RESUMO
Hippocampal neuronal plasticity is a fundamental process underpinning learning and memory formation and requiring elaborate molecular mechanisms that result in the dynamic remodelling of synaptic connectivity. The neurotrophic properties of midkine (Mdk) have been implicated in the development and repair of the nervous system, while Mdk knockout resulted in deficits in the formation of certain types of memory. The role of Mdk in the process of memory-associated neuronal plasticity, however, remains poorly understood. We investigated the learning-induced regulation of Mdk in spatial navigation and association learning using the water maze and the odour reward association learning paradigms, characterising a temporal profile of Mdk protein expression post-learning. Both learning events revealed similar patterns of upregulation of expression of the protein in the rat hippocampal dentate gyrus, which were rapid and transient. Moreover, administration of recombinant Mdk during the endogenous Mdk upregulation following learning enhanced memory in the water maze task revealing a pro-cognitive action of Mdk. We further show that, within the adult hippocampus, Mdk mRNA is predominantly expressed in granular and pyramidal neurons and that hippocampal neuronal Mdk expression is regulated by the canonical plasticity-associated neurotransmitter glutamate. Finally, we confirm that the positive action of Mdk on neurite outgrowth previously noted in cortical and cerebellar neurons extends to hippocampal neurons. Together, our findings suggest a role for Mdk in glutamate-mediated hippocampal neuronal plasticity important for long-term memory consolidation.
Assuntos
Hipocampo , Memória , Midkina , Recompensa , Regulação para Cima , Animais , Midkina/metabolismo , Masculino , Regulação para Cima/fisiologia , Ratos , Hipocampo/metabolismo , Memória/fisiologia , Aprendizagem por Associação/fisiologia , Aprendizagem em Labirinto/fisiologia , Plasticidade Neuronal/fisiologia , Aprendizagem Espacial/fisiologia , Ratos Sprague-DawleyRESUMO
The primary pathological change in Parkinson's disease (PD) is the progressive degeneration of dopaminergic neurons in the substantia nigra. Additionally, excessive microglial activation and synaptic loss are also typical features observed in PD samples. Exercise trainings have been proven to improve PD symptoms, delay the disease progression as well as affect excessive microglial synaptic phagocytosis. In this study, we established a mouse model of PD by injecting mouse-derived α-synuclein preformed fibrils (M-α-syn PFFs) into the substantia nigra, and demonstrated that treadmill exercise inhibits microglial activation and synaptic phagocytosis in striatum. Using RNA-Seq and proteomics, we also found that PD involves excessive activation of the complement pathway which is closely related to over-activation of microglia and abnormal synaptic function. More importantly, exercise training can inhibit complement levels and complement-mediated microglial phagocytosis of synapses. It is probably triggered by CD55, as we observed that CD55 in the striatum significantly increased after exercise training and up-regulation of that molecule rescued motor deficits of PD mice, accompanied with reduced microglial synaptic phagocytosis in the striatum. This research elucidated the interplay among microglia, complement, and synapses, and analyzed the effects of exercise training on these factors. Our work also suggested CD55 as a complement-relevant candidate molecule for developing therapeutic strategies of PD.
