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1.
J Biosci Bioeng ; 131(1): 90-97, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32950383

RESUMO

Decreased cell viability resulting from a severe condition of nutrients deprivation and hypoxia has been the major obstacle in three-dimensional (3D) tissue construction. Therefore, technical improvement which prevents cell death caused by starvation and low oxygen is desired for the development of large, thick tissues. We focused on the anti-glycolytic effect of resveratrol (RSV), a naturally-occurring polyphenol known as a caloric restriction mimetic, and investigated its cytoprotective effect in two-dimensional (2D) and 3D-cell culture using H9c2 rat myoblast cells. Glucose deprivation by culturing with low glucose media caused time- and dose-dependent cell death in H9c2 cells. In contrast, RSV treatment at 100 µM significantly increased the cell viability by preventing cell death. RSV showed anti-glycolytic effect associated with a down-regulation of glycolytic genes (GLUT1, PKM2) and glucose uptake activity, and increased the activation of AMP-activated protein kinase (AMPK), an essential cellular energy sensor activated in the condition of energy deprivation. RSV treatment markedly improved the viability of myoblast cells cultured in a hypoxic, low glucose condition and attenuated the up-regulation of glycolytic genes by hypoxic response. In 3D-cultured model, spheroids constructed with RSV-treated cells showed improved cell viability and intact histological appearance compared with control. These results suggest that glycolytic inhibition by RSV decreases the glucose usage of myoblast cells, therefore, prevents cell death caused by nutrient deprivation and hypoxic condition. Our finding provides useful information to improve cell viability in a condition that nutrients and oxygen are low in supply, and be a possible application to the 3D-tissue construction.


Assuntos
Hipóxia Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Glucose/deficiência , Glicólise/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Resveratrol/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Mioblastos/metabolismo , Ratos , Engenharia Tecidual
2.
Life Sci ; 265: 118806, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33249098

RESUMO

AIMS: Neuronal apoptosis acts as the pivotal pathogenesis of cerebral ischemia/reperfusion (I/R) injury after ischemic stroke. PAQR3 (progestin and adipoQ receptor family member 3) is a crucial player who participates in the regulation of cell death. We aim to explore the specific function and the underlying mechanism of PAQR3 in cerebral I/R induced neuronal injury. MAIN METHODS: We established a mouse middle cerebral artery occlusion/reperfusion (MCAO/R) model and rat adrenal pheochromocytoma (PC12) cell oxygen-glucose deprivation/reperfusion (OGD/R) model to detect the expression and of PAQR3 after I/R treatment in vivo and in vitro. We used lentivirus to knockdown PAQR3 and investigated the function of PAQR3 in I/R induced neuronal apoptosis. KEY FINDINGS: PAQR3 expression is markedly increased in the ischemic hemisphere of C57BL/6 mice and PC12 cells after I/R stimulation. Knockdown PAQR3 can attenuate neuronal apoptosis induced by I/R in PC12 cells and exerts neuroprotective effects. PAQR3 deficiency can significantly raise cell viability and suppress LDH leakage under I/R treatment. Silencing PAQR3 attenuates neuronal apoptosis remarkably with fewer TUNEL-positive cells and lower apoptosis rate under I/R treatment. Mechanistically, knockdown of PAQR3 can inhibit the apoptosis pathway through inducing anti-apoptotic proteins and inhibiting pro-apoptotic proteins. Besides, PI3K/AKT signaling suppression with LY294002 abolished the neuroprotective functions induced by silencing PAQR3. SIGNIFICANCE: Our results elucidate that silencing PAQR3 can protect PC12 from OGD/R injury via activating PI3K/AKT pathway. And therefore, provide a novel therapeutic target for the prevention of cerebral I/R injury.


