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1.
Gene ; 728: 144285, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31838253

RESUMO

Stroke has serious implications on patients and a huge impact on society. The current treatment regimens with drug for acute cerebral infarction are unsatisfactory. Here, we explore whether the two long non-coding RNA (lncRNA) candidates from preliminary research regulate apoptosis after cerebral infarction, and evaluate the underlying mechanism of action. Bioinformatics analysis of the lncRNA microarray in the preliminary research of our group was performed. Changes in the expression of candidate lncRNAs in SH-SY5Y cells were detected by quantitative polymerase chain reaction (qPCR) after treatment with seven different oxygen and glucose deprivation (OGD) methods. The changes were detected after transfection of cells with six small-interfering RNAs (siRNAs). Cell models were established by OGD after transfection with siRNAs. Cell viability was evaluated with the cell counting kit 8 (CCK8) assay, while TUNEL staining and flow cytometry analysis were performed to determine apoptosis. Changes in the expression and phosphorylation of three proteins were detected by western blotting after the knockdown of NR_120420. Changes in the expression and phosphorylation of P65 protein were detected by western blotting after this cell model was treated with PDTC. Cells were transfected with siNR_120420 and treated with and without PDTC, followed by analysis of cell viability and apoptosis. Bioinformatics analysis revealed that the differentially expressed lncRNAs after acute cerebral infarction were mainly involved in nuclear factor kappa B (NF-κB) and apoptosis. Expression of the two lncRNA candidates in SH-SY5Y cells was the maximum after incubation under the OGD condition for 8 h. The knockdown efficiency was more than 60% for four of the six siRNAs, and knockdown of NR_120420 increased the cell viability and decreased the percentage of TUNEL-positive cells and apoptotic cells. Knockdown of lnc-GCH1-2:3 resulted in none of these effects. Phosphorylation of NF-κB (P65) decreased significantly after the knockdown of NR_120420. Expression and phosphorylation of P65 was significantly reduced after it was treated with PDTC. The inhibitor of NF-κB (PDTC) could abolish the effect of NR_120420 on the regulation of apoptosis in this cell model. Both NR_120420 and lnc-GCH1-2:3 had significant changes in this cell model. Knockdown of NR_120420 inhibited the apoptosis of cells, while NR_120420 knockdown inhibited apoptosis after cerebral infarction by downregulating the phosphorylation of a subunit of NF-κB (P65). This study may provide new idea for improving drug treatment of acute cerebral infarction.


Assuntos
Apoptose , Infarto Cerebral/patologia , Glucose/deficiência , NF-kappa B/metabolismo , Neuroblastoma/patologia , Oxigênio/metabolismo , RNA Longo não Codificante/genética , Doença Aguda , Idoso , Estudos de Casos e Controles , Hipóxia Celular , Proliferação de Células , Infarto Cerebral/genética , Infarto Cerebral/metabolismo , Feminino , Humanos , Masculino , Análise em Microsséries , NF-kappa B/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fosforilação , Transdução de Sinais , Células Tumorais Cultivadas
2.
Biosci Biotechnol Biochem ; 84(1): 134-142, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31490096

RESUMO

Plumbagin (PLB), an alkaloid obtained from the roots of the plants of Plumbago genus, is an inhibitor of NADPH oxidase 4 (NOX4). This study aimed to investigate the beneficial effect of PLB against oxygen-glucose deprivation/reoxygenation (OGDR)-induced neuroinjury in human SH-SY5Y neuronal cultures. Our results showed that OGD/R stimulated NOX4 protein expression and reactive oxygen species (ROS) production in SH-SY5Y cells, whereas increased 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) production, resulting in the activation of the NLRP3 inflammasome. And PLB pretreatment reduced the ROS production by regulating the expression of NOX4 and downregulated NF-κB signaling which was induced by OGDR. Furthermore, PLB inhibited OGDR induced NLRP3 inflammasome activation but not PARP1. Overall, PLB improved OGDR induced neuroinjury by inhibiting NOX4-derived ROS-activated NLRP3 inflammasome.


