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1.
Front Immunol ; 13: 989503, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36131917

RESUMO

Intracerebral hemorrhage (ICH) is a highly harmful neurological disorder with high rates of mortality, disability, and recurrence. However, effective therapies are not currently available. Secondary immune injury and cell death are the leading causes of brain injury and a poor prognosis. Pyroptosis is a recently discovered form of programmed cell death that differs from apoptosis and necrosis and is mediated by gasdermin proteins. Pyroptosis is caused by multiple pathways that eventually form pores in the cell membrane, facilitating the release of inflammatory substances and causing the cell to rupture and die. Pyroptosis occurs in neurons, glial cells, and endothelial cells after ICH. Furthermore, pyroptosis causes cell death and releases inflammatory factors such as interleukin (IL)-1ß and IL-18, leading to a secondary immune-inflammatory response and further brain damage. The NOD-like receptor protein 3 (NLRP3)/caspase-1/gasdermin D (GSDMD) pathway plays the most critical role in pyroptosis after ICH. Pyroptosis can be inhibited by directly targeting NLRP3 or its upstream molecules, or directly interfering with caspase-1 expression and GSDMD formation, thus significantly improving the prognosis of ICH. The present review discusses key pathological pathways and regulatory mechanisms of pyroptosis after ICH and suggests possible intervention strategies to mitigate pyroptosis and brain dysfunction after ICH.


Assuntos
Lesões Encefálicas , Piroptose , Lesões Encefálicas/patologia , Caspase 1/metabolismo , Hemorragia Cerebral , Células Endoteliais/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-18/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR
2.
Methods Mol Biol ; 2543: 179-189, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36087268

RESUMO

Pyroptosis is a highly regulated inflammatory form of cell death that plays a role in many different diseases, including cancer. Pyroptosis was initially described to be mediated by caspase-1, which is activated by innate immune signaling complexes called inflammasomes. Inflammasomes trigger caspase-dependent activation of the pore-forming protein, gasdermin D, and plasma membrane disruption. In this protocol, we describe a method to simultaneously detect two hallmarks of inflammasome-mediated pyroptosis. Using a fluorescently tagged inflammasome adaptor protein (ASC-Citrine) and membrane-impermeable nuclear dyes, we can track inflammasome formation and plasma membrane disruption over time in the same cell population.


Assuntos
Inflamassomos , Piroptose , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caspase 1/metabolismo , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
3.
Sci Rep ; 12(1): 15443, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104389

RESUMO

Many psychrophilic microorganisms synthesize ice-binding proteins (IBPs) to survive the cold. The functions of IBPs are evaluated by the effect of the proteins on the nonequilibrium water freezing-point depression, which is called "thermal hysteresis (TH)", and the inhibitory effect of the proteins on the growth of larger ice crystals, which is called "ice recrystallization inhibition (IRI)". To obtain mechanical insight into the two activities, we developed a modified method of ice affinity purification and extracted two new IBP isoforms from Psychromyces glacialis, an Arctic glacier fungus. One isoform was found to be an approximately 25 kDa protein (PsgIBP_S), while the other is a 28 kDa larger protein (PsgIBP_L) that forms an intermolecular dimer. Their TH activities were less than 1 °C at millimolar concentrations, implying that both isoforms are moderately active but not hyperactive IBP species. It further appeared that both isoforms exhibit high IRI activity even at submicromolar concentrations. Furthermore, the isoforms can bind to the whole surface of a hemispherical single ice crystal, although such ice-binding was generally observed for hyperactive IBP species. These results suggest that the binding ability of IBPs to whole ice crystal surfaces is deficient for hyperactivity but is crucial for significant IRI activity.


Assuntos
Proteínas Anticongelantes , Proteínas de Transporte , Adsorção , Proteínas Anticongelantes/química , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Camada de Gelo , Compostos Organotiofosforados
4.
PLoS One ; 17(9): e0274057, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36048803

