RESUMO
Tumor immune microenvironment (TIME) spatial organization predicts outcome and therapy response in triple-negative breast cancer (TNBC). An immunosuppressive TIME containing elevated tumor-associated macrophages (TAM) and scarce CD8+ T cells is associated with poor outcome, but the regulatory mechanisms are poorly understood. Here we show that ETS1-driven caspase-1 expression, required for IL1ß processing and TAM recruitment, is negatively regulated by estrogen receptors alpha (ERα) and a defining feature of TNBC. Elevated tumoral caspase-1 is associated with a distinct TIME characterized by increased pro-tumoral TAMs and CD8+ T cell exclusion from tumor nests. Mouse models prove the functional importance of ERα, ETS1, caspase-1 and IL1ß in TIME conformation. Caspase-1 inhibition induces an immunoreactive TIME and reverses resistance to immune checkpoint blockade, identifying a therapeutically targetable mechanism that governs TNBC spatial organization.
Assuntos
Linfócitos T CD8-Positivos , Caspase 1 , Receptor alfa de Estrogênio , Imunoterapia , Interleucina-1beta , Proteína Proto-Oncogênica c-ets-1 , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Macrófagos Associados a Tumor , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Animais , Feminino , Humanos , Microambiente Tumoral/imunologia , Camundongos , Imunoterapia/métodos , Caspase 1/metabolismo , Receptor alfa de Estrogênio/metabolismo , Linhagem Celular Tumoral , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Interleucina-1beta/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Regulação Neoplásica da Expressão GênicaRESUMO
Minocycline (Min), as an antibiotic, possesses various beneficial properties such as anti-inflammatory, antioxidant, and anti-apoptotic effects. Despite these known qualities, the precise cardioprotective effect and mechanism of Min in protecting against sepsis-induced cardiotoxicity (SIC) remain unspecified. To address this, our study sought to assess the protective effects of Min on the heart. Lipopolysaccharide (LPS) was utilized to establish a cardiotoxicity model both in vivo and in vitro. Min was pretreated in the models. In the in vivo setting, evaluation of heart tissue histopathological injury was performed using hematoxylin and eosin (H&E) staining and TUNEL. Immunohistochemistry (IHC) was employed to evaluate the expression levels of NLRP3 and Caspase-1 in the heart tissue of mice. During in vitro experiments, the viability of H9c2 cells was gauged utilizing the CCK8 assay kit. Intracellular ROS levels in H9c2 cells were quantified using a ROS assay kit. Both in vitro and in vivo settings were subjected to measurement of oxidative stress indexes, encompassing glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. Additionglly, myocardial injury markers like lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) activity were quantified using appropriate assay kits. Western blotting (WB) analysis was conducted to detect the expression levels of NOD-like receptor protein-3 (NLRP3), caspase-1, IL-18, and IL-1ß, alongside apoptosis-related proteins such as Bcl-2 and Bax, and antioxidant proteins including superoxide dismutase-1 (SOD-1) and antioxidant proteins including superoxide dismutase-1 (SOD-2), both in H9c2 cells and mouse heart tissues. In vivo, Min was effective in reducing LPS-induced inflammation in cardiac tissue, preventing cell damage and apoptosis in cardiomyocytes. The levels of LDH and CK-MB were significantly reduced with Min treatment. In vitro studies showed that Min improved the viability of H9C2 cells, reduced apoptosis, and decreased ROS levels in these cells. Further analysis indicated that Min decreased the protein levels of NLRP3, Caspase-1, IL-18, and IL-1ß, while increasing the levels of SOD-1 and SOD-2 both in vivo and in vitro. Min alleviates LPS-induced SIC by suppressing the NLRP3/Caspase-1 signalling pathway in vivo and in vitro.
