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1.
PLoS Pathog ; 15(9): e1007951, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31479498

RESUMO

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from µs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between non-toxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens.


Assuntos
Oomicetos/genética , Oomicetos/metabolismo , Doenças das Plantas/parasitologia , Sequência de Aminoácidos , Etilenos/metabolismo , Necrose/metabolismo , Peptídeos/metabolismo , Peronospora/genética , Proteínas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Cell Physiol Biochem ; 53(3): 496-507, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486324

RESUMO

BACKGROUND/AIMS: Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. METHODS: Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. RESULTS: Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. CONCLUSION: Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.


Assuntos
Antígenos CD59/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Necrose/metabolismo , Adjuvantes Imunológicos , Animais , Apoptose/fisiologia , Bancos de Sangue , Western Blotting , Morte Celular/genética , Morte Celular/fisiologia , Células Cultivadas , Hemólise/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/genética , Fosforilação/fisiologia , Espécies Reativas de Oxigênio/metabolismo
3.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340541

RESUMO

Apoptotic cell death is usually a response to the cell's microenvironment. In the kidney, apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury, but does not trigger an inflammatory response. What distinguishes necrosis from apoptosis is the rupture of the plasma membrane, so necrotic cell death is accompanied by the release of unprocessed intracellular content, including cellular organelles, which are highly immunogenic proteins. The relative contribution of apoptosis and necrosis to injury varies, depending on the severity of the insult. Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis. Recent advances have enhanced the most revolutionary concept of regulated necrosis. Several modalities of regulated necrosis have been described, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial permeability transition-dependent regulated necrosis. We review the different modalities of apoptosis, necrosis, and regulated necrosis in kidney injury, focusing particularly on evidence implicating cell death in ectopic renal calcification. We also review the evidence for the role of cell death in kidney injury, which may pave the way for new therapeutic opportunities.


Assuntos
Lesão Renal Aguda/metabolismo , Proteínas Reguladoras de Apoptose/genética , Calcinose/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Necrose/metabolismo , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/tratamento farmacológico , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/classificação , Proteínas Reguladoras de Apoptose/metabolismo , Calcinose/genética , Calcinose/patologia , Calcinose/prevenção & controle , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , /genética , Regulação da Expressão Gênica , Humanos , /genética , Rim/efeitos dos fármacos , Rim/patologia , /genética , /genética , Necrose/genética , Necrose/patologia , Necrose/prevenção & controle , Substâncias Protetoras/farmacologia , Piroptose/efeitos dos fármacos , Piroptose/genética , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
5.
Redox Biol ; 26: 101279, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31349119

RESUMO

The phagocyte NADPH oxidase (the NOX2 complex) generates superoxide, the precursor to reactive oxygen species (ROS). ROS possess both antimicrobial and immunoregulatory function. Inactivating mutations in alleles of the NOX2 complex cause chronic granulomatous disease (CGD), characterized by an enhanced susceptibility to infections and autoimmune diseases such as Systemic lupus erythematosus (SLE). The latter is characterized by insufficient removal of dead cells, resulting in an autoimmune response against components of the cell's nucleus when non-cleared apoptotic cells lose their membrane integrity and present autoantigenic molecules in an inflammatory context. Here we aimed to shed light on the role of the NOX2 complex in handling of secondary necrotic cells (SNECs) and associated consequences for inflammation and autoimmunity during lupus. We show that individuals with SLE and CGD display accumulation of SNECs in blood monocytes and neutrophils. In a CGD phenotypic mouse strain (Ncf1** mice) build-up of SNECs in Ly6CHI blood monocytes was connected with a delayed degradation of the phagosomal cargo and accompanied by production of inflammatory mediators. Treatment with H2O2 or activators of ROS-formation reconstituted phagosomal abundance of SNECs to normal levels. Induction of experimental lupus further induced increased antibody-dependent uptake of SNECs into neutrophils. Lupus-primed Ncf1** neutrophils took up more SNECs than wild type neutrophils, whereas SNEC-accumulation in regulatory Ly6C-/LO monocytes was lower in Ncf1**mice. We deduce that the inflammatory rerouting of immune-stimulatory necrotic material into inflammatory phagocyte subsets contributes to the connection between low ROS production by the NOX2 complex and SLE.


