância Magnética , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Índice de Gravidade de Doença , Sequenciamento Completo do Genoma , alfa-Defensinas/metabolismo
11.
BMC Complement Altern Med ; 19(1): 216, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412862

RESUMO

BACKGROUND: Breast cancer is still the most common malignant tumor that threatens the female's life in the world, especially triple-negative breast cancer (TNBC), one of the most difficult subtypes. Lack of targeted therapies brings about urgent demand for novel treatments. In this study we aim to investigate the anti-tumor activity of Berberine (BBR), a Chinese plant-derived alkaloid, against the TNBC cell line MDA-MB-231 and elucidate its mechanism referring to anti-inflammation. METHODS: Cell inhibition rate was measured by Cell Proliferation Assay, the cytotoxic effects was detected by Lactate dehydrogenase (LDH) leakage assay, the colony formation and migration potential were evaluated by colony formation assay and wound healing assay, the release of inflammatory cytokines was detected by EMD multifactor detection, and alterations of proteins and genes related to the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway were analyzed using western blotting and real-time Polymerase Chain Reaction (PCR). RESULTS: BBR reduce the viability of MDA-MB-231 cells and increased the release of LDH from the cells in a dose-dependent manner, with and inhibition of colony formation potential and migration of the cells. BBR also caused a marked reduction in the secretion of proinflammatory cytokines, Interleukin-1α (IL-1α), Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Besides, a down-regulated behavior was observed with the expression of P2X purinoceptor 7 (P2X7), NLRP3, pro-caspase-1, apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (ASC), caspase-1 p20, Interleukin-18 (IL-18), IL-1ß proteins and NLRP3, Caspase-1 and ASC mRNAs in the NLRP3 inflammasome cascade. CONCLUSIONS: Our results confirmed that BBR can effectively affect both tumor outgrowth and spontaneous metastasis in TNBC, and that we identified a new mechanism associated with inhibition the NLRP3 inflammasome pathway, suggesting its potential therapeutic relevance in clinical use.


Assuntos
Berberina/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Apoptose/efeitos dos fármacos , Caspase 1/genética , Caspase 1/imunologia , Feminino , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/fisiopatologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
12.
Artif Cells Nanomed Biotechnol ; 47(1): 3448-3455, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31411068

RESUMO

Ventilator has been widely used for life support, but ventilator-induced lung injury (VILI) is still a major problem. Oxidative stress has been considered as a key contributor for VILI, but the specific mechanism remains unclear. The expression of NLRP3 inflammasome in cells and inflammatory factors in the supernatant were measured. Mitochondrial ROS and TRPM2 channel currents were investigated using flow cytometry and Patch-clamp technique, respectively. TRPM2-/- and NLRP3-/- mice were used for animal experiments. Lung tissues were stained by HE and the wet-dry ratio, bronchoalveolar lavage fluid (BALF) protein, MPO (marrow peroxidase), NLRP3 inflammasome were also investigated. Knockdown of NLRP3 or Caspase-1 or treatments with SS-31 or YVAD inhibited the expression of the NLRP3 inflammasome, and reduced IL-1ß and IL-18 levels in cell supernatant. These treatments suppressed the production of ROS and lowered the TRPM2 channel currents, but Rotenone exerted an opposite effect. High-tidal volume ventilation significantly increased the levels of IL-1ß, IL-18, NLRP3 inflammasome, wet-dry ratio of lung, MPO and BALF protein. However, these parameters were down-regulated in TRPM2-/- and NLRP3-/- mice. These parameters were suppressed in TRPM2-/- and NLRP3-/- mice indicate that oxidative stress might promote VILI through activating NLRP3 inflammasome and TRPM2 channel.


Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Oxidativo , Canais de Cátion TRPM/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Animais , Técnicas de Silenciamento de Genes , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/genética
13.
EMBO J ; 38(21): e101365, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31468569

RESUMO

Inflammasomes are potent innate immune signalling complexes that couple cytokine release with pro-inflammatory cell death. However, pathogens have evolved strategies to evade this cell autonomous system. Here, we show how antibodies combine with innate sensors in primary human macrophages to detect viral infection and activate the inflammasome. Our data demonstrate that antibody opsonisation of virions can activate macrophages in multiple ways. In the first, antibody binding of adenovirus causes lysosomal damage, activating NLRP3 to drive inflammasome formation and IL-1ß release. Importantly, this mechanism enhances virion capture but not infection and is accompanied by cell death, denying the opportunity for viral replication. Unexpectedly, we also find that antibody-coated viruses, which successfully escape into the cytosol, trigger a second system of inflammasome activation. These viruses are intercepted by the cytosolic antibody receptor TRIM21 and the DNA sensor cGAS. Together, these sensors stimulate both NLRP3 inflammasome formation and NFκB activation, driving dose-dependent IL-1ß and TNF secretion, without inducing cell death. Our data highlight the importance of cooperativity between multiple sensing networks to expose viruses to the inflammasome pathway, which is particularly important for how our innate immune system responds to infection in the presence of pre-existing immunity.


Assuntos
Infecções por Adenoviridae/imunologia , Anticorpos Antivirais/imunologia , Inflamassomos/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotidiltransferases/metabolismo , Ribonucleoproteínas/metabolismo , Replicação Viral/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Animais , Células Cultivadas , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nucleotidiltransferases/genética , Ribonucleoproteínas/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
14.
Int J Mol Sci ; 20(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461911

RESUMO

The purpose of this study is to investigate whether nicotinamide riboside (NR) can improve inflammation and cognitive function in diabetic mice. ICR male mice were fed for 14 weeks with either high-fat chow diet (HF, 60% kcal fat) or standard chow diet (CON, 10% kcal fat). HF, streptozotocin, and nicotinamide were used to induce hyperglycemia. NR or vehicle was delivered via stomach gavage for six weeks. Oral glucose tolerance test, Y-maze test, and nest construction test were conducted before and after the NR treatment period. NR treatment induced down-regulation of NLRP3, ASC, and caspase-1. NR reduced IL-1 expression significantly by 50% in whole brains of hyperglycemic mice. Other inflammatory markers including TNF-α and IL-6 were also attenuated by NR. Brain expression of amyloid-ß precursor protein and presenilin 1 were reduced by NR. In addition, NR induced significant reduction of amyloid-ß in whole brains of diabetic mice. NR treatment restored hyperglycemia-induced increases in brain karyopyknosis to the levels of controls. Nest construction test showed that NR improved hippocampus functions. Spatial recognition memory and locomotor activity were also improved by NR supplementation. These findings suggest that NR may be useful for treating cognitive impairment by inhibiting amyloidogenesis and neuroinflammation.


Assuntos
Anti-Inflamatórios/uso terapêutico , Encéfalo/efeitos dos fármacos , Disfunção Cognitiva/tratamento farmacológico , Diabetes Mellitus Experimental/complicações , Niacinamida/análogos & derivados , Animais , Anti-Inflamatórios/farmacologia , Encéfalo/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Cognição , Disfunção Cognitiva/etiologia , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos ICR , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo
15.
Life Sci ; 233: 116696, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31351969

RESUMO

AIMS: To explore the mechanism of how LSD1 regulates autophagy and the correlation between LSD1 and Ox-LDL-induced inflammation. MAIN METHODS: RAW264.7 cells were used during the whole study. Firstly, the effect of Ox-LDL-stimulation on LSD1 expression was detected. Through loss-of-function assay, the associations between LSD1 interference and SESN2 expression, autophagy, NLRP3 inflammasome and inflammatory cytokines were explored. Finally, the function of LSD1 exerted on activation of PI3K/Akt/mTOR signal pathway was detected using western blotting assay. KEY FINDINGS: The expression of LSD1 was significantly elevated in Ox-LDL-treated RAW264.7 cells. Inhibition of LSD1 promoted autophagy, inhibited inflammation and activated NLRP3 inflammasome. SESN2 was elevated by LSD1 inhibition, and thus activate the PI3K/Akt/mTOR signal pathway. What' more, Knockdown of SESN2 or deactivate the PI3K/Akt/mTOR signal pathway partly reversed the effect of LSD1 inhibition on autophagy. SIGNIFICANCE: Our present study drew the finding that the knockdown of LSD1 meliorated Ox-LDL-stimulated NLRP3 activation and inflammation through promoting autophagy via SESN2-mediated PI3K/Akt/mTOR pathway.


