Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26.915
Filtrar
1.
Methods Mol Biol ; 2519: 53-63, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066709

RESUMO

Many apoptosis assays are available since there are many proteins regulated at multiple points and involved in apoptosis signaling cascade. To detect apoptosis accurately, two or more assays should be used since there are many overlapped features between apoptosis and necrosis. There are six major groups of available assays to detect apoptosis: membrane alteration, mitochondrial assays, cytomorphological alterations, DNA fragmentation, detection of caspase, cleaved substrate, inhibitors and regulators, and detection of apoptosis in whole mounts. Among those assay, early apoptosis could be detected through annexin V, which is based on the loss of the cellular membrane integrity. Also, there are many assays that can detect midphase of apoptosis using caspase activation and molecular processing including PARP degradation. Late phase of apoptosis could be detected with DNA fragmentation assays. Combinations of these assays allow us to identify the mechanisms of apoptosis induction after specific stimulus. This chapter will introduce three apoptosis detection assays including annexin assay, DNA/chromatin condensation assays, and TUNEL assay.


Assuntos
Apoptose , Caspases , Anexina A5/metabolismo , Caspases/metabolismo , Fragmentação do DNA , Humanos , Necrose
2.
Dis Markers ; 2022: 4688203, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36046381

RESUMO

Objective: To explore the impact of genistein (Gen) on the apoptosis of neuronal cells in naturally aged rats and its mechanism. Methods: Fifty SD male rats were allocated into five groups at random, including youth group (3M group), natural aging group (24M group), and Gen low-, medium-, and high-dose groups. Starting from 18 months of age, Gen 10, 30, and 60 mg-kg-1 were administered via gavage to the Gen low-, medium-, and high-dose groups, respectively, while the rats in the natural aging group was given saline by gavage until 24 months of age, and the drug was stopped for 1 d per week for 6 months. The protein expression of target genes was examined using western blotting. Results: In contrast to the 3M group, the 24M group rats showed disturbed neuronal cell arrangement and massive cell degeneration. After 6 months of Gen intervention, in contrast to the 24M group, the neural cell pathology in the CA3 area of the hippocampus improved and cell apoptotic decreased observably. In contrast to the 3M group, the protein expression of c-Jun amino-terminal kinase (p-JNK), C/EBP homologous protein (CHOP), inflammatory vesicle 3-associated factor (NLRP3), cysteine protease-1 (Caspase-1), and apoptosis-related punctate protein (ASC) and downstream inflammatory factors in the hippocampus was obviously increased in the 24M group. In contrast to the 24M group, the protein expression of p-JNK, CHOP, NLRP3, Caspase-1, and ASC and downstream inflammatory factors in the hippocampus was observably declined in Gen groups. Conclusion: Gen has a protective effect on hippocampal neurons in aging rat brain tissue via the inhibition of the ERS apoptotic signaling pathway and NLRP3 inflammatory vesicle activation.


Assuntos
Isoflavonas , Envelhecimento , Animais , Apoptose , Caspases , Genisteína/farmacologia , Isoflavonas/farmacologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos
3.
Curr Protoc ; 2(9): e525, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36069669

RESUMO

Unicellular eukaryotic organisms such as yeast and protozoa serve as useful models for studying the impact of chemicals on cell physiology, cellular growth, and genome duplication. The yeast Saccharomyces cerevisiae has been widely used to assess apoptosis induced by chemicals due to its genetic tractability, ease of evaluation, and readily available impact assessment tools. Apoptosis in S. cerevisiae is characterized by many features, including increased cell death, loss of membrane integrity, release of caspases, chromatin condensation, and nuclear fragmentation, which are similar to the ones observed in mammalian cells. Current methods of apoptosis assessment typically require specialized equipment and reagents, which limits wide adoption. Here, we describe a rapid, inexpensive, and easy-to-perform assay in yeast for the analysis of late-stage apoptotic features in cells treated with a chemical. We describe a protocol for assessing loss of cell survival and changes in the nucleus. We demonstrate the approach by using acetic acid and hydrogen peroxide as test chemicals. This assay for the study of late-stage apoptotic features in S. cerevisiae can be performed reliably and rapidly by any laboratory with basic equipment and may be extended for studying apoptosis in similar single-cell organisms after treatment with toxicological agents. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culture of Saccharomyces cerevisiae, treatment with acetic acid or hydrogen peroxide, and semi-quantitative growth assay Basic Protocol 2: DAPI staining and fluorescence microscopy for the assessment of change in nucleus-to-cytoplasm ratio and nuclear integrity.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácido Acético/metabolismo , Animais , Apoptose/fisiologia , Caspases/metabolismo , Peróxido de Hidrogênio/metabolismo , Mamíferos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
4.
J Oleo Sci ; 71(9): 1375-1385, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36047243