Assuntos
Isquemia Encefálica/metabolismo , Glucose/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas de Membrana/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Apoptose/fisiologia , Isquemia Encefálica/genética , Isquemia Encefálica/prevenção & controle , Hipóxia Celular/fisiologia , Inativação Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Células PC12 , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/prevenção & controle
3.
Science ; 370(6518): 853-856, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33184215

RESUMO

Shutoff of global protein synthesis is a conserved response to cellular stresses. This general phenomenon is accompanied by the induction of distinct gene programs tailored to each stress. Although the mechanisms driving repression of general protein synthesis are well characterized, how cells reprogram the translation machinery for selective gene expression remains poorly understood. Here, we found that the noncanonical 5' cap-binding protein eIF3d was activated in response to metabolic stress in human cells. Activation required reduced CK2-mediated phosphorylation near the eIF3d cap-binding pocket. eIF3d controls a gene program enriched in factors important for glucose homeostasis, including members of the mammalian target of rapamycin (mTOR) pathway. eIF3d-directed translation adaptation was essential for cell survival during chronic glucose deprivation. Thus, this mechanism of translation reprogramming regulates the cellular response to metabolic stress.


Assuntos
Fator de Iniciação 3 em Eucariotos/biossíntese , Glucose/deficiência , Biossíntese de Proteínas , Estresse Fisiológico , Adaptação Fisiológica , Sobrevivência Celular , Fator de Iniciação 3 em Eucariotos/genética , Células HEK293 , Humanos , Fosforilação , Serina-Treonina Quinases TOR/metabolismo
4.
J Stroke Cerebrovasc Dis ; 29(11): 105210, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33066952

RESUMO

OBJECTIVE: We attempt to investigate the role of TNFRSF1A and its underlying mechanism in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in rat pheochromocytoma PC12 cells. METHODS: Public datasets GSE61616 and GSE106680 were downloaded from GEO database. PC12 cells were used to construct OGD/R models. QRT-PCR and western blot were implemented to test the relative mRNA and protein levels, respectively. The miRNA online prediction website TargetScan was used to predict TNFRSF1A upstream regulated miRNAs, which were then confirmed by luciferase reporter assay. The changes in cell viability and apoptosis were evaluated using cell counting kit 8 (CCK-8), lactose dehydrogenase (LDH), and flow cytometry assays. RESULTS: Bioinformatics analysis demonstrated that the expression of TNFRSF1A was upregulated in CI/RI and middle cerebral artery occlusion models compared with control, respectively. And a significant upregulation was also observed in OGD/R-damaged PC12 cells. Depletion of TNFRSF1A can notably enhance the cells proliferation after OGD/R treatment, while enlargement of TNFRSF1A presented the opposite outcomes. Moreover, miR-29a-3p was shown to be the upstream regulatory miRNA of TNFRSF1A. The levels of TNFRSF1A were inversely mediated by miR-29a-3p. Overexpression of miR-29a-3p can raise the cell viability, decrease the LDH activity, and reduce the apoptotic ratio in OGD/R-treated cells. Besides, TNFRSF1A can attenuate the protective effect of miR-29a-3p on OGD/R-treated cells. Furthermore, miR-29a-3p mimic inhibited, while overexpression of TNFRSF1A promoted the activation of NF-κB signaling pathway, and TNFRSF1A can attenuate the suppressive effect of miR-29a-3p on the NF-κB pathway. CONCLUSION: Our research illustrated that the potential regulatory role of miR-29a-3p/TNFRSF1A axis in neurons cells suffered from OGD/R, and their effects on NF-κB signaling pathway, providing a possible bio-target for protecting cells from OGD/R damage .


Assuntos
MicroRNAs/metabolismo , NF-kappa B/metabolismo , Neurônios/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Apoptose , Hipóxia Celular , Proliferação de Células , Sobrevivência Celular , Bases de Dados Genéticas , Glucose/deficiência , Humanos , MicroRNAs/genética , Neurônios/patologia , Células PC12 , Fosforilação , Ratos , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Regulação para Cima
5.
Life Sci ; 260: 118393, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32898527

