RESUMO
Retinal degeneration, characterized by Müller cell gliosis and photoreceptor apoptosis, is considered an early event in diabetic retinopathy (DR). Our previous study proposed that GMFB may mediate diabetic retinal degeneration. This study identified GMFB as a sensitive and functional gliosis marker for DR. Compared to the wild type (WT) group, Gmfb knockout (KO) significantly improved visual function, attenuated gliosis, reduced the apoptosis of neurons, and decreased the mRNA levels of tumor necrosis factor α (Tnf-α) and interleukin-1ß (Il-1ß) in diabetic retinas. Tgf-ß3 was enriched by hub genes using RNA sequencing in primary WT and KO Müller cells. Gmfb KO significantly upregulated the transforming growth factor (TGF)-ß3 protein level via the AKT pathway. The protective effect of TGF-ß3 in the vitreous resulted in significantly improved visual function and decreased the number of apoptotic cells in the diabetic retina. The protection of Gmfb KO in primary Müller cells against high glucose (HG)-induced photoreceptor apoptosis was partially counteracted by TGF-ß3 antibody and administration of TGFBR1/2 inhibitors. Nuclear receptor subfamily 3 group C member 1 (NR3C1) binds to the promoter region of Gmfb and regulates Gmfb mRNA at the transcriptional level. NR3C1 was increased in the retinas of early diabetic rats but decreased in the retinas of late diabetic rats. N'-[(1E)-(3-Methoxyphenyl)Methylene]-3-Methyl-1H-Pyrazole-5-Carbohydrazide (DS-5) was identified as an inhibitor of GMFB, having a protective role in DR. We demonstrated that GMFB/AKT/TGF-ß3 mediated early diabetic retinal degeneration in diabetic rats. This study provides a novel therapeutic strategy for treating retinal degeneration in patients with DR.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Degeneração Retiniana , Humanos , Ratos , Animais , Degeneração Retiniana/patologia , Células Ependimogliais/metabolismo , Estreptozocina/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta3/efeitos adversos , Fator de Crescimento Transformador beta3/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Gliose/patologia , Retina/metabolismo , Retinopatia Diabética/patologia , RNA Mensageiro/metabolismoRESUMO
Aggregation of amyloid-ß (Aß) peptides is characteristic of Alzheimer's disease (AD), which is the most common neurodegenerative disorder. Increasing evidence shows that Aß oligomers, the intermediates during aggregation, rather than the fully mature fibrils are the most toxic species of Aß and the key contributors to neurodegeneration. Aß oligomers have been considered as both biomarkers and drug targets for the diagnosis and treatment of AD. However, the high heterogeneity and metastability of oligomers make it difficult to determine their exact pathogenic mechanisms. Recent developments in Aß oligomer-targeting agents and techniques have provided great opportunities for overcoming the existing limitations. This review introduces the formation, structure, and toxicity of Aß oligomers and categorizes the Aß oligomer-targeting agents based on their chemical biological applications, including recognition and detection of Aß oligomers for diagnosis, intervention of Aß oligomerization for treatment, and stabilization of Aß oligomers for pathogenic studies. The design strategies and working mechanisms of the representative examples published in the past five years are highlighted. Finally, future development directions and challenges of Aß oligomer targeting are tentatively proposed.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/tratamento farmacológico , Biologia , Fragmentos de Peptídeos/uso terapêuticoRESUMO
To investigate the effect of rapamycin on mitochondrial dynamic balance in diabetic rats subjected to cerebral ischemia-reperfusion injury. Male Sprague Dawley (SD) rats (n = 78) were treated with high fat diet combined with streptozotocin injection to construct diabetic model in rats. Transient middle cerebral artery occlusion (MCAO) of 2 hours was induced and the brains were harvested after 1 and 3 days of reperfusion. Rapamycin was injected intraperitoneally for 3 days prior to and immediately after operation, once a day. The neurological function was assessed, infarct volumes were measured and HE staining as well as immunohistochemistry were performed. The protein of hippocampus was extracted and Western blotting were performed to detect the levels of mTOR, mitochondrial dynamin related proteins (DRP1, p-DRP1, OPA1), SIRT3, and Nix/BNIP3L. Diabetic hyperglycemia worsened the neurological function performance (p < 0.01), enlarged infarct size (p < 0.01) and increased ischemic neuronal cell death (p < 0.01). The increased damage was associated with elevations of p-mTOR, p-S6, and p-DRP1; and suppressions of SIRT3 and Nix/BNIP3L. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and reversed the biomarker alterations caused by diabetes. High glucose activated mTOR pathway and caused mitochondrial dynamics toward fission. The protective effect of rapamycin against diabetes-enhanced ischemic brain damage was associated with inhibiting mTOR pathway, redressing mitochondrial dynamic imbalance, and elevating SIRT3 and Nix/BNIP3L expression.
