RESUMEN
The absent in melanoma 2 (AIM2) inflammasome contributes to ischemic brain injury by inducing cell pyroptosis and inflammatory responses. Our research group has previously demonstrated that ATP-sensitive potassium channels (KATP channels) openers can modulate neuronal synaptic plasticity post-ischemic stroke for neuroprotection. However, the specific mechanisms of KATP channels in the inflammatory response following ischemic stroke remain unclear. Here, we assessed cellular damage by observing changes in BV-2 morphology and viability. 2,3,5-Triphenyl tetrazolium chloride (TTC) staining, mNSS scoring, Nissl staining, and TdT-mediated dUTP nick end labeling (TUNEL) staining were used to evaluate behavioral deficits, brain injury severity, and neuronal damage in mice subjected to middle cerebral artery occlusion (MCAO). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to measure cell pyroptosis and nuclear factor-kappaB (NF-κB) activation in vivo and in vitro. We observed that AIM2 protein expression was upregulated and localized within the cytoplasm of BV-2 cells. Notably, low-dose Nicorandil treatment reduced inflammatory cytokine secretion and pyroptosis-related protein expression, including AIM2, cleaved cysteinyl aspartate-specific protease-1 (cleaved caspase-1), and Gasdermin D N-terminal (GSDMD-NT). Further investigations revealed that the KATP channel inhibitor 5-HD upregulated p-NF-κB p65, NF-κB p65, and p-IκBα expression, reversing Nicorandil's neuroprotective effect in vivo. In summary, our results suggest that Nicorandil may serve as a potential therapeutic option for ischemic stroke. Targeting AIM2 and NF-κB represents effective strategies for inhibiting neuroinflammation.
Asunto(s)
Accidente Cerebrovascular Isquémico , Canales KATP , Ratones Endogámicos C57BL , FN-kappa B , Enfermedades Neuroinflamatorias , Nicorandil , Animales , Nicorandil/farmacología , Ratones , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/patología , FN-kappa B/metabolismo , FN-kappa B/antagonistas & inhibidores , Masculino , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Canales KATP/metabolismo , Canales KATP/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Transducción de Señal/efectos de los fármacos , Proteínas de Unión a Fosfato/metabolismo , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patologíaRESUMEN
Neuroinflammation has been proven to exert an important effect on brain injury after intracerebral hemorrhage (ICH). Previous studies reported that Didymin possessed anti-inflammatory properties after acute hepatic injury, hyperglycemia-induced endothelial dysfunction, and death. However, the role of Didymin in microglial pyroptosis and neuroinflammation after ICH is unclear. The current study aimed to investigate the effect of Didymin on neuroinflammation mediated by microglial pyroptosis in mouse models of ICH and shed some light on the underlying mechanisms. In this study, we observed that Didymin treatment remarkably improved neurobehavioral performance and decreased BBB disruption and brain water content. Microglial activation and neutrophil infiltration in the peri-hematoma tissue after ICH were strikingly mitigated by Didymin as well. At the molecular level, administration of Didymin significantly unregulated the expression of Rkip and downregulated the expression of pyroptotic molecules and inflammatory cytokines such as Nlrp3 inflammasome, GSDMD, caspase-1, and mature IL-1ß, TNF-α, and MPO after ICH. Besides, Didymin treatment decreased the number of Caspase-1-positive microglia and GSDMD-positive microglia after ICH. Inversely, Locostatin, an Rkip-specific inhibitor, significantly abolished the anti-pyroptosis and anti-neuroinflammation effects of Didymin. Moreover, Rkip binding with Asc could interrupt the activation and assembly of the inflammasome. Mechanistically, inhibition of Caspase-1 by VX-765 attenuated brain injury and suppressed microglial pyroptosis and neuroinflammation by downregulation of GSDMD, mature IL-1ß, TNF-α, and MPO based on Locostatin-treated ICH. Taken together, Didymin alleviated microglial pyroptosis and neuroinflammation, at least in part through the Asc/Caspase-1/GSDMD pathway via upregulating Rkip expression after ICH. Therefore, Didymin may be a potential agent to attenuate neuroinflammation via its anti-pyroptosis effect after ICH.