Assuntos
Hipóxia Celular/efeitos dos fármacos , Glucose/deficiência , Inflamassomos/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Naftoquinonas/farmacologia , Neurônios/metabolismo , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Raízes de Plantas/química , Plumbaginaceae/química
4.
Mol Cell Biochem ; 462(1-2): 185-194, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31482388

RESUMO

Circular RNAs (circRNAs) are effector molecules that exert functions in cardiovascular diseases. Nevertheless, the effects of circRNAs on myocardial ischemia remain uninvestigated. This paper aimed to explore the functions of circ_0010729 in oxygen-glucose-deprivation (OGD)-caused injury of human cardiomyocytes (HCM). HCM were exposed to OGD environment for 4 h. Then the expression of circ_0010729 was evaluated by RT-qPCR. After transfection, cell viability, apoptosis, and migration were examined to evaluate the impact of overexpression and knockdown of circ_0010729 on OGD-induced cell injury. The regulation between circ_0010729 and microRNA-145-5p (miR-145-5p) was verified. After miR-145-5p inhibitor transfection, whether aberrant miR-145-5p expression affected the modulation of circ_0010729 in OGD-induced cell injury was measured. Western blot was utilized to analyze mTOR and MEK/ERK pathway-related proteins. OGD treatment enhanced circ_0010729 expression and evoked cell injury in HCM. Moreover, OGD-induced injury was aggrandized by circ_0010729 overexpression via suppressing cell growth and migration in HCM. Knockdown of circ_0010729 attenuated OGD-induced injury. In addition, circ_0010729 negatively regulated miR-145-5p expression. MiR-145-5p inhibition reversed the effects of silencing circ_0010729 on OGD-induced injury and mTOR and MEK/ERK pathways. We demonstrated that silencing circ_0010729 activated mTOR and MEK/ERK pathways by up-regulating miR-145-5p, thereby protecting HCM from OGD-induced injury.


Assuntos
Cardiotônicos/metabolismo , Inativação Gênica , Glucose/deficiência , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo , Regulação para Cima/genética , Humanos , Sistema de Sinalização das MAP Quinases , Serina-Treonina Quinases TOR/metabolismo
5.
Mol Cells ; 42(9): 672-685, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31486328

RESUMO

Currently, liver transplantation is the only available remedy for patients with end-stage liver disease. Conservation of transplanted liver graft is the most important issue as it directly related to patient survival. Carbonyl reductase 1 (CBR1) protects cells against oxidative stress and cell death by inactivating cellular membrane-derived lipid aldehydes. Ischemia-reperfusion (I/R) injury during living-donor liver transplantation is known to form reactive oxygen species. Thus, the objective of this study was to investigate whether CBR1 transcription might be increased during liver I/R injury and whether such increase might protect liver against I/R injury. Our results revealed that transcription factor Nrf2 could induce CBR1 transcription in liver of mice during I/R. Pre-treatment with sulforaphane, an activator of Nrf2, increased CBR1 expression, decreased liver enzymes such as aspartate aminotransferase and alanine transaminase, and reduced I/R-related pathological changes. Using oxygenglucose deprivation and recovery model of human normal liver cell line, it was found that oxidative stress markers and lipid peroxidation products were significantly lowered in cells overexpressing CBR1. Conversely, CBR1 knockdown cells expressed elevated levels of oxidative stress proteins compared to the parental cell line. We also observed that Nrf2 and CBR1 were overexpressed during liver transplantation in clinical samples. These results suggest that CBR1 expression during liver I/R injury is regulated by transcription factor Nrf2. In addition, CBR1 can reduce free radicals and prevent lipid peroxidation. Taken together, CBR1 induction might be a therapeutic strategy for relieving liver I/R injury during liver transplantation.


Assuntos
Carbonil Redutase (NADPH)/metabolismo , Transplante de Fígado , Fator 2 Relacionado a NF-E2/metabolismo , Traumatismo por Reperfusão/terapia , Regulação para Cima , Adulto , Oxirredutases do Álcool/genética , Animais , Biópsia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Glucose/deficiência , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/patologia , Doadores Vivos , Luteolina/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Oxigênio , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
6.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370282

RESUMO

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia.