RESUMO

Glioblastoma multiforme or GBM is a destructive malignancy of the central nervous system and is accountable for leading cause of cancer related mortality. Inadequate success rate of surgical interventions and development of resistance towards the current therapeutical regime provides impetus for exploring novel therapeutical interventions against the disease. Recently, several epidemiological studies have explored the plausible utility of natural, dietary compounds in influencing the development, progression, and cancer metastasis. Recently, different phytoconstituents of Cassia angustifolia were found to be associated with anti-microbial, anti-cancer and anti-inflammatory effects. Therefore, the aim of the present study was to evaluate the anti-proliferative efficacy of ethanolic leaf extract of C. angustifolia (LCaEt-OH) against rat derived glioblastoma C6 cells. Briefly, the anti-proliferative potential of LCaEt-OH was assessed using MTT assay, quantitative estimation of ROS, and evaluation of mitochondrial membrane potential (ΔΨm). Moreover, the activity of caspases involved in intrinsic apoptotic pathways was also investigated using colorimetric kit followed by quantitative RT-PCR evaluation of modulation in gene expressions triggered due to LCaEt-OH treatment. Treatment of LCaEt-OH on C6 cells elucidated substantial dose-dependent decline in cellular viability. Furthermore, LCaEt-OH showed its efficacy in substantially enhancing intracellular ROS. LCaEt-OH also incited apoptosis in C6 cells by instigating nuclear condensation and dissipation of ΔΨm. In addition, LCaEt-OH mediated instigation of apoptosis was directly influenced by increased activity of caspases indispensable for intrinsic apoptotic pathway. These conclusive evidences indicate towards anticancer efficacy of LCaEt-OH against C6 cells.


Assuntos
Glioblastoma , Animais , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Extratos Vegetais/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo
5.
Pharm Biol ; 60(1): 1801-1811, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36121296

RESUMO

CONTEXT: Acute promyelocytic leukaemia (APL) is a malignant hematological tumour characterized by the presence of promyelocytic leukaemia-retinoic acid receptor A (PML-RARA) fusion protein. Cinobufagin (CBG) is one of the main effective components of toad venom with antitumor properties. However, only a few reports regarding the CBG treatment of APL are available. OBJECTIVE: We explored the effect and mechanism of action of CBG on NB4 and NB4-R1 cells. MATERIALS AND METHODS: We evaluated the viability of NB4 and NB4-R1 cells treated with 0, 20, 40, and 60 nM CBG for 12, 24, and 48 h. After treatment with CBG for 24 h, Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl-2), ß-catenin, cyclin D1, and c-myc expression was detected using western blotting and real-time polymerase chain reaction. Caspase-3 and PML-RARA expression levels were detected using western blotting. RESULTS: CBG inhibited the viability of NB4 and NB4-R1 cells. The IC50 values of NB4 and NB4-R1 cells treated with CBG for 24 h were 45.2 nM and 37.9 nM, respectively. CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner and inhibited the ß-catenin signalling pathway. DISCUSSION AND CONCLUSION: CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway. This study proposes a novel treatment strategy for patients with APL, particularly those with ATRA-resistant APL.


Assuntos
Venenos de Anfíbios , Leucemia Promielocítica Aguda , Venenos de Anfíbios/farmacologia , Apoptose , Bufanolídeos , Gluconato de Cálcio/farmacologia , Caspase 3 , Caspases , Ciclina D1 , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/farmacologia , Receptores do Ácido Retinoico , Proteína X Associada a bcl-2 , beta Catenina
6.
Circ Heart Fail ; 15(9): e009693, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36126144