Assuntos
Lesões Encefálicas , Isquemia Encefálica , Diabetes Mellitus Experimental , Traumatismo por Reperfusão , Sirtuína 3 , Ratos , Masculino , Animais , Ratos Sprague-Dawley , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Dinâmica Mitocondrial , Diabetes Mellitus Experimental/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Sirtuína 3/metabolismo , Encéfalo/metabolismo , Lesões Encefálicas/complicações , Isquemia Encefálica/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/complicações , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
Age-related macular degeneration (AMD) is one of the most common leading causes of irreversible blindness, and there is no effective treatment for it. It has been reported that aging is the greatest risk factor for AMD, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells plays an important role in the pathogenesis of AMD. To clarify the relationship between senescence and EMT in RPE cells, we used the replicative senescence model, H2O2- and/or Nutlin3a-induced senescence model, and low-density and/or TGF-ß-induced EMT model to detect the expression of senescence-, RPE- and EMT-related genes, and assessed the motility of cells by using a scratch wound migration assay. The results showed that replicative senescence of RPE cells was accompanied by increased expression of EMT markers. However, senescent RPE cells themselves did not undergo EMT, as the H2O2and Nutlin3a treated cells showed no increase in EMT characteristics, including unchanged or decreased expression of EMT markers and decreased motility. Furthermore, conditioned medium (CM) from senescent cells induced EMT in presenescent RPE cells, and EMT accelerated the process of senescence. Importantly, dasatinib plus quercetin, which selectively eliminates senescent cells, inhibited low-density-induced EMT in RPE cells. These findings provide a better understanding of the interconnection between senescence and EMT in RPE cells. Removal of senescent cells by certain methods such as senolytics, might be a promising potential approach to prevent or delay the progression of RPE-EMT-related retinal diseases such as AMD.
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Transição Epitelial-Mesenquimal , Degeneração Macular , Senescência Celular , Meios de Cultivo Condicionados/farmacologia , Dasatinibe/farmacologia , Células Epiteliais/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Degeneração Macular/metabolismo , Quercetina/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
AIMS/HYPOTHESIS: Microglial activation in diabetic retinopathy and the protective effect of erythropoietin (EPO) have been extensively studied. However, the regulation of microglia in the retina and its relationship to inner blood-retinal barrier (iBRB) maintenance have not been fully characterised. In this study, we investigated the role of microglia in iBRB breakdown in diabetic retinopathy and the protective effects of EPO in this context. METHODS: Male Sprague Dawley rats were injected intraperitoneally with streptozotocin (STZ) to establish the experimental model of diabetes. At 2 h after STZ injection, the right and left eyes were injected intravitreally with EPO (16 mU/eye, 2 µl) and an equivalent volume of normal saline (NaCl 154 mmol/l), respectively. The rats were killed at 2 or 8 weeks after diabetes onset. Microglia activation was detected by ionised calcium binding adaptor molecule (IBA)-1 immunolabelling. Leakage of the iBRB was evaluated by albumin staining and FITC-dextran permeability assay. BV2 cells and primary rat microglia under hypoxic conditions were used to model microglial activation in diabetic retinopathy. Phagocytosis was examined by confocal microscopy in flat-mounted retina preparations and in microglia and endothelial cell cocultures. Protein levels of IBA-1, CD11b, complement component 1r (C1r), and Src/Akt/cofilin signalling pathway components were assessed by western blotting. RESULTS: In diabetic rat retinas, phagocytosis of endothelial cells by activated microglia was observed at 8 weeks, resulting in an increased number of acellular capillaries (increased by 426.5%) and albumin leakage. Under hypoxic conditions, activated microglia transmigrated to the opposite membrane of the transwell, where they disrupted the endothelial cell monolayer by engulfing endothelial cells. The activation and phagocytic activity of microglia was blocked by intravitreal injection of EPO. In vitro, IBA-1, CD11b and C1r protein levels were increased by 50.9%, 170.0% and 135.5%, respectively, by hypoxia, whereas the phosphorylated proteins of Src/Akt/cofilin signalling pathway components were decreased by 74.2%, 47.8% and 39.7%, respectively, compared with the control; EPO treatment abrogated these changes. CONCLUSIONS/INTERPRETATION: In experimental diabetic retinopathy, activated microglia penetrate the basement membrane of the iBRB and engulf endothelial cells, leading to iBRB breakdown. EPO exerts a protective effect that preserves iBRB integrity via activation of Src/Akt/cofilin signalling in microglia, as demonstrated in vitro. These data support a causal role for activated microglia in iBRB breakdown and highlight the therapeutic potential of EPO for the treatment of diabetic retinopathy. Graphical abstract.