Assuntos
Autofagia/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Oxigênio/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Hipóxia Celular , Linhagem Celular , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Expressão Gênica/efeitos dos fármacos , Glucose/deficiência , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
7.
Artif Cells Nanomed Biotechnol ; 47(1): 3511-3516, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31432688

RESUMO

The aim of this study was to investigate whether lncRNA TUG1 could mediate the progression of ischemia-reperfusion injury following acute myocardial infraction. Mouse cardiomyocytes HL-1 cells were subjected to oxygen glucose deprivation followed by reperfusion (OGD/R) to induce myocardial I/R injury. The expression of TUG1 was detected by real-time PCR. Overexpression or down expression of TUG1 was performed in mouse HL-1 cardiomyocytes. The myocardial cell viability and apoptosis were respectively detected. In addition, the expression levels of inflammatory factors, apoptosis-related proteins and HMGB1 proteins were detected. Besides, an inhibitor of HMGB1 was used to treat cells to verify the relationship between TUG1 and HMGB1 protein. The expression of TUG1 was significantly up-regulated in OGD/R-induced myocardial HL-1 cells. The overexpression of TUG1-induced inflammation and apoptosis in OGD-R-induced myocardial HL-1 cells. Knock down of TUG1 protected OGD/R-induced myocardial I/R injury by inhibiting HMGB1 expression. Suppression of lncRNA TUG1 may prevent myocardial I/R injury following acute myocardial infarction via inhibiting HMGB1 expression.


Assuntos
Proteína HMGB1/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia , RNA Longo não Codificante/genética , Animais , Apoptose/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Glucose/deficiência , Camundongos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo
8.
Artif Cells Nanomed Biotechnol ; 47(1): 3492-3499, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31432699

RESUMO

Background: Many lncRNAs have been recognized as critical regulatory factors in acute myocardial infarction (AMI). Herein, we further tested the influence of long non-coding RNA urothelial carcinoma associated 1 (UCA1) on cardiomyocytes injury in AMI, along with the role of microRNA-122 (miR-122) in this influence. Methods: Cardiomyocytes H9c2 was subjected to oxygen-glucose deprivation (OGD) stimulation. Cell viability and apoptosis were assessed. The UCA1 and miR-122 expressions were measured by qRT-PCR. Plasmids and miRNAs transfection were utilized to elevate UCA1 and miR-122 expressions. Subsequently, the influences of UCA1 and/or miR-122 overexpression on OGD-aroused H9c2 cell viability inhibition and apoptosis were probed. The AKT/mTOR and JNK/p38MAPK pathways in cells were analyzed. Results: OGD aroused H9c2 cell injury by suppressing cell viability and elevating cell apoptosis. Followed by OGD stimulation, the UCA1 expression was lowered in H9c2 cells. Overexpression of UCA1 weakened H9c2 cell injury aroused by OGD and declined miR-122 expression. Moreover, miR-122 attended to the influence of UCA1 overexpression on OGD-aroused H9c2 cell injury. Overexpression of UCA1 weakened OGD-aroused AKT/mTOR pathway inactivation and JNK/p38MAPK pathway activation by declining miR-122. Conclusion: UCA1-relieved OGD-aroused H9c2 cell injury might be achieved via declining miR-122 and then promoting AKT/mTOR pathway and suppressing JNK/p38MAPK pathway.


Assuntos
Glucose/deficiência , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo , RNA Longo não Codificante/genética , Linhagem Celular , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Artif Cells Nanomed Biotechnol ; 47(1): 2980-2988, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31322008