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Proteínas Adaptadoras de Señalización CARD/inmunología , Caspasa 1/inmunología , Microglía/efectos de los fármacos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas de Unión a Fosfatidiletanolamina/inmunología , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Animales , Células Cultivadas , Hemorragia Cerebral , Flavonoides/farmacología , Glicósidos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , Enfermedades Neuroinflamatorias/inmunología , Proteínas de Unión a Fosfato/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunologíaRESUMEN
OBJECTIVE: Trimetazidine (TMZ) exerts a strong inhibitory effect on ischemia/reperfusion (I/R) injury. Inflammation plays a key role in I/R injury. We hypothesized that TMZ may protect cardiomyocytes from I/R injury by inhibiting inflammation. METHODS: The left anterior descending coronary artery was ligated for 30 min followed by 6 h of reperfusion to establish a model of I/R injury. H9c2 cardiomyocytes were subjected to 2 h of hypoxia and 3 h of normoxic conditions to establish a model of hypoxia/reoxygenation (H/R) injury. We monitored the change in pyroptosis by performing Western blot analysis, microscopy and ELISA. RESULTS: I/R and H/R treatment stimulated gasdermin D-N domain (GSDMD-N) expression in cardiomyocytes (sham onefold vs. I/R 2.5-fold; control onefold vs. H/R 2.0-fold). Moreover, TMZ increased the viability of H9c2 cardiomyocytes subjected to H/R treatment (H/R 65.0% vs. H/R + TMZ 85.3%) and reduced the infarct size in vivo (I/R 47.0% vs. I/R + TMZ 28.3%). H/R and I/R treatment increased the levels of TLR4, MyD88, phospho-NF-κB p65 and the NLRP3 inflammasome; however, TMZ reduced the expression of these proteins. Additionally, TMZ inhibited noncanonical inflammasome signaling induced by I/R injury. CONCLUSIONS: In summary, TMZ alleviated pyroptosis induced by myocardial I/R injury through the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, TMZ represents an alternative treatment for myocardial I/R injury.
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Daño por Reperfusión Miocárdica/tratamiento farmacológico , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Trimetazidina/farmacología , Animales , Masculino , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , FN-kappa B/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Proteínas de Unión a Fosfato/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/fisiologíaRESUMEN
Coronavirus disease 2019 (Covid-19) is a worldwide infectious disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). In severe SARS-CoV-2 infection, there is severe inflammatory reactions due to neutrophil recruitments and infiltration in the different organs with the formation of neutrophil extracellular traps (NETs), which involved various complications of SARS-CoV-2 infection. Therefore, the objective of the present review was to explore the potential role of NETs in the pathogenesis of SARS-CoV-2 infection and to identify the targeting drugs against NETs in Covid-19 patients. Different enzyme types are involved in the formation of NETs, such as neutrophil elastase (NE), which degrades nuclear protein and release histones, peptidyl arginine deiminase type 4 (PADA4), which releases chromosomal DNA and gasdermin D, which creates pores in the NTs cell membrane that facilitating expulsion of NT contents. Despite of the beneficial effects of NETs in controlling of invading pathogens, sustained formations of NETs during respiratory viral infections are associated with collateral tissue injury. Excessive development of NETs in SARS-CoV-2 infection is linked with the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) due to creation of the NETs-IL-1ß loop. Also, aberrant NTs activation alone or through NETs formation may augment SARS-CoV-2-induced cytokine storm (CS) and macrophage activation syndrome (MAS) in patients with severe Covid-19. Furthermore, NETs formation in SARS-CoV-2 infection is associated with immuno-thrombosis and the development of ALI/ARDS. Therefore, anti-NETs therapy of natural or synthetic sources may mitigate SARS-CoV-2 infection-induced exaggerated immune response, hyperinflammation, immuno-thrombosis, and other complications.