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/fisiopatologia , Eritropoetina/administração & dosagem , Microglia/fisiologia , Fagocitose/efeitos dos fármacos , Fatores de Despolimerização de Actina/metabolismo , Animais , Barreira Hematorretiniana/fisiopatologia , Hipóxia Celular , Técnicas de Cocultura , Células Endoteliais/metabolismo , Eritropoetina/uso terapêutico , Humanos , Injeções Intravítreas , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismoRESUMO
Deep mining of the molecular mechanisms underlying diabetic retinopathy (DR) is critical for the development of novel therapeutic targets. This study aimed to identify key molecular signatures involved in experimental DR on the basis of integrated bioinformatics analysis. Four datasets consisting of 37 retinal samples were downloaded from the National Center of Biotechnology Information Gene Expression Omnibus. After batch-effect adjustment, bioinformatics tools such as Networkanalyst, Enrichr, STRING, and Metascape were used to evaluate the differentially expressed genes (DEGs), perform enrichment analysis, and construct protein-protein interaction networks. The hub genes were identified using Cytoscape software. The DEGs of interest from the meta-analysis were confirmed by quantitative reverse transcription-polymerase chain reaction in diabetic rats and a high-glucose-treated retinal cell model, respectively. A total of 743 DEGs related to lens differentiation, insulin resistance, and high-density lipoprotein (HDL) cholesterol metabolism were obtained using the meta-analysis. Alterations of dynamic gene expression in the chloride ion channel, retinol metabolism, and fatty acid metabolism were involved in the course of DR in rats. Importantly, H3K27m3 modifications regulated the expression of most DEGs at the early stage of DR. Using an integrated bioinformatics approach, novel molecular signatures were obtained for different stages of DR progression, and the findings may represent distinct therapeutic strategies for DR patients.
Assuntos
Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Regulação da Expressão Gênica , Mapas de Interação de Proteínas/genética , Animais , Linhagem Celular , Bases de Dados Factuais , Diabetes Mellitus Experimental/genética , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Perfilação da Expressão Gênica/métodos , Glucose/farmacologia , Histonas/genética , Histonas/metabolismo , Masculino , Ratos Sprague-DawleyRESUMO
Photoreceptor (PR) dysfunction or death is the key pathological change in retinal degeneration (RD). The death of PRs might be due to a primary change in PRs themselves or secondary to the dysfunction of the retinal pigment epithelium (RPE). Poly(ADP-ribose) polymerase (PARP) was reported to be involved in primary PR death, but whether it plays a role in PR death secondary to RPE dysfunction has not been determined. To clarify this question and develop a new therapeutic approach, we studied the changes in PAR/PARP in the RCS rat, a RD model, and tested the effect of PARP intervention when given alone or in combination with RPE cell transplantation. The results showed that poly(ADP-ribosyl)ation of proteins was increased in PRs undergoing secondary death in RCS rats, and this result was confirmed by the observation of similar changes in sodium iodate (SI)-induced secondary RD in SD rats. The increase in PAR/PARP was highly associated with increased apoptotic PRs and decreased visual function, as represented by lowered b-wave amplitudes on electroretinogram (ERG). Then, as we expected, when the RCS rats were treated with subretinal injection of the PARP inhibitor PJ34, the RD process was delayed. Furthermore, when PJ34 was given simultaneously with subretinal ARPE-19 cell transplantation, the therapeutic effects were significantly improved and lasted longer than those of ARPE-19 or PJ34 treatment alone. These results provide a potential new approach for treating RD.