Assuntos
Cardiotoxicidade , Caspase 1 , Lipopolissacarídeos , Minociclina , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Caspase 1/metabolismo , Cardiotoxicidade/metabolismo , Cardiotoxicidade/tratamento farmacológico , Camundongos , Minociclina/farmacologia , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Apoptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , RatosRESUMO
No single treatment significantly reduces the mortality rate and improves neurological outcomes after intracerebral haemorrhage (ICH). New evidence suggests that pyroptosis-specific proteins are highly expressed in the perihaematomal tissues of patients with ICH and that the disulfiram (DSF) inhibits pyroptosis. An ICH model was established in C57BL/6 mice by intracranial injection of collagenase, after which DSF was used to treat the mice. Cell model of ICH was constructed, and DSF was used to treat the cells. HE, TUNEL, Nissl, FJC and IF staining were performed to evaluate the morphology of brain tissues; Western blotting and ELISA were performed to measure the protein expression of NOD-like receptor protein 3 (NLRP3)/Caspase-1/gasdermin D (GSDMD) classical pyroptosis pathway and Toll-likereceptor4 (TLR4)/nuclear factor-kappaB (NF-κB) inflammatory signaling pathway and bloodâbrain barrier-associated factoes, and the wet/dry weight method was used to determine the brain water content. The expression of proteins related to the NLRP3/Caspase-1/GSDMD pathway and the TLR4/NF-κB pathway was upregulated in tissues surrounding the haematoma compared with that in control tissues; Moreover, the expression of the blood-brain barrier structural proteins occludin and zonula occludens-1 (ZO-1) was downregulated, and the expression of Aquaporin Protein-4 (AQP4) and matrix metalloprotein 9 (MMP-9) was upregulated. DSF significantly inhibited these changes, reduced the haematoma volume, decreased the brain water content, reduced neuronal death and degeneration and improved neurological function after ICH. ICH activated the classical pyroptosis pathway and TLR4/NF-κB inflammatory pathway, disruped the expression of blood-brain barrier structural proteins, and exacerbated brain injury and neurological dysfunction. DSF inhibited these changes and exerted the therapeutic effects on pathological changes and dysfunction caused by ICH.
Assuntos
Barreira Hematoencefálica , Dissulfiram , Camundongos Endogâmicos C57BL , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Piroptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Dissulfiram/farmacologia , Transdução de Sinais/efeitos dos fármacos , Masculino , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Modelos Animais de Doenças , Caspase 1/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Hemorragias Intracranianas/tratamento farmacológico , Hemorragias Intracranianas/metabolismo , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Humanos , GasderminasRESUMO
OBJECTIVE: Traumatic brain injury (TBI)-induced anxiety is a common but under-investigated disorder, for which neuroinflammation is a significant contributor. Here we aim to investigate the protective effects of genistein, a plant-derived anti-inflammatory drug, against TBI-induced anxiety, and the underlying mechanisms. METHODS: A rat model of TBI was constructed using the lateral fluid percussion injury method. Genistein at the doses of 5, 10, and 20 mg/kg were used to treat rats at 30 min, 12 h, 24 h, 48 h, and 72 h up to 14 days after TBI. The evaluation of neurological deficit was performed preoperatively, on days 1, 3, 7, and 14 after TBI. The elevated plus maze test was carried out to assess anxiety and explorative behaviours, and the open field test was performed to assess locomotive activities. Brain injury was assessed by measuring brain water content and TdT-mediated dUTP Nick-End Labeling staining. Inflammatory responses were examined using enzyme-linked immunosorbent assay. The mRNA and protein expression were analysed using real-time polymerase chain reaction and Western blot, respectively. RESULTS: In the behavioural level, genistein treatment alleviated TBI-induced anxiety behaviours and neurological deficit in rats. In the meanwhile, brain oedema was also reduced by genistein treatment, showing alleviating effects of genistein at the pathological level. TUNEL staining also showed reduced apoptosis in rats treated with genistein. Genistein also inhibited Nlrp3/caspase-1 signalling, unveiling the effects of genistein in altering molecular pathways in brains with TBI. CONCLUSION: Genistein alleviates anxiety-like behaviours in TBI rats, which may be mediated via inhibiting Nlrp/caspase-1 signalling pathway.
Assuntos
Ansiedade , Lesões Encefálicas Traumáticas , Caspase 1 , Genisteína , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos Sprague-Dawley , Transdução de Sinais , Animais , Genisteína/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/psicologia , Caspase 1/metabolismo , Caspase 1/efeitos dos fármacos , Ratos , Masculino , Ansiedade/tratamento farmacológico , Ansiedade/etiologia , Transdução de Sinais/efeitos dos fármacos , Modelos Animais de Doenças , Comportamento Animal/efeitos dos fármacosRESUMO
Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes.
