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1.
J Cell Physiol ; : e31366, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38958520

RESUMO

Autophagy is a lysosome-mediated self-degradation process of central importance for cellular quality control. It also provides macromolecule building blocks and substrates for energy metabolism during nutrient or energy deficiency, which are the main stimuli for autophagy induction. However, like most biological processes, autophagy itself requires ATP, and there is an energy threshold for its initiation and execution. We here present the first comprehensive review of this often-overlooked aspect of autophagy research. The studies in which ATP deficiency suppressed autophagy in vitro and in vivo were classified according to the energy pathway involved (oxidative phosphorylation or glycolysis). A mechanistic insight was provided by pinpointing the critical ATP-consuming autophagic events, including transcription/translation/interaction of autophagy-related molecules, autophagosome formation/elongation, autophagosome fusion with the lysosome, and lysosome acidification. The significance of energy-dependent fine-tuning of autophagic response for preserving the cell homeostasis, and potential implications for the therapy of cancer, autoimmunity, metabolic disorders, and neurodegeneration are discussed.

2.
Molecules ; 28(8)2023 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-37110604

RESUMO

Recent data indicate the link between the number and function of T regulatory cells (Treg) in the gut immune tissue and initiation and development of autoimmunity associated with type 1 diabetes (T1D). Since type 3 innate lymphoid cells (ILC3) in the small intestine are essential for maintaining FoxP3+ Treg and there are no data about the possible role of ILC3 in T1D pathogenesis, the aim of this study was to explore ILC3-Treg link during the development of T1D. Mature diabetic NOD mice had lower frequencies of IL-2-producing ILC3 and Treg in small intestine lamina propria (SILP) compared to prediabetic NOD mice. Similarly, in multiple low doses of streptozotocin (MLDS)-induced T1D in C57BL/6 mice, hyperglycemic mice exhibited lower numbers of ILC3, IL-2+ ILC3 and Treg in SILP compared to healthy controls. To boost T1D severity, mice were treated with broad-spectrum antibiotics (ABX) for 14 days prior to T1D induction by MLDS. The higher incidence of T1D in ABX-treated mice was associated with significantly lower frequencies of IL-2+ ILC3 and FoxP3+ Treg in SILP compared with mice without ABX treatment. The obtained findings show that the lower proportions of IL-2-expressing ILC3 and FoxP3+ Treg in SILP coincided with diabetes progression and severity.


Assuntos
Diabetes Mellitus Tipo 1 , Camundongos , Animais , Diabetes Mellitus Tipo 1/patologia , Camundongos Endogâmicos NOD , Linfócitos T Reguladores , Interleucina-2 , Imunidade Inata , Camundongos Endogâmicos C57BL , Linfócitos/patologia , Fatores de Transcrição , Intestino Delgado/patologia , Fatores de Transcrição Forkhead/genética
3.
Int Endod J ; 55(1): 64-78, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34614243