Assuntos
Modelos Animais de Doenças , Fenantrenos/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Poli Adenosina Difosfato Ribose/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/transplante , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Transplante de Células , Células Cultivadas , Eletrorretinografia , Marcação In Situ das Extremidades Cortadas , Células Fotorreceptoras de Vertebrados/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos Mutantes , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologiaRESUMO
MicroRNAs (miRNAs) have been shown to play critical roles in the pathogenesis and progression of degenerative retinal diseases like age-related macular degeneration (AMD). In this study, we first demonstrated that miR-24 plays an important role in maintaining retinal structure and visual function of rats by targeting chitinase-3-like protein 1 (CHI3L1). In the retinal pigment epithelial (RPE) cells of Royal College of Surgeons (RCS) rats, an animal model of genetic retinal degeneration (RD), miR-24 was found lower and CHI3L1 level was higher in comparison with those in Sprague-Dawley (SD) rats. Other changes in the eyes of RCS rats include activated AKT/mTOR and ERK pathways and abnormal autophagy in the RPE cells. Such roles of miR-24 and CHI3L1 were further confirmed in RCS rats by subretinal injection of agomiR-24, which decreased CHI3L1 level and preserved retinal structure and function. Upstream, NF-κB was identified as the regulator of miR-24 in the RPE cells of these rats. On the other hand, in SD rats, intraocular treatment of antagomiR-24 induced pathological changes similar to those in RCS rats. The results revealed the protective roles for miR-24 to RPE cells and a mechanism for RD in RCS rats was proposed: extracellular stress stimuli first activate the NF-κB signaling pathway, which lowers miR-24 expression so that CHI3L1 increased. CHI3L1 sequentially results in aberrant autophagy and RPE dysfunction by activating AKT/mTOR and ERK pathways. Taken together, although the possibility, that the therapeutic effects in RCS rats are caused by other transcriptional changes regulated by miR-24, cannot be excluded, these findings indicate that miR-24 protects rat retina by targeting CHI3L1. Thus, miR-24 and CHI3L1 might be the targets for developing more effective therapy for degenerative retinal diseases like AMD.
Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , MicroRNAs/fisiologia , Retina/metabolismo , Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/metabolismo , Animais , Autofagia , Western Blotting , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Eletrorretinografia , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Ratos Mutantes , Ratos Sprague-Dawley , Retina/fisiopatologia , Degeneração Retiniana/enzimologia , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/fisiopatologia , Transdução de SinaisRESUMO
Retinitis pigmentosa (RP) is a group of genetically heterogeneous retinal diseases with more than 80 identified causative genes to date. Mutations in the RHO (rhodopsin, OMIM, 180380) are the most common cause of autosomal dominant RP (adRP) worldwide. RHO is also one of the few RP genes that can cause autosomal recessive RP (arRP). To explore the frequency of RP mutations in Chinese populations, panel-based NGS (next-generation sequencing) screening and Sanger sequencing validation were performed for RP patients from 72 unrelated Chinese families. Here we reported the identified mutations only in the RHO gene. Our results showed that 4 mutations in RHO were detected in 5 (6.94%) of the 72 RP families, including two known missense mutations, c.158Câ¯>â¯G (p.P53R) and c.551Aâ¯>â¯C (p.Q184P), and two novel mutations, c.34delC (p.P12NA) and c.82Câ¯>â¯T (p.Q28X). The c.34delC (p.P12NA) mutation was detected in heterozygous state in one patient with intermediate RP phenotype. The c.82Câ¯>â¯T (p.Q28X) mutation was found in a homozygous state in one proband with advanced RP phenotype at the age of 32. Clinical examination of the heterozygous carriers of c.82Câ¯>â¯T (p.Q28X) in that family showed that the father at the age of 60s experienced no symptoms of RP and normal fundus examinations but displayed reduced electroretinography (ERG) and abnormal visual field. The sister and brother at the age of 30s showed no typical aspects of RP phenotypes. Our results not only expand the mutation spectrum of the RHO gene, but also suggest that the 2 null mutations might play minor dominant effects, leading to less severe and slower retinal degeneration in heterozygous state and more severe phenotype in homozygous state.