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Lesión Pulmonar Aguda/inmunología , Antiinflamatorios/farmacología , COVID-19/inmunología , Síndrome de Liberación de Citoquinas/inmunología , Trampas Extracelulares/inmunología , Lesión Pulmonar Aguda/prevención & control , Lesión Pulmonar Aguda/virología , Antiinflamatorios/uso terapéutico , COVID-19/complicaciones , COVID-19/virología , Síndrome de Liberación de Citoquinas/prevención & control , Síndrome de Liberación de Citoquinas/virología , Trampas Extracelulares/efectos de los fármacos , Trampas Extracelulares/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Arginina Deiminasa Proteína-Tipo 4/antagonistas & inhibidores , Arginina Deiminasa Proteína-Tipo 4/metabolismo , SARS-CoV-2/inmunología , Tratamiento Farmacológico de COVID-19RESUMEN
Objectives: Combination therapy with mycophenolate mofetil, tacrolimus and steroids are effective in achieving complete remission in lupus nephritis (LN). Combination therapy uniquely downregulated caspase-1 compared with monotherapies, which can cleave gasdermin D (GSDMD) and was recently identified as the pyroptosis executioner. We therefore investigated whether combination therapy enabled the suppression of caspase-1/GSDMD-mediated pyroptosis in LN. Methods: Expression and activation of GSDMD were detected in kidney specimens of the human and mouse with LN using immunohistochemical staining and immunoblotting. Primary podocytes isolated from MRL/lpr mice were incubated with LPS+ATP, and pretreated with monotherapy or combination therapy. Inhibition of caspase-1/GSDMD-induced pyroptosis by combination therapy were assessed in MRL/lpr mice and human specimens. Pyroptosis was examined using a FAM caspase-1 kit and flow cytometry. The correlation between pyroptosis in peripheral blood and the systemic lupus erythematosus disease activity index (SLEDAI) was analyzed. Results: Kidney tissue specimens from LN patients and mice exhibited greatly increased expression levels and cleavage of GSDMD. In cultured podocytes, combination treatment significantly suppressed the activation of NLRP3 and caspase-1 and reduced GSDMD N-terminal levels. Combination therapy repressed disease progression through inhibition of caspase-1/GSDMD-mediated pyroptosis in both humans and MRL/lpr mice. Caspase-1/PI positive cell numbers in peripheral blood were positively correlated with SLE-DAI. LN patients with complete remission and partial remission had remarkably reduced caspase-1/PI positive cell numbers compared to baseline. Ac-FLTD-CMK, a GSDMD-derived inhibitor, prevented the development of LN. Conclusion: Combination therapy suppressed caspase-1/GSDMD-mediated pyroptosis in vitro and in vivo and reduced disease progression.
Asunto(s)
Inhibidores de Caspasas/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Nefritis Lúpica/tratamiento farmacológico , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Animales , Inhibidores de la Calcineurina/administración & dosificación , Caspasa 1/efectos de los fármacos , Células Cultivadas , Estudios de Cohortes , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Persona de Mediana Edad , Ácido Micofenólico/administración & dosificación , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/patología , Prednisona/administración & dosificación , Piroptosis/efectos de los fármacos , Tacrolimus/administración & dosificación , Adulto JovenRESUMEN
Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery.
Asunto(s)
Unión Neuromuscular/metabolismo , Proteínas de Unión a Fosfato/fisiología , Podosomas/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Neuromuscular/efectos de los fármacos , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Podosomas/efectos de los fármacos , Densidad Postsináptica/efectos de los fármacos , Densidad Postsináptica/metabolismo , ARN Interferente Pequeño/farmacología , Sinapsis/efectos de los fármacosRESUMEN
ABSTRACT: Cardiotoxicity has been well documented as a side effect of cisplatin (CDDP) treatment. The inflammatory response plays a crucial role in the pathological process of CDDP-induced cardiotoxicity. Wogonin is a natural flavonoid compound that possesses cardioprotective and anti-inflammatory qualities. Knowledge of the pharmacological effect and mechanism of wogonin could reveal an efficient way to identify therapeutic strategies. In this study, the potential of wogonin to antagonize CDDP-induced cardiotoxicity was evaluated in C57BL/6 mice in vivo and in H9c2 cells in vitro. The results showed that wogonin protected against CDDP-induced cardiac dysfunction, myocardial injury, and pyroptosis in vivo. Using a Gasdermin D expression plasmid, we revealed that wogonin dramatically reduced CDDP-induced pyroptosis by modulating the Gasdermin D protein in H9c2 cells. In conclusion, wogonin has great potential in attenuating CDDP-induced cardiotoxicity. In addition, greater emphasis should be placed on the antipyroptotic effects of wogonin for the treatment of other diseases.