Assuntos
Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Ligação a Fosfato , Piroptose , Anticorpos de Domínio Único , Humanos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/química , Inflamassomos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Células HEK293 , Caspase 1/metabolismo , Caspase 3/metabolismo , Membrana Celular/metabolismo , Multimerização Proteica , Apoptose/efeitos dos fármacos , GasderminasRESUMO
Chinese giant salamander (Andrias davidianus) is the largest extant urodela species and has unique evolutionary position. Studying the immune system of Chinese giant salamander contributes to understanding the evolution of immune systems of vertebrates. The NLR-related protein 3 (NLRP3) inflammasome comprised of NLRP3, ASC and caspase-1 play important roles in the host innate immunity. However, little is know about the NLRP3 inflammasome components in Chinese giant salamander. In this study, the NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1 (adaNLRP3, adaASC and adaCaspase-1) were characterized from Chinese giant salamander. The proteins of these three genes shared similar motifs and structures with their mammalian counterparts, with a PYD motif, a nucleotide-binding domain (NACHT) motif, and four leucine-rich repeat domain (LRR) motifs identified in adaNLRP3, a pyrin domain (PYD) motif and a caspase recruitment domain (CARD) motif in adaASC, and a CARD motif and a CASc motif in adaCaspase-1. These three genes were constitutively expressed in the skin, heart, lung, kidney, muscle, brain, spleen, and liver of Chinese giant salamander. Following Aeromonas hydrophia infection, all the three genes were up-regulated in various tissues. Molecular docking analysis revealed that the key residues involved in forming the adaNLRP3/adaASC complex were located in the PYD motifs, and that involved in forming the adaASC/adaCaspase-1 complex were located in the CARD motifs. Further analysis revealed that the hydrogen bonds and salt bridges had crucial roles in the formation of adaNLRP3/acaASC and adaASC/adaCaspase-1 complexes. To the best of our knowledge, this is the first report on the NLRP3 inflammasome components in Chinese giant salamander which will be helpful in further understanding the function of the NLRP3 inflammasome and in elucidating its role in the immune response to microbes.
Assuntos
Imunidade Inata , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Urodelos , Animais , Urodelos/imunologia , Urodelos/genética , Inflamassomos/metabolismo , Inflamassomos/imunologia , Inflamassomos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Espécies em Perigo de Extinção , Proteínas de Anfíbios/metabolismo , Proteínas de Anfíbios/genética , Caspase 1/metabolismo , Caspase 1/genética , FilogeniaRESUMO
Cleavage of the innate immune receptor NLRP1B by various microbial proteases causes the proteasomal degradation of its N-terminal fragment and the subsequent release of a C-terminal fragment that forms an inflammasome. We reported previously that metabolic stress caused by intracellular bacteria triggers NLRP1B activation, but the mechanism by which this occurs was not elucidated. Here we demonstrate that TLR4 signaling in metabolically stressed macrophages promotes the formation of a TRIF/RIPK1/caspase-8 complex. Caspase-8 activity, induced downstream of this TLR4 pathway or through a distinct TNF receptor pathway, causes cleavage and activation of NLRP1B, which facilitates the maturation of both pro-caspase-1 and pro-caspase-8. Thus, our findings indicate that caspase-8 and NLRP1B generate a positive feedback loop that amplifies cell death processes and promotes a pro-inflammatory response through caspase-1. The ability of NLRP1B to detect caspase-8 activity suggests that this pattern recognition receptor may play a role in the defense against a variety of pathogens that induce apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose , Caspase 8 , Inflamassomos , Macrófagos , Caspase 8/metabolismo , Caspase 8/genética , Inflamassomos/metabolismo , Animais , Camundongos , Macrófagos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Transdução de Sinais , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/metabolismo , Caspase 1/metabolismo , Humanos , Camundongos Knockout , ApoptoseRESUMO
Sepsis is a common and severe infectious disease, and its associated coagulation dysfunction can cause disseminated intravascular coagulation (DIC) and organ failure, leading to a significant increase in mortality. Pyroptosis is a form of programmed cell death mediated by caspase-1 in the classical pathway and caspase-4/caspase-5/caspase-11 in the non-classical pathway, along with the effector molecule gasdermin (GSDM) family. Recent studies have shown that pyroptosis plays an important role in the development of coagulation disorders in sepsis. Pyroptosis leads to the formation of cytoplasmic membrane pores, cell swelling and membrane rupture, as well as the release and enhanced activity of procoagulant contents, strongly promoting the development of systemic coagulation activation and DIC in sepsis. Therefore, exploring the role and molecular mechanisms of pyroptosis in sepsis-related coagulation disorders is of great significance for the prevention and treatment of sepsis. This article provides a review of the mechanisms involved in pyroptosis and coagulation disorders in sepsis, as well as the role and mechanisms of pyroptosis in sepsis-associated coagulation disorders to provide new ideas for sepsis related research.