RESUMO

AIM: To investigate the influence of strain differences in immune responses on the pathogenesis of experimental periapical lesions in Dark Agouti (DA) and Albino Oxford (AO) inbred strains of rats. METHODOLOGY: Periapical lesions were induced in male DA and AO rats by pulp exposure of the first mandibular right molars to the oral environment. Animals were killed 21 days after pulp exposure. The mandibular jaws were retrieved and prepared for radiographic, pathohistological, immunohistochemical analysis, real-time PCR and flow cytometry. Blood samples and the supernatant of periapical lesions were collected for measurement of cytokines and oxidative stress marker levels. Statistical analysis was performed using the Kruskal-Wallis H and Mann-Whitney U non-parametric tests or parametric One-Way anova and Independent Samples T-test to determine the differences between groups depending on the normality of the data. A significant difference was considered when p values were <.05. RESULTS: DA rats developed significantly larger (p < .05) periapical lesions compared to AO rats as confirmed by radiographic and pathohistological analysis. The immunohistochemical staining intensity for CD3 was significantly greater in periapical lesions of DA rats compared to AO rats (p < .05). In DA rats, periapical lesions had a significantly higher (p < .05) percentage of CD3+ cells compared to AO rats. Also, the percentage of INF-γ, IL-17 and IL-10 CD3+CD4+ cells was significantly higher in DA rats (p < .05). DA rats had a significantly higher Th17/Th10 ratio. RT-PCR expression of IL-1ß, INF-γ and IL-17 genes was significantly higher in periapical lesions of DA compared to AO rats (p < .05). The receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin ratio was higher in DA compared to AO rats with periapical lesions (p < .05). Systemic levels of TNF-α and IL-6 were significantly higher in DA compared to AO rats (p < .05). Levels of lipid peroxidation measured as thiobarbituric acid reactive substances and reduced glutathione were significantly higher (p < .05) in the supernatant in the periapical lesions of DA rats. CONCLUSION: After pulp exposure, DA rats developed much larger periapical lesions compared to AO rats. Genetically determined differences in immunopathology have been demonstrated to be a significant element defining the severity of periapical lesions.


Assuntos
Conservadores da Densidade Óssea , Fator de Necrose Tumoral alfa , Animais , Masculino , Ratos , Ratos Endogâmicos
4.
J Pharmacol Sci ; 147(1): 156-167, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34294367

RESUMO

We investigated the effect of 3-methyladenine (3MA), a class III phosphatidylinositol 3-kinase (PI3K)-blocking autophagy inhibitor, on cancer cell death induced by simultaneous inhibition of glycolysis by 2-deoxyglucose (2DG) and mitochondrial respiration by rotenone. 2DG/rotenone reduced ATP levels and increased mitochondrial superoxide production, causing mitochondrial swelling and necrotic death in various cancer cell lines. 2DG/rotenone failed to increase proautophagic beclin-1 and autophagic flux in melanoma cells despite the activation of AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin complex 1 (mTORC1). 3MA, but not autophagy inhibition with other PI3K and lysosomal inhibitors, attenuated 2DG/rotenone-induced mitochondrial damage, oxidative stress, ATP depletion, and cell death, while antioxidant treatment mimicked its protective action. The protection was not mediated by autophagy upregulation via class I PI3K/Akt inhibition, as it was preserved in cells with genetically inhibited autophagy. 3MA increased AMPK and mTORC1 activation in energy-stressed cells, but neither AMPK nor mTORC1 inhibition reduced its cytoprotective effect. 3MA reduced JNK activation, and JNK pharmacological/genetic suppression mimicked its mitochondria-preserving and cytoprotective activity. Therefore, 3MA prevents energy stress-triggered cancer cell death through autophagy-independent mechanisms possibly involving JNK suppression and decrease of oxidative stress. Our results warrant caution when using 3MA as an autophagy inhibitor.


Assuntos
Adenina/análogos & derivados , Autofagia/efeitos dos fármacos , Melanoma/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Adenina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma/metabolismo , Melanoma Experimental , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Dilatação Mitocondrial , Necrose , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Rotenona/farmacologia
5.
Molecules ; 25(18)2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32916780

RESUMO

Ethyl pyruvate (EP), a stable form of pyruvate, has shown beneficial effects in animal models of shock, ischemia/reperfusion injury, and sepsis due to its potent anti-oxidant and anti-inflammatory properties. Our recent study demonstrated that EP application prevented the clinical manifestation of type 1 diabetes in mice by augmenting regulatory T cell (Treg) number and function. Our present study shows that EP increases Treg proliferation and suppressive function (perforin and IL-10 expression) during in vitro differentiation from conventional CD4+CD25- T cells. Enhanced expansion of Treg after EP treatment correlated with increased ATP levels and relied on increased glycolysis. Inhibition of oxidative phosphorylation did not attenuate EP stimulatory effects, suggesting that this metabolic pathway was not mandatory for EP-driven Treg proliferation. Moreover, EP lowered the expression of carnitine palmitoyltransferase I, an enzyme involved in fatty acid oxidation. Further, the stimulatory effect of EP on Treg proliferation was not mediated through inhibition of the mTOR signaling pathway. When given in vivo either intraperitoneally or orally to healthy C57BL/6 mice, EP increased the number of Treg within the peritoneal cavity or gut-associated lymphoid tissue, respectively. In conclusion, EP promotes in vitro Treg proliferation through increased glycolysis and enhances Treg proliferation when administered in vivo.