Assuntos
Antígenos CD55 , Proteínas do Sistema Complemento , Camundongos Endogâmicos C57BL , Doença de Parkinson , Fagocitose , Condicionamento Físico Animal , Sinapses , Regulação para Cima , Animais , Fagocitose/fisiologia , Camundongos , Condicionamento Físico Animal/fisiologia , Condicionamento Físico Animal/métodos , Regulação para Cima/fisiologia , Sinapses/metabolismo , Sinapses/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Proteínas do Sistema Complemento/metabolismo , Antígenos CD55/metabolismo , Microglia/metabolismo , Masculino , alfa-Sinucleína/metabolismo , Modelos Animais de DoençasRESUMO
AIMS: Physical exercise (PE) can accelerate post-stroke recovery. This study investigated contributions of circRNAs to PE-induced improvements in post-stroke neurological function. METHODS: Rats subjected to transient middle cerebral artery occlusion were left sedentary or provided running-wheel access for 4 weeks during recovery. CircRNAs from peri-infarct cortex were identified by high-throughput sequencing, and interactions with miRNAs by immunoprecipitation, fluorescence in suit hybridization, and dual-luciferase reporter assays. In vivo circRNA knockdown was achieved using shRNA-AAVs and in vitro overexpression by plasmid transfection. Transmission electron microscopy, western blotting, and TUNEL assays were conducted to explore circRNA contributions to endoplasmic reticulum (ER) stress and neuronal apoptosis. CircRNA levels were measured in plasma from stroke patients by qRT-PCR and associations with neurological scores assessed by Pearson's correlation analysis. RESULTS: PE upregulated circAnks1b, reduced infarct volume, and mitigated neurological dysfunction, while circAnks1b knockdown exacerbated neurological dysfunction and increased infarct size despite PE. CircAnks1b sponged miR-130b-5p, thereby disinhibiting Pak2 expression. Conversely, Pak2 downregulation disrupted PE-mediated protective ER stress, leading to reduced IRE1/XBP1 and heightened apoptosis. Plasma circAnks1b was higher in stroke patients receiving PE than sedentary patients and correlated negatively with neurological scores. CONCLUSIONS: CircAnks1b upregulation may be an effective therapeutic strategy for post-stroke recovery.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , AVC Isquêmico , MicroRNAs , Condicionamento Físico Animal , RNA Circular , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para Cima , Animais , Apoptose/fisiologia , MicroRNAs/metabolismo , MicroRNAs/genética , Ratos , Estresse do Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/genética , RNA Circular/metabolismo , RNA Circular/genética , Masculino , AVC Isquêmico/metabolismo , AVC Isquêmico/genética , AVC Isquêmico/patologia , Regulação para Cima/fisiologia , Humanos , Condicionamento Físico Animal/fisiologia , Condicionamento Físico Animal/métodos , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/genética , Modelos Animais de Doenças , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The aim of this study was to develop an animal model to investigate whether prolonged intensive endurance exercise induces RV remodeling, taking into account the involvement of Wnt/ß-catenin signaling. METHODS: Four-week-old male Wistar rats (100 to 125 g) were assigned to four groups (n = 8/group): 1) sixteen weeks of intensive (36 m/min) exercise (INT), 2) twelve weeks of the intensive exercise followed by four weeks of moderate intensity (18 m/min) exercise (INT + MOD), 3) twelve weeks of the intensive exercise followed by four weeks of detraining (INT + DT), and 4) sedentary rats (SED). The exercise protocols were performed five days a week for one h/day. Echocardiography, real-time PCR, western blotting, and histological staining were performed at the end of week sixteen. RESULTS: INT rats developed concentric hypertrophy without diastolic dysfunction compared to SED (p = 0.006) and INT + DT (p = 0.035). Wnt1, ß-catenin and CyclinD1 proteins in the training groups were significantly higher than SED rats (p < 0.001). Interestingly, INT rats had higher protein levels than INT + DT and INT + MOD (p < 0.001), with higher gene expression compared to SED rats (p < 0.05). There was also a significant increase in collagen deposition in INT rats compared to SED (p = 0.046) and INT + DT (p = 0.034). Furthermore, INT + MOD and INT + DT rats did not show any adverse structural, functional, or histological changes. CONCLUSIONS: Long-term intensive endurance training seems to be associated with increased collagen deposition and wall thickness in the RV through Wnt/ß-catenin signaling (which is concentration dependent), without changes in diastolic function. CLINICAL PERSPECTIVE: Over the past decades, there has been an ongoing debate about whether the structural and functional adaptations of the cardiovascular system in trained endurance athletes are benign physiological responses to training or potentially pathological changes related to disease. While the adaptations of the left heart are well-documented, the remodeling of the right heart remains a subject of discussion. To gain insights into the ability of sustained high-intensity exercise to cause adverse right ventricular (RV) remodeling, we conducted an experimental study in which male rats were trained to run vigorously for 1 h daily over a 16-week period and compared them to a parallel group of sedentary control rats. Our findings revealed that intense long-term exercise induced morphological changes along with fibrosis affecting the RV. These fibrotic changes were a result of the 16-week vigorous exercise training regimen. If these results are confirmed in humans, they suggest that prolonged high-intensity endurance exercise training may lead to adverse cardiac remodeling. Our findings have important potential implications for the assessment of cardiac remodeling in individuals engaged in high-level exercise training.