Assuntos
Transtornos da Coagulação Sanguínea , Coagulação Intravascular Disseminada , Piroptose , Sepse , Sepse/metabolismo , Sepse/complicações , Sepse/fisiopatologia , Humanos , Transtornos da Coagulação Sanguínea/etiologia , Coagulação Intravascular Disseminada/etiologia , Caspases/metabolismo , Caspase 1/metabolismo , AnimaisRESUMO
Background: IL-1ß plays a critical role in the pathophysiology of neuroinflammation. The presence of cleaved IL-1ß (cIL-1ß) in the neurons of the dorsal root ganglion (DRG) implicates its function in biological signaling arising from the sensory neuron. This study was conducted to analyze the role of IL-1ß in nociceptive transduction after tissue injury. Methods: A plantar incision was made in C57BL/6 mice, following which immunohistochemistry and RNA scope in situ hybridization were performed at various time points to analyze cIL-1ß, caspase-1, and IL-1 receptor 1 (IL-1R1) expression in the DRG. The effect of intrathecal administration of a caspase-1 inhibitor or regional anesthesia using local anesthetics on cIL-1ß expression and pain hypersensitivity was analyzed by immunohistochemistry and behavioral analysis. ERK phosphorylation was also analyzed to investigate the effect of IL-1ß on the activity of spinal dorsal horn neurons. Results: cIL-1ß expression was significantly increased in caspase-1-positive DRG neurons 5 min after the plantar incision. Intrathecal caspase-1 inhibitor treatment inhibited IL-1ß cleavage and pain hypersensitivity after the plantar incision. IL-1R1 was also detected in the DRG neurons, although the majority of IL-1R1-expressing neurons lacked cIL-1ß expression. Regional anesthesia using local anesthetics prevented cIL-1ß processing. Plantar incision-induced phosphorylation of ERK was inhibited by the caspase-1 inhibitor. Conclusion: IL-1ß in the DRG neuron undergoes rapid cleavage in response to tissue injury in an activity-dependent manner. Cleaved IL-1ß causes injury-induced functional activation of sensory neurons and pain hypersensitivity. IL-1ß in the primary afferent neurons is involved in physiological nociceptive signal transduction.
Assuntos
Gânglios Espinais , Interleucina-1beta , Animais , Masculino , Camundongos , Caspase 1/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Hiperalgesia/metabolismo , Interleucina-1beta/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Células do Corno Posterior/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/metabolismoRESUMO
NLRP3 has an important role in the immune response and viral infection as an essential inflammasome component. However, it is unclear whether the grouper immune system is regulated by NLRP3 inflammasome. In this study, we cloned the NLRP3 gene from Epinephelus coioides. Ec-NLRP3 encodes 893 amino acids and contains two major structural domains, the NACHT domain (69-234aa) and the LRR domain (477-893aa). Tissue distribution analysis showed that Ec-NLRP3 was expressed in all tissues tested, with the spleen exhibiting the highest expression. Additionally, after being infected with SGIV, the expression of the Ec-NLRP3 gene was significantly increased. The results of subcellular localization revealed that Ec-NLRP3 was distributed throughout GS cells. In addition, Ec-NLRP3 co-localized with Ec-ASC and was observed as a cytosolic speck. Ec-NLRP3 overexpression significantly inhibited SGIV infection, which was further inhibited by co-overexpression of Ec-NLRP3 and Ec-ASC. Further studies revealed that overexpression of Ec-NLRP3 significantly upregulated caspase-1 activity, and co-overexpression of Ec-NLRP3 and Ec-ASC further upregulated caspase-1 activity. In addition, inhibition of Caspase-1 activity with VX-765 significantly increased the infection of SGIV. Furthermore, the NLRP3 inflammasome activator Nigericin was able to inhibit the infection of SGIV significantly. The above findings suggest that Ec-NLRP3 inhibits SGIV infection by upregulating caspase-1 activity.
Assuntos
Bass , Caspase 1 , Doenças dos Peixes , Proteínas de Peixes , Regulação da Expressão Gênica , Proteína 3 que Contém Domínio de Pirina da Família NLR , Filogenia , Alinhamento de Sequência , Regulação para Cima , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Bass/imunologia , Bass/genética , Alinhamento de Sequência/veterinária , Regulação da Expressão Gênica/imunologia , Caspase 1/genética , Caspase 1/imunologia , Caspase 1/metabolismo , Sequência de Aminoácidos , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Imunidade Inata/genética , Perfilação da Expressão Gênica/veterinária , IridoviridaeRESUMO
The NLRP3 inflammasome is dysregulated in autoinflammatory disorders caused by inherited mutations and contributes to the pathogenesis of several chronic inflammatory diseases. In this study, we discovered that disulfiram, a safe US Food and Drug Administration (FDA)-approved drug, specifically inhibits the NLRP3 inflammasome but not the NLRC4 or AIM2 inflammasomes. Disulfiram suppresses caspase-1 activation, ASC speck formation, and pyroptosis induced by several stimuli that activate NLRP3. Mechanistically, NLRP3 is palmitoylated at cysteine 126, a modification required for its localization to the trans-Golgi network and inflammasome activation, which was inhibited by disulfiram. Administration of disulfiram to animals inhibited the NLRP3, but not NLRC4, inflammasome in vivo. Our study uncovers a mechanism by which disulfiram targets NLRP3 and provides a rationale for using a safe FDA-approved drug for the treatment of NLRP3-associated inflammatory diseases.