Assuntos
NADPH Oxidase 2/metabolismo , Fagócitos/metabolismo , Animais , Autoanticorpos/imunologia , Citocinas/metabolismo , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/metabolismo , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , NADPH Oxidase 2/genética , Necrose/genética , Necrose/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagócitos/imunologia , Fagocitose/genética , Fagocitose/imunologia , Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/metabolismo
6.
Biomed Res Int ; 2019: 2121357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31080811

RESUMO

Background: cIAP2 is involved in necroptosis as a key upstream regulation factor. We aimed to investigate the role of cIAP2 in ARDS/ALI induced by H7N9 virus through regulating the RIPK1/3 necroptosis pathway. Methods: Lung tissues of 11 patients who died from ARDS-complicated H7N9 infection between 2013 and 2016 were obtained as the H7N9-ARDS group. Lung tissues near benign lung nodules were acquired as the control group. Histological changes were evaluated by H&E staining. Protein levels of cIAP2, RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL in the lung tissues were detected by Western Blot. The mRNA levels of cIAP2, RIPK1, and RIPK3 were detected by real-time PCR. Results: H7N9 virus infection had a high mortality, with ARDS being the leading cause of death. The protein level of cIAP2 in the experimental group was lower than that in the control group (P<0.05). However, the experimental group showed higher RIPK1, RIPK3, and p-RIPK3 protein levels than the control group (P<0.05), as well as the expression level of MLKL and p-MLKL protein, which is a key downstream protein in necroptosis (P<0.05). Conclusion: In tissues from patients with fatal H7N9, downregulation of cIAP2 and induction of necroptosis was observed. We could speculate that necroptosis of the pulmonary epithelium is associated with severe H7N9 infection leading to ARDS. Thus, necroptosis inhibition may be a novel therapy for H7N9 influenza virus.


Assuntos
Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Síndrome do Desconforto Respiratório do Adulto/virologia , Adulto , Idoso , Animais , Células Cultivadas , Regulação para Baixo/fisiologia , Feminino , Humanos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Camundongos , Pessoa de Meia-Idade , Necrose/metabolismo , Necrose/virologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia
7.
Drug Des Devel Ther ; 13: 1461-1472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118580

RESUMO

Background: Random skin flap is frequently used in plastic and reconstructive surgery, but its distal part often occurs ischemia and necrosis. Pravastatin (Prava) with bioactivities of pro-angiogenesis, anti-apoptosis and anti-oxidative stress, may be beneficial for flap survival. Materials and methods: A modified McFarlane flap model was performed in Sprague-Dawley rats. The animals were divided into the Control and Prava groups and treated as follows: the Prava group was injected intraperitoneally with 2 mg/kg Prava for consecutive 7 days, and the Control group received an equal volume of vehicle daily. On day 7, the necrosis skin flaps were observed, while visualization of blood flow below the tissue surface was performed by Laser Doppler blood flow imaging (LDBFI). Then animals were euthanized, and levels of angiogenesis, apoptosis and oxidative stress were analyzed. Results: Prava decreased necrosis and edema of skin flaps compared with the Control group, with more blood flow in the flap under LDBFI. Prava treatment increased the mean vessels density, elevated the expression levels of angiogenic proteins (matrix metallopeptidase 9, vascular endothelial growth factor, Cadherin5) and antioxidant proteins (superoxide dismutase 1 (SOD1), endothelial nitric oxide synthase, heme oxygenase), and decreased the expression of apoptotic factors (BAX, CYC, Caspase3). In addition, malondialdehyde content was reduced, and glutathione level and SOD activity were increased in the skin flaps after treatment with Prava. Conclusion: Prava promotes survival of random skin flap through induction of angiogenesis, and inhibition of apoptosis and oxidative stress.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Necrose/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pravastatina/farmacologia , Retalhos Cirúrgicos , Animais , Relação Dose-Resposta a Droga , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Injeções Intraperitoneais , Masculino , Necrose/metabolismo , Necrose/patologia , Pravastatina/administração & dosagem , Ratos , Ratos Sprague-Dawley
8.
J Ethnopharmacol ; 239: 111898, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31028855