RESUMO

BACKGROUND: The TOPCAT trial (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial) suggested clinical benefits of spironolactone treatment among patients with heart failure with preserved ejection fraction enrolled in the Americas. However, a comprehensive assessment of biologic pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction has not been performed. METHODS: We conducted aptamer-based proteomic analysis utilizing 5284 modified aptamers to 4928 unique proteins on plasma samples from TOPCAT participants from the Americas (n=164 subjects with paired samples at baseline and 1 year) to identify proteins and pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction. Mean percentage change from baseline was calculated for each protein. Additionally, we conducted pathway analysis of proteins altered by spironolactone. RESULTS: Spironolactone therapy was associated with proteome-wide significant changes in 7 proteins. Among these, CARD18 (caspase recruitment domain-containing protein 18), PKD2 (polycystin 2), and PSG2 (pregnancy-specific glycoprotein 2) were upregulated, whereas HGF (hepatic growth factor), PLTP (phospholipid transfer protein), IGF2R (insulin growth factor 2 receptor), and SWP70 (switch-associated protein 70) were downregulated. CARD18, a caspase-1 inhibitor, was the most upregulated protein by spironolactone (-0.5% with placebo versus +66.5% with spironolactone, P<0.0001). The top canonical pathways that were significantly associated with spironolactone were apelin signaling, stellate cell activation, glycoprotein 6 signaling, atherosclerosis signaling, liver X receptor activation, and farnesoid X receptor activation. Among the top pathways, collagens were a consistent theme that increased in patients receiving placebo but decreased in patients randomized to spironolactone. CONCLUSIONS: Proteomic analysis in the TOPCAT trial revealed proteins and pathways altered by spironolactone, including the caspase inhibitor CARD18 and multiple pathways that involved collagens. In addition to effects on fibrosis, our studies suggest potential antiapoptotic effects of spironolactone in heart failure with preserved ejection fraction, a hypothesis that merits further exploration.


Assuntos
Produtos Biológicos , Insuficiência Cardíaca , Insulinas , Apelina/farmacologia , Apelina/uso terapêutico , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Caspases/farmacologia , Caspases/uso terapêutico , Humanos , Insulinas/uso terapêutico , Receptores X do Fígado , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Proteínas de Transferência de Fosfolipídeos/farmacologia , Proteínas de Transferência de Fosfolipídeos/uso terapêutico , Proteoma , Proteômica , Espironolactona/efeitos adversos , Volume Sistólico/fisiologia , Resultado do Tratamento
7.
JCI Insight ; 7(18)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36048544

RESUMO

Pseudomonas aeruginosa is one of the most common nosocomial infections worldwide, and it frequently causes ventilator-associated acute pneumonia in immunocompromised patients. Abundant neutrophil extracellular traps (NETs) contribute to acute lung injury, thereby aggravating ventilator-induced lung damage. While pattern recognition receptors (PRRs) TLR4 and TLR5 are required for host defense against P. aeruginosa invasion, the PRR responsible for P. aeruginosa-induced NET formation, proinflammatory cytokine release, and acute lung injury remains unclear. We found that myeloid C-type lectin domain family 5 member A (CLEC5A) interacts with LPS of P. aeruginosa and is responsible for P. aeruginosa-induced NET formation and lung inflammation. P. aeruginosa activates CLEC5A to induce caspase-1-dependent NET formation, but it neither causes gasdermin D (GSDMD) cleavage nor contributes to P. aeruginosa-induced neutrophil death. Blockade of CLEC5A attenuates P. aeruginosa-induced NETosis and lung injury, and simultaneous administration of anti-CLEC5A mAb with ciprofloxacin increases survival rate and decreases collagen deposition in the lungs of mice challenged with a lethal dose of P. aeruginosa. Thus, CLEC5A is a promising therapeutic target to reduce ventilator-associated lung injury and fibrosis in P. aeruginosa-induced pneumonia.


Assuntos
Lesão Pulmonar Aguda , Pneumonia , Animais , Caspases , Ciprofloxacina , Citocinas , Lectinas Tipo C/genética , Lipopolissacarídeos/toxicidade , Camundongos , Pseudomonas aeruginosa , Receptores de Superfície Celular , Receptor 4 Toll-Like , Receptor 5 Toll-Like
8.
Biosens Bioelectron ; 216: 114648, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36055132