RESUMO

AIMS: The aim of this study was to investigate the mechanism of pro-inflammatory phenotype transformation of microglia induced by oxygen-glucose deprivation (OGD), and how salvianolate regulates the polarization of microglia to exert neuroprotective effects. MAIN METHODS: The immunofluorescence and western blot experiments were used to verify the injury effect on neuronal cells after inflammatory polarization of microglia. Secondly, immunofluorescence staining and western blot were analyzed inflammatory phenotype of microglia and TLR4 signaling pathway after salvianolate treatment. RT-qPCR and ELISA assays were showed the levels of RNA and proteins of inflammatory factors in microglia. Finally, flow cytometry and western blot assay proved that salvianolate had a certain protective effect on neuronal injury after inhibiting the phenotype of microglia. KEY FINDINGS: The OGD condition could promote inflammation and activate of TLR4 signal pathway in microglia, and the polarization of microglia triggered caspase-3 signal pathway of neuronal cell. The optimal concentrations of salvianolate were incubated with microglia under OGD condition, which could reduce the reactive oxygen species (ROS) expression (P = 0.002) and also regulate the activity of SOD, CAT and GSH-px enzymes (P < 0.05). Moreover, salvianolate treatment could inhibit TLR4 signal pathway (P = 0.012), suppress the pro-inflammatory phenotype of microglia in OGD condition (P = 0.018), and reduce the expression of IL-6 and TNF-α (P < 0.05). Finally, neuronal damage induced by microglia under OGD condition was reversed after administration of the microglia supernatant after salvianolate treatment. SIGNIFICANCE: Salvianolate, as an antioxidant, plays a neuroprotective role by inhibiting the pro-inflammatory phenotype and decreasing the expression of ROS in microglia.


Assuntos
Apoptose , Glucose/deficiência , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oxigênio/metabolismo , Extratos Vegetais/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Microglia/metabolismo , Microglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
6.
J Stroke Cerebrovasc Dis ; 29(10): 105126, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32912499

RESUMO

BACKGROUND: Long non-coding RNAs (LncRNAs) have been reported to play important roles in the pathogenesis and development of many diseases, including cerebral ischemia and reperfusion (I/R) injury. In this study, we aimed to investigate the role of LncRNA-Potassium Voltage-Gated Channel Subfamily Q Member 1 opposite strand/antisense transcript 1 (KCNQ1OT1) in cerebral I/R induced neuronal injury, and its underlying mechanisms. METHODS: Primary mouse cerebral cortical neurons treated with oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro and mice subjected to middle cerebral artery occlusion (MCAO) and reperfusion were used to mimic cerebral I/R injury. Small inference RNA (siRNA) was used to knockdown KCNQ1OT1 or microRNA-153-3p (miR-153-3p). Dual-luciferase assay was performed to detect the interaction between KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and Fork head box O3a (Foxo3). Flow cytometry analysis was performed to detect neuronal apoptosis. qRT-PCR and Western blotting were performed to detect RNA and protein expressions. RESULTS: KCNQ1OT1 and Foxo3 expressions were significantly increased in neurons subjected to I/R injury in vitro and in vivo, and miR-153-3p expression were significantly decreased. Knockdown of KCNQ1OT1 or overexpression of miR-153-3p weakened OGD/R-induced neuronal injury and regulated Foxo3 expressions. Dual-luciferase analysis showed that KCNQ1OT1 directly interacted with miR-153-3p and Foxo3 is a direct target of miR-153-3p. CONCLUSIONS: Our results indicate that LncRNA-KCNQ1OT1 promotes OGD/R-induced neuronal injury at least partially through acting as a competing endogenous RNA (ceRNA) for miR-153-3p to regulate Foxo3a expression, suggesting LncRNA-KCNQ1OT1 as a potential therapeutic target for cerebral I/R injury.


Assuntos
Córtex Cerebral/metabolismo , Proteína Forkhead Box O3/metabolismo , Infarto da Artéria Cerebral Média/terapia , MicroRNAs/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/metabolismo , Reperfusão/efeitos adversos , Animais , Hipóxia Celular , Células Cultivadas , Córtex Cerebral/patologia , Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica , Glucose/deficiência , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurônios/patologia , RNA Longo não Codificante/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
7.
PLoS Pathog ; 16(8): e1008695, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750090

RESUMO

The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida's ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections.


Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Glucose/deficiência , Interações Hospedeiro-Patógeno/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Animais , Células 3T3 BALB , Candida albicans/metabolismo , Candidíase/metabolismo , Candidíase/microbiologia , Caspase 1/fisiologia , Caspases Iniciadoras/fisiologia , Feminino , Hifas , Inflamação/metabolismo , Inflamação/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/fisiologia , Piroptose
8.
Yonsei Med J ; 61(8): 660-669, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32734729

RESUMO

PURPOSE: Neonatal hypoxic ischemic encephalopathy (HIE) is an essential factor underlying neonatal death and disability. This study sought to explore the role of miR-146b-5p in regulating neonatal HIE. MATERIALS AND METHODS: In vitro and in vivo HIE models were established in PC12 cells and 10-day neonatal Sprague Dawley rats, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess miR-146b-5p expression and inflammatory factors [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] in brain lesions and PC12 cells, while enzyme-linked immunosorbent assay was employed to detect the expression of oxidative stress factors (SOD and GSH-Px). Gain- and loss-assays of miR-146b-5p were conducted to verify its role in modulating the viability and apoptosis of PC12 cells under oxygen-glucose deprivation (OGD) treatment. Expression of TLR4, IRAK1, TRAF6, TAK1, and NF-κB were examined by qRT-PCR and/or Western blot. Dual luciferase activity assay was conducted to identify relationships between miR-146b-5p and IRAK1. RESULTS: In the HIE models, significant oxidative stress and inflammatory responses emerged upon upregulation of TLR4/IRAK1/TRAF6/TAK1/NF-κB signaling. Overexpression of miR-146b-5p greatly inhibited OGD-induced PC12 cell injury, inflammatory responses, and oxidative stress. Inhibiting miR-146b-5p, however, had the opposite effects. IRAK1 was found to be a target of miR-146b-5p, and miR-146b-5p overexpression suppressed the activation of IRAK1/TRAF6/TAK1/NF-κB signaling. CONCLUSION: This study demonstrated that miR-146b-5p overexpression alleviates HIE-induced neuron injury by inhibiting the IRAK1/TRAF6/TAK1/NF-κB pathway.


Assuntos
Hipóxia-Isquemia Encefálica/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , MAP Quinase Quinase Quinases/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/genética , Sequência de Bases , Modelos Animais de Doenças , Regulação para Baixo/genética , Glucose/deficiência , Inflamação/patologia , MicroRNAs/genética , Estresse Oxidativo , Oxigênio , Células PC12 , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
9.
J Stroke Cerebrovasc Dis ; 29(9): 105037, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32807449

RESUMO

BACKGROUND: Cerebral ischemia/reperfusion (I/R) injury after ischemic stroke is usually accompanied with the activation of inflammasome which seriously impairs neurological function. MiR-139 has been reported to be associated with inflammatory regulation in multiple diseases. However, its effect and mechanism on inflammation regulation after cerebral I/R injury are still poorly understood. METHODS: An in vitro model of cerebral I/R injury was constructed with oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. TargetScan bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the targeted relationship between miR-139 and c-Jun. Cell pyroptosis was verified by flow cytometry and Caspase-1 Detection Kit. qRT-PCR assay was performed to detect the expression levels of miR-139, c-Jun, NLRP3 and ASC. Western blotting was applied to measure the protein levels of c-Jun and pyroptosis-related markers NLRP3, ASC, caspase-1, GSDMDNterm. The ELISA assay was applied to measure the release of IL-1ß, IL-18 and LDH. RESULTS: MiR-139 was significantly downregulated whereas c-Jun was obviously upregulated after OGD/R treatment. TargetScan analysis predicted that c-Jun was a potential target of miR-139, which was verified by the dual-luciferase reporter assay. Also, overexpression of miR-139 repressed c-Jun expression. Furthermore, miR-139 inhibited OGD/R-induced cell pyroptosis and the upregulation of NLRP3, caspase-1, ASC, GSDMDNterm, and the release of IL-1ß, IL-18 and LDH, while miR-139 inhibition exerted the opposite effects. However, overexpression of c-Jun aggravated OGD/R-induced nerve injury and partly abolished the neuroprotective effect of miR-139. CONCLUSION: Upregulation of miR-139 exerted neuroprotection against OGD/R-induced nerve injury by negatively regulating c-Jun/NLRP3 inflammasome signaling. This study offered insights for providing potential therapeutic targets for treating cerebral I/R injury.