Asunto(s)
Flavanonas/farmacología , Cardiopatías/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Sustancias Protectoras/farmacología , Piroptosis/efectos de los fármacos , Animales , Cardiotoxicidad , Línea Celular , Cisplatino , Modelos Animales de Enfermedad , Cardiopatías/inducido químicamente , Cardiopatías/metabolismo , Cardiopatías/patología , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ratas , Transducción de SeñalRESUMEN
BACKGROUND: Gasdermins (GSDMs)-mediated pyroptosis is widely involved in activating anti-tumor immunity and suppressing tumor growth. However, whether gasdermin D (GSDMD)-mediated pyroptosis affects patient prognosis in pan-cancer remains unknown. METHODS: We performed analyses of the RNA expression, genetic alteration, prognosis and immune infiltration of GSDMD in pan-cancer. In order to explore the relationship between pyroptosis and tumors, we calculated the correlation between GSDMD and pyroptosis key genes in pan-cancer. We also investigated the enrichment pathway of GSDMD-related genes. RESULTS: GSDMD was differentially expressed in the vast majority of cancer, and could be used as a prognostic marker in adrenocortical carcinoma (ACC), kidney renal clear cell carcinoma (KIRC), brain lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), skin cutaneous melanoma (SKCM) and rectum adenocarcinoma (READ). Strong evidence indicated the significant correlation of GSDMD with almost all immune checkpoints and immune cells. Pyroptosis-related genes strongly associated with GSDMD in ACC, KIRC, LGG, LIHC and SKCM, suggesting that GSDMD-mediated pyroptosis might play a critical role in the five cancers. CONCLUSION: All the evidence supported the potential role of GSDMD-mediated pyroptosis in cancer. Our results provided new insights into GSDMD as a prognostic marker and potential therapeutic target for cancer.
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Biomarcadores de Tumor/genética , Neoplasias/inmunología , Proteínas de Unión a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Piroptosis/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Conjuntos de Datos como Asunto , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/mortalidad , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Pronóstico , Piroptosis/efectos de los fármacos , Piroptosis/inmunología , Análisis de SupervivenciaRESUMEN
Inhibition of ruminal microbial urease is of particular interest due to its crucial role in regulating urea-N utilization efficiency and nitrogen pollution in the livestock industry. Acetohydroxamic acid (AHA) is currently the only commercially available urease inhibitor, but it has adverse side effects. The urease accessory protein UreG, which facilitates the functional incorporation of the urease nickel metallocentre, has been proposed in developing urease inhibitor through disrupting urease maturation. The objective of this study was to screen natural compounds as potential urease inhibitors by targeting UreG in a predominant ruminal microbial urease. In silico screening and in vitro tests for potential inhibitors were performed using molecular docking and an assay for the GTPase activity of UreG. Chelerythrine chloride was selected as a potential urease inhibitor of UreG with an inhibition concentration IC50 value of 18.13 µM. It exhibited mixed inhibition, with the Ki value being 26.28 µM. We further explored its inhibition mechanism using isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy, and we found that chelerythrine chloride inhibited the binding of nickel to UreG and induced changes in the secondary structure, especially the α-helix and ß-sheet of UreG. Chelerythrine chloride formed a pi-anion interaction with the Asp41 residue of UreG, which is an important residue in initiating the conformational changes of UreG. In conclusion, chelerythrine chloride exhibited a potential inhibitory effect on urease, which provided new evidence for strategies to develop novel urease inhibitors targeting UreG to reduce nitrogen excretion from ruminants.
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Proteínas Bacterianas/antagonistas & inhibidores , Benzofenantridinas/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Rumen/microbiología , Amoníaco/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Benzofenantridinas/química , Sitios de Unión , Bovinos , Inhibidores Enzimáticos/química , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Proteínas de Unión a Fosfato/química , Proteínas de Unión a Fosfato/metabolismo , Unión ProteicaRESUMEN
Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.