Assuntos
Condicionamento Físico Animal , Ratos Wistar , Remodelação Ventricular , Via de Sinalização Wnt , Animais , Masculino , Ratos , beta Catenina/metabolismo , Treino Aeróbico , Condicionamento Físico Animal/fisiologia , Resistência Física/fisiologia , Regulação para Cima/fisiologia , Remodelação Ventricular/fisiologia , Via de Sinalização Wnt/fisiologiaRESUMO
Central nervous system oxygen toxicity (CNS-OT) is a complication of hyperbaric oxygen (HBO) treatment, with limited prevention and treatment options available. In this study, we aimed to explore the effect of polyethylene glycol 300 (PEG300) on CNS-OT and underlying mechanisms. Motor and cognitive functions of mice in normobaric conditions were evaluated by Morris water maze, passive active avoidance, and rotarod tests. HBO was applied at 6 atmospheres absolute (ATA) for 30 min after drug administration. The latency period of convulsion in mice was recorded, and hippocampal tissues were extracted for biochemical experiments. Our experimental results showed that PEG300 extended the convulsion latencies in CNS-OT mice, reduced oxidative stress and inflammation levels in hippocampal tissues. Furthermore, PEG300 preserved mitochondrial integrity and maintained mitochondrial membrane potential in hippocampal tissue by upregulating Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha (PGC-1α). This protective effect was enhanced following the administration of ZLN005, an agonist of PGC-1a. Hence, our study suggests that PEG300 might exert protective effects by upregulating PGC-1α expression and preserving mitochondrial health, offering promising prospects for CNS-OT treatment.
Assuntos
Hipocampo , Mitocôndrias , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Polietilenoglicóis , Regulação para Cima , Animais , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Masculino , Polietilenoglicóis/toxicidade , Polietilenoglicóis/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Oxigênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologiaRESUMO
NADPH-oxidase (NOX) is a multi-subunit enzyme complex. The upregulation of NOX causes massive production of superoxide (O2¯), which avidly reacts with nitric oxide (NO) and increases cellular reactive oxygen/nitrogen species (ROS/RNS). Increased ROS/RNS plays pivotal role in the sporadic Alzheimer's disease (sAD) development and brain damage following impaired insulin signaling. Hence, this study aimed to examine early-time course of changes in NOX and NOS expression, and apoptotic proteins in the rats hippocampi following insulin signaling impairment [induced by STZ injection; intraperitoneal (IP) or in cerebral ventricles (ICV)]. Early effects (1, 3, or 6 weeks) on the NOX activity, translocation of NOX subunits from cytosol to the membrane, NO-synthases [neuronal-, inducible- and endothelial-NOS; nNOS, iNOS and eNOS], The Rac-1 protein expression, levels of NO and O2¯, cytochrome c release, caspase-3 and 9 activations (cleavage) were studied. STZ injection (in both models) increased NOX activity, O2¯ production, and enhanced cytosolic subunits translocation into membrane. The iNOS but not nNOS and eNOS expression and NO levels were increased in STZ treated rats. Finally, STZ injection increased cytochrome c release, caspase-3 and 9 activations in a manner that was significantly associated with levels of O2¯ and NO in the hippocampus. ICV-STZ administration resulted in significant profound changes over the IP route. In conclusion, impairment in insulin function induces early changes in ROS/RNS contents through NOX and iNOS upregulation and neuronal apoptosis in the hippocampus. Our results could mechanistically explain the role of impaired insulin function in the development of sAD.
Assuntos
Doença de Alzheimer , Apoptose , Hipocampo , Insulina , NADPH Oxidases , Óxido Nítrico Sintase Tipo II , Animais , Masculino , Ratos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Apoptose/fisiologia , Caspase 3/metabolismo , Citocromos c/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Insulina/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Estreptozocina , Regulação para Cima/fisiologiaRESUMO
Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.