RESUMO

Background: Myocardial ischemia is a troublesome disease. Bilobalide possesses multiple biological functions. We researched the consequents of bilobalide in OGD-irritated H9c2 cells. Methods: OGD-stimulated H9c2 cells were treated by bilobalide, and/or transfected with miR-27a inhibitor or negative control. Use CCK-8 and flow cytometry to test cell activity and apoptosis, respectively. Luciferase activity experiment was to test targeting link between miR-27a and Tmub1. Levels of cell-cycle and apoptosis relative proteins and phosphorylation of PI3K/AKT and Wnt/ß-catenin related proteins were detected through western blot. Results: OGD stimulation reduced cell activity and negatively regulated the expression of CDK4, CDK6 and CyclinD1. Cell apoptosis was increased and its related proteins were affected by OGD. Bilobalide administration reversed all the results above caused by OGD. OGD negatively regulated miR-27a while bilobalide upregulated miR-27a. miR-27a's target gene was Tmub1. The protection consequents of bilobalide were suppressed when cells were transfected with a miR-27a inhibitor that cell activity was reduced and apoptosis was raised. Attenuation in the phosphorylation level of PI3K, AKT and ß-catenin by OGD was reversed by bilobalide, whereas there were opposite results after transfected with miR-27a inhibitor. Conclusion: Bilobalide relieved OGD-caused H9c2 cell damage, raising cell activity and attenuating apoptosis via upregulating miR-27a and activating of PI3K/AKT and Wnt/ß-catenin signal pathway. Highlights Bilobalide alleviates OGD-induced H9c2 cell injury. Bilobalide upregulates miR-27a expression in OGD-stimulated H9c2 cells. Bilobalide alleviates cell injury by upregulation of miR-27a. Bilobalide actuates PI3K/AKT and Wnt/ß-catenin pathways.


Assuntos
Ciclopentanos/farmacologia , Citoproteção/efeitos dos fármacos , Furanos/farmacologia , Ginkgolídeos/farmacologia , Glucose/deficiência , MicroRNAs/genética , Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Via de Sinalização Wnt/efeitos dos fármacos
10.
J Mol Neurosci ; 69(1): 49-59, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31187440

RESUMO

The destruction of the blood-brain barrier (BBB) contributes to a spectrum of neurological diseases such as stroke, and the hyperpermeability of endothelial cells is one of the characters of stroke, which is possibly exacerbated after reperfusion. However, the underlying mechanisms involving hyperpermeability after reperfusion between the endothelial cells remain poorly understood. Therefore, in the present study, the human microvascular endothelial cells (HBMECs) were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) to mimic ischemic stroke condition in vitro with the aim to investigate the potential mechanisms induced by OGD/R. The permeability of cultured HBMECs was measured using FITC-labeled dextran in a Transwell system and transendothelial electrical resistance (TEER), while the RhoA activity was detected by pull-down assay. In addition, the phosphorylation of MYPT1, which reflects the activation of ROCK and the internalization of VE-cadherin, was detected by Western blot. It showed that OGD/R treatment significantly increased the permeability of HBMEC monolayers and facilitated the internalization of VE-cadherin in HBMEC monolayers. Pull-down assay showed that RhoA activation was obviously enhanced after OGD/R treatment, while RhoA and ROCK inhibitor significantly reversed OGD/R-induced HBMEC monolayers hyperpermeability and the internalization of VE-cadherin. Meanwhile, the knockdown assay showed that RhoA small interfering RNA (siRNA) led to similar effects. The inactivation of the downstream effector protein ROCK was also examined. Intriguingly, ROCK2 rather than ROCK1 exerted its adverse effects on HBMEC monolayer integrity, since ROCK2 knockdown markedly reverses the injury of OGD/R in HBMEC monolayers. In conclusion, the present study provides evidence that OGD/R may induce HBMEC monolayer hyperpermeability via RhoA/ROCK2-mediated VE-cadherin internalization, which may provide an impetus for the development of therapeutics targeting BBB damage in ischemic stroke.


Assuntos
Caderinas/metabolismo , Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Glucose/deficiência , Oxigênio/metabolismo , Encéfalo/irrigação sanguínea , Hipóxia Celular , Células Cultivadas , Endocitose , Endotélio Vascular/citologia , Humanos , Microvasos/citologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
11.
Artif Cells Nanomed Biotechnol ; 47(1): 2274-2281, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31184214