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Trampas Extracelulares/genética , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Insuficiencia Multiorgánica/genética , Proteínas de Unión a Fosfato/genética , Sepsis/genética , Inhibidores del Acetaldehído Deshidrogenasa/uso terapéutico , Traslado Adoptivo , Anciano , Animales , Células Cultivadas , Disulfiram/uso terapéutico , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Insuficiencia Multiorgánica/patología , Insuficiencia Multiorgánica/terapia , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Sepsis/patología , Sepsis/terapiaRESUMEN
Context: Hypoxia-induced injury is a classic symptom of obstructive sleep apnea hypopnea syndrome (OSAHS), which is a risk factor of various diseases, such as hypertension, heart failure and stroke. However, there is no effective therapy for hypoxia-induced injury or OSAHS due to the elusive mechanism involved.Objective: This study aimed to assess the effects of paeoniflorin on hypoxia-induced injury and explore the underlying mechanism.Materials and methods: Hypoxic models of SD rats and CTX-TNA2 cells were used to assess the effect of paeoniflorin, and the expressions of hif1a, miR-210, caspase1 and GSDMD were detected using western blots and RT-PCR. Plasmid transfection was performed to explore the role of miR-210 in the effect of paeoniflorin.Results: Firstly, we confirmed that hypoxia induced severe neuronal injury and an enhancement of inflammation in the rat brain, with elevated expression of caspase1, IL1b and IL18. In addition, the results showed an activation of astrocytes and an increased level of pyroptosis under hypoxic conditions, which suggested a critical role of pyroptosis in hypoxiainduced injury of the brain. Furthermore, we found that compared with the controls, paeoniflorin treatment improved hypoxia-induced pyroptosis in astrocytes. Moreover, we detected the activation of hif1a/miR-210 signaling in the effects of paeoniflorin on astrocytes. As expected, the expression of hif1a and miR-210 was significantly upregulated in astrocytes when exposed to hypoxia, while paeoniflorin treatment reversed these enhancements. After transfection of miR-210 mimics, the attenuation of pyroptosis induced by paeoniflorin was suppressed, which was accompanied by an increase of ROS levels, as well as LDH release, indicating a critical role of miR-210 in pyroptosis in astrocytes.Conclusions: Our findings demonstrated that paeoniflorin improved hypoxia-induced pyroptosis in astrocytes via depressing hif1a/miR-210/caspase1/GSDMD signaling, providing robust evidence for the treatment of hypoxic injury and OSAHS.HighlightsHypoxia induces severe injury and inflammatory response in the rat brain;Hypoxia enhanced pyroptotic level and led to an activation of astrocytes.;Paeoniflorin alleviates hypoxia-induced pyroptosis in astrocytes;Transfection of miR-210 mimics suppressed the effects of paeoniflorin on hypoxia-induced pyroptosis in astrocytes.
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Antiinflamatorios no Esteroideos/uso terapéutico , Caspasa 1/metabolismo , Glucósidos/uso terapéutico , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MicroARNs/metabolismo , Monoterpenos/uso terapéutico , Proteínas de Unión a Fosfato/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/metabolismo , Células Cultivadas , Glucósidos/farmacología , Hipoxia/tratamiento farmacológico , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Masculino , MicroARNs/antagonistas & inhibidores , Monoterpenos/farmacología , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Stroke is a major cause of death and disability worldwide that triggers a variety of neuropathological conditions, leading to the initiation of several pro-inflammatory mediators and neuronal damage. Neuroinflammation has been considered the potential therapeutic target and contributes to the pathology of ischemia and reperfusion. Pyroptosis is an inflammatory form of programmed cell death that plays an important role in immune protection against stroke. Gasdermin D (GSDMD) is the final executor of pyroptosis upon cleavage by caspases-1/4/5/11, followed by canonical and noncanonical inflammasome activation, leading to a series of inflammatory responses. GSDMD N-terminal domain assembles plasma membrane as well as organelle membrane pores to induce cytolysis, thereby triggering cytokine release and inflammatory-related cell death. In our review, we concisely summarized and highlighted the potential role of GSDMD-regulated pyroptosis and the biological characteristic of GSDMD as a therapeutic target in ischemic stroke. A better understanding of the roles of GSDMD may provide a theoretical basis for the design of novel therapeutic interventions for the treatment of ischemic stroke.