RESUMO

Multimodal imaging probes have shown promise in both biomedical research and clinical diagnosis. However, the development of molecular probes capable of being switched on multimodality imaging signals in response to a biomarker of interest remains challenging. In this paper, we report a caspase-3-activatable small-molecule bimodal probe (Gd-IR780) for photoacoustic imaging (PAI) and magnetic resonance imaging (MRI) of tumor apoptosis via caspase-3-triggered intramolecular macrocyclization and in situ self-assembly process. Upon interaction with caspase-3, Gd-IR780 can be efficiently converted into a macrocyclic product, Gd-IR780-MC, which subsequently self-assembles into near-infrared absorptive and paramagnetic nanoparticles (Gd-IR780-NPs), allowing to concurrently switch on PAI (∼4.3-fold at 855 nm) and MRI (r1 relaxivity increases from 7.98 ± 0.03 mM-1 s-1 to 19.66 ± 0.7 mM-1 s-1 at 0.5 T) bimodal signals in caspase buffer. Noninvasive in vivo imaging results show that Gd-IR780 can enter apoptotic U87MG tumor tissues after systemic administration, and produce markedly enhanced PAI and MRI signals for high sensitivity and spatial-resolution visualization of caspase-3 activity in the doxorubicin-treated apoptotic U87MG tumor tissues. Gd-IR780 holds a good potential to report tumor apoptosis via combined PAI and MRI, which is beneficial for the early evaluation of antitumor efficacy in vivo.


Assuntos
Técnicas Biossensoriais , Nanopartículas , Neoplasias , Técnicas Fotoacústicas , Apoptose , Caspase 3 , Caspases , Doxorrubicina , Humanos , Imageamento por Ressonância Magnética/métodos , Sondas Moleculares , Neoplasias/diagnóstico por imagem , Técnicas Fotoacústicas/métodos
9.
J Agric Food Chem ; 70(37): 11560-11571, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36094400

RESUMO

In this study, 10 metabolites were obtained by collecting and extracting fecal samples after oral administration of panaxadiol (PD). Of these 10 metabolites, M7 (3ß,21ß,22α-hydroxy-24-norolean-12-ene), M8 (21ß,22α-hydroxy-24-norolean-12-ene-3-one), M9 (3ß,30α-hydroxy-24-norolean-22,30-epoxy-12-ene), and M10 (3ß,21ß-hydroxy-24-norolean-12-ene) were new compounds. MTT screening of the isolated compounds revealed that the inhibitions of cancer cells by M2, M4, M7, M8, and M10 were significantly stronger than that by the mother drug M0, with the activity of M2 being the most significant. Further, we investigated the anticancer mechanism of M2. The results showed that M2 significantly increased the level of ROS in cells; regulated the expressions of Bax, Bcl-2, and Cyt-C through the mitochondrial pathway; triggered the caspase cascade; and induced apoptosis. M2 could also induce G1 phase arrest and significantly regulate cell cycle-related proteins. In conclusion, the experimental results provide data for further study on the metabolic mechanism of PD in vivo and the potential of developing new anti-cancer drugs.


Assuntos
Antineoplásicos , Apoptose , Antineoplásicos/farmacologia , Caspases , Linhagem Celular Tumoral , Proliferação de Células , Fase G1 , Ginsenosídeos , Espécies Reativas de Oxigênio , Proteína X Associada a bcl-2
10.
FASEB J ; 36(10): e22557, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36125006

RESUMO

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin (ß-PFT). It exhibits potent hemolytic activity against erythrocytes that appears to be a direct outcome of its pore-forming functionality. However, VCC-mediated cell-killing mechanism is more complicated in the case of nucleated mammalian cells. It induces apoptosis in the target nucleated cells, mechanistic details of which are still unclear. Furthermore, it has never been explored whether the ability of VCC to trigger programmed cell death is stringently dependent on its pore-forming activity. Here, we show that VCC can evoke hallmark features of the caspase-dependent apoptotic cell death even in the absence of the pore-forming ability. Our study demonstrates that VCC mutants with abortive pore-forming hemolytic activity can trigger apoptotic cell death responses and cytotoxicity, similar to those elicited by the wild-type toxin. VCC as well as its pore formation-deficient mutants display prominent propensity to translocate to the target cell mitochondria and cause mitochondrial membrane damage. Therefore, our results for the first time reveal that VCC, despite being an archetypical ß-PFT, can kill target nucleated cells independent of its pore-forming functionality. These findings are intriguing for a ß-PFT, whose destination is generally expected to remain limited on the target cell membranes, and whose mode of action is commonly attributed to the membrane-damaging pore-forming ability. Taken together, our study provides critical new insights regarding distinct implications of the two important virulence functionalities of VCC for the V. cholerae pathogenesis process: hemolytic activity for iron acquisition and cytotoxicity for tissue damage by the bacteria.