Assuntos
Inflamassomos/metabolismo , MicroRNAs/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Piroptose , Traumatismo por Reperfusão/prevenção & controle , Caspase 1/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Glucose/deficiência , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , L-Lactato Desidrogenase/metabolismo , MicroRNAs/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neurônios/patologia , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais
10.
Nat Metab ; 2(8): 703-716, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32747793

RESUMO

CD8+ effector T (TE) cell proliferation and cytokine production depends on enhanced glucose metabolism. However, circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE cells metabolically primes effector functions and enhances tumour clearance in mice. Tumour-specific TGR CD8+ TE cells co-cultured with tumour spheroids in replete conditions display enhanced effector molecule expression, and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumour clearance. Mechanistically, TE cells treated with TGR undergo metabolic remodelling that, after glucose re-exposure, supports enhanced glucose uptake, increased carbon allocation to the pentose phosphate pathway (PPP) and a cellular redox shift towards a more reduced state-all indicators of a more anabolic programme to support their enhanced functionality. Thus, metabolic conditioning could be used to promote efficiency of T-cell products for adoptive cellular therapy.


Assuntos
Transferência Adotiva/métodos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Animais , Carbono/metabolismo , Linhagem Celular , Citocinas/biossíntese , Glucose/deficiência , Glucose/farmacologia , Memória Imunológica , Ativação Linfocitária , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Oxirredução , Via de Pentose Fosfato , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Metab ; 2(9): 893-901, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719541

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.


Assuntos
Fosfato de Di-Hidroxiacetona/fisiologia , Glucose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Di-Hidroxiacetona/metabolismo , Fosfato de Di-Hidroxiacetona/biossíntese , Metabolismo Energético , Frutose-Bifosfato Aldolase/metabolismo , Glucose/deficiência , Glicólise , Células HEK293 , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Serina-Treonina Quinases TOR/genética
12.
Nat Metab ; 2(7): 635-647, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32694789

RESUMO

T cells undergo metabolic rewiring to meet their bioenergetic, biosynthetic and redox demands following antigen stimulation. To fulfil these needs, effector T cells must adapt to fluctuations in environmental nutrient levels at sites of infection and inflammation. Here, we show that effector T cells can utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose in vitro. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. We demonstrate that the ribose subunit of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors, and that cancer cells display diverse capacities to utilize inosine as a carbon source. Moreover, the supplementation with inosine enhances the anti-tumour efficacy of immune checkpoint blockade and adoptive T-cell transfer in solid tumours that are defective in metabolizing inosine, reflecting the capability of inosine to relieve tumour-imposed metabolic restrictions on T cells.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Carbono/metabolismo , Glucose/deficiência , Inosina/metabolismo , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Hipoxantina/metabolismo , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nutrientes , Purina-Núcleosídeo Fosforilase/metabolismo , Ribose/metabolismo
13.
Nucleic Acids Res ; 48(13): 7307-7320, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32484543

RESUMO

Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly, each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses, we characterized global transcription patterns of modification mutants with defects in either tRNAGlnUUG or tRNALysUUU function. We observe that the mutants share inappropriate induction of multiple starvation responses in exponential growth phase, including derepression of glucose and nitrogen catabolite-repressed genes. In addition, autophagy is prematurely and inadequately activated in the mutants. We further demonstrate that improper induction of individual starvation genes as well as the propensity of the tRNA modification mutants to form protein aggregates are diminished upon overexpression of tRNAGlnUUG or tRNALysUUU, the tRNA species that lack the modifications of interest. Hence, our data suggest that global alterations in mRNA translation and proteostasis account for the transcriptional stress signatures that are commonly triggered by loss of anticodon modifications in different tRNAs.