Assuntos
Metilenotetra-Hidrofolato Desidrogenase (NADP) , Doença de Newcastle , Vírus da Doença de Newcastle , Replicação Viral , Animais , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Doença de Newcastle/enzimologia , Doença de Newcastle/fisiopatologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Nucleotídeos/metabolismo , Serina/metabolismo , Replicação Viral/genética , Linhagem Celular , Células A549 , Humanos , Mesocricetus , Técnicas de Silenciamento de Genes , Transporte Proteico/genética , Mitocôndrias/enzimologia , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: The protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway regulates early follicular activation and follicular pool maintenance in female germline cells. Fragile X mental retardation 1 (FMR1) regulates folliculogenesis and it is variably expressed in patients with Premature Ovary Insufficiency. FMR1 expression is supposed to be linked to AKT/mTOR signaling in an ovarian response dependent manner as demonstrated in recent in vitro and in vivo studies in the female germline in vitro and in vivo. METHODS: We evaluated changes in the expression of AKT/mTOR signaling pathway genes by real time PCR in the peripheral blood of 74 patients with Premature Ovarian Insufficiency and 56 fertile controls and correlated their expression with FMR1 expression. RESULTS: Expression of the genes AKT1, TSC2, mTOR, and S6K was significantly more abundant in patients with POI than in the controls. For AKT1, TSC2 and mTOR, gene expression was not affected by FMR1-CGG repeat number in the 5´-untranslated region. FMR1 and S6K expression levels, however, were significantly upregulated in patients with POI and an FMR1 premutation. Independent of a premutation, expression of mTOR, S6K, and TSC2 was significantly correlated with that of FMR1 in all patients. Furthermore, when grouped according to ovarian reserve, this effect remained significant only for mTOR and S6K, with higher significance note in patients with Premature Ovarian Insufficiency than in the controls. CONCLUSIONS: In Premature ovarian insufficiency patients, activation of AKT/mTOR signaling pathway is remarkable and putatively pathognomonic. Additionally, it seems to be triggered by an FMR1/mTOR/S6K linkage mechanism, most relevant in premutation carriers.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Insuficiência Ovariana Primária , Proteínas Proto-Oncogênicas c-akt , Serina-Treonina Quinases TOR , Adulto , Estudos de Casos e Controles , Feminino , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Regulação da Expressão Gênica , Humanos , Reserva Ovariana/genética , Insuficiência Ovariana Primária/sangue , Insuficiência Ovariana Primária/genética , Proteínas Proto-Oncogênicas c-akt/sangue , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/sangue , Serina-Treonina Quinases TOR/genética , Regulação para Cima/fisiologiaRESUMO
Sp1 transcription factor regulates genes involved in various phenomena of tumor progression. Vasculogenic mimicry (VM) is the alternative neovascularization by aggressive tumor cells. However, there is no evidence of the relationship between Sp1 and VM. This study investigated whether and how Sp1 plays a crucial role in the process of VM in human prostate cancer (PCa) cell lines, PC-3 and DU145. A cell viability assay and three-dimensional culture VM tube formation assay were performed. Protein and mRNA expression levels were detected by Western blot and reverse transcriptase-polymerase chain reaction, respectively. The nuclear twist expression was observed by immunofluorescence assay. A co-immunoprecipitation assay was performed. Mithramycin A (MiA) and Sp1 siRNA significantly decreased serum-induced VM, whereas Sp1 overexpression caused a significant induction of VM. Serum-upregulated vascular endothelial cadherin (VE-cadherin) protein and mRNA expression levels were decreased after MiA treatment or Sp1 silencing. The protein expression and the nuclear localization of twist were increased by serum, which was effectively inhibited after MiA treatment or Sp1 silencing. The interaction between Sp1 and twist was reduced by MiA. On the contrary, Sp1 overexpression enhanced VE-cadherin and twist expressions. Serum phosphorylated AKT and raised matrix metalloproteinase-2 (MMP-2) and laminin subunit 5 gamma-2 (LAMC2) expressions. MiA or Sp1 silencing impaired these effects. However, Sp1 overexpression upregulated phosphor-AKT, MMP-2 and LAMC2 expressions. Serum-upregulated Sp1 was significantly reduced by an AKT inhibitor, wortmannin. These results demonstrate that Sp1 mediates VM formation through interacting with the twist/VE-cadherin/AKT pathway in human PCa cells.