Assuntos
Toxinas Biológicas , Vibrio cholerae , Animais , Caspases/metabolismo , Morte Celular , Citotoxinas/metabolismo , Ferro/metabolismo , Mamíferos/metabolismo , Toxinas Biológicas/metabolismo , Vibrio cholerae/metabolismo
11.
Cell ; 185(18): 3356-3374.e22, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36055199

RESUMO

Drug-tolerant persister cells (persisters) evade apoptosis upon targeted and conventional cancer therapies and represent a major non-genetic barrier to effective cancer treatment. Here, we show that cells that survive treatment with pro-apoptotic BH3 mimetics display a persister phenotype that includes colonization and metastasis in vivo and increased sensitivity toward ferroptosis by GPX4 inhibition. We found that sublethal mitochondrial outer membrane permeabilization (MOMP) and holocytochrome c release are key requirements for the generation of the persister phenotype. The generation of persisters is independent of apoptosome formation and caspase activation, but instead, cytosolic cytochrome c induces the activation of heme-regulated inhibitor (HRI) kinase and engagement of the integrated stress response (ISR) with the consequent synthesis of ATF4, all of which are required for the persister phenotype. Our results reveal that sublethal cytochrome c release couples sublethal MOMP to caspase-independent initiation of an ATF4-dependent, drug-tolerant persister phenotype.


Assuntos
Citocromos c , Neoplasias/tratamento farmacológico , Animais , Apoptose , Proteínas de Transporte , Caspases/metabolismo , Citocromos c/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Mitocôndrias/metabolismo
12.
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
13.
BMC Neurosci ; 23(1): 50, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945502

RESUMO

BACKGROUND: Evidences indicate that inflammasome compounds participate in amyotrophic lateral sclerosis (ALS), a fatal progressive motoneuron degenerative disease. Researchers have observed the expressions of nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) related inflammasome components in specific regions of the central nervous system in different ALS models, but the cellular spatiotemporal evolution of this canonical inflammasome pathway and pyroptosis during ALS progression are unclear. METHODS: The spinal cords of hSOD1G93A mice (ALS mice) and age-matched littermates (CON mice) were dissected at pre-symptomatic stage (60 d), early- symptomatic stage (95 d), symptomatic stage (108 d) and late-symptomatic stage (122 d) of the disease. By using Nissl staining, double immunofluorescence labelling, qRT-PCR or western blot, we detected morphology change and the expression, cellular location of GSDMD, NLRP3, caspase-1 and IL-1ß in the ventral horn of lumbar spinal cords over the course of disease. RESULTS: Neural morphology changes and GSDMD+/NeuN+ double positive cells were observed in ventral horn from ALS mice even at 60 d of age, even though there were no changes of GSDMD mRNA and protein expressions at this stage compared with CON mice. With disease progression, compared with age-matched CON mice, increased expressions of GSDMD, NLRP3, activated caspase-1 and IL-1ß were detected. Double immunofluorescence labeling revealed that NLRP3, caspase-1, IL-1ß positive signals mainly localized in ventral horn neurons at pre- and early-symptomatic stages. From symptomatic stage to late-symptomatic stage, robust positive signals were co-expressed in reactive astrocytes and microglia. CONCLUSIONS: Early activation of the canonical NLRP3 inflammasome induced pyroptosis in ventral horn neurons, which may participate in motor neuron degeneration and initiate neuroinflammatory processes during ALS progression.


Assuntos
Esclerose Amiotrófica Lateral , Inflamassomos , Esclerose Amiotrófica Lateral/genética , Animais , Caspases , Modelos Animais de Doenças , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Superóxido Dismutase , Superóxido Dismutase-1/genética
15.
Front Immunol ; 13: 898819, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35928825

RESUMO

Molecular mechanisms underlying auto-antibody-induced acantholysis in pemphigus vulgaris are subject of current research to date. To decipher the discrepancy between ubiquitous antibody binding to the epidermal desmosomes, but discontinuous disease manifestation, we were able to identify Ultraviolet A (UVA) as a cofactor for acantholysis. UVA induces interleukin (IL)-1 secretion in keratinocytes, mirroring innate immune system activation. In an in vitro keratinocyte dissociation assay increased fragmentation was observed when UVA was added to anti-Desmoglein 3 Immunoglobulins (anti-Dsg3 IgG). These results were confirmed in skin explants where UVA enhanced anti-Dsg3-mediated loss of epidermal adhesion. The UVA-mediated effect was blocked in vitro by the pan-caspase-inhibitor zVAD-fmk. Thus, we introduce UVA as a caspase-dependent exogenous cofactor for acantholysis which suggests that local innate immune responses largely contribute to overt clinical blister formation upon autoantibody binding to epidermal cells in pemphigus vulgaris.