RESUMO

The morphological feature of apoptosis is induced by oxygen and glucose deprivation (OGD) in cardiomyocytes H9c2 cells. Salvianolic acid B (Sal-B) has been studied in several pathological progresses, whereas it is still unclear whether maternally expressed gene 3 (MEG3) is an intermediate regulator during this progress. After pre-incubation with Sal-B and stimulation with OGD, viability and apoptosis of were examined in MEG3-overexpressed H9c2 cells. Cyclin D1, apoptosis-correlated proteins and regulators of signalling pathways were quantified with Western blot assay. MEG3 was detected by quantitative reverse transcription PCR (qRT-PCR). Sal-B was implicated in the enhancement of cell viability and suppression of apoptosis in OGD-treated H9c2 cells by repressing MEG3. In addition, MEG3 overexpression exerted an inhibitory effect on murine double minute 2 (MDM2) expression while aggrandized p53 expression in OGD-treated H9c2 cells which were pre-incubated with Sal-B. Furthermore, MEG3 overexpression abolished the up-regulative effect of Sal-B on phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in OGD-treated H9c2 cells. These results indicated that cardio-protective function of Sal-B might be ascribed to its down-regulatory property on MEG3 expression which hence blocks p53 and triggers AMPK activation in OGD-treated cells.


Assuntos
Benzofuranos/farmacologia , Cardiotônicos/farmacologia , Glucose/deficiência , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Camundongos , Miócitos Cardíacos/citologia , RNA Longo não Codificante/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
12.
Chem Biol Interact ; 309: 108705, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31199929

RESUMO

MicroRNAs have emerged as critical mediators of cerebral ischaemia/reperfusion injury. Recent studies have demonstrated that microRNA-302b-3p (miR-302b-3p) plays an important role in regulating apoptosis and oxidative stress in various cells. However, whether miR-302b-3p is involved in regulating cerebral ischaemia/reperfusion injury-induced neuronal apoptosis and oxidative stress remains unknown. In the present study, we explored the potential function and molecular mechanism of miR-302b-3p in oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced neuronal injury, using an in vitro model of cerebral ischaemia/reperfusion injury. We found that miR-302b-3p expression was up-regulated by OGD/R treatment in neurons. The inhibition of miR-302b-3p improved cell viability, and reduced apoptosis and the production of reactive oxygen species, showing a protective effect against OGD/R-induced injury. Interestingly, miR-302b-3p was shown to target and modulate murine fibroblast growth factor 15 (FGF15). Moreover, our results showed that miR-302b-3p down-regulation contributed to the promotion of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE)-mediated antioxidant signaling associated with the inactivation of glycogen synthase kinase-3ß. However, the knockdown of FGF15 significantly reversed the miR-302b-3p inhibition-mediated protective effect in OGD/R-treated neurons. Overall, these results demonstrated that miR-302b-3p inhibition confers a neuroprotective effect in OGD/R-treated neurons by up-regulating Nrf2/ARE antioxidant signaling via targeting FGF15, providing a novel target for neuroprotection in cerebral ischaemia/reperfusion injury.


Assuntos
Hipóxia Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Glucose , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Elementos de Resposta Antioxidante/genética , Linhagem Celular , Sobrevivência Celular , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/genética , Glucose/deficiência , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Neurônios/citologia , Neurônios/metabolismo , Neuroproteção , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Regulação para Cima
13.
Biomed Pharmacother ; 117: 108941, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200256

RESUMO

Ischemic stroke represents a major cause of adult physical disability, which is triggered by cerebral artery occlusion induced blood flow blockage. MiR-874-3p has been reported to be down-regulated in the brain injury induced by ischemia-reperfusion (I/R), but the direct evidence associated with injury of I/R remains unknown. In this study, we found that miR-874-3p levels significantly decreased in rat I/R brain induced by middle cerebral artery occlusion/reperfusion (MCAO/R) and SH-SY5Y cells following oxygen-glucose deprivation and reperfusion (OGD/R) treatment. Upregulation of miR-874-3p reduced infarct volumes and cell apoptosis in the in vivo I/R stroke model using TTC and TUNEL staining, as well as increased proliferation and inhibited apoptosis in OGD/R induced SH-SY5Y cells by CCK-8, Edu staining and flow cytometry analysis. Mechanistically, bioinformatics analysis and luciferase reporter assay confirmed BCL-2-modifying factor (BMF) and Bcl-2 family protein Bcl-rambo (BCL2L13) were the direct targets of miR-874-3p. Furthermore, BMF or BCL2L13 knockdown also provided significant protection against OGD/R induced injury, while their overexpression reversed the protective effects of miR-874-3p on SH-SY5Y cells following OGD/R. In summary, our results suggest that miR-874-3p attenuated ischemic injury by negatively regulating BMF and BCL2L13, highlighting a novel therapeutic target for ischemic stroke.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Isquemia Encefálica/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismo por Reperfusão/genética , Regulação para Cima/genética , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/genética , Sequência de Bases , Isquemia Encefálica/complicações , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo/genética , Glucose/deficiência , Humanos , Masculino , MicroRNAs/metabolismo , Oxigênio , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Traumatismo por Reperfusão/complicações
14.
Biol Pharm Bull ; 42(5): 837-839, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061328