Assuntos
Dissulfiram , Inflamassomos , Lipoilação , Proteína 3 que Contém Domínio de Pirina da Família NLR , United States Food and Drug Administration , Dissulfiram/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Animais , Humanos , Camundongos , Lipoilação/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Estados Unidos , Caspase 1/metabolismo , Células HEK293 , Aprovação de Drogas , Piroptose/efeitos dos fármacosRESUMO
Cardiovascular complications pose a significant burden in type 2 diabetes mellitus (T2DM), driven by the intricate interplay of chronic hyperglycemia, insulin resistance, and lipid metabolism disturbances. Myocardial ischemia/reperfusion (MI/R) injury during cardiopulmonary bypass (CPB) exacerbates cardiac vulnerability. This study aims to probe the role of Caspase-1-dependent pyroptosis in global ischemia/reperfusion injury among T2DM rats undergoing CPB, elucidating the mechanisms underlying heightened myocardial injury in T2DM. This study established a rat model of T2DM and compared Mean arterial pressure (MAP), heart rate (HR), and hematocrit (Hct) between T2DM and normal rats. Myocardial cell morphology, infarction area, mitochondrial ROS and caspase-1 levels, NLRP3, pro-caspase-1, caspase-1 p10, GSDMD expressions, plasma CK-MB, cTnI, IL-1ß, and IL-18 levels were assessed after reperfusion in both T2DM and normal rats. The role of Caspase-1-dependent pyroptosis in myocardial ischemia/reperfusion injury during CPB in T2DM rats was examined using the caspase-1 inhibitor VX-765 and the ROS scavenger NAC. T2DM rats demonstrated impaired glucose tolerance but stable hemodynamics during CPB, while showing heightened vulnerability to MI/R injury. This was marked by substantial lipid deposition, disrupted myocardial fibers, and intensified cellular apoptosis. The activation of caspase-1-mediated pyroptosis and increased reactive oxygen species (ROS) production further contributed to tissue damage and the ensuing inflammatory response. Notably, myocardial injury was mitigated by inhibiting caspase-1 through VX-765, which also attenuated the inflammatory cascade. Likewise, NAC treatment reduced oxidative stress and partially suppressed ROS-mediated caspase-1 activation, resulting in diminished myocardial injury. This study proved that Caspase-1-dependent pyroptosis significantly contributes to the inflammation and injury stemming from global MI/R in T2DM rats under CPB, which correlate with the surplus ROS generated by oxidative stress during reperfusion.
Assuntos
Ponte Cardiopulmonar , Caspase 1 , Diabetes Mellitus Tipo 2 , Traumatismo por Reperfusão Miocárdica , Piroptose , Espécies Reativas de Oxigênio , Animais , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Ponte Cardiopulmonar/efeitos adversos , Caspase 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicações , Ratos , Masculino , Espécies Reativas de Oxigênio/metabolismo , para-Aminobenzoatos/farmacologia , Ratos Sprague-Dawley , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicações , Interleucina-18/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , DipeptídeosRESUMO
Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (CIB-1476) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, CIB-1476 directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1ß production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of CIB-1476 with caspase-1. CIB-1476 showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in Casp1-/- mice. These results warrant further development of CIB-1476 along with its analogues as a novel strategy for caspase-1 inhibitors.
Assuntos
Caspase 1 , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Piroptose/efeitos dos fármacos , Caspase 1/metabolismo , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Inflamassomos/metabolismo , Camundongos , Humanos , Camundongos Endogâmicos C57BL , Inibidores de Caspase/farmacologia , Inibidores de Caspase/química , Isotiocianatos/farmacologia , Isotiocianatos/química , Camundongos Knockout , Descoberta de DrogasRESUMO
Noisy tinnitus is a common auditory system disease characterized by persistent tinnitus symptoms. The TLR4/NF - κ B/NLRP3 signaling pathway plays an important role in neuroinflammatory response. Select 6 control and 6 noise exposed mice for transcriptome sequencing analysis in the hippocampus, conduct high-throughput data analysis, identify differentially expressed genes, and screen for pathways. Auditory brainstem response (ABR) detection was performed to understand the hearing changes, and the modeling effect was evaluated using the GPIAS% inhibition experiment of auditory startle reflex. Morphological observation of the basement membrane was performed to determine whether the inner hair cells were damaged. Immunohistochemistry and immunofluorescence were used to determine the activation of microglia in the hippocampus of noise induced tinnitus mice. Finally, qPCR and Western Blot were used to detect the expression of TLR4, NF kB, NLRP3, caspase-1, and IL-1 ß in the hippocampus of each group of mice. Through high-throughput data analysis, it was found that there was no significant difference in the auditory threshold of the three groups of mice; After 2 h of exposure to 100 dB SPL noise, the GPIAS% of mice decreased significantly compared to before exposure, and membrane construction was successful. After 7 days, the GPIAS% of the drug intervention group increased. After noise exposure, mice developed tinnitus, and hippocampus neuroinflammation. Roflupram can inhibit neuroinflammation and improve tinnitus through the TLR4/NF kB/NLRP3/caspase-1/IL-1 ß signaling pathway.