RESUMO

OBJECTIVE: The present study explored whether pyroptosis is involved in the injury process of PC12 cells induced by glucocorticoid (GC) and the regulatory relationship between endoplasmic reticulum stress (ERS) and pyrolysis. METHODS: LDH leakage of PC12 cells was detected by LDH assay. The number of dead cells was detected by SYTOX green nucleic acid staining. The levels of IL-1ß and IL-18 in the supernatants was detected by ELSIA assay. The expression levels of glucose regulated protein 78 (GRP78), cleaved gasdermin D-NT (cleaved-GSDMD-NT), NLR-pyrin domain-containing 3 (NLRP3) and cleaved-caspase-1 were observed by immunofluorescence staining and western blot. RESULTS: The LDH assay revealed that GC exposure significantly increased the release of LDH. The results of SYTOX green acid staining showed that GC exposure significantly increased the number of SYTOX green acid-positive cells. The ELSIA assay revealed that GC exposure significantly increased the levels of IL-1ß and IL-18 in the supernatants. The results of immunofluorescence staining and western blot showed that GC exposure significantly increased the expression of GRP78, cleaved-GSDMD-NT, NLRP3 and cleaved caspase-1. Treatment with the ERS inhibitor tauroursodeoxycholate (TUDCA) and siRNA GSDMD attenuated related damage and downregulated the expression of the abovementioned proteins. CONCLUSION: The present study clearly demonstrated that GC exposure can induce GSDMD-dependent pyrolysis, and ERS is involved in the above damage process.


Assuntos
Interleucina-18 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Caspase 1/metabolismo , Estresse do Retículo Endoplasmático , Glucocorticoides , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células PC12 , Pirólise , Piroptose , Ratos
5.
Pharm Biol ; 60(1): 1812-1818, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36121248

RESUMO

CONTEXT: Astragaloside IV (AS-IV) is extracted from Astragalus membranaceus (Fisch.) Bunge (Fabaceae). However, its effects on endothelial cell injury remain unclear. OBJECTIVE: To investigate the mechanisms underlying the effects of AS-IV on lipopolysaccharide (LPS)-induced endothelial injury in vitro. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were pre-treated with AS-IV (100 µmol/mL), 4-hydroxy-3-methoxyacetophenone (APO, 10 µmol/mL), N-acetylcysteine (NAC, 50 µmol/mL) and Ac-YVAD-cmk (AC, 5 µmol/mL) for 2 h before 1 µg/mL LPS 24 h exposure. Untreated cells cultured without any exposure were used as controls. Cell viability, reactive oxygen species (ROS) and pyroptosis assays were performed. The pyroptosis related proteins were detected by western blot. RESULTS: The rate in late pyroptosis (Q2-2) of AS-IV (13.65 ± 0.74%), APO (13.69 ± 0.67%) and NAC (15.87 ± 0.46%) groups was lower than the LPS group (21.89 ± 0.66%, p < 0.05), while the rate in early pyroptosis (Q2-4) of AS-IV group (12.00 ± 0.26%) was lower than other groups (p < 0.05). The expression of NOX4, GSDMD, NLRP3, ASC and caspase-1 decreased after AS-IV, NAC or AC intervention (p < 0.05). The ROS production in AS-IV (4664 ± 153.20), APO (4094 ± 78.37), NAC (5103 ± 131.10) and AC (3994 ± 102.50) groups was lower than the LPS (5986 ± 127.30) group, while the mitochondrial BCL2/BAX protein expression ratio increased in AS-IV, APO and NAC groups (p < 0.05). DISCUSSION AND CONCLUSIONS: AS-IV suppressed pyroptosis in LPS-activated HUVECs by inducing ROS/NLRP3-mediated inhibition of the inflammatory response, providing a scientific basis for clinical applications of AS-IV.


Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Acetilcisteína/farmacologia , Caspase 1/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipopolissacarídeos/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Espécies Reativas de Oxigênio/metabolismo , Saponinas , Triterpenos , Proteína X Associada a bcl-2
6.
Int J Mol Sci ; 23(17)2022 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-36077522

RESUMO

Mangiferin (MF), a xanthone that extensively exists in many herbal medicines, processes significant activities of anti-inflammation and immunomodulation. The potential regulatory effect and mechanism of mangiferin on cell pyroptosis remain unclear. In this study, mouse bone-marrow-derived macrophages (BMDMs) were stimulated with 1 µg/mL LPS to induce cell pyroptosis and were treated with 10, 50, or 100 µg/mL MF for regulating pyroptosis. The cell supernatants TNF-α, IL-1ß, IL-6, and IL-18 were detected by enzyme-linked immunosorbent assay (ELISA); gene expression of TNF-α, IL-1ß, IL-6, IL-18, Caspase-1, Caspase-11, and gasdermin D (GSDMD) was tested by real-time polymerase chain reaction (RT-PCR), and protein expression levels of apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), nod-like receptor protein-3 (NLRP3), caspase-1, caspase-11, GSDMD, and NF-κB were detected by Western blot. The results showed that MF significantly inhibited the secretion and gene expression of TNF-α, IL-6, IL-1ß, and IL-18 that were elevated by LPS. Moreover, MF significantly suppressed the gene expression of Caspase-1, Caspase-11, and GSDMD, and decreased the protein levels of NLRP3, caspase-1, caspase-11, full-length GSDMD (GSDMD-FL), GSDMD N-terminal (GSDMD-N), and NF-κB. In conclusion, mangiferin has a multi-target regulating effect on inflammation and pyroptosis by inhibiting the NF-κB pathway, suppressing inflammatory caspase-mediated pyroptosis cascades, and reducing GSDMD cleavage in LPS-induced BMDMs.


Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Xantonas , Animais , Anti-Inflamatórios/farmacologia , Caspase 1/metabolismo , Caspases , Inflamassomos/metabolismo , Interleucina-18 , Interleucina-6 , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR , Fator de Necrose Tumoral alfa , Xantonas/farmacologia
7.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36077562

RESUMO

Antibacterial delivery emulsions are potential materials for treating bacterial infections. Few studies have focused on the role and mechanism of emulsions in inflammation relief. Therefore, based on our previous analysis, in which the novel and natural Pickering emulsions stabilized by antimicrobial peptide nanoparticles were prepared, the regulation effect of emulsion on inflammasome was explored in silico, in vitro and in vivo. Firstly, the interactions between inflammasome components and parasin I or Pickering emulsion were predicted by molecular docking. Then, the inflammasome stimulation by different doses of the emulsion was tested in RAW 264.7 and THP-1 cells. Finally, in Kunming mice with peritonitis, NLRP3 and IL-1ß expression in the peritoneum were evaluated. The results showed that the Pickering emulsion could combine with ALK, casp-1, NEK7, or NLRP3 to affect the assembly of the NLRP3 and further relieve inflammation. LPNE showed a dose-dependent inhibition effect on the release of IL-1ß and casp-1. With the concentration of parasin I increased from 1.5 mg/mL to 3 mg/mL, the LDH activity decreased in the chitosan peptide-embedded nanoparticles emulsion (CPENE) and lipid/peptide nanoparticles emulsion (LPNE) groups. However, from 1.5 to 6 mg/mL, LPNE had a dose-dependent effect on the release of casp-1. The CPENE and parasin I-conjugated chitosan nanoparticles emulsion (PCNE) may decrease the release of potassium and chloride ions. Therefore, it can be concluded that the LPNE may inhibit the activation of the inflammasome by decreasing LDH activity, potassium and chloride ions through binding with compositions of NLRP3.


Assuntos
Quitosana , Nanopartículas , Animais , Caspase 1/metabolismo , Cloretos , Emulsões/química , Emulsões/farmacologia , Inflamassomos/metabolismo , Inflamação , Camundongos , Simulação de Acoplamento Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nanopartículas/química , Potássio
8.
Sci Rep ; 12(1): 15123, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36068262

RESUMO

Brucellosis is a zoonotic disease caused by Brucella abortus. An efficient immune response is crucial for curing brucellosis. The inflammasome plays a significant role in the immune response. It is unclear which inflammasome is active in acute and chronic brucellosis and how its levels relate to inflammatory cytokines. A total of 40 patients with acute or chronic brucellosis and 20 healthy volunteers had peripheral blood samples collected. The expression levels of AIM2, NLRP3, ASC, and Caspase-1 were determined by a real-time polymerase chain reaction from RNA and serum samples, and IL-1ß, IL-18, and IFN-γ were measured by enzyme-linked immunosorbent assay. In the acute brucellosis group, AIM2 expression was significantly higher, while ACS expression was significantly lower than that of healthy volunteers. In patients with chronic brucellosis, AIM2 expression was significantly lower, while Caspase-1 expression was significantly higher than that of healthy volunteers. Serum IL-18 and IFN-γ levels were significantly higher in patients with acute brucellosis than in healthy controls. The IFN-γ level was also significantly higher in patients with chronic brucellosis than in healthy controls. The inflammasome responds differently in different stages of brucellosis. The inflammasome may be the site of action of immune escape in brucellosis.