Assuntos
Pênfigo , Acantólise/metabolismo , Caspases , Humanos , Imunidade Inata , Imunoglobulina G
16.
Int J Nanomedicine ; 17: 3583-3599, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35974872

RESUMO

Purpose: In recent years, a variety of nanoparticles with excellent anticancer and delivery properties have emerged for cancer therapy. However, potential toxicity, high production cost and complex preparation procedures have been obstacles to their use in biomedicine. Here, we obtained cucumber-derived nanovesicles (CDNVs) at high yield and low cost by simple juicing and ultracentrifugation. The anticancer effects of CDNVs were evaluated in vitro and in vivo. Methods: Transmission electron microscope, nanoparticle tracking analysis and laser particle size analysis were used to characterize the morphology, diameter and zeta potential of CDNVs, respectively. The anticancer effects of CDNVs in vitro were evaluated by MTT and apoptosis assays. The mechanism was further explored by measuring the protein levels of signal transducer and activator of transcription 3 pathway, reactive oxygen species, cell cycle distribution and caspase activity. In-vivo anticancer efficacy was evaluated by measuring tumor volume and weight of mice in three different treatment groups (CDNVs, cucurbitacin B and PBS). Results: CDNVs inhibited proliferation of human non-small cell lung cancer cells by suppressing signal transducer and activator of transcription 3 activation, generating reactive oxygen species, promoting cell cycle arrest, and activating the caspase pathway. These CDNVs exhibited strong anticancer effects both in vitro and in vivo, and reduced the rate of tumor growth without obvious toxicity to mouse visceral organs. Compared with an equivalent dose of cucurbitacin B, CDNVs exerted stronger anticancer effects in vitro and in vivo. Conclusion: These results demonstrate that CDNVs suppress tumor growth. This study addresses the development of cancer therapeutic drugs using plant-derived nanovesicles that are cost-efficient, simple to produce in high yields, and provide an alternative approach to drug isolation that may help advance sustainability of medicinal plants.


Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Cucumis sativus , Neoplasias Pulmonares , Triterpenos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
17.
World J Microbiol Biotechnol ; 38(10): 182, 2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-35953631

RESUMO

Biomolecules from Streptomyces spp. are emerging sources of natural drugs and have been focused on over the decade. The discovery of bioactive chemotherapeutic molecules from soil Streptomyces spp. has opened the medium for the search for natural drugs. In the current study, 8-HOQ was extracted and purified from soil Streptomyces spp. and was evaluated on A549 and BEAS cell lines. The apoptotic and caspase mediated pathways were evaluated using cell proliferation, dual fluorescent staining, migration, invasion and mRNA as well as protein quantification of apoptotic markers. In vitro cytotoxicity test revealed that 8-HOQ possesses potent cytotoxicity activities with IC50 values of 26 µM, 5 µM, 7.2 µM at 24 h, 48 h, and 72 h respectively against A549 lung cancer cell lines. The result also demonstrated that 8-HOQ from Streptomyces spp significantly inhibited the A549 lung cancer cell lines and activated the intrinsic pathways of apoptosis. The caspase-3 and caspase-8 activities were potentially elevated in 8-HOQ treated A549 cell lines and confirmed that 8-HOQ mediated A549 cancer cell death through the intrinsic pathway. The results explored caspase-mediated apoptosis as a mechanism underlying the inhibition of cancer cell viability in a dose-dependent manner. The expression of P53, BCL2 and STAT3 were inhibited in A549 cell lines and confirmed the metastasis inhibitory potential of 8-HOQ by blocking migration and invasion in A549 cell lines. These results indicated that 8-HOQ from Streptomyces spp. potentially inhibited growth and migration of A549 lung cancer cell lines.