Assuntos
Autofagia , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/metabolismo , Inflamação/patologia , Lipoproteínas LDL/efeitos adversos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Inflamação/etiologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas Nucleares/genética , Peroxidases , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
16.
Nat Commun ; 10(1): 3238, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324763

RESUMO

Leucine-rich repeat (LRR) domains are evolutionarily conserved in proteins that function in development and immunity. Here we report strict exonic modularity of LRR domains of several human gene families, which is a precondition for alternative splicing (AS). We provide evidence for AS of LRR domain within several Nod-like receptors, most prominently the inflammasome sensor NLRP3. Human NLRP3, but not mouse NLRP3, is expressed as two major isoforms, the full-length variant and a variant lacking exon 5. Moreover, NLRP3 AS is stochastically regulated, with NLRP3 ∆ exon 5 lacking the interaction surface for NEK7 and hence loss of activity. Our data thus reveals unexpected regulatory roles of AS through differential utilization of LRRs modules in vertebrate innate immunity.


Assuntos
Processamento Alternativo , Éxons/genética , Inflamassomos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Células HEK293 , Humanos , Imunidade Inata/genética , Inflamassomos/química , Inflamassomos/metabolismo , Macrófagos/metabolismo , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processos Estocásticos , Suínos
17.
Int J Mol Sci ; 20(14)2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319469

RESUMO

Activation of the nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome cascade has a role in the pathogenesis of ischemia/reperfusion (IR) injury. There is growing evidence indicating cytochrome p450 (CYP450)-derived metabolites of n-3 and n-6 polyunsaturated fatty acids (PUFAs) possess both adverse and protective effects in the heart. CYP-derived epoxy metabolites are rapidly hydrolyzed by the soluble epoxide hydrolase (sEH). The current study hypothesized that the cardioprotective effects of inhibiting sEH involves limiting activation of the NLRP3 inflammasome. Isolated hearts from young wild-type (WT) and sEH null mice were perfused in the Langendorff mode with either vehicle or the specific sEH inhibitor t-AUCB. Improved post-ischemic functional recovery and better mitochondrial respiration were observed in both sEH null hearts or WT hearts perfused with t-AUCB. Inhibition of sEH markedly attenuated the activation of the NLRP3 inflammasome complex and limited the mitochondrial localization of the fission protein dynamin-related protein-1 (Drp-1) triggered by IR injury. Cardioprotective effects stemming from the inhibition of sEH included preserved activities of both cytosolic thioredoxin (Trx)-1 and mitochondrial Trx-2 antioxidant enzymes. Together, these data demonstrate that inhibiting sEH imparts cardioprotection against IR injury via maintaining post-ischemic mitochondrial function and attenuating a detrimental innate inflammatory response.


Assuntos
Epóxido Hidrolases/genética , Deleção de Genes , Inflamassomos/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Epóxido Hidrolases/metabolismo , Inflamassomos/genética , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética
18.
Invest Ophthalmol Vis Sci ; 60(8): 3034-3045, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31311035