Assuntos
Brucelose , Inflamassomos , Caspase 1/metabolismo , China , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Imunidade Inata , Inflamassomos/metabolismo , Interleucina-18 , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
9.
Dis Markers ; 2022: 2329904, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36061354

RESUMO

Ulcerative colitis (UC) is a chronic inflammatory disease. Intestinal mucosal injury is a significant factor in UC. Pyroptosis is a kind of programmed cell death induced by inflammatory caspases. Proteasome 20S subunit beta 5 (PSMB5) promotes cell viability. The purpose of this study was to determine the impact of PSMB5 on intestinal mucosal injury and to elucidate the underlying processes in dextran sulfate sodium- (DSS-) induced UC mice. Kunming (KM) mice received 3% DSS for 5 days to induce UC. We collected clinical symptoms, body weight, colon length, and histological changes. MDA (malondialdehyde) and SOD (superoxide dismutase) levels were determined using an ELISA assay. RT-PCR was used to assess the expression of IL-1ß and IL-18. PSMB5 demonstrated a significant effect against UC by increasing body weight and colon length and decreasing DAI (disease activity index), colon macroscopic damage index (CMDI), histological injury scores, and reactive oxygen species (ROS), MDA, and SOD levels, thereby alleviating histopathological changes and inhibiting oxidative stress. HIEC-6 cells were exposed to lipopolysaccharide (LPS) condition with or without PSMB5, along with caspase-1 inhibitor (Z-VAD-FMK), NLRP3 inhibitor (MCC950), and ROS scavenger N-acetylcysteine (NAC). The viability of the cells, the release of lactate dehydrogenase (LDH), and intracellular ROS generation were determined using assay kits. Western blot analysis was used to determine the levels of NLRP3, ASC, cleaved caspase-1 (p20), pro-IL-1ß, IL-1ß, pro-IL-18, and IL-18. PSMB5 overexpression enhanced the inflammatory damage in LPS-treated HIEC-6 cells by activating the NLRP3 inflammasome and mediating pyroptosis, as demonstrated by increased LDH release and lower cell viability, as well as increased expression of NLRP3, ASC, cleaved caspase-1 (p20), IL-1, and IL-18. Meanwhile, NAC protected HIEC-6 cells from LPS-induced damage by reversing the activation of the NLRP3 inflammasome-mediated pyroptosis. In conclusion, PSMB5 may lower HIEC-6 cell susceptibility to LPS and ameliorate UC-induced HIEC-6 cell damage by decreasing ROS generation and hence inhibiting NLRP3-mediated pyroptosis.


Assuntos
Colite Ulcerativa , Inflamassomos , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Peso Corporal , Caspase 1/metabolismo , Caspase 1/farmacologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Inflamassomos/metabolismo , Inflamassomos/farmacologia , Interleucina-18/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase
10.
Front Immunol ; 13: 918507, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36045672

RESUMO

Chronic pulmonary inflammation and chronic obstructive pulmonary disease (COPD) are major health issues largely due to air pollution and cigarette smoke (CS) exposure. The role of the innate receptor NLRP3 (nucleotide-binding domain and leucine-rich repeat containing protein 3) orchestrating inflammation through formation of an inflammasome complex in CS-induced inflammation or COPD remains controversial. Using acute and subchronic CS exposure models, we found that Nlrp3-deficient mice or wild-type mice treated with the NLRP3 inhibitor MCC950 presented an important reduction of inflammatory cells recruited into the bronchoalveolar space and of pulmonary inflammation with decreased chemokines and cytokines production, in particular IL-1ß demonstrating the key role of NLRP3. Furthermore, mice deficient for Caspase-1/Caspase-11 presented also decreased inflammation parameters, suggesting a role for the NLRP3 inflammasome. Importantly we showed that acute CS-exposure promotes NLRP3-dependent cleavage of gasdermin D in macrophages present in the bronchoalveolar space and in bronchial airway epithelial cells. Finally, Gsdmd-deficiency reduced acute CS-induced lung and bronchoalveolar space inflammation and IL-1ß secretion. Thus, we demonstrated in our model that NLRP3 and gasdermin D are key players in CS-induced pulmonary inflammation and IL-1ß release potentially through gasdermin D forming-pore and/or pyroptoctic cell death.


Assuntos
Fumar Cigarros , Pneumonia , Doença Pulmonar Obstrutiva Crônica , Animais , Caspase 1/metabolismo , Fumar Cigarros/efeitos adversos , Células Epiteliais/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Tabaco/metabolismo
11.
Front Immunol ; 13: 832306, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091026

RESUMO

Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.