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Plant-specific fungus of natural compound of Ascochyta viciae has traditionally been used in the treatment of sleeping sickness and tumors. The anti-tumor activities of the compounds obtained from Pisum sativum L were evaluated in this study. AIM OF THE STUDY: In this study, during the prolonged incubation, treatment of the LPS-stimulated tumor-like macrophage RAW 264.7 cells with ASC exhibited the shift of anti-inflammatory behavior to a type of necroptotic cell death named necroptosis. MATERIALS AND METHODS: Ascochlorin (ASC) purified from plant-specific fungus Ascochyta viciae is a natural compound with the trimethyl oxocyclohexyl structure and an anti-cancer and antibiotic agent. The fungus contributes to the Ascochyta blight disease complex of pea (Pisum sativum L). RAW 264.7 cells have been stimulated with LPS and treated with ASC. Cell viability of the LPS-treated RAW 264.7 cells and bone marrow-derived macrophage (BMDM) cells were examined. Flow cytometry analysis with 7AAD and Annexin V was examined for the apoptotic or necroptosis/late-apoptosis. Cleaved caspase-3, -7 and -8 as well as cleaved PARP were assessed with a caspase inhibitor, z-VAD-fmk. LPS-responsible human leukemic U937 and colon cancer SW480 and HT-29 cells were also examined for the cell viabilities. RESULTS: Flow cytometry analysis after Annexin V and 7AAD double staining showed that ASC alone induces apoptosis in RAW 264.7 cells, while it induces necroptosis/late-apoptosis in LPS-treated RAW 264.7 cells. 7AAD and Annexin V positive populations were increased in the LPS-treated cells with ASC. Although viability of LPS-treated cells with ASC was decreased, the amounts of cleaved caspase-3, -7 and -8 as well as cleaved PARP were reduced when compared with ASC-treated cells. Upon ASC treatment, the cleaved caspase-8 level was not changed, however, cleaved caspase-3, -7, and PARP were reduced in LPS-stimulated RAW 264.7 cells treated with ASC, claiming a caspase-8 independent necroptosis of ASC. Furthermore, ASC and LPS-cotreated cells which a caspase inhibitor, z-VAD-fmk, was pretreated, showed the decreased cell viability compared with control cells without the inhibitor. Cell viability of RAW 264.7 cells co-treated with ASC and LPS when treated with z-VAD was decreased. In the LPS-responsible human leukemic U937 and colon cancer SW480 and HT-29 cells, cell viabilities were decreased by 10 µM ASC. CONCLUSION: Prolonged stimulation of ASC with LPS induces the necroptosis in RAW cells. Activated immune cells may share the susceptibility of antitumor agents with the cancer cells.


Assuntos
Alcenos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Necrose/induzido quimicamente , Fenóis/farmacologia , Animais , Caspases/metabolismo , Linhagem Celular Tumoral , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Necrose/metabolismo , Células RAW 264.7
9.
Cell Biol Int ; 43(6): 582-592, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30958602

RESUMO

Cell death was once believed to be the result of one of two distinct processes, apoptosis (also known as programmed cell death) or necrosis (uncontrolled cell death); in recent years, however, several other forms of cell death have been discovered highlighting that a cell can die via a number of differing pathways. Apoptosis is characterised by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme-dependent biochemical processes. The result being the clearance of cells from the body, with minimal damage to surrounding tissues. Necrosis, however, is generally characterised to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof. Failure of apoptosis and the resultant accumulation of damaged cells in the body can result in various forms of cancer. An understanding of the pathways is therefore important in developing efficient chemotherapeutics. It has recently become clear that there exists a number of subtypes of apoptosis and that there is an overlap between apoptosis, necrosis and autophagy. The goal of this review is to provide a general overview of the current knowledge relating to the various forms of cell death, including apoptosis, necrosis, oncosis, pyroptosis and autophagy. This will provide researchers with a summary of the major forms of cell death and allow them to compare and contrast between them.