Assuntos
Povo Asiático/genética , Mutação , Retinose Pigmentar/genética , Rodopsina/genética , Adulto , China/epidemiologia , Códon sem Sentido , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Mutação da Fase de Leitura , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Retina/fisiopatologia , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/fisiopatologia , Transtornos da Visão/fisiopatologia , Campos Visuais/fisiologia , Adulto JovemRESUMO
PURPOSE: To explore the mechanisms of erythropoietin (EPO) in maintaining outer blood-retinal barrier (BRB) in diabetic rats. METHODS: Sprague-Dawley rats were rendered diabetic with intraperitoneal injection of streptozotocin, and then followed by intravitreal injection of EPO. Two and four weeks later, the permeability of outer BRB was examined with FITC-dextran leakage assay, following a method to demarcate the inner and outer retina based on retinal blood supply. The glyoxal-treated ARPE-19 cells, incubated with EPO, soluble EPO receptor (sEPOR), Gö6976, or digoxin, were studied for cell viability and barrier function. The expressions of ZO-1, occludin, VEGFR2, HIF-1α, MAPKs, and AKT were examined with Western blot and immunofluorescence. RESULTS: The major Leakage of FITC-dextran was detected in the outer nuclear layer in both 2- and 4-week diabetic rats. The leakage was largely ameliorated in EPO-treated diabetic rats. The protein expressions of ZO-1 and occludin in the RPE-Bruch's membrane choriocapillaris complex were significantly decreased, whereas HIF-1α and JNK pathways were activated, in 4-week diabetic rats. These changes were prevented by EPO treatment. The in vitro study with ARPE-19 cells confirmed these changes, and the protective effect of EPO was abolished by sEPOR. Gö6976 and digoxin rescued the tight junction and barrier function in glyoxal-treated ARPE-19 cells. CONCLUSIONS: In early diabetic rats, the outer BRB might be more severely damaged and its breakdown is the major factor for retinal oedema. EPO maintains the outer BRB integrity through down-regulation of HIF-1α and JNK signallings, and thus up-regulating ZO-1 and occludin expressions in RPE cells.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Eritropoetina/administração & dosagem , Ocludina/metabolismo , Vasos Retinianos/fisiopatologia , Regulação para Cima , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Western Blotting , Diabetes Mellitus Experimental , Retinopatia Diabética/metabolismo , Retinopatia Diabética/fisiopatologia , Injeções Intravítreas , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Vasos Retinianos/efeitos dos fármacosRESUMO
The pathological change of retinal pigment epithelial (RPE) cells is one of the main reasons for the development of age-related macular degeneration (AMD). Thus, cultured RPE cells are a proper cell model for studying the etiology of AMD in vitro. However, such cultured RPE cells easily undergo epithelial-mesenchymal transition (EMT) that results in changes of cellular morphology and functions of the cells. To restore and maintain the mesenchymal-epithelial transition (MET) of the cultured RPE cells, we cultivated dedifferentiated porcine RPE (pRPE) cells and compared their behaviors in four conditions: 1) in cell culture dishes with DMEM/F12 containing FBS (CC dish-FBS), 2) in petri dishes with DMEM/F12 containing FBS (Petri dish-FBS), 3) in cell culture dishes with DMEM/F12 containing N2 and B27 supplements (CC dish-N2B27), and 4) in petri dishes with DMEM/F12 containing N2 and B27 (Petri dish-N2B27). In addition to observing the cell morphology and behavior, RPE specific markers, as well as EMT-related genes and proteins, were examined by immunostaining, quantitative real-time PCR and Western blotting. The results showed that dedifferentiated pRPE cells maintained EMT in CC dish-FBS, Petri dish-FBS and CC dish-N2B27 groups, whereas MET was induced when the dedifferentiated pRPE cells were cultured in Petri dish-N2B27. Such induced pRPE cells showed polygonal morphology with increased expression of RPE-specific markers and decreased EMT-associated markers. Similar results were observed in induced pluripotent stem cell-derived RPE cells. Furthermore, during the re-differentiation of those dedifferentiated pRPE cells, Petri dish-N2B27 reduced the activity of RhoA and induced F-actin rearrangement, which promoted the nuclear exclusion of transcriptional co-activator with PDZ-binding motif (TAZ) and TAZ target molecule zinc finger E-box binding protein (ZEB1), both of which are EMT inducing factors. This study provides a simple and reliable method to reverse dedifferentiated phenotype of pRPE cells into epithelialized phenotype, which is more appropriate for studying AMD in vitro, and suggests that MET of other cell types might be induced by a similar approach.
