Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.632
Filtrar
1.
Int J Mol Med ; 47(4): 1, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33537804

RESUMEN

Quercetin (Quer) is a typical antioxidant flavonoid from plants that is involved in bone metabolism, as well as in the progression of inflammatory diseases. Elevated levels of tumor necrosis factor­α (TNF­α), a typical pro­inflammatory cytokine, can affect osteogenesis. In the present study, TNF­α was used to establish an in vitro model of periodontitis. The effects of Quer on, as well as its potential role in the osteogenic response of human periodontal ligament stem cells (hPDLSCs) under TNF­α­induced inflammatory conditions and the underlying mechanisms were then investigated. Within the appropriate concentration range, Quer did not exhibit any cytotoxicity. More importantly, Quer significantly attenuated the TNF­α induced the suppression of osteogenesis­related genes and proteins, alkaline phosphatase (ALP) activity and mineralized matrix in the hPDLSCs. These findings were associated with the fact that Quer inhibited the activation of the NF­κB signaling pathway, as well as the expression of NLRP3 inflammation­associated proteins in the inflammatory microenvironment. Moreover, the silencing of NLRP3 by small interfering RNA (siRNA) was found to protect the hPDLSCs against TNF­α­induced osteogenic damage, which was in accordance with the effects of Quer. On the whole, the present study demonstrates that Quer reduces the impaired osteogenesis of hPDLSCs under TNF­α­induced inflammatory conditions by inhibiting the NF­κB/NLRP3 inflammasome pathway. Thus, Quer may prove to be a potential remedy against periodontal bone defects.


Asunto(s)
Inflamasomas/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Osteogénesis/efectos de los fármacos , Ligamento Periodontal/patología , Quercetina/farmacología , Células Madre/patología , Factor de Necrosis Tumoral alfa/toxicidad , Adolescente , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Adulto Joven
2.
Mol Cell ; 81(3): 423-425, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33545058

RESUMEN

Recent studies provide evidence that two chemically and mechanistically distinct signals activate the human NLRP1 inflammasome, challenging the concept that it-like other mammalian inflammasomes characterized thus far-evolved to detect and respond to a single danger-associated molecular pattern.


Asunto(s)
Inflamasomas , Péptido Hidrolasas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Cisteína Endopeptidasas , Humanos , Inflamasomas/metabolismo , Proteínas Virales
3.
Nat Commun ; 12(1): 188, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33420028

RESUMEN

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells-a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Inflamasomas/química , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Adaptadoras de Señalización CARD/genética , Caspasa 1/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Inflamasomas/genética , Inflamación , Simulación del Acoplamiento Molecular , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Transducción de Señal
4.
Nat Commun ; 12(1): 189, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33420033

RESUMEN

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD-caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ancirinas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Ancirinas/química , Apoptosis , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/genética , Caspasa 1/metabolismo , Dominio de Reclutamiento y Activación de Caspasas , Microscopía por Crioelectrón , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Células HEK293 , Humanos , Inflamasomas/química , Inflamasomas/ultraestructura , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal
5.
Ecotoxicol Environ Saf ; 207: 111306, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-32949934

RESUMEN

Although studies have demonstrated that fine particulate matter (PM2.5) induces ocular surface damage, PM2.5 exposure causes cornea toxicity is not entirely clear. The aim of this study is to investigate the role of the nod-like receptor family pyrin domain containing three (NLRP3) inflammasome-mediated pyroptosis in PM2.5-related corneal toxicity. Human corneal epithelial cells (HCECs) were exposed to different concentrations of PM2.5, and the cell viability, expressions of NLRP3 inflammasome mediated pyroptosis axis molecules and intracellular reactive oxygen species (ROS) formation were measured in HCECs. Animal experiments were undertaken to topically apply PM2.5 suspension to mouse eyes for three months and the pyroptosis related molecules in the mouse corneas were measured. RESULTS: Our results showed a dose-dependent decrease of HCEC viability in the PM2.5-treated cells. NLRP3 inflammasome-mediated pyroptosis axis (NLRP3, ASC, GSDMD, caspase-1, IL-1ß, and IL-18) were activated in the PM2.5-treated HCECs, accompanied by increased ROS formation. Further in vivo study confirmed the activation of this pathway in the mouse corneas exposed to PM2.5. In conclusion, this study provids novel evidence that PM2.5 induces corneal toxicity by triggering cell pyroptosis.