Assuntos
Regulação Fúngica da Expressão Gênica , Glucose/deficiência , Nitrogênio/deficiência , RNA de Transferência/metabolismo , Autofagia , Glucose/metabolismo , Mutação , Nitrogênio/metabolismo , RNA de Transferência/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Nature ; 582(7812): 395-398, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494010

RESUMO

Neuroprotectant strategies that have worked in rodent models of stroke have failed to provide protection in clinical trials. Here we show that the opposite circadian cycles in nocturnal rodents versus diurnal humans1,2 may contribute to this failure in translation. We tested three independent neuroprotective approaches-normobaric hyperoxia, the free radical scavenger α-phenyl-butyl-tert-nitrone (αPBN), and the N-methyl-D-aspartic acid (NMDA) antagonist MK801-in mouse and rat models of focal cerebral ischaemia. All three treatments reduced infarction in day-time (inactive phase) rodent models of stroke, but not in night-time (active phase) rodent models of stroke, which match the phase (active, day-time) during which most strokes occur in clinical trials. Laser-speckle imaging showed that the penumbra of cerebral ischaemia was narrower in the active-phase mouse model than in the inactive-phase model. The smaller penumbra was associated with a lower density of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive dying cells and reduced infarct growth from 12 to 72 h. When we induced circadian-like cycles in primary mouse neurons, deprivation of oxygen and glucose triggered a smaller release of glutamate and reactive oxygen species, as well as lower activation of apoptotic and necroptotic mediators, in 'active-phase' than in 'inactive-phase' rodent neurons. αPBN and MK801 reduced neuronal death only in 'inactive-phase' neurons. These findings suggest that the influence of circadian rhythm on neuroprotection must be considered for translational studies in stroke and central nervous system diseases.


Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Modelos Animais de Doenças , Neurônios/patologia , Neuroproteção , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controle , Animais , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/prevenção & controle , Glucose/deficiência , Humanos , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Acidente Vascular Cerebral/fisiopatologia , Pesquisa Médica Translacional , Falha de Tratamento
15.
Life Sci ; 254: 117793, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32416164

RESUMO

AIMS: Oxygen and glucose deprivation and reperfusion (OGD/R) injury contributes to the pathophysiology after ischemic stroke, which needs to urgently develop treatment strategies. Previous studies have demonstrated that autophagy in reperfusion period exerted adverse effects on the cerebral ischemic injury. Ginsenoside monomer compound K (CK) is the main intestinal metabolite of ginseng that exerts the pharmacological activities and has a protective effect against cerebral OGD/R injury. However, the specific molecular mechanism of CK protects against OGD/R injury in neurons is still unclear. MATERIALS AND METHODS: In this study, cell viability, reactive oxygen species (ROS) generation, Ca2+ overload, mitochondrial membrane potential depolarization, autophagy and apoptosis were investigated in OGD/R-induced neuronal cells injury after pretreatment with CK and in combination with BML-275 or rapamycin. KEY FINDINGS: Our study found that pretreatment with CK protected neurons against OGD/R injury by increasing cell viability and decreasing the ROS generation, mitochondrial damage, and Ca2+ overload. Moreover, CK cut down autophagy-mediated apoptosis via promoting the process of forming autophagosomes into phagocytic precursors. Furthermore, our study clarified the neuroprotective of CK against OGD/R-induced neural autophagy and apoptosis through the regulation of the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway. SIGNIFICANCE: Taken together, our study provides credible experimental evidence and explains the potential molecular mechanism of CK as one of the main bioactive ingredients of ginseng for the treatment of cerebral ischemia/reperfusion injury.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Morte Celular Autofágica/efeitos dos fármacos , Ginsenosídeos/farmacologia , Glucose/deficiência , Hipóxia/metabolismo , Traumatismo por Reperfusão/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hipóxia/complicações , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Traumatismo por Reperfusão/complicações , Transdução de Sinais/efeitos dos fármacos
16.
Proc Natl Acad Sci U S A ; 117(22): 12239-12248, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32430326

RESUMO

The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant-although lower-level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics to characterize the cells' response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells' immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, both bulk autophagy, a process that frees amino acids, and lipid degradation via ß-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and ß-oxidation as important energy providers during acute glucose starvation.