Assuntos
Morte Celular/genética , Morte Celular/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Autofagia/genética , Autofagia/fisiologia , Humanos , Necrose/genética , Necrose/metabolismo , Transdução de Sinais
10.
Environ Toxicol Pharmacol ; 68: 155-163, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30986632

RESUMO

OBJECTIVE: To observe the effects of prolonged exposure to high concentrations of PM2.5 on the trachea and lungs of mice and to determine whether the damages to the trachea and lung are induced by necroptosis. METHODS: Six- to eight-week-old female Balb/C mice of PM group were restrained in an animal restraining device using a nose-only "PM2.5 online enrichment system" for 8 weeks, in Shijiazhuang, Hebei, China. Anti -Fas group was exposed to PM2.5 inhalation and anti-Fas treatment via intranasal instillation. The mice in the control group inhaled filtered clean air. PM2.5 sample was collected and analyzed. Airway Hyperresponsiveness (AHR) was tested. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for Hematoxylin and eosin (HE) staining, electron microscopy, cellular inflammation, cytokines, Tunel, Fas, RIPK3 and MLKL expression. RESULTS: Compared to the other two groups, PM group displayed significantly increased AHR, neutrophils in BALF, significant bronchitis and alveolar epithelial hyperplasia and inflammation and necroptosis which were indicated by increased TUNEL, Fas, RIPK3 and MLKL measure. CONCLUSION: Our findings suggest that PM2.5 can enhance AHR and these changes are induced by necroptosis-related inflammation.


Assuntos
Poluentes Atmosféricos/toxicidade , Material Particulado/toxicidade , Hipersensibilidade Respiratória/induzido quimicamente , Fenômenos Fisiológicos Respiratórios/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Feminino , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiologia , Pulmão/ultraestrutura , Camundongos Endogâmicos BALB C , Necrose/induzido quimicamente , Necrose/metabolismo , Necrose/patologia , Necrose/fisiopatologia , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Hipersensibilidade Respiratória/fisiopatologia , Traqueia/efeitos dos fármacos , Traqueia/patologia , Traqueia/fisiologia , Traqueia/ultraestrutura
11.
Methods Mol Biol ; 1981: 149-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016653

RESUMO

Necroptosis is emerging as a critical pathogenic mechanism in several liver diseases, including cholestatic disorders. Necroptosis was recently described as a novel cell death subroutine, activated downstream of death receptor stimulation and dependent on receptor-interacting serine/threonine-protein kinase 3 activity and mixed lineage kinase domain-like oligomerization and translocation to cell membrane. Here, we describe a combination of methods to evaluate necroptosis triggering in in vitro and in vivo models of cholestasis. Particularly, we detail alternative protocols to isolate total and soluble/insoluble protein extracts from tissues and cell cultures, as well as in vitro receptor-interacting serine/threonine-protein kinase 3 kinase activity assays, and subsequent Western blot analysis.


Assuntos
Colestase/metabolismo , Colestase/patologia , Animais , Apoptose/fisiologia , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Necrose/metabolismo , Necrose/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/fisiologia
12.
FEBS Open Bio ; 9(3): 446-456, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30868053

RESUMO

One of the major roles of professional phagocytes is the removal of dead cells in the body. We know less about the clearance of necrotic cells than apoptotic cell phagocytosis, despite the fact that both types of dead cells need to be cleared together and necrotic cells appear often in pathological settings. In the present study, we examined phagocytosis of heat- or H2O2-killed necrotic and apoptotic thymocytes by mouse bone marrow-derived macrophages (BMDMs) in vitro and found that the two cell types are engulfed at equal efficiency and compete with each other when added together to BMDMs. Phagocytosis of both apoptotic and necrotic thymocytes was decreased by (a) blocking phosphatidylserine on the surface of dying cells; (b) inhibition of Mer tyrosine kinase, Tim-4, integrin ß3 receptor signaling, or Ras-related C3 botulinum toxin substrate 1 activity; or (c) using BMDMs deficient for transglutaminase 2. Stimulation of liver X, retinoid X, retinoic acid or glucocorticoid nuclear receptors in BMDMs enhanced not only apoptotic, but also necrotic cell uptake. Electron microscopic analysis of the engulfment process revealed that the morphology of phagosomes and the phagocytic cup formed during the uptake of dying thymocytes is similar for apoptotic and necrotic cells. Our data indicate that apoptotic and necrotic cells are cleared via the same mechanisms, and removal of necrotic cells in vivo can be facilitated by molecules known to enhance the uptake of apoptotic cells.