Assuntos
Neovascularização Patológica/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fator de Transcrição Sp1/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Neovascularização Patológica/patologia , Células PC-3 , RNA Mensageiro/metabolismo , Regulação para Cima/fisiologiaRESUMO
Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Endométrio/metabolismo , Proteoma/metabolismo , Adulto , Antígenos de Diferenciação/metabolismo , Regulação para Baixo/fisiologia , Endométrio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Oncogenes/fisiologia , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Regulação para Cima/fisiologiaRESUMO
AIM: Aging-related dysfunction of retinal pigment epithelium (RPE) is the main pathogenic factors for pathological angiogenesis due to dysregulated vascular endothelial growth factor (VEGF) in retinal vascular diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). However, the molecular mechanism behind the up-regulation of VEGF in senescent RPE is still blurred. MATERIALS AND METHODS: As oxidative damage is the key cause of RPE dysfunction, we employed a model of oxidative stress-induced premature senescence of ARPE-19 to explore the effect of senescent RPE on VEGF. KEY FINDINGS: We reported that senescent ARPE-19 up-regulated VEGF expression under both short-term and prolonged H2O2 treatment, accompanying with increased HIF-1α, the key mediator of VEGF. STING signaling, which could be activated by oxidative stress-damaged DNA, was also observed to be increased in senescent ARPE-19 treated with H2O2. And the inhibition of STING significantly reduced HIF-1α expression to alleviate the up-regulation of VEGF. NF-κB was also shown to be involved in the regulation of VEGF in senescent ARPE-19 in response to STING signaling. Furthermore, oxidative stress impaired the lysosomal clearance of damaged DNA to enhance STING signaling, thereby up-regulating VEGF expression in senescent RPE. SIGNIFICANCE: Our data provide evidence that STING plays an important role in VEGF regulation in senescent RPE induced by oxidative stress.
Assuntos
Senescência Celular/fisiologia , Degeneração Macular/metabolismo , Proteínas de Membrana/biossíntese , Estresse Oxidativo/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Senescência Celular/efeitos dos fármacos , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Peróxido de Hidrogênio/toxicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Degeneração Macular/patologia , NF-kappa B/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Endothelial defects significantly contribute to cardiovascular pathology in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Using an endothelium-specific progeria mouse model, we identify a novel, endothelium-specific microRNA (miR) signature linked to the p53-senescence pathway and a senescence-associated secretory phenotype (SASP). Progerin-expressing endothelial cells exert profound cell-non-autonomous effects initiating senescence in non-endothelial cell populations and causing immune cell infiltrates around blood vessels. Comparative miR expression analyses revealed unique upregulation of senescence-associated miR34a-5p in endothelial cells with strong accumulation at atheroprone aortic arch regions but also, in whole cardiac- and lung tissues as well as in the circulation of progeria mice. Mechanistically, miR34a-5p knockdown reduced not only p53 levels but also late-stage senescence regulator p16 with no effect on p21 levels, while p53 knockdown reduced miR34a-5p and partially rescued p21-mediated cell cycle inhibition with a moderate effect on SASP. These data demonstrate that miR34a-5p reinforces two separate senescence regulating branches in progerin-expressing endothelial cells, the p53- and p16-associated pathways, which synergistically maintain a senescence phenotype that contributes to cardiovascular pathology. Thus, the key function of circulatory miR34a-5p in endothelial dysfunction-linked cardiovascular pathology offers novel routes for diagnosis, prognosis and treatment for cardiovascular aging in HGPS and potentially geriatric patients.