RESUMO

In solid tumors, cancer cells are exposed to microenvironment stress, including hypoxia and insufficient nutrients. An acidic microenvironment in tumors is facilitated by the increase in synthesis of lactic acid; this is known as Warburg effect. We previously showed that B16F10 melanoma cells were induced autophagic cell death by glucose-deprivation stress, and lactic acid suppressed the cell death through the inhibition of autophagy. In this study, effects of lactic acid on cell death of B16F10 cells under hypoxic and glucose-depleted double stress conditions were investigated. The double stress promoted autophagic cell death earlier than glucose-depleted stress alone. Lactic acid repressed the double stress-induced cell death by inhibiting autophagy. These results suggest that lactic acid serves for cell survival under microenvironmental stress conditions in B16F10 melanoma cells.


Assuntos
Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Glucose/deficiência , Ácido Láctico/farmacologia , Melanoma Experimental , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Melanoma Experimental/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo
15.
J Mol Neurosci ; 69(1): 123-132, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31127537

RESUMO

Previous studies found that miR-374a-5p was decreased in infants suffering from the hypoxic-ischemic encephalopathy (HIE) compared with healthy control infants. However, the molecular mechanism of miR-374a-5p in the development of HIE remained unknown. This study is aimed to investigate the potential molecular pathway for shedding light on the treatment of HIE. An in vitro ischemia model in PC12 cells was established by oxygen/glucose deprivation (OGD). Reverse-transcription quantitative polymerase chain reaction and western blot were used to determine the levels of related genes or proteins in the OGD model or cells obtained from infants with HIE. Flow cytometry was conducted to quantify the apoptosis level of PC12 cells after OGD treatment. The TargetScan prediction algorithm was used to identify the potentially functional targets of miR-374a-5p. A dual-luciferase reporter assay was adopted to elucidate the sequences of miR-374a-5p binding to the 3'-UTR of potential target-PTEN. miR-374a-5p was downregulated in cells derived from human newborns with HIE, rat model with HIE, and PC12 cells after the OGD treatment. Inhibition of miR-374a-5p increased the expression of apoptotic markers and the apoptosis percentage of PC12 cells induced by OGD treatment while overexpression rescued the apoptosis. Meanwhile, PTEN expression was increased and suppressed after miR-374a-5p silence or overexpression, respectively. Upregulation of PTEN reversed the inhibitory effect of apoptotic markers elevation and PC12 cells apoptosis by the overexpression of miR-374a-5p after OGD treatment. PI3K pathway was required for the apoptosis effect caused by PTEN overexpression. We found that overexpression of miR-374a-5p reduced cell apoptosis through inhibiting PTEN/PI3K pathway in PC12 cells treated by OGD.


Assuntos
Apoptose , Hipóxia-Isquemia Encefálica/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Hipóxia Celular , Células Cultivadas , Glucose/deficiência , Humanos , Hipóxia-Isquemia Encefálica/genética , Recém-Nascido , Camundongos , MicroRNAs/metabolismo , Oxigênio/metabolismo , Células PC12 , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ratos
16.
Cell Mol Biol (Noisy-le-grand) ; 65(4): 63-68, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31078154