Assuntos
Apoptose , Macrófagos/metabolismo , Necrose/metabolismo , Fosfatidilserinas/metabolismo , Timócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilserinas/antagonistas & inibidores , Timócitos/efeitos dos fármacos
13.
Toxins (Basel) ; 11(3)2019 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832306

RESUMO

Pterocephalus hookeri (C. B. Clarke) Höeck, recorded in the Chinese Pharmacopoeia (2015 version) as a Tibetan medicine for the treatment of various diseases, especially rheumatoid arthritis, was believed to possess a slight toxicity. However, hardly any research has been carried out about it. The present study aimed to evaluate the toxicity in vivo and in vitro. Toxicity was observed by the evaluation of mice weight loss and histopathological changes in the liver. Then, the comparison research between ethyl acetate extract (EAE) and n-butanol extract (BUE) suggested that liver toxicity was mainly induced by BUE. The mechanical study suggested that BUE-induced liver toxicity was closely associated with necrosis detected by MTT and propidium iodide (PI) staining, via releasing lactate dehydrogenase (LDH), reducing the fluidity, and increasing the permeability of the cell membrane. Western blot analysis confirmed that the necrosis occurred molecularly by the up-regulation of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3), as well as the activation of the nuclear factor-kappa-gene binding (NF-κB) signaling pathway in vivo and in vitro. This finding indicated that the liver toxicity induced by BUE from P. hookeri was mainly caused by necrosis, which provides an important theoretical support for further evaluation of the safety of this folk medicine.


Assuntos
Caprifoliaceae , Doença Hepática Induzida por Substâncias e Drogas , Fígado/efeitos dos fármacos , Necrose/induzido quimicamente , Extratos Vegetais/toxicidade , Animais , Carragenina , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Edema/induzido quimicamente , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , NF-kappa B/metabolismo , Necrose/metabolismo , Necrose/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
14.
Proc Natl Acad Sci U S A ; 116(12): 5675-5680, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30837313

RESUMO

Demyelination in the central nervous system (CNS) underlies many human diseases, including multiple sclerosis (MS). We report here the findings of our study of the CNS demyelination process using immune-induced [experimental autoimmune encephalomyelitis (EAE)] and chemical-induced [cuprizone (CPZ)] mouse models of demyelination. We found that necroptosis, a receptor-interacting protein 3 (RIP3) kinase and its substrate mixed lineage kinase domain-like protein (MLKL)-dependent cell death program, played no role in the demyelination process, whereas the MLKL-dependent, RIP3-independent function of MLKL in the demyelination process initially discovered in the peripheral nervous system in response to nerve injury, also functions in demyelination in the CNS in these models. Moreover, a receptor-interacting protein 1 (RIP1) kinase inhibitor, RIPA-56, blocked disease progression in the EAE-induced model but showed no effect in the CPZ-induced model. It does so most likely at a step of monocyte elevation downstream of T cell activation and myelin-specific antibody generation, although upstream of breakdown of the blood-brain barrier. RIP1-kinase dead knock-in mice shared a similar result as mice treated with the RIP1 inhibitor. These results indicate that RIP1 kinase inhibitor is a potential therapeutic agent for immune-mediated demyelination diseases that works by prevention of monocyte elevation, a function previously unknown for RIP1 kinase.


Assuntos
Encefalomielite Autoimune Experimental/genética , Proteínas Quinases/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Apoptose/fisiologia , Morte Celular , Doenças Desmielinizantes/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Esclerose Múltipla/genética , Necrose/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais
15.
Toxicol In Vitro ; 57: 226-232, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30853489