Assuntos
Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/fisiologia , Lamina Tipo A/metabolismo , MicroRNAs/metabolismo , Progéria/metabolismo , Regulação para Cima/fisiologia , Envelhecimento , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aterosclerose/metabolismo , Senescência Celular , Regulação para Baixo , Lamina Tipo A/genética , Camundongos , MicroRNAs/genética , Comunicação Parácrina/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Thymic stromal lymphopoietin (TSLP), a type I cytokine belonging to the IL-2 cytokine family, promotes Th2-mediated inflammatory responses. The aim of this study is to investigate whether TSLP increases inflammatory responses via induction of autophagy using a murine T cell lymphoma cell line, EL4 cells, and lipopolysaccharide (LPS)-injected mice. TSLP increased expression levels of autophagy-related factors, such as Beclin-1, LC3-II, p62, Atg5, and lysosome associated membrane protein 1/2, whereas these factors increased by TSLP disappeared by neutralization of TSLP in EL4 cells. TSLP activated JAK1/JAK2/STAT5/JNK/PI3K, while the blockade of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways reduced the expression levels of Beclin-1, LC3-II, and p62 in TSLP-stimulated EL4 cells. In addition, TSLP simultaneously increased levels of inflammatory cytokines via induction of autophagy by activation of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways. In an LPS-induced acute liver injury (ALI) mouse model, exogenous TSLP increased expression levels of Beclin-1 and LC3-II, whereas functional deficiency of TSLP by TSLP siRNA resulted in lower expression of Beclin-1, LC3-II, and inflammatory cytokines, impairing their ability to form autophagosomes in ALI mice. Thus, our findings show a new role of TSLP between autophagy and inflammatory responses. In conclusion, regulating TSLP-induced autophagy may be a potential therapeutic strategy for inflammatory responses.
Assuntos
Autofagia/fisiologia , Citocinas/metabolismo , Inflamação/metabolismo , Células Th2/metabolismo , Animais , Células Cultivadas , Hepatopatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Linfopoietina do Estroma do TimoRESUMO
BACKGROUND: Bladder cancer (BC) is a common cause of cancer-relevant deaths globally. This study is designed to delve into expressions, biological functions and molecular mechanisms of circ_0000515 in BC. METHODS: Quantitative real-time polymerase chain reaction was accomplished to examine circ_0000515, miR-542-3p and integrin-linked kinase (ILK) mRNA expressions in BC tissues and cell lines. In RT-4 and RT-112 cells with circ_0000515 depletion and UMUC3 and BIU-87 cells with this circ RNA overexpression, a cell counting kit-8 assay was adopted to monitor the viability. Besides, transwell assay was conducted to detect cell migration and aggressiveness, and luciferase reporter gene assay was applied to probe the interplay among circ_0000515, miR-542-3p and ILK mRNA. Additionally, Besides, the regulatory function of circ_0000515 on miR-542-3p expression was under the assay of quantitative real-time polymerase chain reaction, and western blot was fulfilled to determine the regulative function of circ_0000515/miR-542-3p axis on ILK protein expressions. A xenograft animal was modeled to examine lung metastasis in vivo. RESULTS: Circ_0000515 and ILK expressions were significantly elevated in BC tissues and cell lines, while that of miR-542-3p was dramatically suppressed. Knocking down circ_0000515 could significantly repress the growth, migration and aggressiveness of BC cells while overexpression of circ_0000515 showed opposite effects. Moreover, circ_0000515 knockdown inhibited pulmonary metastasis in vivo. Circ_0000515 was validated to adsorb miR-542-3p, and ILK was testified as the downriver target of miR-542-3p. Circ_0000515 could ascend ILK expression through repressing that of miR-542-3p. CONCLUSIONS: Circ_0000515, as a tumor promoter, strengthens the viability, migration and aggressiveness of BC cells via modulating miR-542-3p/ILK axis.