Asunto(s)
Isquemia Encefálica/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/efectos de los fármacos , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Proteínas de Unión a Fosfato/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Piroptosis/fisiologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: After cerebral ischemia/reperfusion injury, pro-inflammatory M1 and anti-inflammatory M2 phenotypes of microglia are involved in neuroinflammation, in which activation of NLRP3 inflammasome and subsequent pyroptosis play essential roles. Salvianolic Acids for Injection (SAFI) is Chinese medicine injection which composed of multiple phenolic acids extracted from Radix Salviae Miltiorrhizae, and has been reported to generate neuroprotective effects after cerebral ischemic insult in clinical and animal studies. AIM OF THE STUDY: The present study was designed to investigate whether SAFI exerts neuroprotective effects by switching microglial phenotype and inhibiting NLRP3 inflammasome/pyroptosis axis in microglia. MATERIALS AND METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats and oxygen-glucose deprivation/reoxygenation (OGD/R) model in co-cultured primary neurons and primary microglia were utilized. The neuroprotective effect of SAFI was evaluated through measuring neurological deficit scores, neuropathological changes, inflammatory factors, cell phenotype markers, and related proteins of NLRP3 inflammasome/pyroptosis axis. RESULTS: The results showed that SAFI treatment was able to: (1) produce a significant increase in neurological deficit scores and decrease in infarct volumes, and alleviate histological injury and neuronal apoptosis in cerebral cortex in MCAO/R model; (2) increase neuronal viability and reduce neuronal apoptosis in the OGD model; (3) reshape microglial polarization patterns from M1-like phenotype to M2-like phenotype; (4) inhibit the activation of the NLRP3 inflammasome and the expression of proteins related to NLRP3 inflammasome/pyroptosis axis in vivo and in vitro. CONCLUSION: These findings indicate that SAFI exert neuroprotective effect, probably via reducing neuronal apoptosis, switching microglial phenotype from M1 towards M2, and inhibiting NLRP3 inflammasome/pyroptosis axis in microglia.
Asunto(s)
Alquenos/farmacología , Antiinflamatorios no Esteroideos/administración & dosificación , Inflamasomas/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Polifenoles/farmacología , Piroptosis/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/metabolismo , Animales , Apoptosis/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Proteínas de Unión al Calcio/metabolismo , Caspasa 1/genética , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Inyecciones Intraperitoneales , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Proteínas de Unión a Fosfato/genética , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismoRESUMEN
Gasdermin D (GSDMD) plays a causal role in NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis eruption, which has been regarded as a potential therapeutic target for pyroptosis-related diseases including acute gouty arthritis. In the present study, the synthesized PEI-Chol (cholesterol grafted polyethylenimine) was assembled with GSDMD small interfering RNA (siRNA) to form PEI-Chol/siGSDMD polyplexes, which provided high transfection efficiency for siRNA-mediated GSDMD knockdown. Then we evaluated the effect of GSDMD siRNA-loaded PEI-Chol on inflammatory cascades in bone-marrow-derived macrophages (BMDMs) and acute gouty arthritis animal models under MSU exposure. When accompanied by pyroptosis blockade and decreased release of interleukin-1 beta (IL-1ß), NLRP3 inflammasome activation was also suppressed by GSDMD knockdown in vivo and in vitro. Moreover, in MSU-induced acute gouty arthritis mice, blocking GSDMD with siRNA significantly improved ankle swelling and inflammatory infiltration observed in histopathological analysis. Furthermore, investigation using a mouse air pouch model verified the effect of siGSDMD-loaded PEI-Chol on pyroptosis of recruited macrophages and related signaling pathways in response to MSU. These novel findings exhibited that GSDMD knockdown relieved acute gouty arthritis through inhibiting pyroptosis, providing a possible therapeutic approach for MSU-induced acute gouty arthritis molecular therapy using PEI-Chol as a nucleic acid delivery carrier.
Asunto(s)
Artritis Gotosa/tratamiento farmacológico , Portadores de Fármacos/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , ARN Interferente Pequeño/administración & dosificación , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Gotosa/inducido químicamente , Artritis Gotosa/inmunología , Artritis Gotosa/patología , Células Cultivadas , Colesterol , Técnicas de Silenciamiento del Gen/métodos , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Polietileneimina/química , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Ácido Úrico/administración & dosificación , Ácido Úrico/toxicidadRESUMEN
Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.