Assuntos
Neutrófilos , Serina Proteases , Autofagia , Caspase 1/metabolismo , Humanos , Interleucina-1beta/metabolismo , Neutrófilos/metabolismo , Serina Endopeptidases , Serina Proteases/genética
12.
ACS Chem Neurosci ; 13(17): 2579-2598, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-35947794

RESUMO

Suppression of excessive microglial overactivation can prevent the progression of multiple sclerosis (MS). Histone deacetylases 3 inhibitor (HDAC3i) has been demonstrated to exert anti-inflammatory effects by suppressing microglia (M1-liked) activation. Here, we demonstrate that the RGFP966 (a selective inhibitor of HDAC3) protects white matter after cuprizone-induced demyelination, as shown by reductions in neurological behavioral deficits and increases in myelin basic protein. Moreover, in this study, we found that RGFP966 caused a significant reduction in the levels of inflammatory cytokines, including IL-1ß, TNF-α, as well as iNOS, and inhibited microglial (M1-liked) activation in the experimental cuprizone model and LPS-stimulated BV2 cells. Meanwhile, RGFP966 alleviated apoptosis of LPS-induced BV2 cells in vitro. Furthermore, RGFP966 suppressed the expression of P2X7R, NLRP3, ASC, IL-18, IL-1ß, and caspase-1, inhibited the ratio of phosphorylated-STAT3/STAT3 and phosphorylated NF-κB p65/NF-κB p65, as well as increased acetylated NF-κB p65 in vitro and in vivo. Furthermore, we confirmed that brilliant blue G (antagonists of P2X7R) suppressed the expression of microglial NLRP3, IL-18, IL-1ß, caspase-1, NF-κB p65 (including phosphorylated NF-κB p65), and STAT3 (including phosphorylated STAT3) in vitro. These findings demonstrated that RFFP966 alleviated the inflammatory response and exerted a neuroprotective effect possibly by modulating P2X7R/STAT3/NF-κB65/NLRP3 signaling pathways. Thus, HDAD3 might be considered a promising intervention target for neurodegenerative diseases, such as MS.


Assuntos
Cuprizona , Inibidores Enzimáticos/farmacologia , Histona Desacetilases/metabolismo , NF-kappa B , Acrilamidas , Animais , Caspase 1/metabolismo , Cuprizona/metabolismo , Cuprizona/toxicidade , Modelos Animais de Doenças , Interleucina-18/metabolismo , Interleucina-18/farmacologia , Lipopolissacarídeos/toxicidade , Camundongos , Microglia , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doenças Neuroinflamatórias , Fenilenodiaminas
13.
Int J Mol Sci ; 23(15)2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35955441

RESUMO

BACKGROUND: Pyroptosis is a catabolic process relevant to periodontal disorders for which interleukin-1ß (IL-1ß) inflammation is central to the pathophysiology of the disease. Despite platelet-rich fibrin (PRF) anti-inflammatory properties and its application to support periodontal regeneration, the capacity of PRF to modulate pyroptosis, specifically the production and release of IL-1ß, remains unknown. The question arises whether PRF could regulate IL-1ß release from macrophages in vitro. METHODS: To answer this question, RAW 264.7 macrophages and primary macrophages obtained from murine bone marrow were primed with PRF before being challenged by lipopolysaccharide (LPS). Cells were then analysed for the pyroptosis signalling components by gene expression analyses and IL-1ß secretion at the protein level. The release of mitochondrial reactive oxygen species (ROS) was also detected. RESULTS: PRF lowered the LPS-induced expression of IL-1ß and NLRP3 inflammasome, caspase-11 and IL-18 in primary macrophages, and IL-1ß and caspase-11 in RAW 264.7 cells. Additionally, PRF diminished the secretion of IL-1ß at the protein level in LPS-induced RAW 264.7 cells. This was shown through immunoassays performed with the supernatant and further confirmed by analysing the lysates of permeabilised cells. Furthermore, PRF reduced the ROS release provoked by LPS in RAW 264.7 cells. Finally, to enhance IL-1ß release from the LPS-primed macrophages, we introduced a second signal with adenosine triphosphate (ATP). In this setting, PRF significantly reduced IL-1ß release in RAW 264.7 cells and a trend to diminish IL-1ß release in primary macrophages. CONCLUSION: These findings suggest that PRF can reduce IL-1ß release and, at least in part, inhibit pyroptosis-related factors in LPS-challenged macrophages.