RESUMO

To investigate the expressions and roles of semaphorin3A (Sema3A) and vascular endothelial growth factor 165 (VEGF165) in cultured rat cortical neurons and vascular endothelial cells after oxygen glucose deprivation (OGD) stimulation. Cultured cortical neurons (NC) and vascular endothelial cells (VEC) of Sprague Dawley (SD) rats (SPF grade) were randomly divided into control group and OGD treatment group. Western blot assay, immunofluorescent staining and immunohistochemical methods were used to determine the expressions of VEGF165, Sema3A and neuropilin-1 (Nrp-1) protein. Cell migration was determined by Transwell, while TUNEL assay was used to measure apoptosis. The expressions of Sema3A, Nrp-1 and VEGF165 in NC and VEC cells after OGD treatment were up-regulated, when compared with the control group. With transfection of Sema3A shRNA, apoptosis of neurons decreased significantly after 2 h of OGD treatment, but the apoptosis of VEC cells was not obvious. The migration rate of VEC cells in the treatment group was significantly increased, relative to that of the control group. Stimulation with OGD induces neuronal expression of VEGF165 and regulates the migration of vascular endothelial cells, thereby enhancing their participation in angiogenesis, which may involve Sema3A.


Assuntos
Córtex Cerebral/patologia , Células Endoteliais/metabolismo , Glucose/deficiência , Neurônios/metabolismo , Oxigênio/metabolismo , Semaforina-3A/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Movimento Celular , Forma Celular , Células Cultivadas , Células Endoteliais/patologia , Neurônios/patologia , Neuropilina-1/metabolismo , Substâncias Protetoras/metabolismo , Ratos Sprague-Dawley
17.
Artif Cells Nanomed Biotechnol ; 47(1): 1797-1807, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31062620

RESUMO

This study aimed to investigate the potential effect of Scutellaria barbata D. Don (SBD) on oxygen glucose deprivation/reperfusion (OGD/R)-injured PC12 cells. PC12 cells were pretreated with various concentrations of 0.1-0.8 mg/ml SBD for indicated times (12-48 h) and then subjected to OGD/R injury. Cell viability, apoptosis and proliferation were detected using MTT assay, flow cytometry, Ki67 staining and western blot. Oxidative damage was assessed by detecting MDA content, SOD activity and GSH levels. The mitochondrial membrane potential (Δψm) was measured by Rh123 staining. Western blot was performed to assess the expression levels of Nrf2 and PI3K/AKT pathway-related proteins. We found that SBD pretreatment promoted cell viability and proliferation but inhibited apoptosis of OGD/R-injured PC12 cells in dosage- and time-dependent manner. Meanwhile, SBD attenuated oxidative damage and restored mitochondria dysfunction, as evidenced by the reduced MDA content, the increased SOD and GSH levels, and the increased Δψm. Furthermore, SBD induced the expression of Nrf2 in a PI3K/AKT-dependent signalling. Knockdown of Nrf2 blocked the protective effects of SBD on PC12 cells. In conclusion, this study demonstrates that SBD pretreatment protects PC12 cells against OGD/R-induced injury. The potential mechanism may be through up-regulating the expression of Nrf2 in a PI3K/AKT-dependent pathway.


Assuntos
Glucose/deficiência , Fator 2 Relacionado a NF-E2/metabolismo , Oxigênio/metabolismo , Extratos Vegetais/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Scutellaria/química , Regulação para Cima/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
18.
Oxid Med Cell Longev ; 2019: 8739730, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31073355

RESUMO

Transplantation of neural stem cells (NSCs) is a promising therapy for ischemic stroke. However, the effectiveness of this approach is limited by grafted cell death. Breast cancer susceptibility protein 1 (BRCA1) could suppress apoptosis in neural progenitors and modulate oxidative stress in neurons. In this study, we found that BRCA1 was upregulated by oxygen-glucose deprivation/reoxygenation (OGD/R). Overexpression of BRCA1 in NSCs reduced cell apoptosis and oxidative stress after OGD/R insult. The molecule overexpression also stimulated cellular proliferation in OGD/R NSCs and increased the survival rate of grafted cells. Further, the transplantation of BRCA1-transfected NSCs into mice with ischemic stroke increased brain-derived neurotropic factor and nerve growth factor expression in the brain and elicited neurological function improvement. In addition, we found that RING finger domain and BRCT domain of BRCA1 could physically interact with p53 in NSCs. The cross talk between BRCA1 RING finger domain and p53 was responsible for p53 ubiquitination and degradation. Our findings indicate that modification with BRCA1 could enhance the efficacy of NSCs transplantation in ischemic stroke.