Assuntos
Técnicas de Cultura de Células/métodos , Transição Epitelial-Mesenquimal/fisiologia , Epitélio Pigmentado da Retina/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Desdiferenciação Celular/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Reação em Cadeia da Polimerase , Epitélio Pigmentado da Retina/metabolismo , SuínosRESUMO
miRs play critical roles in oxidative stress-related retinopathy pathogenesis. miR-365 was identified in a previously constructed library from glyoxal-treated rat Müller cell. This report explores epigenetic alterations in Müller cells under oxidative stress to develop a novel therapeutic strategy. To examine the miR-365 expression pattern, in situ hybridization and quantitative RT-PCR were performed. Bioinformatical analysis and dual luciferase report assay were applied to identify and confirm target genes. Streptozotocin (STZ)-treated rats were used as the diabetic retinopathy (DR) model. Lentivirus-mediated anti-miR-365 was delivered subretinally and intravitreally into the rats' eyes. The functional and structural changes were evaluated by electroretinogram (ERG), histologically, and through examination of expression levels of metallopeptidase inhibitor 3 (Timp3), glial fibrillary acidic protein (Gfap), recoverin (Rcvrn) and vascular endothelia growth factor A (Vegfa). Oxidative stress factors and pro-inflammatory cytokines were analyzed. miR-365 expression was confirmed in the glyoxal-treated rat Müller cell line (glyoxal-treated rMC-1). In the retina, miR-365 mainly localized in the inner nuclear layer (INL). The increased miR-365 participated in Müller cell gliosis through oxidative stress aggravation, as observed in glyoxal-treated rMC-1 and DR rats before 6 weeks. Timp3 was a target and negatively regulated by miR-365. When miR-365 was inhibited, Timp3 expression was upregulated, Müller cell gliosis was alleviated, and retinal oxidative stress was attenuated. Visual function was also partially rescued as detected by ERG. miR-365 was found to be highly expressed in the retina and the abnormality of miR-365/Timp3 pathway is closely related to the pathology, like Müller gliosis, and the visual injury in DR. The mechanism might be through oxidative stress, and miR-365/Timp3 could be a potential therapeutic target for treating DR.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/fisiopatologia , MicroRNAs/fisiologia , Estresse Oxidativo/fisiologia , Retina/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Análise de Variância , Animais , Far-Western Blotting , Células Cultivadas , Eletrorretinografia , Células Ependimogliais/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Macrophages play critical roles in wound healing process. They switch from "classically activated" (M1) phenotype in the early inflammatory phase to "alternatively activated" (M2) phenotype in the later healing phase. However, the dynamic process of macrophage phenotype switching in diabetic wounds burdened with bacteria is unclear. In this report, Pseudomonas aeruginosa, frequently detected in diabetic foot ulcers, was inoculated into cutaneous wounds of db/db diabetic mice to mimic bacterium-infected diabetic wound healing. We observed that P. aeruginosa infection impaired diabetic wound healing and quickly promoted the expression of pro-inflammatory genes (M1 macrophage markers) tumor necrosis factor-α (tnf-α), interleukin-1ß (il-1ß) and il-6 in wounds. The expression of markers of M2 macrophages, including il-10, arginase-1, and ym1 were also upregulated. In addition, similar gene expression patterns were observed in macrophages isolated directly from wounds. Immunostaining showed that P. aeruginosa infection increased both the ratios of M1 and M2 macrophages in wounds compared with that in control groups, which was further confirmed by in vitro culturing macrophages with P. aeruginosa and skin fibroblast conditioned medium. However, the ratios of the expression levels of pro-inflammatory genes to anti-inflammatory gene il-10 was increased markedly in P. aeruginosa infected wounds and macrophages compared with that in control groups, and P. aeruginosa prolonged the presence of M1 macrophages in the wounds. These data demonstrated that P. aeruginosa in diabetic wounds activates a mixed M1/M2 macrophage phenotype with an excessive activation of M1 phenotype or relatively inadequate activation of M2 phenotype.