Assuntos
Proliferação de Células/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Piruvatos/farmacologia , Linfócitos T Reguladores/citologia , Trifosfato de Adenosina/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Diferenciação Celular , Ácidos Graxos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Baço/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos
6.
J Biol Chem ; 291(44): 22936-22948, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27587392

RESUMO

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy.


Assuntos
Desoxiglucose/farmacologia , Glioma/metabolismo , Glicólise/efeitos dos fármacos , Imidazóis/farmacologia , Lisossomos/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sinergismo Farmacológico , Glioma/tratamento farmacológico , Glioma/fisiopatologia , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos
7.
Eur J Immunol ; 45(2): 531-43, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25359399

RESUMO

Galectin-3 (Gal-3), an endogenous lectin, exhibits pro- and anti-inflammatory effects in various disease conditions. In order to explore the role of Gal-3 in NKT-cell-dependent pathology, we induced hepatitis in C57BL/6 WT and Gal-3-deficient mice by using specific ligand for NKT cells: α-galactosylceramide, glycolipid Ag presented by CD1d. The injection of α-galactosylceramide significantly enhanced expression of Gal-3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal-3 (induced by Gal-3-inhibitor TD139) abrogated the susceptibility to NKT-cell-dependent hepatitis. Blood levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal-3 alleviated influx of inflammatory CD11c(+) CD11b(+) DCs in the liver and favored tolerogenic phenotype and IL-10 production of liver NKT and DCs. Deletion of Gal-3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro-inflammatory cytokines in vitro. Gal-3-deficient DCs failed to optimally stimulate production of pro-inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal-3 regulates the capacity of DCs to support NKT-cell-mediated liver injury, playing an important pro-inflammatory role in acute liver injury.


Assuntos
Antígenos CD1d/genética , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Células Dendríticas/imunologia , Galactosilceramidas/toxicidade , Galectina 3/genética , Células Matadoras Naturais/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos CD1d/imunologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Movimento Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Células Dendríticas/patologia , Galectina 3/deficiência , Galectina 3/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Células Matadoras Naturais/patologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
8.
Biomed Microdevices ; 18(2): 37, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27106025

RESUMO

Indian spice curcumin is known for its anticancer properties, but the anticancer mechanisms of nanoparticulate curcumin have not been completely elucidated. We here investigated the in vitro anticancer effect of blue light (470 nm, 1 W)-irradiated curcumin nanoparticles prepared by tetrahydrofuran/water solvent exchange, using U251 glioma, B16 melanoma, and H460 lung cancer cells as targets. The size of curcumin nanocrystals was approximately 250 nm, while photoexcitation induced their oxidation and partial agglomeration. Although cell membrane in the absence of light was almost impermeable to curcumin nanoparticles, photoexcitation stimulated their internalization. While irradiation with blue light (1-8 min) or nanocurcumin (1.25-10 µg/ml) alone was only marginally toxic to tumor cells, photoexcited nanocurcumin displayed a significant cytotoxicity depending both on the irradiation time and nanocurcumin concentration. Photoexcited nanocurcumin induced phosphorylation of c-Jun N-terminal kinase (JNK), mitochondrial depolarization, caspase-3 activation, and cleavage of poly (ADP-ribose) polymerase, indicating apoptotic cell death. Accordingly, pharmacologial inhibition of JNK and caspase activity rescued cancer cells from photoexcited nanocurcumin. On the other hand, antioxidant treatment did not reduce photocytotoxicity of nanocurcumin, arguing against the involvement of oxidative stress. By demonstrating the ability of photoexcited nanocurcumin to induce oxidative-stress independent, JNK- and caspase-dependent apoptosis, our results support its further investigation in cancer therapy.