Asunto(s)
Córnea/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Material Particulado/toxicidad , Piroptosis/efectos de los fármacos , Animales , Caspasa 1/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Córnea/patología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Inflamación , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo
6.
Phytomedicine ; 80: 153398, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33130474

RESUMEN

BACKGROUND: Celastrol, a pentacyclic triterpenoid quinonemethide isolated from several spp. of Celastraceae family, exhibits anti-inflammatory activities in a variety of diseases including arthritis. PURPOSE: This study aims to investigate whether the inhibition of NLRP3 inflammasome is engaged in the anti-inflammatory activities of celastrol and delineate the underlying mechanism. METHODS: The influence of celastrol on NLRP3 inflammasome activation was firstly studied in lipopolysaccharide (LPS)-primed mouse bone marrow-derived macrophages (BMDMs) and phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells treated with nigericin. Reconstituted inflammasome was also established by co-transfecting NLRP3, ASC, pro-caspase-1 and pro-IL-1ß in HEK293T cells. The changes of inflammasome components including NLRP3, ASC, pro-caspase-1/caspase-1 and pro-IL-1ß/IL-1ß were examined by enzyme-linked immunosorbent assay (ELISA), western blotting and immunofluorescence. Furthermore, Propionibacterium acnes (P. acnes)/LPS-induced liver injury and monosodium urate (MSU)-induced gouty arthritis in mice were employed in vivo to validate the inhibitory effect of celastrol on NLRP3 inflammasome. RESULTS: Celastrol significantly suppressed the cleavage of pro-caspase-1 and pro-IL-1ß, while not affecting the protein expressions of NLRP3, ASC, pro-caspase-1 and pro-IL-1ß in THP-1 cells, BMDMs and HEK293T cells. Celastrol suppressed NLRP3 inflammasome activation and alleviated P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis. Mechanism study revealed that celastrol could interdict K63 deubiquitination of NLRP3, which may concern interaction of celastrol and BRCA1/BRCA2-containing complex subunit 3 (BRCC3), and thereby prohibited the formation of NLRP3, ASC and pro-caspase-1 complex to block the generation of mature IL-1ß. CONCLUSION: Celastrol suppresses NLRP3 inflammasome activation in P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3, which presents a novel insight into inhibition of celastrol on NLRP3 inflammasome and provides more evidences for its application in the therapy of inflammation-related diseases.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Artritis Gotosa/tratamiento farmacológico , Hígado/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Triterpenos/farmacología , Animales , Artritis Gotosa/inducido químicamente , Artritis Gotosa/metabolismo , Células HEK293 , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Lipopolisacáridos/toxicidad , Hígado/microbiología , Hígado/patología , Lisina/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Propionibacterium acnes/patogenicidad , Células THP-1 , Ubiquitinación/efectos de los fármacos , Ácido Úrico/toxicidad
7.
J Exp Med ; 218(3)2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33231615

RESUMEN

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.


Asunto(s)
/patología , Inflamasomas/metabolismo , Índice de Severidad de la Enfermedad , Apoptosis , Comorbilidad , Citocinas/biosíntesis , Humanos , Pulmón/patología , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Cambios Post Mortem , Resultado del Tratamiento
8.
Cell Prolif ; 54(2): e12973, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33382502

RESUMEN

OBJECTIVES: NLRP3 inflammasome is a critical part of the innate immune system and plays an important role in a variety of inflammatory diseases. However, the effects of NLRP3 inflammasome on periodontitis have not been fully studied. MATERIALS AND METHODS: We used ligature-induced periodontitis models of NLRP3 knockout mice (NLRP3KO ) and their wildtype (WT) littermates to compare their alveolar bone phenotypes. We further used Lysm-Cre/RosanTnG mouse to trace the changes of Lysm-Cre+ osteoclast precursors in ligature-induced periodontitis with or without MCC950 treatment. At last, we explored MCC950 as a potential drug for the treatment of periodontitis in vivo and in vitro. RESULTS: Here, we showed that the number of osteoclast precursors, osteoclast differentiation and alveolar bone loss were reduced in NLRP3KO mice compared with WT littermates, by using ligature-induced periodontitis model. Next, MCC950, a specific inhibitor of the NLRP3 inflammasome, was used to inhibit osteoclast precursors differentiation into osteoclast. Further, we used Lysm-Cre/RosanTnG mice to demonstrate that MCC950 decreases the number of Lysm-Cre+ osteoclast precursors in ligature-induced periodontitis. At last, treatment with MCC950 significantly suppressed alveolar bone loss with reduced IL-1ß activation and osteoclast differentiation in ligature-induced periodontitis. CONCLUSION: Our findings reveal that NLRP3 regulates alveolar bone loss in ligature-induced periodontitis by promoting osteoclastic differentiation.