Assuntos
Fibrina Rica em Plaquetas , Piroptose , Animais , Caspase 1/metabolismo , Caspases/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
14.
Front Immunol ; 13: 898039, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35911682

RESUMO

The NLRP3 inflammasome plays an important role in intestinal homeostasis as well as inflammation. However, in vivo studies investigating the role of the NLRP3 inflammasome in inflammatory bowel disease (IBD) report contrasting results, leaving it unclear if the NLRP3 inflammasome augments or attenuates intestinal inflammation. To investigate the role of the NLRP3/caspase-1 pathway in a model of acute intestinal inflammation, we modified a previously established in vitro triple culture model of the healthy and inflamed intestine (Caco-2/HT29-MTX-E12/THP-1). Using THP-1 knockout cell lines, we analyzed how the NLRP3 inflammasome and its downstream enzyme caspase-1 (CASP1) affect inflammatory parameters including barrier integrity and cytotoxicity, as well as gene expression and secretion of pro-inflammatory cytokines and mucus. Furthermore, we investigated differences in inflammation-mediated cytotoxicity towards enterocyte-like (Caco-2) or goblet-like (HT29-MTX-E12) epithelial cells. As a complementary approach, inflammation-related cytotoxicity and gene expression of cytokines was analyzed in intestinal tissue explants from wildtype (WT) and Nlrp3-/- mice. Induction of intestinal inflammation impaired the barrier, caused cytotoxicity, and altered gene expression of pro-inflammatory cytokines and mucins in vitro, while the knockout of NLRP3 and CASP1 in THP 1 cells led to attenuation of these inflammatory parameters. The knockout of CASP1 tended to show a slightly stronger attenuating effect compared to the NLRP3 knockout model. We also found that the inflammation-mediated death of goblet-like cells is NLRP3/caspase-1 dependent. Furthermore, inflammation-related cytotoxicity and upregulation of pro-inflammatory cytokines was present in ileal tissue explants from WT, but not Nlrp3-/- mice. The here presented observations indicate a pro-inflammatory and adverse role of the NLRP3 inflammasome in macrophages during acute intestinal inflammation.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Células CACO-2 , Caspase 1/genética , Caspase 1/metabolismo , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Intestinos/patologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células THP-1
15.
Food Funct ; 13(18): 9459-9469, 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-35979800

RESUMO

This study aimed to identify and prepare peptides from selenium (Se)-enriched soybeans and determine whether dietary Se-enriched soybean peptides (Se-SPep) could inhibit lung injury in mice induced by fine particulate matter 2.5 (PM2.5). BALB/c mice were randomly divided into six groups. The mice in the prevention groups were pretreated with 378 mg kg-1 of Se-SPep, soybean peptides (SPep), and Se-enriched soybean protein (Se-SPro), respectively, for four weeks. The mice in the PM2.5 exposure group received concentrated PM2.5 (15 µg per day mice) for 1 h daily from the third week for two weeks. The results showed that the leukocyte and cytokine (IL-1ß, IL-6, TNF-α) levels in the bronchoalveolar lavage fluid (BALF) of the PM2.5 exposure group were higher than those in the control group. Se-SPep pretreatment decreased the IL-1ß, IL-6, and TNF-α levels compared with the PM2.5 exposure group. Additionally, Se-SPep pretreatment inhibited TLR4/NF-κB/IκBα and NLRP3/ASC/caspase-1 protein expression in the lungs. In conclusion, Se-SPep pretreatment may protect the lungs of the mice against PM2.5-induced inflammation, suggesting that Se-SPep represents a potential preventative agent to inhibit PM2.5-induced lung injury.


Assuntos
Lesão Pulmonar , Selênio , Animais , Caspase 1/metabolismo , Citocinas/metabolismo , Inflamassomos/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmão/metabolismo , Lesão Pulmonar/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Material Particulado/toxicidade , Selênio/farmacologia , Transdução de Sinais , Proteínas de Soja/metabolismo , Soja/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
16.
BMC Immunol ; 23(1): 39, 2022 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-35965334