Assuntos
Apoptose/efeitos dos fármacos , Curcumina/química , Curcumina/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Luz , Nanopartículas/química , Solventes/química , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Transporte Biológico/efeitos da radiação , Caspase 3/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Curcumina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Tamanho da Partícula
9.
Exp Cell Res ; 335(2): 248-57, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26026740

RESUMO

Chemoresistance is a severe limitation to glioblastoma (GBM) therapy and there is a strong need to understand the underlying mechanisms that determine its response to different chemotherapeutics. Therefore, we induced resistance in C6 rat glioma cell line, which considerably resembles the characteristics of human GBM. The resistant phenotype was developed by 3-bis (2-chloroethyl)-1-nitrosourea (BCNU), one of the most commonly used therapeutic drug in the course of GBM treatment. After confirmation of the cross-resistance to cisplatin (CPt) and temozolomide (TMZ) in newly established RC6 cell line, we examined cell death induction and DNA damage by these drugs. Resistance to apoptosis and deficiency in forming DNA double-strand breaks was followed by significant decrease in the mRNA expression of pro-apoptotic and anti-apoptotic genes. The development of drug resistance was associated with significant increase in reactive oxygen species (ROS) and decrease in oxidized to reduced gluthatione ratio in RC6 cell line indicating a reduced level of oxidative stress. The mRNA expression levels of manganese superoxid dismutase (MnSOD), inducible nitric oxide synthase (iNOS) and gluthatione peroxidase (GPx) were increased while hypoxia-inducible factor 1-α (HIF-1α) was decreased in RC6 compared to C6 cells. This was in line with obtained changes in ROS content and increased antioxidative capacity of RC6 cells. Importantly, RC6 cells demonstrated collateral sensitivity to doxorubicin (DOX). The analysis of this phenomenon revealed increased accumulation of DOX in RC6 cells due to their adaptation to high ROS content and acidification of cytoplasm. In conclusion, newly established RC6 rat glioma cell line could be used as a starting material for the development of allogenic animal model and preclinical evaluation of new antiglioma agents. Collateral sensitivity to DOX obtained after BCNU treatment may prompt new studies aimed to find efficient delivery of DOX to the glioma site in brain.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Carmustina/farmacologia , Doxorrubicina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Dano ao DNA , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Glioma , Concentração Inibidora 50 , Estresse Oxidativo , Ratos , Temozolomida
10.
J Neurochem ; 135(1): 125-38, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26083644

RESUMO

Arylpiperazine-based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2 /5-HT1A ligands, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl}-phenyl]-picolinamide (6a) and N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate-immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5-HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0-7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL-6, IL-1, and GM-CSF, TH 1 cytokine IFN-γ, TH 17 cytokine IL-17, as well as the signature transcription factors of TH 1 (T-bet) and TH 17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti-apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN-93 and neuronal cell line PC12 from mitogen-activated normal T cells or myelin basic protein-activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation. Arylpiperazine dopaminergic/serotonergic ligands reduce neurological symptoms of acute autoimmune encephalomyelitis in rats without affecting the activation of autoreactive immune response, through mechanisms involving a decrease in CNS immune infiltration, as well as direct protection of CNS from immune-mediated damage. These data indicate potential usefulness of arylpiperazine-based compounds in the treatment of neuroinflammatory disorders such as multiple sclerosis.