Asunto(s)
Pérdida de Hueso Alveolar/patología , Diferenciación Celular , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Osteoclastos/citología , Periodontitis/patología , Pérdida de Hueso Alveolar/metabolismo , Pérdida de Hueso Alveolar/prevención & control , Animales , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Periodontitis/tratamiento farmacológico , Periodontitis/etiología , Células Madre/citología , Células Madre/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéutico
9.
Life Sci ; 267: 118918, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33352170

RESUMEN

The NLRP3 inflammasome regulates innate immune and inflammatory responses by promoting pro-inflammatory cytokines such as IL-18 and IL-1ß. NLRP3 is one of the main factors restricting the activation of the inflammasome, which is closely related to the abundance and localization of NLRP3. A substantial number of studies have focused on specifically targeting NLRP3 to develop inhibitors against NLRP3 inflammasome. Here, we succinctly review the regulation of NLRP3 expression at DNA/chromosome, transcriptional, post-transcriptional, and translation levels. These are critical for the fine regulation of the NLRP3 inflammasome.


Asunto(s)
Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Animales , Citocinas/metabolismo , Humanos , Inmunidad Innata/inmunología , Inflamasomas/metabolismo , Inflamasomas/fisiología , Inflamación/inmunología , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal
10.
Zhonghua Yi Xue Za Zhi ; 100(48): 3863-3869, 2020 Dec 29.
Artículo en Chino | MEDLINE | ID: mdl-33371632

RESUMEN

Objective: To investigate the role and regulation mechanism of X box binding protein 1 (XBP1) for hypoxia/reoxygenation(H/R) injury in mouse renal tubular epithelial cells (TCMK-1) through thioredoxin interacting protein (TXNIP)-nucleotide-binding domain (NOD)-like receptor protein (TXNIP-NLRP3) signaling pathway. Methods: The cells were divided into 4 groups: si-NC group transfected with negative control siRNA (si-NC), si-XBP1 group transfected with siRNA targeting XBP1 (si-XBP1), si-NC+H/R group transfected with si-NC and exposed to H/R, and si-XBP1+H/R group transfected with si-XBP1 and exposed to H/R. The Annexin Ⅴ/PI double-staining method was used to detect cell apoptosis; The mitochondrial membrane potential (MMP) was determined by using JC-1 dye; The mitochondrial reactive oxygen species (mROS) was assessed by using MitoSOX™ dye. The interference efficiency of XBP1 was tested by Western blotting and quantitative real-time polymerase chain reaction. The expression levels of TXNIP, NLRP3 and IL-1ß protein were detected by Western blotting. The colocalization of mitochondria and TXNIP was detected by double-labeling immunofluorescent staining. The intergroup difference was compared by using an independent samples t-test. Results: Compared with the si-NC group, more mROS, apoptosis and lower MMP were observed in si-NC+H/R group. Compared with the si-NC+H/R group, less apoptosis (12.08±0.51 vs 19.01±1.80, P<0.05), mROS (34.63±0.64 vs 48.17±1.84, P<0.01) and higher MMP (1.03±0.11 vs 0.45±0.08, P<0.05) were observed in si-XBP1+H/R group. Down-regulation of XBP1U (protein: 1.31±0.18 vs 0.23±0.02, P<0.01; mRNA: 1.12±0.07 vs 0.38±0.01, P<0.001) and XBP1S (protein: 1.13±0.17 vs 0.28±0.07, P<0.01; mRNA: 8.39±0.63 vs 2.45±0.22, P<0.001) inhibited expression of TXNIP (0.15±0.02 vs 0.04±0.01, P<0.01), NLRP3 (1.13±0.12 vs 0.51±0.12, P<0.05) and IL-1ß (1.02±0.04 vs 0.19±0.06, P<0.001) during H/R. Meanwhile, TXNIP exhibited significantly much less colocalization with mitochondria in the si-XBP1+H/R group. Conclusion: Supression of XBP1 expression can effectively alleviate H/R-induced TCMK-1 cells injury, whose mechanism may be inhibition of TXNIP-induced NLRP3 inflammasome activation.


Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Proteínas Portadoras , Células Epiteliales/metabolismo , Hipoxia , Inflamasomas/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Tiorredoxinas/metabolismo , Proteína 1 de Unión a la X-Box/genética
11.
Nutrients ; 13(1)2020 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-33375692

RESUMEN

Inflammasomes are intracellular protein complexes that form in response to a variety of stress signals and that serve to catalyze the proteolytic conversion of pro-interleukin-1ß and pro-interleukin-18 to active interleukin-1ß and interleukin-18, central mediators of the inflammatory response; inflammasomes can also promote a type of cell death known as pyroptosis. The NLRP3 inflammasome has received the most study and plays an important pathogenic role in a vast range of pathologies associated with inflammation-including atherosclerosis, myocardial infarction, the complications of diabetes, neurological and autoimmune disorders, dry macular degeneration, gout, and the cytokine storm phase of COVID-19. A consideration of the molecular biology underlying inflammasome priming and activation enables the prediction that a range of nutraceuticals may have clinical potential for suppressing inflammasome activity-antioxidants including phycocyanobilin, phase 2 inducers, melatonin, and N-acetylcysteine, the AMPK activator berberine, glucosamine, zinc, and various nutraceuticals that support generation of hydrogen sulfide. Complex nutraceuticals or functional foods featuring a number of these agents may find utility in the prevention and control of a wide range of medical disorders.


Asunto(s)
Antioxidantes/uso terapéutico , Suplementos Dietéticos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , /metabolismo , Animales , /metabolismo , Humanos
12.
Phytomedicine ; 79: 153328, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33007730

RESUMEN

BACKGROUND: Chaiqin chengqi decoction (CQCQD) is a Chinese herbal formula derived from dachengqi decoction. CQCQD has been used for the management of acute pancreatitis (AP) in the West China Hospital for more than 30 years. Although CQCQD has a well-established clinical efficacy, little is known about its bioactive ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory effects. PURPOSE: Toll-like receptor 4 (TLR4) and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pro-inflammatory signaling pathways, play a central role in AP in determining the extent of pancreatic injury and systemic inflammation. In this study, we screened the bioactive ingredients using a pharmacological sub-network analysis based on the TLR4/NLRP3 signaling pathways followed by experimental validation. METHODS: The main CQCQD bioactive compounds were identified by UPLC-QTOF/MS. The TLR4/NLRP3 targets in AP for CQCQD active ingredients were confirmed through a pharmacological sub-network analysis. Mice received 7 intraperitoneal injections of cerulein (50 µg/kg; hourly) to induce AP (CER-AP), while oral gavage of CQCQD (5, 10, 15 and 20 g/kg; 3 doses, 2 hourly) was commenced at the 3rd injection of cerulein. Histopathology and biochemical indices were used for assessing AP severity, while polymerase chain reaction, Western blot and immunohistochemistry analyses were used to study the mechanisms. Identified active CQCQD compounds were further validated in freshly isolated mouse pancreatic acinar cells and cultured RAW264.7 macrophages. RESULTS: The main compounds from CQCQD belonged to flavonoids, iridoids, phenols, lignans, anthraquinones and corresponding glycosides. The sub-network analysis revealed that emodin, rhein, baicalin and chrysin were the compounds most relevant for directly regulating the TLR4/NLRP3-related proteins TLR4, RelA, NF-κB and TNF-α. In vivo, CQCQD attenuated the pancreatic injury and systemic inflammation of CER-AP and was associated with reduced expression of TLR4/NLRP3-related mRNAs and proteins. Emodin, rhein, baicalin and chrysin significantly diminished pancreatic acinar cell necrosis with varied effects on suppressing the expression of TLR4/NLRP3-related mRNAs. Emodin, rhein and chrysin also decreased nitric oxide production in macrophages and their combination had synergistic effects on alleviating cell death as well as expression of TLR4/NLRP3-related proteins. CONCLUSIONS: CQCQD attenuated the severity of AP at least in part by inhibiting the TLR4/NLRP3 pro-inflammatory pathways. Its active ingredients, emodin, baicalin, rhein and chrysin contributed to these beneficial effects.


Asunto(s)
Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Pancreatitis/tratamiento farmacológico , Células Acinares/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Ceruletida/toxicidad , Emodina/farmacología , Flavonoides/farmacología , Inflamasomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Pancreatitis/inducido químicamente , Pancreatitis/metabolismo , Pancreatitis/patología , Células RAW 264.7 , Receptor Toll-Like 3/antagonistas & inhibidores
13.
Molecules ; 25(20)2020 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-33066442