Assuntos
Proteína BRCA1/metabolismo , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/terapia , Células-Tronco Neurais/transplante , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia , Animais , Apoptose , Isquemia Encefálica/complicações , Isquemia Encefálica/patologia , Proliferação de Células , Sobrevivência Celular , Glucose/deficiência , Masculino , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Estresse Oxidativo , Oxigênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/patologia , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
19.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091659

RESUMO

Dysregulation of cellular energy metabolism is closely linked to cancer development and progression. Calorie or glucose restriction (CR or GR) inhibits energy-dependent pathways, including IGF-1/PI3K/Akt/mTOR, in cancer cells. However, alterations in proton dynamics and reversal of the pH gradient across the cell membrane, which results in intracellular alkalinization and extracellular acidification in cancer tissues, have emerged as important etiopathogenic factors. We measured glucose, lactate, and ATP production after GR, plant-derived CR-mimetic curcumin treatment, and curcumin plus GR in human hepatoma cells. Intracellular pH regulatory effects, in particular, protein-protein interactions within mTOR complex-1 and its structural change, were investigated. Curcumin treatment or GR mildly inhibited Na+/H+ exchanger-1 (NHE1). vATPase, monocarboxylate transporter (MCT)-1, and MCT4 level. Combination treatment with curcumin and GR further enhanced the inhibitory effects on these transporters and proton-extruding enzymes, with intracellular pH reduction. ATP and lactate production decreased according to pH change. Modeling of mTOR protein revealed structural changes upon treatments, and curcumin plus GR decreased binding of Raptor and GßL to mTOR, as well as of Rag A and Rag B to Raptor. Consequently, 4EBP1 phosphorylation was decreased and cell migration and proliferation were inhibited in a pH-dependent manner. Autophagy was increased by curcumin plus GR. In conclusion, curcumin treatment combined with GR may be a useful supportive approach for preventing intracellular alkalinization and cancer progression.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Curcumina/farmacologia , Glucose/deficiência , Neoplasias Hepáticas/metabolismo , Álcalis/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Glucose/metabolismo , Células Hep G2 , Humanos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
20.
Neurochem Res ; 44(8): 1807-1817, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31093905

RESUMO

Cerebral ischemic injury is a leading cause of human mortality and disability, seriously threatening human health in the world. Activin A (Act A), as a well-known neuroprotective factor, could alleviate ischemic brain injury mainly through Act A/Smads signaling. In our previous study, a noncanonical Act A/Smads signal loop with self-amplifying property was found, which strengthened the neuroprotective effect of Act A. However, this neuroprotective effect was limited due to the self-limiting behavior mediated by Smad anchor for receptor activation (SARA) protein. It was reported that microRNA-17-5p (miR-17-5p) could suppress the expression of SARA in esophageal squamous cell carcinoma. Thus we proposed that knockdown of miR-17-5p could strengthen the neuroprotective effect of Act A/Smads signal loop through SARA. To testify this hypothesis, oxygen-glucose deficiency (OGD) was introduced to highly differentiated rattus pheochromocytoma (PC12) cells. After the transfection of miR-17-5p mimic or inhibitor, the activity of Act A signal loop was quantified by the expression of phosphorylated Smad3. The results showed that suppression of miR-17-5p up-regulated the expression of SARA protein, which prolonged and strengthened the activity of Act A signaling through increased phosphorylation of downstream Smad3 and accumulation of Act A ligand. Further luciferase assay confirmed that SARA was a direct target gene of miR-17-5p. These practical discoveries will bring new insight on the endogenous neuroprotective effects of Act A signal loop by interfering a novel target: miR-17-5p.


Assuntos
Subunidades beta de Inibinas/metabolismo , MicroRNAs/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Hipóxia Celular , Técnicas de Silenciamento de Genes , Glucose/deficiência , Isquemia/genética , Isquemia/metabolismo , Neuroproteção , Células PC12 , Ratos , Transdução de Sinais , Proteína Smad3/metabolismo , Regulação para Cima
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