Assuntos
Dopaminérgicos/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Animais , Dopamina/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-6/metabolismo , Ligantes , Esclerose Múltipla/imunologia , Células PC12 , Ratos , Serotonina/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
11.
Arch Toxicol ; 89(3): 437-50, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24770553

RESUMO

Hepatotoxicity of the antidiabetic drug metformin has been reported, but the underlying mechanisms remain unclear. We here investigated the effect of metformin in immune-mediated liver damage. While not hepatotoxic alone, metformin (200 mg/kg) aggravated concanavalin A (Con A, 12 mg/kg)-induced hepatitis, an experimental model of T cell-mediated liver injury, in both relatively resistant BALB/c and highly susceptible C57Bl/6 mice. Metformin + Con A-treated mice had elevated serum levels of pro-inflammatory cytokines TNF-α and IFN-γ, accompanied by a massive mononuclear cell infiltration in the liver. This was associated with the higher numbers of CD4(+) T cells producing TNF-α, IFN-γ and IL-17, CD4(+) T cells expressing chemokine receptor CXCR3 and activation marker CD27, CD4(+)CD62L(-)CCR7(-) and CD8(+)CD62L(-)CCR7(-) effector memory cells, IFN-γ producing NK cells, IL-4 and IL-17 producing NKT cells and IL-12 producing macrophages/dendritic cells. The percentage of CD4(+)CXCR3(+)Tbet(+)IL-10(+) and CD4(+)CD69(+)CD25(-) regulatory T cells was reduced. Metformin stimulated inducible nitric oxide synthase (iNOS) expression in the liver and spleen, and genetic deletion of iNOS attenuated the hepatotoxicity of metformin. Metformin increased the autophagic light chain 3 conversion and mRNA expression of important autophagy-inducing (beclin-1, Atg5 and GABARAP) and pro-apoptotic (p21, p27, Puma, Noxa, Bax, Bad, Bak1, Bim and Apaf1), but not anti-apoptotic molecules (Bcl-xL, survivin and XIAP), which correlated with the apoptotic caspase-3/PARP cleavage in the liver. The autophagy inhibitor chloroquine (20 mg/kg) prevented liver injury and apoptotic changes induced by metformin. Therefore, metformin aggravates immune-mediated hepatitis by promoting autophagy and activation of immune cells, affecting effector, as well as liver-specific regulatory T cells and iNOS expression.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Concanavalina A/toxicidade , Hipoglicemiantes/toxicidade , Fígado/efeitos dos fármacos , Metformina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/imunologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Citocinas/imunologia , Citometria de Fluxo , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/patologia , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
12.
Neurobiol Dis ; 63: 1-11, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24269733

RESUMO

In the present study, we investigated the role of the main intracellular energy sensor, AMP-activated protein kinase (AMPK), in the in vitro neurotoxicity of α-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinson's disease. The loss of viability in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells inducibly overexpressing wild-type ASYN was associated with the reduced activation of AMPK and its activator LKB1, as well as AMPK target Raptor. ASYN-overexpressing rat primary neurons also displayed lower activity of LKB1/AMPK/Raptor pathway. Restoration of AMPK activity by metformin or AICAR reduced the in vitro neurotoxicity of ASYN overexpression, acting independently of the prosurvival kinase Akt or the induction of autophagic response. The conditioned medium from ASYN-overexpressing cells, containing secreted ASYN, as well as dopamine-modified or nitrated recombinant ASYN oligomers, all inhibited AMPK activation in differentiated SH-SY5Y cells and reduced their viability, but not in the presence of metformin or AICAR. The RNA interference-mediated knockdown of AMPK increased the sensitivity of SH-SY5Y cells to the harmful effects of secreted ASYN. AMPK-dependent protection from extracellular ASYN was also observed in rat neuron-like pheochromocytoma cell line PC12. These data demonstrate the protective role of AMPK against the toxicity of both intracellular and extracellular ASYN, suggesting that modulation of AMPK activity may be a promising therapeutic strategy in Parkinson's disease.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neurônios/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Fragmentação do DNA , Embrião de Mamíferos , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Neuroblastoma/patologia , Neuroblastoma/ultraestrutura , Neurônios/ultraestrutura , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ribonucleotídeos/farmacologia , Tretinoína/farmacologia , alfa-Sinucleína/genética
13.
Immunol Lett ; 267: 106855, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38537720

RESUMO

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3- (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1ß, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.