Asunto(s)
Disulfiram/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Sepsis/tratamiento farmacológico , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Línea Celular Tumoral , Disulfiram/uso terapéutico , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Femenino , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Liposomas , Ratones , Mutagénesis Sitio-Dirigida , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepsis/inmunología , Células Sf9 , SpodopteraRESUMEN
Pyroptosis, a proinflammatory form of programmed cell death, plays important roles in the pathogenesis of many diseases. Inflammasome activation, which has been shown in hepatic ischemia-reperfusion injury (IRI), is demonstrated to be closely associated with pyroptosis, indicating that pyroptosis may occur and perform functions in hepatic IRI. However, there is no direct evidence showing the function of pyroptosis in hepatic IRI. In this study, by detecting the pyroptosis markers, we showed that pyroptosis may be induced during hepatic IRI. Furthermore, by adopting caspase-1 inhibitors, we showed that inhibition of pyroptosis could significantly ameliorate liver injury and suppress inflammatory response during hepatic IRI. Interestingly, caspase-1 inhibitors have no protective effects on in vitro hepatocytes under hypoxic reoxygenation condition. To investigate pyroptosis induced in which specific cell types may affect hepatic IRI, we generated hepatocyte-specific Gsdmd-knockout (Hep-Gsdmd-/-) and myeloid-specific Gsdmd-knockout (LysmCre+Gsdmdf/f) mice. Functional experiments showed that compared to control mice (Gsdmdf/f), there were alleviated liver injury and inflammation in LysmCre+Gsdmdf/f mice, but not in AlbCre+Gsdmdf/f mice. In parallel in vitro studies, cytokine expression and production decreased in bone-marrow-derived macrophages and Kupffer cells from LysmCre+Gsdmdf/f mice compared to their controls. Our findings demonstrated that pyroptosis in innate immune cells aggravates hepatic IRI and implied that hepatic IRI could be protected by blocking pyroptosis, which may become a potential therapeutic target in the clinic.
Asunto(s)
Hepatocitos/metabolismo , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Daño por Reperfusión/prevención & control , Animales , Humanos , Hígado , Ratones , Ratones Noqueados , Daño por Reperfusión/genéticaRESUMEN
WDrepeat domain phosphoinositideinteracting protein 2 (WIPI2) is a protein that regulates the assembly of multiprotein complexes by presenting a betapropeller platform for simultaneous and reversible proteinprotein interactions. This study was designed to investigate the association between the expression of WIPI2 and the growth of hepatocellular carcinoma (HCC). Publiclyavailable data from the UALCAN platform revealed that WIPI2 is upregulated in tumor tissues compared with that noted in normal tissues in many types of tumors especially in HCC, and high WIPI2 expression predicts a poor patient prognosis. WIPI2 expression was significantly higher in tumor tissues compared with that in the corresponding adjacent normal tissues. Depletion of WIPI2 inhibited the proliferation and promoted the apoptosis both in HCC Huh7 and Hep3B cells. In order to explore the mechanisms of WIPI2 in HCC, WIPI2 was depleted in HCC cell lines and a gene microarray was constructed. The bioinformatic analysis showed that WIPI2 regulated the proliferation of HCC cells mainly through the AMPK signaling pathway. Further analysis indicated that the downstream factors of the AMPK signaling pathway were downregulated after WIPI2 depletion. Collectively, our study revealed that WIPI2 plays an important role in the pathogenesis of HCC mainly through the AMPK signaling pathway.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , ARN Interferente Pequeño/farmacología , Regulación hacia Arriba , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Persona de Mediana Edad , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Pronóstico , Transducción de Señal , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BT-11 is a new oral, gut-restricted, first-in-class investigational drug for Crohn disease (CD) and ulcerative colitis (UC) that targets the lanthionine synthetase C-like 2 (LANCL2) pathway and immunometabolic mechanisms. Oral BT-11 was assessed for safety, tolerability, and pharmacokinetics (PK) in normal healthy volunteers (n = 70) in a randomized, double-blind, placebo-controlled trial. Subjects (n = 70) were randomly assigned to one of five single ascending dose cohorts (up to 100 mg/kg, p.o.) and three multiple ascending dose cohorts [up to 100 mg/kg daily (QD) for seven days, orally]. Safety and tolerability were assessed