RESUMO

Alcoholic liver disease (ALD), featured by excessive hepatocyte death and inflammation, is a prevalent disease that causes heavy health burdens worldwide. Hepatocyte necroptosis is a central event that promotes inflammation in ALD. At molecular levels, inhibition of nuclear factor (erythroid - derived 2) - like 2 (NRF2) was an important trigger for cell necroptosis. The protective effects of gallic acid (GA) on liver diseases caused by multiple factors have been elucidated, however, the role of GA in ALD remained unclear. Therefore, this study was aimed to investigate the anti-ALD effects of GA and further reveal the molecular mechanisms. Results showed that GA could effectively recover cell viability and reduce the release of aspartate transaminase, alanine transaminase, and lactic dehydrogenase by ethanol-stimulated hepatocytes. More importantly, GA limited hepatocyte necroptosis under ethanol stimulation, which was characterized by reduced expression of distinct necroptotic signals receptor-interacting protein 1 (RIP1) and RIP3 and release of high mobility group box protein 1. Mechanistically, GA could induce NRF2 expression in ethanol-incubated hepatocytes, which was a molecular basis for GA to suppress ethanol-induced hepatocyte necroptosis. In conclusion, this study demonstrated that GA improved ethanol-induced hepatocyte necroptosis in vitro. Further, NRF2 activation might be requisite for GA to exert its protective effects.


Assuntos
Etanol/toxicidade , Ácido Gálico/farmacologia , Hepatócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Substâncias Protetoras/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fator 2 Relacionado a NF-E2/genética , Necrose/induzido quimicamente , Necrose/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
16.
Life Sci ; 221: 168-177, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30738043

RESUMO

AIMS: To investigate whether mucin 1 (MUC1) downregulation reduced the sensitivity of tumor necrosis factor-alpha (TNF-α)-induced bronchial epithelial cells to glucocorticoid-mediated necroptosis and explore the underlying mechanisms. MAIN METHODS: The human lung bronchial epithelial cell line (16HBE) was transfected with small interfering RNA (siRNA) against MUC1 and then stimulated by TNF-α, where some cells were pretreated with dexamethasone. Flow cytometry was performed to analyze necroptosis in 16HBE cells, and western blot analysis was used to detect protein expression levels of MUC1, glucocorticoid receptor (GR)α, GRß, NF-κB p65, phospho-p65 (p-p65), and histone deacetylase-2 (HDAC2). Additionally, nuclear translocation of MUC1 and GRα was assessed by immunofluorescence. KEY FINDINGS: We observed that MUC1 downregulation by siRNA significantly augmented TNF-α-induced necroptosis in 16HBE cells, and that dexamethasone showed impaired anti-necroptotic effects of MUC1 downregulation. Furthermore, we found that GRα nuclear translocation was inhibited in 16HBE cells with MUC1 downregulation, and that dexamethasone-mediated inhibition of p65 phosphorylation was lower in cells transfected with MUC1-siRNA compared to those transfected with negative control siRNA. SIGNIFICANCE: Impaired GRα nuclear translocation and inhibited p-p65 expression might contribute to glucocorticoid resistance caused by MUC1 deficiency in TNF-α-induced necroptosis in 16HBE cells, and should be considered as a potential target for the development of novel therapeutics for asthma.


Assuntos
Células Epiteliais/metabolismo , Mucina-1/metabolismo , Brônquios/metabolismo , Linhagem Celular , Dexametasona , Regulação para Baixo , Glucocorticoides/metabolismo , Humanos , Erros Inatos do Metabolismo , Mucina-1/genética , Necrose/metabolismo , RNA Interferente Pequeno , Receptores de Glucocorticoides/deficiência , Fator de Transcrição RelA/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo
17.
J Nanobiotechnology ; 17(1): 24, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30722792