RESUMEN

The activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome and/or its components is associated with the physio-pathogenesis of many respiratory diseases including asthma, COPD (chronic obstructive pulmonary disease), SARS Cov-2 (severe acute respiratory syndrome coronavirus 2), and in several autoimmune diseases. Hibiscus noldeae Baker f. has been widely reported to be traditionally used in the treatment of different ailments, some of which are of inflammatory background such as asthma, wounds, headache, etc. However, the claims have not been supported by evidence at the molecular and functional levels. Here, we report on the bio-guided fractionation of H. noldeae and assessment of the inhibitory properties of some fractions and purified compounds on NLRP3 inflammasome and Interleukin 6 (IL-6). The activation of the NLRP3 inflammasome was determined by detecting the activity of caspase-1 and the production of Interleukin 1ß (IL-1ß) in Lipopolysaccharide (LPS) and ATP-stimulated Tamm-Horsfall Protein 1 (THP-1) macrophages, while the production of IL-6 was studied in LPS-stimulated RAW264.7 mouse macrophages. It was observed that hexane and ethyl acetate fractions of the crude extract of the aerial parts of H. noldeae, as well as caffeic acid, isoquercetin, and ER2.4 and ER2.7 fractions revealed significant inhibitory effects on Caspase-1 activities, and on IL-1ß and IL-6 production. The ER2.4 and ER2.7 fractions downregulated the production of IL-1ß and IL-6, in a similar range as the caspase-1 inhibitor AC-YVAD-CHO and the drug Dexamethasone, both used as controls, respectively. Overall, our work does provide the very first scientific based evidence for Hibiscus noldeae anti-inflammatory effects and widespread use by traditional healers in Rwanda for a variety of ailments.


Asunto(s)
Antiinflamatorios/farmacología , Hibiscus/química , Inflamasomas/efectos de los fármacos , Inflamación/tratamiento farmacológico , Interleucina-6/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Extractos Vegetales/farmacología , Animales , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucina-6/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células RAW 264.7
14.
J Toxicol Sci ; 45(10): 625-637, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33012731

RESUMEN

NOD-like receptor protein 3 (NLRP3) is involved in acute lung injury (ALI), but its exact role in phosgene-induced ALI is not clearly understood. The aim of the study is to explore the potential therapeutic effect of NLRP3 inflammasome modulation in the management of phosgene-induced ALI. ALI was induced in rats by phosgene exposure at 8.33 g/m3 for 5 min, 30 hr before intravenous injection of adenovirus-NLRP3 shRNA (Ad/NLRP3-shRNA). The histological changes in the lung were evaluated. Bronchoalveolar lavage fluid (BALF) neutrophils were counted (smear), and protein content was measured using the BCA assay. The wet/dry ratio of lung tissue (W/D) was measured. TUNEL staining for DNA damage was used to indirectly assess pyroptosis. NLRP3 inflammasome was assessed by immunohistochemistry, RT-PCR, western blotting. Cytokines were measured by ELISA. Histological analyses revealed reduced severity in phosgene-induced ALI with Ad/NLRP3-shRNA pretreatment. TUNEL staining indicated decreased pyroptosis in Psg-Ad/NLRP3-shRNA rats. Decreased mRNA and protein levels of NLRP3 and caspase-1 (all P < 0.05), but not ASC (P > 0.05), were found in Psg-Ad/NLRP3-shRNA rats. Immunohistochemistry revealed that Ad/NLRP3-shRNA pretreatment inhibited NLRP3 inflammasome activation. Reduced level of pro-inflammatory interleukin (IL)-1ß, IL-18, IL-33, and tumor necrosis factor (TNF)-α (all P < 0.05), but not of anti-inflammatory IL-4 and IL-10 (all P > 0.05), were found in serum and BALF from Ad/NLRP3-shRNA rats. NLRP3 gene silencing exerts beneficial effects on phosgene-induced lung injury by inhibiting NLRP3 inflammasome activation and pro-inflammatory factors, but not anti-inflammatory factors. Disruption of NLRP3 inflammasome activation might be used as a therapeutic modality for the treatment of phosgene-induced ALI.


Asunto(s)
Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/terapia , Silenciador del Gen , Terapia Genética/métodos , Inflamasomas/genética , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fosgeno/envenenamiento , ARN Interferente Pequeño/administración & dosificación , Lesión Pulmonar Aguda/diagnóstico , Animales , Biomarcadores/metabolismo , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Inyecciones Intravenosas , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Masculino , Ratas Sprague-Dawley
15.
Clín. investig. arterioscler. (Ed. impr.) ; 32(5): 193-199, sept.-oct. 2020. graf
Artículo en Español | IBECS | ID: ibc-196742