Assuntos
Caspase 1 , Interleucina-1beta , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ruído , Transdução de Sinais , Zumbido , Receptor 4 Toll-Like , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor 4 Toll-Like/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Caspase 1/metabolismo , NF-kappa B/metabolismo , Interleucina-1beta/metabolismo , Zumbido/tratamento farmacológico , Zumbido/metabolismo , Zumbido/patologia , Ruído/efeitos adversos , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Masculino , Modelos Animais de Doenças , SulfonamidasRESUMO
OBJECTIVE: To explore the effect of miR-370-3p on LPS triggering, in particular its involvement in disease progression by targeting the TLR4-NLRP3-caspase-1 cellular pyroptosis pathway in macrophages. METHODS: Human macrophage RAW264.7 was divided into 6 groups: control, LPS, LPS + inhibitor-NC, LPS + miR-370-3p inhibitor, LPS + mimics-NC and LPS + miR-370-3p mimics. RT-qPCR was used to detect the expression level of miR-370-3p and analyzed comparatively. CCK-8 and flow cytometry assays were used to detect cell viability and apoptosis. ELISA assay was used to detect the levels of IL-1ß and TNF-α in the supernatant of the cells. The WB assay was used to detect TLR4, NLRP3, Caspase-1 and GSDMD levels. RESULTS: After LPS induction, macrophage miR-370-3p levels decreased, cell viability decreased, and apoptosis increased. At the same time, the levels of TLR4, NLRP3, Caspase-1 and GSDMD increased in the cells, and the levels of IL-1ß and TNF-α increased in the cell supernatant. Compared with the LPS group, the significantly higher expression level of miR-370-3p in the cells of the LPS + miR-370-3p mimics group was accompanied by significantly higher cell viability, significantly lower apoptosis rate, significantly lower levels of TLR4, NLRP3, Caspase-1, and GSDMD in the cells, and significantly lower levels of IL-1ß and TNF-α in the cell supernatant. CONCLUSION: MiR-370-3p may be involved in anti-infective immune responses by targeting and inhibiting the macrophage TLR4-NLRP3-caspase-1 cellular pyroptosis pathway.
Assuntos
Caspase 1 , Lipopolissacarídeos , Macrófagos , MicroRNAs , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Receptor 4 Toll-Like , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Humanos , Caspase 1/metabolismo , Caspase 1/genética , Camundongos , Células RAW 264.7 , Animais , Transdução de Sinais , Interleucina-1beta/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Infecções Bacterianas/imunologiaRESUMO
OBJECTIVE: To investigate the changes and clinical significance of NOD like receptor protein 3 (NLRP3) inflammasomes and related factors in patients with spinal fractures complicated with acute spinal cord injury (SCI). METHODS: Eighty-six spinal fracture patients complicated with acute SCI admitted to hospital from June 2019 to March 2022 were selected as SCI group, There were 48 males and 38 females, with an average age of (43.48±6.58) years old. And 100 healthy volunteers who underwent physical examination during the same time were selected as control group, including 56 males patients and 44 females patients, with an average age of (45.13±6.43) years old. Peripheral blood mononuclear cell (PBMC) were collected, and the mRNA expressions of NLRP3 and Caspase-1 were detected. Serum was collected and the levels of interleukin (IL)- 1ß, IL-18 were detected. According to Frankel's grade, the SCI group was divided into complete injury patients and incomplete injury patients, and according to the Japanese Orthopedic Society (JOA) grade, the SCI group was divided into good prognosis group and poor prognosis group. The difference of NLRP3, Caspase-1, IL-1ß, IL-18 among groups were compared, the influencing factors for poor prognosis in SCI patients was analyzed by Logistic regression. RESULTS: The mRNA expression levels of NLRP3 (1.41±0.33) and Caspase-1 (1.44±0.35) in PBMC and the levels of IL-1ß(45.34±13.22) pg·ml-1, IL-18(40.95±8.77) pg·ml-1 in serum of SCI group were higher than those of the control group[(1.00±0.19), (1.00±0.16), (16.58±4.24) pg·ml-1, (12.57±3.68) pg·ml-1] (P<0.05). The mRNA expression levels of NLRP3(1.63±0.34) and Caspase-1 (1.67±0.27) in PBMC and the levels of IL-1ß(51.09±11.10) pg·ml-1, IL-18 (47.65±7.93) pg·ml-1 in serum of patients with complete injury in the SCI group were higher than those of patients with incomplete injury [(1.31±0.27), (1.34±0.33), (42.85±13.36) pg·ml-1, (38.05±7.48) pg·ml-1](P<0.05). The mRNA expression levels of NLRP3 (1.66±0.31) and Caspase-1 (1.72±0.31)in PBMC and the levels of IL-1ß(51.21±11.31) pg·ml-1, IL-18 (45.70±7.25) pg·ml-1 in serum, the proportion of complete injury(21 patients), and the proportion of spinal cord edema or bleeding of patients(15 patients) with poor prognosis in the SCI group were higher than those of patients with good prognosis[(1.28±0.26), (1.37±0.36), (42.79±13.25) pg·ml-1ã(38.90±8.63) pg·ml-1, 5ã20 cases](P<0.05). Complete injury and the mRNA expression of NLRP3 in PBMC were the influencing factors for poor prognosis in the SCI group (P<0.05). CONCLUSION: The activation of NLRP3 inflammasomes in patients with spinal fractures complicated with acute SCI is associated with worsening injury and poor prognosis, and NLRP3 expression can serve as a marker for evaluating prognosis.