RESUMO

BACKGROUND: Silica nanoparticles (SiNPs) are widely used for biosensing and diagnostics, and for the targeted delivery of therapeutic agents. Safety concerns about the biomedical and clinical applications of SiNPs have been raised, necessitating analysis of the effects of their intrinsic properties, such as sizes, shapes, and surface physicochemical characteristics, on human health to minimize risk in biomedical applications. In particular, SiNP size-associated toxicological effects, and the underlying molecular mechanisms in the vascular endothelium remain unclear. This study aimed to elucidate the detailed mechanisms underlying the cellular response to exposure to trace amounts of SiNPs and to determine applicable size criteria for biomedical application. METHODS: To clarify whether these SiNP-mediated cytotoxicity due to induction of apoptosis or necrosis, human ECs were treated with SiNPs of four different non-overlapping sizes under low serum-containing condition, stained with annexin V and propidium iodide (PI), and subjected to flow cytometric analysis (FACS). Two types of cell death mechanisms were assessed in terms of production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress induction, and autophagy activity. RESULTS: Spherical SiNPs had a diameter of 21.8 nm; this was further increased to 31.4, 42.9, and 56.7 nm. Hence, we investigated these effects in human endothelial cells (ECs) treated with these nanoparticles under overlap- or agglomerate-free conditions. The 20-nm SiNPs, but not SiNPs of other sizes, significantly induced apoptosis and necrosis. Surprisingly, the two types of cell death occurred independently and through different mechanisms. Apoptotic cell death resulted from ROS-mediated ER stress. Furthermore, autophagy-mediated necrotic cell death was induced through the PI3K/AKT/eNOS signaling axis. Together, the present results indicate that SiNPs within a diameter of < 20-nm pose greater risks to cells in terms of cytotoxic effects. CONCLUSION: These data provide novel insights into the size-dependence of the cytotoxic effects of silica nanoparticles and the underlying molecular mechanisms. The findings are expected to inform the applicable size range of SiNPs to ensure their safety in biomedical and clinical applications.


Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Nanopartículas/toxicidade , Necrose/patologia , Transdução de Sinais/efeitos dos fármacos , Dióxido de Silício , Autofagia/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Nanopartículas/química , Necrose/metabolismo , Tamanho da Partícula , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/química , Dióxido de Silício/toxicidade
18.
Int J Mol Sci ; 20(3)2019 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-30744021

RESUMO

Autophagy is an evolutionarily conserved cellular process, through which damaged organelles and superfluous proteins are degraded, for maintaining the correct cellular balance during stress insult. It involves formation of double-membrane vesicles, named autophagosomes, that capture cytosolic cargo and deliver it to lysosomes, where the breakdown products are recycled back to cytoplasm. On the basis of degraded cell components, some selective types of autophagy can be identified (mitophagy, ribophagy, reticulophagy, lysophagy, pexophagy, lipophagy, and glycophagy). Dysregulation of autophagy can induce various disease manifestations, such as inflammation, aging, metabolic diseases, neurodegenerative disorders and cancer. The understanding of the molecular mechanism that regulates the different phases of the autophagic process and the role in the development of diseases are only in an early stage. There are still questions that must be answered concerning the functions of the autophagy-related proteins. In this review, we describe the principal cellular and molecular autophagic functions, selective types of autophagy and the main in vitro methods to detect the role of autophagy in the cellular physiology. We also summarize the importance of the autophagic behavior in some diseases to provide a novel insight for target therapies.


Assuntos
Autofagia/efeitos dos fármacos , Autofagia/genética , Suscetibilidade a Doenças , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Doenças Autoimunes , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Humanos , Inflamação , Síndrome Metabólica , Necrose/genética , Necrose/metabolismo , Neoplasias , Doenças Neurodegenerativas
19.
Immunity ; 50(3): 576-590.e6, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30770249

RESUMO

Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.


Assuntos
Apoptose/fisiologia , Inflamação/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Imunidade Inata/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Serina/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismo
20.
BMB Rep ; 52(4): 239-249, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30670153

RESUMO

Membrane-anchored full-length MET stimulated by its ligand HGF/SF induces various biological responses, including survival, growth, and invasion. This panel of responses, referred to invasive growth, is required for embryogenesis and tissue regeneration in adults. On the contrary, MET deregulation is associated with tumorigenesis in many kinds of cancer. In addition to its well-documented ligand-stimulated downstream signaling, the receptor can be cleaved by proteases such as secretases, caspases, and calpains. These cleavages are involved either in MET receptor inactivation or, more interestingly, in generating active fragments that can modify cell fate. For instance, MET fragments can promote cell death or invasion. Given a large number of proteases capable of cleaving MET, this receptor appears as a prototype of proteolytic-cleavage-regulated receptor tyrosine kinase. In this review, we describe and discuss the mechanisms and consequences, both physiological and pathological, of MET proteolytic cleavages. [BMB Reports 2019; 52(4): 239-249].


Assuntos
Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas c-met/fisiologia , Animais , Apoptose , Calpaína/metabolismo , Caspases/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Ligantes , Necrose/metabolismo , Neoplasias/metabolismo , Proteólise , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia
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