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Macrófagos/microbiologia , Fenótipo , Infecções por Pseudomonas/microbiologia , Cicatrização/fisiologia , Animais , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/microbiologia , Expressão Gênica/fisiologia , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Few-shot fault diagnosis is a challenging problem for complex engineering systems due to the shortage of enough annotated failure samples. This problem is increased by varying working conditions that are commonly encountered in real-world systems. Meta-learning is a promising strategy to solve this point, open issues remain unresolved in practical applications, such as domain adaptation, domain generalization, etc. This article attempts to improve domain adaptation and generalization by focusing on the distribution-shift robustness of meta-learning from the task generation perspective. In fact, few-shot fault diagnosis under varying working conditions allows to address the distribution shift problem in a natural way. An unsupervised across-tasks meta-learning strategy with distributional similarity preference is proposed, where the core is the distribution-distance-weighting mechanism. Differently from the naive random meta-train task generation strategy used in existing meta-learning methods, the source instances that present a more similar distribution with respect to the target instances gain larger weightings in the task generation. This strategy leads to a meta-task training set that is enough diverse, and at the same time can be easily learned due to the distribution similarity features of the source tasks. The proposed method introduces the concept of maximum mean discrepancy that is applied to derive the distribution distance of the measurements. Moreover, a model-agnostic meta-learning is applied to realize few-shot fault diagnosis under varying working conditions. The proposed solutions are verified and compared by considering two public datasets used for bearing fault diagnosis. The results show that the proposed strategy outperforms different related few-shot fault diagnosis methods under varying working conditions. Moreover, it is thus proved that, meta-learning with distribution similarity feature represents an effective approach for domain adaptation and generalization.
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The service life of large battery packs can be significantly influenced by only one or two abnormal cells with faster aging rates. However, the early-stage identification of lifetime abnormality is challenging due to the low abnormal rate and imperceptible initial performance deviations. This work proposes a lifetime abnormality detection method for batteries based on few-shot learning and using only the first-cycle aging data. Verified with the largest known dataset with 215 commercial lithium-ion batteries, the method can identify all abnormal batteries, with a false alarm rate of only 3.8%. It is also found that any capacity and resistance-based approach can easily fail to screen out a large proportion of the abnormal batteries, which should be given enough attention. This work highlights the opportunities to diagnose lifetime abnormalities via "big data" analysis, without requiring additional experimental effort or battery sensors, thereby leading to extended battery life, increased cost-benefit, and improved environmental friendliness.
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We herein report a "Fight Aggregation with Aggregation" (FAA) approach for redirection of amyloid-ß peptide (Aß) aggregation using a europium(III) complex (EuL3) that can undergo H-aggregation in aqueous solution under physiological conditions. The H-aggregates of EuL3 may serve as scaffolds that can facilitate the accumulation of Aß to form non-fibrillar co-assemblies. As a result, the Aß aggregation-induced cytotoxicity was inhibited.