Assuntos
MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Circular/metabolismo , Regulação para Cima/fisiologia , Neoplasias da Bexiga Urinária/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Feminino , Regulação da Expressão Gênica , Humanos , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Neoplasias Experimentais , Proteínas Serina-Treonina Quinases/genética , RNA Circular/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
OBJECTIVE: Sepsis is a leading cause of morbidity and mortality after surgery. We aimed to explore the role of long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) sponging microRNA-26a-5p in sepsis-induced myocardial injury by regulating regulator of calcineurin 2 (Rcan2). METHODS: HL-1 cells were incubated with lipopolysaccharide (LPS) to induce in vitro cardiomyocyte injury models, which were then treated with silenced MALAT1 vector, miR-26a-5p mimic or Rcan2 overexpression vector. Next, inflammatory factor level and apoptosis of cells were determined. The in vivo mouse models were constructed by intraperitoneal injection of LPS. The modeled mice were injected with relative oligonucleotides and the pathology, apoptosis, and inflammation in mouse myocardial tissues were assessed. Expression of MALAT1, miR-26a-5p and Rcan2 in vivo and in vitro was evaluated. RESULTS: MALAT1 and Rcan2 were upregulated while miR-26a-5p was downregulated in LPS-treated HL-1 cells and mice. MALAT1 silencing or miR-26a-5p upregulation suppressed LPS-induced inflammation and apoptosis of cardiomyocytes in cellular and animal models. These effects of elevated miR-26a-5p could be reversed by upregulating Rcan2, and MALAT1 knockdown-induced ameliorative impacts could be reversed by miR-26a-5p downregulation. CONCLUSION: MALAT1 silencing elevated miR-26a-5p to ameliorate LPS-induced myocardial injury by reducing Rcan2. Our research may provide novel biomarkers for the treatment of sepsis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/metabolismo , Isquemia Miocárdica/fisiopatologia , RNA Longo não Codificante/metabolismo , Sepse/fisiopatologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/etiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Sepse/complicações , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Diabetic retinopathy (DR) is a disease that can cause blindness. Bone morphogenetic protein-4 (BMP4) was reported be overexpressed in DR model. However, the specific mechanism of BMP4 in DR development has not been explored. MiR-340-5p and BMP4 levels were detected by RT-qPCR in MIO-M1 cells and retinas of mice. Western blot analysis was used to examine GFAP, BMP4 and BRB junction protein levels. Inflammatory cytokine secretion and the retina structure were examined by ELISA and H&E staining, respectively. The interaction between miR-340-5p and BMP4 was identified by luciferase reporter assay. In HG-stimulated MIO-M1 cells, BMP4 was upregulated. Mechanically, BMP4 was targeted by miR-340-5p and negatively regulated by miR-340-5p. In rescue assays, BMP4 countervailed the suppressive effects of miR-340-5p on activation of Müller cells and release of inflammatory cytokines. Additionally, miR-18a-3p overexpression alleviated BRB injury to inhibit DR progression in vivo. In conclusion, miR-340-5p inhibits DR progression by targeting BMP4, which may offer a new pathway for treatment of DR.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Citocinas/metabolismo , Células Ependimogliais/metabolismo , Inflamação/metabolismo , MicroRNAs/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Diabetes Mellitus/metabolismo , Retinopatia Diabética , Células Endoteliais/metabolismo , Glucose/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Retina/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologiaRESUMO
Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Cisplatin (CDDP) has been used as a standard adjuvant therapy for advanced NSCLC patients, while chemoresistance is one of the most challenging problems to limit its clinical application. Our data showed that the expression of visfatin was significantly increased in CDDP resistant NSCLC cells as compared with that in their parental cells, while knockdown of visfatin or its neutralization antibody can restore the CDDP sensitivity of resistant NSCLC cells. The upregulation of visfatin in CDDP resistant NSCLC cells was due to the increased mRNA stability and promoter activity. Further, we found that signal transducer and activator of transcription 3 (STAT3), which was increased in chemoresistant cells, can increase the transcription of visfatin. While tristetraprolin (TTP), which can decease mRNA stability of visfatin, was decreased in chemoresistant cells. Inhibition of STAT3 or over expression of TTP can restore CDDP sensitivity of resistant NSCLC cells. Collectively, our data showed that STAT3 and TTP-regulated expression of visfatin was involved in CDDP resistance of NSCLC cells. It indicated that targeted inhibition of visfatin should be a potential approach to overcome CDDP resistance of NSCLC treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Pulmonares/fisiopatologia , Nicotinamida Fosforribosiltransferase/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Estabilidade de RNA/fisiologia , Fator de Transcrição STAT3/metabolismo , Tristetraprolina/metabolismo , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Acute kidney injury (AKI) is both a consequence and determinant of outcomes in COVID-19. The kidney is one of the major organs infected by the causative virus, SARS-CoV-2. Viral entry into cells requires the viral spike protein, and both the virus and its spike protein appear in the urine of COVID-19 patients with AKI. We examined the effects of transfecting the viral spike protein of SARS-CoV-2 in kidney cell lines. METHODS: HEK293, HEK293-ACE2+ (stably overexpressing ACE2), and Vero E6 cells having endogenous ACE2 were transfected with SARS-CoV-2 spike or control plasmid. Assessment of gene and protein expression, and syncytia formation was performed, and the effects of quercetin on syncytia formation examined. FINDINGS: Spike transfection in HEK293-ACE2+ cells caused syncytia formation, cellular sloughing, and focal denudation of the cell monolayer; transfection in Vero E6 cells also caused syncytia formation. Spike expression upregulated potentially nephrotoxic genes (TNF-α, MCP-1, and ICAM1). Spike upregulated the cytoprotective gene HO-1 and relevant signaling pathways (p-Akt, p-STAT3, and p-p38). Quercetin, an HO-1 inducer, reduced syncytia formation and spike protein expression. INTERPRETATION: The major conclusions of the study are: 1) Spike protein expression in kidney cells provides a relevant model for the study of maladaptive and adaptive responses germane to AKI in COVID-19; 2) such spike protein expression upregulates HO-1; and 3) quercetin, an HO-1 inducer, may provide a clinically relevant/feasible protective strategy in AKI occurring in the setting of COVID-19. FUNDING: R01-DK119167 (KAN), R01-AI100911 (JPG), P30-DK079337; R01-DK059600 (AA).