RESUMO

OBJECTIVE: This study was designed to investigate the role of the nucleotide-binding-domain -and leucine-rich repeat -containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome in the pathogenesis of polymyositis (PM). METHODS: Immunochemistry was performed to analyze the NLRP3, caspase-1 and interleukin-1 beta (IL-1ß) expression in the muscle tissue of PM patients. Rat model of PM and C2C12 cell were used to investigate the potential role of NLRP3 inflammasome in PM. RESULTS: The percentage of CD 68+ macrophages, and the expression levels of NLRP3, caspase-1 and IL-1ß in the muscle tissue were elevated in 27 PM patients. LPS/ATP treatment resulted in activation of NLRP3 inflammasome and secretion of IL-1ß as well as interferons (IFNs) and monocyte chemotactic protein-1 (MCP-1) in the Raw 264.7 macrophages. Meanwhile, LPS/ATP challenged activation of NLRP3 inflammasome induced overexpression of major histocompatibility complex class I (MHC-I), a key molecular of PM in the co-cultured C2C12 cells. The effect was decreased by treatment of NLRP3 inflammasome inhibitor MCC950 or siRNA of NLRP3 inflammasome. These findings suggested certain levels of IL-1ß rather than IFNs up-regulated MHC-I expression in C2C12 cells. IL-1ß blockade using neutralizing IL-1ß monoclonal antibody or siRNA of IL-1ß suppressed MHC-I overexpression. In vivo, NLRP3 inflammasome inhibition by MCC950 reduced the expression of NLRP3, IL-1ß and MHC-I in the muscle tissue of PM modal rats. Also, it attenuated the intensity of muscle inflammation as well as the CRP, CK, and LDH levels in the serum. CONCLUSION: NLRP3/caspase-1/IL-1ß axis may play an important role in the development of PM. Inhibition of NLRP3 activation may hold promise in the treatment of PM.


Assuntos
Inflamassomos , Polimiosite , Trifosfato de Adenosina , Animais , Caspase 1/genética , Caspase 1/metabolismo , Inflamassomos/metabolismo , Lipopolissacarídeos , Complexo Principal de Histocompatibilidade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Polimiosite/genética , RNA Interferente Pequeno , Ratos , Regulação para Cima
17.
J Ethnopharmacol ; 298: 115593, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-35973629

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Psoralea corylifolia (P. corylifolia Linn.) is a traditional Chinese medicinal plant that exhibits significant aphrodisiac, diuretic, and anti-rheumatic effects. However, it has been reported to cause hepatic injury, but the precise mechanisms remain unclear. AIM OF THE STUDY: To evaluate the safety and risk of P. corylifolia and to elucidate the underlying mechanisms of drug-induced liver injury. MATERIALS AND METHODS: Western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, quantitative polymerase chain reaction (Q-PCR), and flow cytometry were used to explore the effect of bakuchiol (Bak), one of the most abundant and biologically active components of P. corylifolia, on the AIM2 inflammasome activation and the underlying mechanism. Furthermore, we used the lipopolysaccharides (LPS)-induced drug-induced liver injury (DILI) susceptible mice model to study the Bak-mediated hepatotoxicity. RESULTS: Bak induced the maturation of caspase-1 P20, and significantly increased the expression of IL-1ß and TNF-α (P < 0.0001) compared with the control group. Moreover, compared to the Bak group, knockdown of AIM2 inhibited Bak-induced caspase-1 maturation and significantly decreased the production of IL-1ß and TNF-α, but knockout of NLRP3 had no effect. Mechanistically, Bak-induced AIM2 inflammasome activation is involved in mitochondrial damage, mitochondrial DNA (mtDNA) release, and subsequent recognition of cytosolic mtDNA. Our in vivo data showed that co-exposure to LPS and non-hepatotoxic doses of Bak significantly increased the levels of ALT, AST, IL-1ß, TNF-α, and IL-18, indicating that Bak can induce severe liver inflammation (P < 0.005). CONCLUSIONS: The result shows that Bak activates the AIM2 inflammasome by inducing mitochondrial damage to release mtDNA, and subsequently binds to the AIM2 receptor, indicating that Bak may be a risk factor for P. corylifolia-induced hepatic injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Inflamassomos , Animais , Caspase 1/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , DNA Mitocondrial , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fenóis , Fator de Necrose Tumoral alfa
18.
Exp Neurol ; 357: 114207, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35985555

RESUMO

Inflammation-induced preterm birth is the leading cause of perinatal mortality and long-term sequelae in surviving children. IL-1ß is a major contributor to inflammation-induced preterm labor and its sequelae. It has recently been demonstrated that the cytokine storm and its progression depend on IL-1ß release into circulation and that the P2X7 receptor (P2X7R) is the key player of the ATP-driven NLRP3/caspase-1 activation, necessary for the cleavage of pro-IL-1ß to its mature form as well as its subsequent secretion. Being a key component to the inflammatory cascade, P2X7R illuminates a new therapeutic avenue to halt progression of inflammation prior to perinatal brain injury. In this review, we summarize the basic role of the P2X7 receptor in the inflammatory signaling cascade and the possibility of it being used as a therapeutic target in perinatal brain injury. We discuss the antagonists and agonists of the receptor as well as its role in other inflammatory diseases, showing the importance of discovering the functions of the receptor.