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Background: To identify key and shared insulin resistance (IR) molecular signatures across all insulin-sensitive tissues (ISTs), and their potential targeted drugs. Methods: Three datasets from Gene Expression Omnibus (GEO) were acquired, in which the ISTs (fat, muscle, and liver) were from the same individual with obese mice. Integrated bioinformatics analysis was performed to obtain the differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) was carried out to determine the "most significant trait-related genes" (MSTRGs). Enrichment analysis and PPI network were performed to find common features and novel hub genes in ISTs. The shared genes of DEGs and genes between DEGs and MSTRGs across four ISTs were identified as key IR therapeutic target. The Attie Lab diabetes database and obese rats were used to verify candidate genes. A medical drug-gene interaction network was conducted by using the Comparative Toxicogenomics Database (CTD) to find potential targeted drugs. The candidate drug was validated in Hepa1-6 cells. Results: Lipid metabolic process, mitochondrion, and oxidoreductase activity as common features were enriched from ISTs under an obese context. Thirteen shared genes (Ubd, Lbp, Hp, Arntl, Cfd, Npas2, Thrsp., Tpx2, Pkp1, Sftpd, Mthfd2, Tnfaip2, and Vnn3) of DEGs across ISTs were obtained and confirmed. Among them, Ubd was the only shared gene between DEGs and MSTRGs across four ISTs. The expression of Ubd was significantly upregulated across four ISTs in obese rats, especially in the liver. The IR Hepa1-6 cell models treated with dexamethasone (Dex), palmitic acid (PA), and 2-deoxy-D-ribose (dRib) had elevated expression of Ubd. Knockdown of Ubd increased the level of p-Akt. A lowing Ubd expression drug, promethazine (PMZ) from CTD analysis rescued the decreased p-Akt level in IR Hepa1-6 cells. Conclusion: This study revealed Ubd, a novel and shared IR molecular signature across four ISTs, as an effective biomarker and provided new insight into the mechanisms of IR. PMZ was a candidate drug for IR which increased p-Akt level and thus improved IR by targeting Ubd and downregulation of Ubd expression. Both Ubd and PMZ merit further clinical translational investigation to improve IR.
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A terbium(III) complex-based time-resolved luminescence probe for selenocysteine can inhibit selenoprotein activity via a selenolate-triggered cleavage reaction of sulfonamide bonds in living cells.
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Selenocisteína , Térbio , Térbio/química , Luminescência , SelenoproteínasRESUMO
The laboratory practice "Primary culture and directional differentiation of rat bone marrow mesenchymal stem cells (BMSCs)" is part of a required course for sophomore medical students at Tongji university, which has been conducted since 2012. Blended learning has been widely applied in medical courses. Based on a student-centered teaching philosophy, we reconstructed a comprehensive stem cell laboratory module with blended learning in 2021, aiming to facilitate students in enhancing their understanding of the multi-lineage differentiation potential of stem cells and improve their experimental skills, self-directed learning ability, and innovative thinking. First, we constructed in-depth online study resources, including videos demonstrating laboratory procedures, a PowerPoint slide deck, and published literature on student self-learning before class. In class, students performed a primary culture of BMSCs, freely chose among adipogenic, osteogenic, or chondrogenic differentiation, and used cytochemical or immunofluorescence staining for identification. After class, the extracurricular part involved performing quantitative polymerase chain reaction to examine the expression of multi-lineage differentiation marker genes, which was designed as an elective. After 2 years of practice, positive feedback was obtained from both students and faculty members who achieved, the learning goal as expected. The reconstructed stem cell laboratory module provides comprehensive practice opportunities for students. Students have a better understanding of BMSC at the molecular, cellular, and functional levels and have improved their experimental skills, which forms a basis for scientific research for medical students. Introducing blended learning into other medical laboratory practices thus seems valuable.
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Diferenciação Celular , Células-Tronco Mesenquimais , Estudantes de Medicina , Humanos , Ratos , Animais , Células-Tronco Mesenquimais/citologia , Universidades , Aprendizagem , Laboratórios , Educação de Graduação em Medicina/métodosRESUMO
BACKGROUND: Glia maturation factor beta (GMFB) is a growth and differentiation factor that acts as an intracellular regulator of signal transduction pathways. The small ubiquitin-related modifier (SUMO) modification, SUMOylation, is a posttranslational modification (PTM) that plays a key role in protein subcellular localization, stability, transcription, and enzymatic activity. Recent studies have highlighted the importance of SUMOylation in the inflammation and progression of numerous diseases. However, the relationship between GMFB and SUMOylation is unclear. RESULTS: Here, we report for the first time that GMFB and SUMO1 are markedly increased in retinal pigment epithelial (RPE) cells at the early stage of diabetes mellitus (DM) under hyperglycemia. The GMFΒ protein could be mono-SUMOylated by SUMO1 at the K20, K35, K58 or K97 sites. SUMOylation of GMFB led to its increased protein stability and subcellular translocation. Furthermore, deSUMOylation of GMFΒ downregulates multiple signaling pathways, including the Jak-STAT signaling pathway, p38 pathway and NF-kappa B signaling pathway. CONCLUSIONS: This work provides novel insight into the role of SUMOylated GMFB in RPE cells and provides a novel therapeutic target for diabetic retinopathy (DR).