Assuntos
Caspase 1 , Inflamassomos , Interleucina-18 , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR , Traumatismos da Medula Espinal , Fraturas da Coluna Vertebral , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Masculino , Feminino , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/sangue , Adulto , Pessoa de Meia-Idade , Interleucina-18/sangue , Interleucina-1beta/sangue , Interleucina-1beta/genética , Caspase 1/sangue , Fraturas da Coluna Vertebral/sangue , Fraturas da Coluna Vertebral/complicações , Leucócitos Mononucleares/metabolismo , Prognóstico , Relevância ClínicaRESUMO
Aberrant activation of the NLRP3 inflammasome is recognized to induce a chronic inflammatory response in the liver, ultimately leading to hepatic fibrosis. HSP90 is suggested to regulate NLRP3 activation and its downstream signaling. This study is the first to explore the potential therapeutic role of pimitespib in mitigating liver fibrosis in rats. The results of the study revealed that pimitespib effectively suppressed hepatic inflammation and fibrogenesis by modulating HSP90's control over the NFκB/NLRP3/caspase-1 axis. In vitro experiments demonstrated that pimitespib reduced LDH levels and increased hepatocyte survival, whereas in vivo, it prolonged the survival of rats with hepatic fibrosis. Additionally, pimitespib exhibited improvements in the function and microscopic characteristics of rat livers. Pimitespib effectively inhibited NFκB, which serves as the priming signal for NLRP3 activation. Pimitespib's inhibitory effect on NLRP3, identified as an HSP90 client protein, plays a central role in the observed anti-fibrotic effect. The simultaneous inhibition of both priming and activation signals of NLRP3 by pimitespib led to a reduction in caspase-1 activity and subsequent suppression of the N-terminal fragment of gasdermin D, ultimately constraining hepatocyte pyroptotic cell death. These diverse effects were associated with a decrease in the transcription of inflammatory mediators IL-1ß, IL-18, and TNF-α, as well as the fibrogenic mediators TGF-ß, TIMP-1, PDGF-BB, and Col1a1. Moreover, pimitespib induced the expression of HSP70, which could further contribute to the repression of fibrosis development. In summary, our findings provide an evolutionary perspective on managing liver fibrosis, positioning pimitespib as a promising candidate for anti-inflammatory and antifibrotic therapy.