Assuntos
Lesões Encefálicas , Nascimento Prematuro , Trifosfato de Adenosina/farmacologia , Caspase 1/metabolismo , Criança , Feminino , Humanos , Recém-Nascido , Inflamação , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Gravidez , Receptores Purinérgicos P2X7
19.
Chem Biol Interact ; 365: 110122, 2022 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-36002070

RESUMO

Psoriasis is a common chronic autoinflammatory/autoimmune skin disease associated with elevated pro-inflammatory cytokines. The pivotal role of interleukin (IL)-1ß and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome in the pathogenesis of psoriasis has been widely described. Accordingly, the suppression of NLRP3-dependent IL-1ß release is a potential therapy for psoriasis. Repurposing marketed drugs is a strategy for identifying new inhibitors of NLRP3 inflammasome activation. Herein, chlorquinaldol (CQD), a historic antimicrobial agent used as a topical treatment for skin and vaginal infections, was found to have a distinct effect by inhibiting NLRP3 inflammasome activation at concentrations ranging from 2 to 6 µM. CQD significantly suppressed apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) oligomerization, NLRP3-ASC interaction, and pyroptosis in macrophages. The levels of cleaved IL-1ß and caspase-1 were reduced by CQD in the cell lysates of macrophages, suggesting that CQD acted on upstream of pore formation in the cell membrane. Mechanistically, CQD reduced mitochondrial reactive oxygen species production but did not affect the nuclear factor-κB (NF-κB) pathway. Intraperitoneal administration of CQD (15 mg/kg) for 6 days was found to improve the skin lesions in the imiquimod-induced psoriatic mouse model (male C57BL/6 mice), while secretion of pro-inflammatory cytokines (IL-17 and IL-1ß) and keratinocyte proliferation were significantly suppressed by CQD. In conclusion, CQD exerted inhibitory effects on NLRP3 inflammasome activation in macrophages and decreased the severity of psoriatic response in vivo. Such findings indicate that the repurposing of the old drug, CQD, is a potential pharmacological approach for the treatment of psoriasis and other NLRP3-driven diseases.


Assuntos
Clorquinaldol , Dermatite , Psoríase , Animais , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Clorquinaldol/efeitos adversos , Citocinas/metabolismo , Feminino , Imiquimode/toxicidade , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotídeos/efeitos adversos , Nucleotídeos/metabolismo , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Domínio Pirina
20.
Proc Natl Acad Sci U S A ; 119(36): e2201494119, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36037355

RESUMO

Pulmonary emphysema is associated with dysregulated innate immune responses that promote chronic pulmonary inflammation and alveolar apoptosis, culminating in lung destruction. However, the molecular regulators of innate immunity that promote emphysema are ill-defined. Here, we investigated whether innate immune inflammasome complexes, comprising the adaptor ASC, Caspase-1 and specific pattern recognition receptors (PRRs), promote the pathogenesis of emphysema. In the lungs of emphysematous patients, as well as spontaneous gp130F/F and cigarette smoke (CS)-induced mouse models of emphysema, the expression (messenger RNA and protein) and activation of ASC, Caspase-1, and the inflammasome-associated PRR and DNA sensor AIM2 were up-regulated. AIM2 up-regulation in emphysema coincided with the biased production of the mature downstream inflammasome effector cytokine IL-1ß but not IL-18. These observations were supported by the genetic blockade of ASC, AIM2, and the IL-1 receptor and therapy with AIM2 antagonistic suppressor oligonucleotides, which ameliorated emphysema in gp130F/F mice by preventing elevated alveolar cell apoptosis. The functional requirement for AIM2 in driving apoptosis in the lung epithelium was independent of its expression in hematopoietic-derived immune cells and the recruitment of infiltrating immune cells in the lung. Genetic and inhibitor-based blockade of AIM2 also protected CS-exposed mice from pulmonary alveolar cell apoptosis. Intriguingly, IL-6 trans-signaling via the soluble IL-6 receptor, facilitated by elevated levels of IL-6, acted upstream of the AIM2 inflammasome to augment AIM2 expression in emphysema. Collectively, we reveal cross-talk between the AIM2 inflammasome/IL-1ß and IL-6 trans-signaling axes for potential exploitation as a therapeutic strategy for emphysema.


Assuntos
Proteínas de Ligação a DNA , Imunidade Inata , Interleucina-1beta , Interleucina-6 , Enfisema Pulmonar , Animais , Apoptose , Caspase 1/metabolismo , Receptor gp130 de Citocina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Enfisema Pulmonar/imunologia
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