Assuntos
Neoplasias Pulmonares , Streptomyces , Células A549 , Apoptose , Caspases , Linhagem Celular Tumoral , Proliferação de Células , Quelantes/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Oxiquinolina/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/uso terapêutico , Solo , Streptomyces/metabolismo
18.
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
19.
Int J Mol Sci ; 23(15)2022 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-35955469

RESUMO

Ciliary neurotrophic factor (CNTF) was identified as a survival factor in various types of peripheral and central neurons, glia and non-neural cells. At present, there is no available data on the expression and localization of CNTF-receptors in cementoblasts as well as on the role of exogenous CNTF on this cell line. The purpose of this study was to determine if cementoblasts express CNTF-receptors and analyze the mechanism of its apoptotic regulation effects on cementoblasts. OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals). Quantified transcriptional (RT-qPCR) and translational (WB) products of CNTFRα, IL-6Rα (CD126), LIFR, p-GP130, GP130, p-ERK1/2, ERK1/2, Caspase-8, -9, -3 and cleaved-caspase-3 were evaluated. Immunofluorescence (IF) staining was applied to visualize the localization of the CNTF-receptors within cells. The apoptosis ratio was measured with an Annexin-V FITC/PI kit. The ERK1/2 antagonist (FR180204, Calbiochem) was added for further investigation by flow cytometry analysis. The CNTF-receptor complex (CNTFRα, LIFR, GP130) was functionally up-regulated in cementoblasts while cultivated with exogenous CNTF. CNTF significantly attenuated cell viability and proliferation for long-term stimulation. Flow cytometry analysis shows that CNTF enhanced the apoptosis after prolonged duration. However, after only a short-term period, CNTF halts the apoptosis of cementoblasts. Further studies revealed that CNTF activated phosphorylated GP130 and the anti-apoptotic molecule ERK1/2 signaling to participate in the regulation of the apoptosis ratio of cementoblasts. In conclusion, CNTF elicited the cellular functions through a notable induction of its receptor complex in cementoblasts. CNTF has an inhibitory effect on the cementoblast homeostasis. These data also elucidate a cellular mechanism for an exogenous CNTF-triggered apoptosis regulation in a mechanism of ERK1/2 and caspase signaling and provides insight into the complex cellular responses induced by CNTF in cementoblasts.


Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar , Fator Neurotrófico Ciliar , Apoptose , Caspases/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Receptor gp130 de Citocina/metabolismo , Cemento Dentário/metabolismo , Sistema de Sinalização das MAP Quinases , Receptor do Fator Neutrófico Ciliar/metabolismo
20.
Proc Natl Acad Sci U S A ; 119(36): e2202577119, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36037361

RESUMO

Calcific aortic valve disease (CAVD) is common in people over the age of 65. Progressive valvular calcification is a characteristic of CAVD and due to chronic inflammation in aortic valve interstitial cells (AVICs) resulting in CAVD progression. IL-38 is a naturally occurring anti-inflammatory cytokine; here, we report lower levels of endogenous IL-38 in AVICs isolated from patients' CAVD valves compared to AVICs from non-CAVD valves. Recombinant IL-38 suppressed spontaneous inflammatory activity and calcium deposition in cultured AVICs. In mice, knockdown of IL-38 enhanced the production of inflammatory mediators in murine AVICs exposed to the proinflammatory stimulant matrilin-2. We also observed that in cultured AVICs matrilin-2 stimulation activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome with procaspase-1 cleavage into active caspase-1. The addition of IL-38 to matrilin-2-treated AVICs suppressed caspase-1 activation and reduced the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, runt-related transcription factor 2, and alkaline phosphatase. Aged IL-38-deficient mice fed a high-fat diet exhibited aortic valve lesions compared to aged wild-type mice fed the same diet. The interleukin-1 receptor 9 (IL-1R9) is the putative receptor mediating the anti-inflammatory properties of IL-38; we observed that IL-1R9-deficient mice exhibited spontaneous aortic valve thickening and greater calcium deposition in AVICs compared to wild-type mice. These data demonstrate that IL-38 suppresses spontaneous and stimulated osteogenic activity in aortic valve via inhibition of the NLRP3 inflammasome and caspase-1. The findings of this study suggest that IL-38 has therapeutic potential for prevention of CAVD progression.


Assuntos
Estenose da Valva Aórtica , Calcinose , Interleucinas , Animais , Anti-Inflamatórios/farmacologia , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/tratamento farmacológico , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/metabolismo , Calcinose/tratamento farmacológico , Cálcio/metabolismo , Caspases/metabolismo , Células Cultivadas , Humanos , Inflamassomos/metabolismo , Interleucina-1 , Interleucinas/genética , Interleucinas/metabolismo , Interleucinas/farmacologia , Proteínas Matrilinas/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Osteogênese , Receptores de Interleucina-9/genética , Proteínas Recombinantes/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...