Assuntos
Aminoácidos/metabolismo , Autofagia , Metabolismo Energético , Glucose/deficiência , Metabolismo dos Lipídeos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Trifosfato de Adenosina/metabolismo , Regulação Fúngica da Expressão Gênica , Metabolômica , Oxirredução , Saccharomyces cerevisiae/metabolismo , Inanição
17.
Biol Res ; 53(1): 17, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312329

RESUMO

BACKGROUND: Inflammation and apoptosis are considered to be two main factors affecting ischemic brain injury and the subsequent reperfusion damage. MiR-19a-3p has been reported to be a possible novel biomarker in ischemic stroke. However, the function and molecular mechanisms of miR-19a-3p remain unclear in cerebral ischemia/reperfusion (I/R) injury. METHODS: The I/R injury model was established in vivo by middle cerebral artery occlusion/reperfusion (MCAO/R) in rats and in vitro by oxygen-glucose deprivation and reperfusion (OGD/R) induced SH-SY5Y cells. The expression of miR-19a-3p was determined by reverse transcription quantitative PCR. The infarction volumes, Neurological deficit scores, apoptosis, cell viability, pro-inflammatory cytokines and apoptosis were evaluated using Longa score, Bederson score, TTC, TUNEL staining, CCK-8, ELISA, flow cytometry assays. Luciferase reporter assay was utilized to validate the target gene of miR-19a-3p. RESULTS: We first found miR-19a-3p was significantly up-regulated in rat I/R brain tissues and OGD/R induced SH-SY5Y cells. Using the in vivo and in vitro I/R injury model, we further demonstrated that miR-19a-3p inhibitor exerted protective role against injury to cerebral I/R, which was reflected by reduced infarct volume, improved neurological outcomes, increased cell viability, inhibited inflammation and apoptosis. Mechanistically, miR-19a-3p binds to 3'UTR region of IGFBP3 mRNA. Inhibition of miR-19a-3p caused the increased expression of IGFBP3 in OGD/R induced SH-SY5Y cells. Furthermore, we showed that IGFBP3 overexpression imitated, while knockdown reversed the protective effects of miR-19a-3p inhibitor against OGD/R-induced injury. CONCLUSIONS: In summary, our findings showed miR-19a-3p regulated I/R-induced inflammation and apoptosis through targeting IGFBP3, which might provide a potential therapeutic target for cerebral I/R injury.


Assuntos
Isquemia Encefálica/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , MicroRNAs/genética , Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Animais , Apoptose , Biomarcadores/metabolismo , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Glucose/deficiência , Masculino , Neurônios/metabolismo , Neuroproteção , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral , Regulação para Cima
18.
Life Sci ; 253: 117730, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32348838

RESUMO

Stem cell therapy is widely employed in treating osteoarthritis (OA), and bone marrow-derived mesenchymal stem cells (BMSCs) has gradually become the most attractive new method for treating OA due to the benefit for cartilage tissue repair. However, the apoptosis in the neural stem cell transplantation severely decreases repairing efficacy. Icariin has been reported to exert multiple effects on BMSCs, including its proliferation, osteogenic, and chondrogenic differentiation. However, its effects on the injury induced by oxygen, glucose and serum deprivation (OGD) remains unknown. We prospectively investigated the role of ICA on rabbit BMSCs under conditions of OGD. Firstly, BMSCs were cultured under conditions of OGD, ICA relieved OGD-induced cell damage by promoting cell proliferation and suppressing apoptosis. Secondly, Markers of endoplasmic reticulum stress (ERs), ER stress IRE-1 pathway, and autophagy were both inhibited by ICA via inhibition of phosphor-extracellular regulated protein kinases (p-ERKs), p-P38, p-c-Jun N-terminal kinase (p-JNK) or si-MAPK. Finally, decrease of ERs marker levels enhanced protective effect of ICA against OGD-induced injury by limiting apoptosis and autophagy. Moreover, an autophagy inhibitor (3-methyladenine: 3-MA) contributed to a synergistic effect in conjunction with ICA, in promoting cell proliferation, suggesting that ICA exerts anti-ERs and anti-autophagy effects in OGD-treated BMSCs. Therefore, ICA protected rabbit BMSCs from OGD-induced apoptosis through inhibitory regulation of ERs-mediated autophagy related to the MAPK signaling pathway, which provided insights for a potential therapeutic strategy in OA.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Flavonoides/farmacologia , Células-Tronco Mesenquimais/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Glucose/deficiência , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oxigênio/metabolismo , Estudos Prospectivos , Coelhos , Transdução de Sinais/efeitos dos fármacos
19.
Exp Mol Pathol ; 115: 104444, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32335082