RESUMO

Purpose: Visual (retinoid) cycle anomalies induce aberrant build-up of all-trans retinal (atRAL) in the retinal pigment epithelium (RPE), which is a cause of RPE atrophy in Stargardt disease type 1 and age-related macular degeneration. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation is implicated in the etiology of age-related macular degeneration. Here, we elucidated the relationship between NLRP3 inflammasome activation and atRAL-induced death of RPE cells. Methods: Cellular toxicities were assessed by MTS or MTT assays. Expression levels of mRNAs and proteins were determined by quantitative reverse transcription-polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. Fluorescence microscopy was used to examine intracellular signals. Ultrastructural features of organelles were examined by transmission electron microscope. Results: Abnormal accumulation of atRAL was associated with a significant increase in the proportion of human ARPE-19 cells exhibiting features of apoptosis and Caspase-3/gasdermin E (GSDME)-mediated pyroptosis. These cells also exhibited elevated expression of NLRP3, ASC, cleaved Caspase-1/poly ADP-ribose polymerase (PARP)/Caspase-3/GSDME, interleukin-1ß (IL-1ß), and IL-18, as well as NLRP3 inflammasome-related genes (IL1B and IL18). After exposure of human ARPE-19 cells to excess atRAL, reactive oxygen species (ROS) (including mitochondrial ROS) and cathepsins released from lysosomes transmitted signals leading to NLRP3 inflammasome activation. Suppressing the production of ROS, NLRP3 inflammasome, Caspase-1, cathepsin B, or cathepsin D protected ARPE-19 cells against atRAL-associated cytotoxicity. Damage to mitochondria, lysosomes, and endoplasmic reticulum in atRAL-exposed ARPE-19 cells was partially alleviated by treatment with MCC950, a selective NLRP3 inflammasome inhibitor. Conclusions: Aberrant build-up of atRAL promotes the death of RPE cells via NLRP3 inflammasome activation.


Assuntos
Morte Celular/genética , Regulação da Expressão Gênica , Inflamassomos/metabolismo , Degeneração Macular/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Epitélio Pigmentado da Retina/ultraestrutura , Western Blotting , Células Cultivadas , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Microscopia Eletrônica de Transmissão , Proteína 3 que Contém Domínio de Pirina da Família NLR/biossíntese , RNA/genética , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais
19.
Life Sci ; 233: 116631, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31278945

RESUMO

AIMS: Prior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice. MAIN METHODS: In order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice. KEY FINDINGS: There was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment. SIGNIFICANCE: The silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R.


Assuntos
Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Calpaína/antagonistas & inibidores , Caspase 1/química , Estresse do Retículo Endoplasmático , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Calpaína/genética , Calpaína/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Inflamassomos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RNA Interferente Pequeno/genética
20.
Toxicol Lett ; 313: 130-136, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276767

RESUMO

We previously demonstrated that based on their potency, contact allergens differently modulate Blimp-1/NLRP12 expression in human keratinocytes, with the extreme allergen 2,4-dinitrochlorobenzene (DNCB) more rapidly upregulating Blimp-1, leading to downregulation of NLRP12, and to the production of interleukin-18 (IL-18). The purpose of this study was to further investigate the effects of DNCB and para-phenylenediamine (PPD) on the expression of the proteins of the inflammasome, namely NLRP3, ASC and caspase 1 by western blot analysis; to define the intracellular localization and co-localization of NLRP3 and NLPR12 by immunoprecipitation and immunohistochemistry; and to define the role of NF-κB in Blimp-1 induction by pharmacological inhibition. The human keratinocyte cell line NCTC2544 was used for all experiments. Dose and time course experiments were performed to evaluate the effect of the selected contact allergens on the parameters investigated. Results indicate, that consistent with previous finding, DNCB more rapidly (3 h) induces NLRP3, ASC protein expression and caspase-1 activation compared to PPD. Immunoprecipitation studies show the recruitment of ASC to the inflammasome following exposure to both allergens, while high level of NLRP12 and less ASC protein were found associated in control cells. By immunohistochemistry, we found increased NLRP3 expression following exposure to contact allergens, and observed a nuclear co-localization of the two proteins, indicating the NLRP12 likely acts preventing the cytosolic localization of NLRP3 and inflammasome assembly. Finally, contact allergen-induced Blimp-1 mRNA and protein expression can be completely blocked by inhibiting NF-κB activation, confirming the central role of NF-κB in contact allergen-induced keratinocyte activation.


Assuntos
Alérgenos/toxicidade , Dermatite Alérgica de Contato/etiologia , Dinitroclorobenzeno/toxicidade , Inflamassomos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fenilenodiaminas/toxicidade , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fatores de Tempo
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