by adverse event (AE) reporting, vital signs, electrocardiogram, hematology, and clinical chemistry. BT-11 did not increase total or gastrointestinal AE rates, as compared with placebo, and no serious adverse events were observed. Oral BT-11 dosing does not result in any clinically significant findings by biochemistry, coagulation, electrocardiogram, hematology, or urinalysis as compared with placebo. Mean fecal concentrations of BT-11 increased linearly with increasing oral doses, with 2.39 mg/g at 7.7 mg/kg on day 7 of the multiple ascending dose (MAD). Analysis of plasma pharmacokinetics indicates that maximum systemic concentrations are approximately 1/6000th of observed concentrations in feces and the distal gastrointestinal tract. Fecal calprotectin levels were lower in BT-11 treated groups as compared to placebo. BT-11 significantly decreases interferon gamma positive (IFNγ+) and tumor necrosis factor alpha positive (TNFα+) cluster of differentiation 4 positive (CD4+) T cells and increases forkhead box P3 positive (FOXP3+) CD4+ T cells in colonic lamina propria mononuclear cells from patients with CD and patients with UC at concentrations of 0.01 µM when treated ex vivo. BT-11 treatment is well-tolerated with no dose-limiting toxicities up to daily oral doses of 100 mg/kg (16 tablets); whereas the efficacious dose is a single tablet (8 mg/kg). Phase II studies in CD and UC patients are ongoing.
Asunto(s)
Bencimidazoles/farmacología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piperazinas/farmacología , Administración Oral , Adolescente , Adulto , Bencimidazoles/farmacocinética , Método Doble Ciego , Drogas en Investigación , Femenino , Voluntarios Sanos , Humanos , Interferón gamma/sangre , Mucosa Intestinal/citología , Masculino , Persona de Mediana Edad , Piperazinas/farmacocinética , Factor de Necrosis Tumoral alfa/sangre , Virginia , Adulto JovenRESUMEN
Hypomagnesemia is a significant risk factor for critically ill patients to develop sepsis, a life-threatening disease with a mortality rate over 25%. Our clinic data analysis showed that hypomagnesemia is associated with a decreased monocyte count in septic patients. At the cellular level, we found that Mg2+ inhibits pyroptosis. Specifically, Mg2+ limits the oligomerization and membrane localization of gasdermin D N-terminal (GSDMD-NT) upon the activation of either the canonical or noncanonical pyroptotic pathway. Mechanistically, we demonstrated that Ca2+ influx is a prerequisite for the function of GSDMD-NT. Mg2+ blocks Ca2+ influx by inhibiting the ATP-gated Ca2+ channel P2X7, thereby impeding the function of GSDMD-NT and inhibiting lipopolysaccharide (LPS)-induced noncanonical pyroptosis. Furthermore, Mg2+ administration protects mice from LPS-induced lethal septic shock. Together, our data reveal the underlying mechanism of how Mg2+ inhibits pyroptosis and suggest potential clinic applications of magnesium supplementation for sepsis prevention and treatment.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Magnesio/farmacología , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Sepsis/tratamiento farmacológico , Animales , Células Cultivadas , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Magnesio/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión a Fosfato/metabolismo , Sepsis/metabolismo , Sepsis/patologíaRESUMEN
The multiple genetic approaches available for molecular diagnosis of human diseases have made possible to identify an increasing number of pathogenic genetic changes, particularly with the advent of next generation sequencing (NGS) technologies. However, the main challenge lies in the interpretation of their functional impact, which has resulted in the widespread use of animal models. We describe here the functional modelling of seven BBS loci variants, most of them novel, in zebrafish embryos to validate their in silico prediction of pathogenicity. We show that target knockdown (KD) of known BBS (BBS1, BB5 or BBS6) loci leads to developmental defects commonly associated with ciliopathies, as previously described. These KD pleiotropic phenotypes were rescued by co-injecting human wild type (WT) loci sequence but not with the equivalent mutated mRNAs, providing evidence of the pathogenic effect of these BBS changes. Furthermore, direct assessment of cilia located in Kupffer's vesicle (KV) showed a reduction of ciliary length associated with all the studied variants, thus confirming a deleterious effect. Taken together, our results seem to prove the pathogenicity of the already classified and unclassified new BBS variants, as well as highlight the usefulness of zebrafish as an animal model for in vivo assays in human ciliopathies.