RESUMO

Intestinal ischemia-reperfusion (I/R) is a life-threatening condition associated with high morbidity and mortality. Dexmedetomidine (DEX), an agonist of α2-adrenoceptor with sedation and analgesia effect, has recently been identified with protective function against I/R injury in multiple organs. However, the mechanism underlying the beneficial effect of DEX on intestine after I/R injury remained poorly understood. In the present study, using in both in vitro and in vivo models, we found that intestinal I/R injury was associated with the activation of p38 MAPK cascade, while DEX was capable of deactivating p38 MAPK and thus protect intestinal cells from apoptosis by inhibiting p38 MAPK-mediated mitochondrial depolarization and cytochrome c (Cyto C) release. Moreover, through inhibiting p38 MAPK activity, the downstream production of pro-inflammatory cytokines-regulated by NF-κB was also suppressed by DEX treatment, leading to the resolution of I/R-induced inflammation in intestine. In general, our study provided evidence that DEX protected intestine from I/R injury by inhibiting p38 MAPK-mediated mitochondrial apoptosis and inflammatory response.


Assuntos
Dexmedetomidina/uso terapêutico , Intestinos/patologia , Sistema de Sinalização das MAP Quinases , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células CACO-2 , Dexmedetomidina/farmacologia , Glucose/deficiência , Humanos , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Oxigênio , Substâncias Protetoras/farmacologia , Ratos Wistar , Traumatismo por Reperfusão/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
20.
Sci Rep ; 10(1): 6596, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313130

RESUMO

Enhancement of endogenous neurogenesis after ischemic stroke may improve functional recovery. We previously demonstrated that medium B, which is a combination with epidermal growth factor (EGF) and fibronectin, can promote neural stem/progenitor cell (NSPC) proliferation and migration. Here, we showed that medium B promoted proliferation and migration of cultured NSPCs onto various 3-dimentional structures. When rat cortical neurons with oxygen glucose deprivation (OGD) were co-cultured with NSPCs, medium B treatment increased neuronal viability and reduced cell apoptosis. In a rat model with transient middle cerebral artery occlusion (MCAO), post-insult intraventricular medium B treatment enhanced proliferation, migration, and neuronal differentiation of NSPCs and diminished cell apoptosis in the infarct brain. In cultured post-OGD neuronal cells and the infarct brain from MCAO rats, medium B treatment increased protein levels of Bcl-xL, Bcl-2, phospho-Akt, phospho-GSK-3ß, and ß-catenin and decreased the cleaved caspase-3 level, which may be associated with the effects of anti-apoptosis. Notably, intraventricular medium B treatment increased neuronal density, improved motor function and reduced infarct size in MCAO rats. In summary, medium B treatment results in less neuronal death and better functional outcome in both cellular and rodent models of ischemic stroke, probably via promotion of neurogenesis and reduction of apoptosis.


Assuntos
Apoptose , Isquemia Encefálica/tratamento farmacológico , Ventrículos Cerebrais/patologia , Fator de Crescimento Epidérmico/uso terapêutico , Fibronectinas/uso terapêutico , Neurogênese , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Isquemia Encefálica/complicações , Isquemia Encefálica/fisiopatologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ventrículos Cerebrais/fisiopatologia , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/farmacologia , Fibronectinas/farmacologia , Glucose/deficiência , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Ventrículos Laterais/patologia , Ventrículos Laterais/fisiopatologia , Masculino , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/ultraestrutura , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oxigênio , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/fisiopatologia
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