Assuntos
Encefalomielite Autoimune Experimental , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Ratos , Resposta a Proteínas não Dobradas/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Modelos Animais de Doenças , Feminino , Citocinas/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia
14.
Front Endocrinol (Lausanne) ; 14: 1134478, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37008919

RESUMO

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.


Assuntos
Diabetes Mellitus , Células Secretoras de Glucagon , Camundongos , Animais , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Insulina/metabolismo , Células Secretoras de Glucagon/metabolismo , Metilação de DNA , Diabetes Mellitus/metabolismo
15.
Cells ; 12(9)2023 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-37174682

RESUMO

As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1ß were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1ß/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation.


Assuntos
COVID-19 , Inflamação , Humanos , Autofagia , COVID-19/patologia , Inflamação/metabolismo , Interleucina-10/sangue , Interleucina-17/sangue , Interleucina-33/sangue , Interleucina-6/sangue , RNA Mensageiro , SARS-CoV-2
16.
J Autoimmun ; 39(4): 332-46, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22647731

RESUMO

Negative regulation of B cell activation by cognate immune complexes plays an important homeostatic role in suppressing B cell hyperactivity and preventing consequent autoimmunity. Immune complexes co-ligate the BCR and FcγRIIB resulting in both growth arrest and apoptosis. We now show that such apoptotic signalling involves induction and activation of p53 and its target genes, the pro-apoptotic Bcl-2 family members, Bad and Bid, as well as nuclear export of p53. Collectively, these events result in destabilisation of the mitochondrial and lysosomal compartments with consequent activation and interplay of executioner caspases and endosomal-derived proteases. In addition, the upregulation of Fas and FasL with consequent activation of caspase 8-dependent death receptor signalling is required to facilitate efficient apoptosis of B cells. Consistent with this role for Fas death receptor signalling, apoptosis resulting from co-ligation of the BCR and FcγRIIB is defective in B cells from Fas-deficient MRL/MpJ-Fas(lpr) mice. As these mice develop spontaneous, immune complex-driven lupus-like glomerulonephritis, targeting this FcγRIIB-mediated apoptotic pathway may therefore have novel therapeutic implications for systemic autoimmune disease.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Linfócitos B/imunologia , Nefrite Lúpica/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de IgG/imunologia , Receptor fas/imunologia , Animais , Complexo Antígeno-Anticorpo/genética , Apoptose , Linfócitos B/metabolismo , Linfócitos B/patologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/imunologia , Caspase 8/genética , Caspase 8/imunologia , Modelos Animais de Doenças , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Nefrite Lúpica/genética , Nefrite Lúpica/patologia , Lisossomos/imunologia , Lisossomos/patologia , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Mitocôndrias/imunologia , Mitocôndrias/patologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de IgG/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/imunologia , Receptor fas/genética
17.
Biomed Res Int ; 2022: 3456958, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36105929

RESUMO

Multiple anthropometric equations have been developed aiming to provide accurate and affordable assessment of body fat composition in male athletes. This study examined correlations of values obtained from seventeen different anthropometric equations to DXA as well as BIA and DXA values. Male athletes (n = 101) from three different combat sports, wrestling (n = 33), judo (n = 35), and kickboxing (n = 33), with an average age of 20.9 ± 4.2 were included. Body fat percentage was estimated using anthropometry, BIA, and DXA. Correlations between anthropometric methods and DXA, as well as BIA and DXA, were determined using Spearman's rank correlation. Sixteen out of seventeen estimates of body fat percentages using existing anthropometric equations showed strong positive correlation with the values derived from DXA measurements (r = 0.569 - 0.909). The highest correlation was observed using the equation derived by Yuhasz, r = 0.909, followed by the equations from Oliver et al., Evans et al., Faulkner, and Thorland et al. (r ≈ 0.9). Statistical analysis of body fat percentages from DXA and BIA measurements also showed high positive correlation (r = 0.710). Correlation of seventeen anthropometric equations with BIA and DXA methods revealed that equations by Yuhasz, Oliver et al., Evans et al., Faulkner, and Thorland et al. are suitable alternative for assessing body fat percentage among male athletes from combat sports, showing even stronger correlation than BIA method.