Assuntos
Caspase 1 , Proteínas de Choque Térmico HSP90 , Cirrose Hepática , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/tratamento farmacológico , Proteínas de Choque Térmico HSP90/metabolismo , NF-kappa B/metabolismo , Masculino , Caspase 1/metabolismo , Transdução de Sinais , Ratos Sprague-Dawley , Inflamassomos/metabolismo , Sulfonamidas/farmacologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/efeitos dos fármacosRESUMO
Objective: This study aimed to evaluate the role of absent in melanoma 2 (AIM2) inflammasome-mediated pyroptosis in the pathogenesis of acute gouty arthritis (AGA) and asymptomatic hyperuricemia(AHU). Methods: A cohort of 30 AGA patients, 30 AHU individuals, and 30 healthy controls (HC) was assembled. Demographic and biochemical data, along with blood samples, were collected. Serum double-stranded DNA (dsDNA) levels were quantified using a fluorescent assay. Transcriptomic and proteomic analysis of AIM2, Caspase-1, GSDMD, IL-1ß, and IL-18 in peripheral blood mononuclear cells was performed using qRT-PCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) was employed to measure serum IL-1ß and IL-18. Spearman correlation analysis was utilized to assess relationships between variables. Results: Both AGA and AHU groups demonstrated elevated metabolic indicators and serum levels of dsDNA, IL-1ß, and IL-18 compared to the HC group. AGA patients exhibited higher inflammatory markers than the AHU group. In the AGA group, there was a significant increase in the mRNA and protein levels of AIM2, Caspase-1, GSDMD, IL-1ß, and IL-18 (P<0.05 to P<0.001). The AHU group showed higher AIM2, Caspase-1, GSDMD, and IL-18 mRNA levels than the HC group (P<0.001 to P<0.01), with a non-significant increase in AIM2, GSDMD, and IL-1ß proteins (P>0.05). In contrast, Caspase-1 and IL-18 proteins were significantly higher in the AHU group (P<0.05). Notable correlations were observed between AIM2 protein expression and levels of Caspase-1 and GSDMD in both AGA and AHU groups. In the AGA group, AIM2 protein correlated with IL-1ß, but not in the AHU group. The AIM2 protein in the AHU group was positively associated with IL-18, with no such correlation in the AGA group. Conclusion: AIM2 inflammasome may play a role in the inflammatory processes of AGA and AHU and that its activation may be related to the pyroptosis pathway.
Assuntos
Artrite Gotosa , Proteínas de Ligação a DNA , Hiperuricemia , Inflamassomos , Piroptose , Humanos , Masculino , Inflamassomos/metabolismo , Artrite Gotosa/imunologia , Artrite Gotosa/sangue , Artrite Gotosa/metabolismo , Pessoa de Meia-Idade , Hiperuricemia/sangue , Hiperuricemia/imunologia , Feminino , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Adulto , Interleucina-18/sangue , Idoso , Estudos de Casos e Controles , Biomarcadores/sangue , Caspase 1/metabolismoRESUMO
OBJECTIVE: The purpose of the present study was to potential effects of forsythiaside A (FA) on Sjogren's syndrome (SS). METHODS: Enzyme linked immunosorbent assay for detecting cytokines and Western blotting was used for detecting related protein expression. RESULTS: FA effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in SS. FA also effectively inhibited the high expression of Grp78 in SS. When Grp78 expression was silenced, it effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. FA effectively inhibit the secretion of inflammatory cytokines induced by overexpression of Grp78, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. CONCLUSION: FA induces the degradation of Grp78 protein, regulates the NF-κB signaling pathway in SS and inhibited NLRP3 inflammasome activation and reduced the release of inflammatory cytokines to alleviate SS.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Síndrome de Sjogren , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Humanos , Chaperona BiP do Retículo Endoplasmático/metabolismo , Inflamassomos/metabolismo , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Feminino , Transdução de Sinais , Citocinas/metabolismo , Caspase 1/metabolismo , Pessoa de Meia-Idade , Masculino , Fator de Transcrição RelA/metabolismo , NF-kappa B/metabolismoRESUMO
Neutrophil extracellular traps (NETs) function to control infectious agents as well as to propagate inflammatory response in a variety of disease conditions. DNA damage associated with chromatin decondensation and NACHT domain-leucine-rich repeat-and pyrin domain-containing protein 3 (NLRP3) inflammasome activation have emerged as crucial events in NET formation, but the link between the two processes is unknown. In this study, we demonstrate that poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair enzyme, regulates NET formation triggered by NLRP3 inflammasome activation in neutrophils. Activation of mouse neutrophils with canonical NLRP3 stimulants LPS and nigericin induced NET formation, which was significantly abrogated by pharmacological inhibition of PARP-1. We found that PARP-1 is required for NLRP3 inflammasome assembly by regulating post-transcriptional levels of NLRP3 and ASC dimerization. Importantly, this PARP-1-regulated NLRP3 activation for NET formation was independent of inflammasome-mediated pyroptosis, because caspase-1 and gasdermin D processing as well as IL-1ß transcription and secretion remained intact upon PARP-1 inhibition in neutrophils. Accordingly, pharmacological inhibition or genetic ablation of caspase-1 and gasdermin D had no effect on NLRP3-mediated NET formation. Mechanistically, PARP-1 inhibition increased p38 MAPK activity, which was required for downmodulation of NLRP3 and NETs, because concomitant inhibition of p38 MAPK with PARP-1 restored NLRP3 activation and NET formation. Finally, mice undergoing bacterial peritonitis exhibited increased survival upon treatment with PARP-1 inhibitor, which correlated with increased leukocyte influx and improved intracellular bacterial clearance. Our findings reveal a noncanonical pyroptosis-independent role of NLRP3 in NET formation regulated by PARP-1 via p38 MAPK, which can be targeted to control NETosis in inflammatory diseases.