Assuntos
COVID-19/metabolismo , Heme Oxigenase-1/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Quercetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Células Vero , Internalização do Vírus/efeitos dos fármacosRESUMO
Abdominal aortic aneurysm (AAA) has been associated with the dysfunction of vascular smooth muscle cells (VSMCs) and extracellular matrix (ECM) remodelling. Runt-related transcription factor 3 (RUNX3) has been reported to be up-regulated in aneurysmal aorta samples compared with normal aorta. However, its function in VSMCs and the mechanism of function remains unknown. Therefore, our study aimed to investigate the role of RUNX3 in ECM remodelling and VSMC function, and further explore the underlying mechanism. Our results verified that RUNX3 was increased in aortic samples of AAA compared with healthy controls. Overexpression vectors of RUNX3 (ov-RUNX3) and siRNA of RUNX3 (si-RUNX3) were transfected into Human aortic smooth muscle cells (HAoSMCs). The results indicated that ov-RUNX3 promoted cell proliferation, migration, and MMP-2/3/9 secretion, and suppressed TIMP-1, collagen I/III, SM22, MYH11 and CNN1 expression in HAoSMCs. The silencing of RUNX3 has the opposite effect. Furthermore, we found that RUNX3 targets TGF-ß1 and suppressed its transcription. The silencing of TGF-ß1 increased cell proliferation, migration and MMP-2/3/9 expression, and inhibited TIMP-1, Collagen I/III, SM22, MYH11 and CNN1 expression. In addition, TGF-ß1 reversed the effect of RUNX3 overexpression on HAoSMCs. Hence, our study indicated that RUNX3 promotes cell proliferation, migration, and ECM remodelling through suppressing TGF-ß1.
Assuntos
Aneurisma da Aorta Abdominal/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Músculo Liso Vascular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/fisiologia , Adulto , Idoso , Aorta/citologia , Aneurisma da Aorta Abdominal/metabolismo , Western Blotting , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasmídeos/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Sepsis manifests as a dysregulated immune response to infection, damaging organs. Skin has a critical role in protecting the body. In sepsis, skin wound healing is impaired. The mechanisms behind it have been poorly studied. In this study, suction blister wounds were induced on intact abdominal skin in 15 septic patients. A single blister wound was biopsied from each patient and from 10 healthy controls. Immunohistochemical staining of growth factors and extracellular matrix (ECM) proteins was performed. Significance (p < 0.05) of the differences was calculated. The following growth factors were overexpressed in the skin of septic patients compared with healthy controls: epithelial growth factor (intact epithelium p = 0.007, migrating epithelium p = 0.038), vascular epithelial growth factor (intact epithelium p < 0.001, migrating epithelium p = 0.011) and transforming growth factor beta (migrating epithelium p = 0.002). The expression of syndecan-1 was upregulated in the skin of septic patients compared with healthy controls (intact epithelium p = 0.048, migrating epithelium p = 0.028). The following ECM proteins had lower expression in the epithelium in septic patients than in healthy controls: tenascin-C (migrating epithelium p = 0.03) and laminin-332 (intact epithelium p = 0.036). In sepsis, growth factor and syndecan expression was enhanced, while ECM and basement membrane proteins were mostly depressed.