RESUMEN

INTRODUCCIÓN Y OBJETIVOS: Se ha demostrado que el inflamasoma NLRP1 es clave en la disfunción endotelial, estando las plaquetas implicadas en las reacciones inflamatorias que la desencadenan. Investigamos la inhibición in vivo de la inflamación plaquetario-dependiente mediante inhibición del receptor P2Y vía ADP comparada con la de la enzima COX sobre la transcripción del NLRP1 en las células endoteliales. MÉTODOS: Estudio prospectivo, aleatorizado, abierto y cruzado con 2 periodos de inhibición plaquetaria en 20 voluntarios sanos, administrando clopidogrel 75mg/día/7días y aspirina 100mg/día/7días de forma cruzada tras un periodo de lavado de una semana. Las células endoteliales aórticas humanas (HAEC) fueron estimuladas 2h con plasma obtenido de los pacientes antes y después de la inhibición plaquetaria. La cuantificación de la expresión de NLRP1 se determinó mediante análisis qRT PCR. RESULTADOS: Las HAEC expuestas a plasma basal de individuos sanos presentaron niveles más elevados del NLRP1 que las expuestas a plasma de los participantes tras la administración de aspirina o clopidogrel [cuantificación relativa (CR), 1,077±0,05 vs. 1,002±0,06; OR, 1,8; IC95, 1,1-2,9; p < 0,01 y 1,077±0,05 vs. 1,04±0,03; OR, 1,7; IC95, 1,2-2,6; p < 0,001, respectivamente]. La expresión del NLRP1 en HAEC expuestas a plasma de los participantes tras la administración de aspirina o clopidogrel fue similar a las HAEC sin exposición a plasma humano (PBS) [CR 1,002±0,06 vs. 1,009±0,03; OR, 0,9; IC95, 0,5-1,4; p = 0,7 y 1,04±0,03 vs. 1,009±0,03; OR, 0,8; IC95, 0,3-1,2; p = 0,5, respectivamente]. No hubo diferencias en el porcentaje de reducción del NLRP1 en las HAEC expuestas al plasma tras la toma de aspirina comparado con la provocada por el plasma de estos mismos sujetos tras clopidogrel (3,8% vs. 2,8%, p = 0,3, respectivamente). CONCLUSIONES: La inhibición plaquetaria por vías P2Y y COX provoca similar efecto en la inhibición del inflamasoma proaterogénico NLRP1 en las HAEC


INTRODUCTION AND OBJECTIVES: NRP1 inflammasome is crucial in endothelial dysfunction. Platelets are mandatory for the inflammation that precedes it. Aspirin could inhibit NLRP1 inflammasome in endothelial cells, and clopidogrel could also provoke a reduction in vascular inflammation. A study was carried out on the influence of platelet inflammatory inhibition by P2Y receptor inhibition versus COX enzyme inhibition on the transcription of NLRP1 inflammasome in endothelial cells. METHODS: An open-label, prospective, randomised crossover study with two periods of platelet inhibition enrolled 20 healthy volunteers. They received clopidogrel 75mg/day/7days and aspirin 100mg/day/7days. A venous blood sample was collected from all participants before and after this period. Human aortic endothelial cells (HAECs) were exposed for 2h in cultures. NLRP1 gene expression was then analysed in these cultures. RESULTS: HAEC cultures that were exposed to baseline plasma showed higher expression of NLRP1 than HAECs exposed to plasma after one week of aspirin or clopidogrel intake [relative quantification (RQ), 1.077±0.05 vs. 1.002±0.06; OR, 1.8; 95% CI, 1.1-2.9; P<.01 and 1.077±0.05 vs. 1.04±0.03; OR, 1.7; 95% CI, 1.2-2.6; P<.001, respectively]. NLRP1 expression in HAEC cultures exposed to plasma after one week of aspirin or clopidogrel was similar to that observed in control HAECs that was no exposed to human plasma (PBS) [RQ; 1.002±0.06 vs. 1.009±0.03; OR, 0.9; 95% CI, 0.5-1.4; P=.7, and 1.04±0.03 vs. 1.009±0.03; OR, 0.8; 95% CI, 0.3-1.2; P=.5, respectively]. No difference was observed in NLRP1 percentage reduction in HAEC after aspirin or clopidogrel exposure (3.8% vs. 2.8%, P=.3, respectively). CONCLUSIONS: Platelet inhibition by P2Y pathway is similar to COX pathway in NLRP1 expression inhibition in HAECs


Asunto(s)
Humanos , Femenino , Adulto Joven , Adulto , Inflamasomas/antagonistas & inhibidores , Aspirina/administración & dosificación , Clopidogrel/administración & dosificación , Células Endoteliales/efectos de los fármacos , Inflamasomas/metabolismo , Estudios Prospectivos , Índice de Masa Corporal , Voluntarios Sanos/estadística & datos numéricos , Factor de Crecimiento Epidérmico/efectos de los fármacos
16.
Nat Protoc ; 15(10): 3284-3333, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32895525