Assuntos
Tecido Adiposo , Artes Marciais , Absorciometria de Fóton/métodos , Tecido Adiposo/diagnóstico por imagem , Adolescente , Adulto , Antropometria/métodos , Atletas , Impedância Elétrica , Humanos , Masculino , Adulto Jovem
18.
Cells ; 11(18)2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36139470

RESUMO

AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolic and immune functions mainly through the inhibition of the mechanistic target of rapamycin (mTOR)-dependent anabolic pathways and the activation of catabolic processes such as autophagy. The AMPK/mTOR signaling pathway and autophagy markers were analyzed by immunoblotting in blood mononuclear cells of 20 healthy control subjects and 23 patients with an acute demyelinating form of Guillain-Barré syndrome (GBS). The activation of the liver kinase B1 (LKB1)/AMPK/Raptor signaling axis was significantly reduced in GBS compared to control subjects. In contrast, the phosphorylated forms of mTOR activator AKT and mTOR substrate 4EBP1, as well as the levels of autophagy markers LC3-II, beclin-1, ATG5, p62/sequestosome 1, and NBR1 were similar between the two groups. The downregulation of LKB1/AMPK signaling, but not the activation status of the AKT/mTOR/4EBP1 pathway or the levels of autophagy markers, correlated with higher clinical activity and worse outcomes of GBS. A retrospective study in a diabetic cohort of GBS patients demonstrated that treatment with AMPK activator metformin was associated with milder GBS compared to insulin/sulphonylurea therapy. In conclusion, the impairment of the LKB1/AMPK pathway might contribute to the development/progression of GBS, thus representing a potential therapeutic target in this immune-mediated peripheral polyneuropathy.


Assuntos
Síndrome de Guillain-Barré , Insulinas , Metformina , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Beclina-1/metabolismo , Regulação para Baixo , Humanos , Insulinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estudos Retrospectivos , Transdução de Sinais , Sirolimo , Serina-Treonina Quinases TOR/metabolismo
19.
Biochim Biophys Acta Mol Cell Res ; 1868(4): 118944, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33383091

RESUMO

To sustain their proliferative and metastatic capacity, tumor cells increase the activity of energy-producing pathways and lysosomal compartment, resorting to autophagolysosomal degradation when nutrients are scarce. Consequently, large fragile lysosomes and enhanced energy metabolism may serve as targets for anticancer therapy. A simultaneous induction of energy stress (by caloric restriction and inhibition of glycolysis, oxidative phosphorylation, Krebs cycle, or amino acid/fatty acid metabolism) and lysosomal stress (by lysosomotropic detergents, vacuolar ATPase inhibitors, or cationic amphiphilic drugs) is an efficient anti-cancer strategy demonstrated in a number of studies. However, the mechanisms of lysosomal/energy stress co-amplification, apart from the protective autophagy inhibition, are poorly understood. We here summarize the established and suggest potential mechanisms and candidates for anticancer therapy based on the dual targeting of lysosomes and energy metabolism.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Lisossomos/metabolismo , Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia , Metabolismo Energético/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Neoplasias/tratamento farmacológico
20.
Free Radic Biol Med ; 177: 167-180, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34678419

RESUMO

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (•OH), superoxide anion (O2•-), and lipid peroxidation. Nonselective antioxidants, •OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing •OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both •OH/NO scavenging and induction of cytoprotective autophagy.


Assuntos
Grafite , Neuroblastoma , Pontos Quânticos , Antioxidantes/farmacologia , Apoptose , Autofagia , Linhagem Celular Tumoral , Humanos , Estresse Oxidativo
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