RESUMEN

Inflammasomes are multimeric heterogeneous mega-Dalton protein complexes that play key roles in the host innate immune response to infection and sterile insults. Assembly of the inflammasome complex following infection or injury begins with the oligomerization of the upstream inflammasome-forming sensor and proceeds through a multistep process of well-coordinated events and downstream effector functions. Together, these steps enable elegant experimental readouts with which to reliably assess the successful activation of the inflammasome complex and cell death. Here, we describe a comprehensive protocol that details several in vitro (in bone marrow-derived macrophages) and in vivo (in mice) strategies for activating the inflammasome and explain how to subsequently assess multiple downstream effects in parallel to unequivocally establish the activation status of the inflammasome and cell death pathways. Our workflow assesses inflammasome activation via the formation of the apoptosis-associated speck-like protein containing a CARD (ASC) speck; cleavage of caspase-1 and gasdermin D; release of IL-1ß, IL-18, caspase-1, and lactate dehydrogenase from the cell; and real-time analysis of cell death by imaging. Analyses take up to ~24 h to complete. Overall, our multifaceted approach provides a comprehensive and consistent protocol for assessing inflammasome activation and cell death.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Inflamasomas/metabolismo , Inflamasomas/fisiología , Animales , Apoptosis , Caspasa 1/metabolismo , Muerte Celular/fisiología , Femenino , Inmunidad Innata , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/metabolismo
17.
Eur Rev Med Pharmacol Sci ; 24(17): 9169-9171, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32965010

RESUMEN

NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome has recently become an intriguing target of several chronic and viral diseases. Here, we argue that targeting NLRP3 inflammasome could be a strategy to prevent cardiovascular outcomes [fulminant myocarditis, heart failure, venous thromboembolism (VTE)] and acute respiratory distress syndrome (ARDS) in patients with SARS-CoV-2 infection. We discuss the rationale for NLRP3 targeting in clinical trials as an effective therapeutic strategy aimed to improve prognosis of COVID-19, analyzing the potential of two therapeutic options (tranilast and OLT1177) currently available in clinical practice.


Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Infecciones por Coronavirus/diagnóstico , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neumonía Viral/diagnóstico , Betacoronavirus/aislamiento & purificación , Ensayos Clínicos como Asunto , Infecciones por Coronavirus/virología , Citocinas/metabolismo , Humanos , Inflamasomas/metabolismo , Miocarditis/prevención & control , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Nitrilos/uso terapéutico , Pandemias , Neumonía Viral/virología , Pronóstico , Tromboembolia Venosa/prevención & control , ortoaminobenzoatos/uso terapéutico
18.
Mol Immunol ; 127: 136-145, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32971400

RESUMEN

Sepsis-induced inflammatory damage is a crucial cause of acute kidney injury (AKI), and AKI is an ecumenical fearful complication in approximately half of patients with sepsis. CCAAT/enhancer-binding protein ß (C/EBPß) plays roles in regulating acute phase responses and inflammation. However, the role and mechanism of C/EBPß in AKI are unclear. LPS combined with ATP-treated renal epithelial cells HK2 and cecal ligation-peferation (CLP)-mice were used as models of AKI in vitro and in vivo. Cell damage, the secretion of interleukin-1 beta (IL-1ß), IL-18 and cysteinyl aspartate specific proteinase 1 (caspase-1) activity were tested by LDH, ELISA assay and flow cytometry analysis, respectively. The expression levels of TFAM, C/EBPß, and pyroptosis-related molecules were tested by qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assessed the interaction between C/EBPß with TFAM. Hematoxylin-Eosin (H&E) staining detected pathological changes of kidney tissues, and immunohistochemistry measured TFAM and C/EBPß in mice kidney tissues. C/EBPß or TFAM were up-regulated in LPS combined with ATP -induced HK2 cells. Knockdown of C/EBPß could suppress cell injury and the secretion of IL-1ß and IL-18 induced by LPS combined with ATP. Furthermore, C/EBPß up-regulated the expression levels of TFAM via directly binding to TFAM promoter. Overexpression of TFAM reversed the effects of C/EBPß deficiency on pyroptosis. Knockdown of C/EBPß could inhibit NLRP3 inflammasome-mediated caspase-1 signaling pathway by inactivating TFAM/RAGE pathway. It was further confirmed in the AKI mice that C/EBPß and TFAM promoted AKI by activating NLRP3-mediated pyroptosis. The interaction of between C/EBPß and TFAM facilitated pyroptosis by activating NLRP3/caspase-1 signal axis, thereby promoting the occurrence of AKI.


Asunto(s)
Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Inflamasomas/metabolismo , Proteínas Mitocondriales/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis , Factores de Transcripción/metabolismo , Adenosina Trifosfato , Animales , Caspasa 1/metabolismo , Línea Celular , Humanos , Inflamación/patología , Lipopolisacáridos , Masculino , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Unión Proteica , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de Señal
19.
Cell Prolif ; 53(10): e12902, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32945585

RESUMEN

OBJECTIVES: Calcium oxalate (CaOx) crystals can activate inflammatory cytokines by triggering inflammasomes, which cause damage to the adhered epithelium, a dysfunctional microenvironment and even renal failure. However, a comprehensive and in-depth understanding of the mechanisms underlying the effects of these crystals on damage and cytokine function in renal tubular epithelial cells (TECs) remains limited and to be explored. MATERIALS AND METHODS: We detected the pyroptosis of TECs induced after exposure to CaOx crystals and demonstrated the significance of cytokine activation in the subsequent inflammatory processes through a proteomic study. We then conducted animal and cell experiments to verify relevant mechanisms through morphological, protein, histological and biochemical approaches. Human serum samples were further tested to help explain the pathophysiological mechanism of H3 relaxin. RESULTS: We verified that crystal-induced extracellular adenosine triphosphate (ATP) upregulation via the membrane purinergic 2X7 receptor (P2X7 R) promotes ROS generation and thereby activates NLRP3 inflammasome-mediated interleukin-1ß/18 maturation and gasdermin D cleavage. Human recombinant relaxin-3 (H3 relaxin) can act on the transmembrane receptor RXFP1 to produce cAMP and subsequently improves crystal-derived damage via ATP consumption. Additionally, endogenous relaxin-3 was found to be elevated in patients with renal calculus and can thus serve as a biomarker. CONCLUSIONS: Our results provide previously unidentified mechanistic insights into CaOx crystal-induced inflammatory pyroptotic damage and H3 relaxin-mediated anti-inflammatory protection and thus suggest a series of potential therapeutic targets and methods for but not limited to nephrocalcinosis.


Asunto(s)
Antiinflamatorios/farmacología , Oxalato de Calcio/farmacología , Piroptosis/efectos de los fármacos , Relaxina/farmacología , Adenosina Trifosfato/metabolismo , Animales , Oxalato de Calcio/química , Línea Celular , AMP Cíclico/metabolismo , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Relaxina/sangre
20.
Life Sci ; 260: 118454, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-32950575

RESUMEN

Acute kidney injury (AKI) is an abrupt and usually reversible decline in renal function. AKI is considered one of the main drawbacks of the use of gentamicin that critically limits its clinical use. In this study, pirfenidone, an oral antifibrotic drug, was given to rats (200 mg/kg, p.o., daily) for seven days alone before the initiation of gentamicin treatment and continued for seven days alongside daily gentamicin injections. In gentamicin group, gentamicin was given to Wistar rats (100 mg/kg, i.p., daily) for seven days to induce AKI. Pirfenidone managed to alleviate gentamicin-induced AKI by improving kidney function parameters including serum creatinine, blood urea nitrogen (BUN), proteinuria, relative kidney-to-body weight ratio and creatinine clearance. Pirfenidone decreased cytotoxicity induced by gentamicin by decreasing lactate dehydrogenase (LDH) activity and improving histologic picture of tubules and glomeruli. Pirfenidone also alleviated oxidative stress induced by gentamicin by reducing malondialdehyde (MDA) and elevating reduced glutathione (GSH). Pirfenidone prevented the upregulated inflammasome pathway markers in the kidney. It succeeded in decreasing toll like recpetor-4 (TLR4), nuclear factor-kappa B (NF-κB), nucleotide-binding oligomerization domain [NOD]-like pyrin domain containing protein 3 (NLRP3), caspase-1, interleukin-1ß (IL-1ß) and IL-18 levels. Additionally, Pirfenidone caused a decrease in macrophage infiltration displayed by reduction in renal monocyte chemoattractant protein-1 (MCP-1) levels. To sum up, pirfenidone can effectively mitigate gentamicin-induced AKI by inhibiting oxidative stress, macrophage infiltration and inflammasome-dependent NLRP3 pathway-induced inflammation.


Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Gentamicinas/efectos adversos , Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piridonas/farmacología , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Peso Corporal , Quimiocina CCL2/metabolismo , Inflamasomas/metabolismo , Pruebas de Función Renal , L-Lactato Deshidrogenasa/sangre , Masculino , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras , Ratas Wistar , Receptor Toll-Like 4/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA