RESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética , Degeneración Retiniana , Humanos , Ratas , Animales , Degeneración Retiniana/patología , Células Ependimogliales/metabolismo , Estreptozocina/toxicidad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Crecimiento Transformador beta3/efectos adversos , Factor de Crecimiento Transformador beta3/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Gliosis/patología , Retina/metabolismo , Retinopatía Diabética/patología , ARN Mensajero/metabolismoRESUMEN
Hepatocyte transplantation can be an effective treatment for patients with certain liver-based metabolic disorders and liver injuries. Hepatocytes are usually infused into the portal vein, from which hepatocytes migrate into the liver and integrate into the liver parenchyma. However, early cell loss and poor liver engraftment represent major hurdles to sustaining the recovery of diseased livers after transplantation. In the present study, we found that ROCK (Rho-associated kinase) inhibitors significantly enhanced in vivo hepatocyte engraftment. Mechanistic studies suggested that the isolation of hepatocytes caused substantial degradation of cell membrane proteins, including the complement inhibitor CD59, probably due to shear stress-induced endocytosis. ROCK inhibition by ripasudil, a clinically used ROCK inhibitor, can protect transplanted hepatocytes by retaining cell membrane CD59 and blocking the formation of the membrane attack complex. Knockdown of CD59 in hepatocytes eliminates ROCK inhibition-enhanced hepatocyte engraftment. Ripasudil can accelerate liver repopulation of fumarylacetoacetate hydrolase-deficient mice. Our work reveals a mechanism underlying hepatocyte loss after transplantation and provides immediate strategies to enhance hepatocyte engraftment by inhibiting ROCK.
Asunto(s)
Hepatopatías , Hígado , Ratones , Animales , Hígado/metabolismo , Hepatocitos/metabolismo , Vena Porta , Hepatopatías/metabolismo , Activación de ComplementoRESUMEN
The rapid development of nanotechnology has aroused considerable attentions toward understanding the effects of engineered nanomaterials (ENMs) on the propagation of antibiotic resistance. Molybdenum disulfide (MoS2) is an extensively used ENM and poses potential risks associated with environmental exposure; nevertheless, the role of MoS2 toward antibiotic resistance genes (ARGs) transfer remains largely unknown. Herein, it was discovered that MoS2 nanosheets accelerated the horizontal transfer of RP4 plasmid across Escherichia coli in a dose-dependent manner (0.5-10 mg/L), with the maximum transfer frequency 2.07-fold higher than that of the control. Integration of physiological, transcriptomics, and metabolomics analyses demonstrated that SOS response in bacteria was activated by MoS2 due to the elevation of oxidative damage, accompanied by cell membrane permeabilization. MoS2 promoted bacterial adhesion and intercellular contact via stimulating the secretion of extracellular polysaccharides. The ATP levels were maximally increased by 305.7 % upon exposure to MoS2, and the expression of plasmid transfer genes was up-regulated, contributing to the accelerated plasmid conjugation and increased ARG abundance in soil. Our findings highlight the roles of emerging ENMs (e.g., MoS2) in ARGs dissemination, which is significant for the safe applications and risk management of ENMs under the development scenarios of nanotechnology.
Asunto(s)
Antibacterianos , Molibdeno , Antibacterianos/farmacología , Molibdeno/farmacología , Genes Bacterianos , Suelo , Transferencia de Gen Horizontal , Farmacorresistencia Microbiana/genética , Escherichia coli , PlásmidosRESUMEN
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.
Asunto(s)
Transición Epitelial-Mesenquimal , Degeneración Macular , Senescencia Celular , Medios de Cultivo Condicionados/farmacología , Dasatinib/farmacología , Células Epiteliales/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Degeneración Macular/metabolismo , Quercetina/farmacología , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/metabolismo , Pigmentos Retinianos/farmacología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Corneal endothelial cells (CECs) play a major role in the maintenance of stromal hydration via the barrier and pump function for clear vision. Adult CECs cannot regenerate after injury. CECs cultured in vitro can undergo mitosis but may undergo corneal endothelial-to-mesenchymal transition (EnMT) and lose their endothelial characteristics. In this study, we examined the effects of CHIR99021 on transforming growth factor beta-1(TGFß1)-induced EnMT in human CECs (hCECs) lines. CHIR99021 kept hCECs in the hexagonal shape and could downregulate the EnMT markers alpha-smooth muscle actin (α-SMA) and fibronectin (FN1), meanwhile maintained the hCECs function markers Na+/K+-ATPase and zonula occludens-1 (ZO-1) at levels comparable to those in the normal control. Interestingly, we found that the combination of CHIR99021 and TGFß1 at appropriate concentrations would significantly promote the proliferation and migration of hCECs. These effects may be related to the inhibition of RhoA or Rac1, as well as the activation of Wnt and Erk pathway, with a calcium homeostasis. Our findings indicate that CHIR99021 inhibit EnMT and that the combination of CHIR99021 and TGFß1 may provide new ideas for corneal endothelial regeneration and wound healing.
Asunto(s)
Células Endoteliales , Endotelio Corneal , Factor de Crecimiento Transformador beta1/farmacología , Adulto , Proliferación Celular , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Transición Epitelial-Mesenquimal , Humanos , Piridinas , PirimidinasRESUMEN
Thorough investigations of the environmental fate and risks are necessary for the safe application of engineered nanomaterials. Nevertheless, the current understanding of potential transformations of MoS2 (an intensively studied two-dimensional nanosheet) upon interactions with ubiquitous environmentally relevant thiols (ERTs) in water is limited. This study revealed that two ERTs, l-cysteine and mercaptoacetic acid, could modify MoS2 by covalently grafting thiol groups on S atoms of 1T phases, improving the colloidal persistence and chemical stability of MoS2. Compared with the pristine form, MoS2-thiols with higher dispersity exhibited significantly mitigated envelopment and ultrastructural damage to microalgae. MoS2-triggered growth inhibition, upregulation of reactive oxygen species, photosynthetic injury, and metabolic perturbation in algae were remarkably attenuated by ERTs. The diminished capability for MoS2 to generate reactive intermediates and glutathione oxidation driven by ERTs caused the weakness of oxidative stress and negative effects. Additionally, molecular dynamics simulations demonstrated that ERTs altered the extent of the influence of MoS2 on the secondary structures and functions of adsorbed intracellular proteins, which also contributed to the lower phytotoxicity of MoS2. Our findings provide evidence for the crucial role of specific organic ligands in the risk of MoS2 in aquatic environments.
Asunto(s)
Molibdeno , Nanoestructuras , Disulfuros , Nanoestructuras/química , Nanoestructuras/toxicidad , Compuestos de SulfhidriloRESUMEN
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.
Asunto(s)
Barrera Hematorretinal/efectos de los fármacos , Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/fisiopatología , Eritropoyetina/administración & dosificación , Microglía/fisiología , Fagocitosis/efectos de los fármacos , Factores Despolimerizantes de la Actina/metabolismo , Animales , Barrera Hematorretinal/fisiopatología , Hipoxia de la Célula , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Eritropoyetina/uso terapéutico , Humanos , Inyecciones Intravítreas , Masculino , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Retinopatía Diabética/genética , Retinopatía Diabética/patología , Regulación de la Expresión Génica , Mapas de Interacción de Proteínas/genética , Animales , Línea Celular , Bases de Datos Factuales , Diabetes Mellitus Experimental/genética , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/patología , Perfilación de la Expresión Génica/métodos , Glucosa/farmacología , Histonas/genética , Histonas/metabolismo , Masculino , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Fenantrenos/farmacología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Poli Adenosina Difosfato Ribosa/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Degeneración Retiniana/terapia , Epitelio Pigmentado de la Retina/trasplante , Animales , Western Blotting , Supervivencia Celular/fisiología , Trasplante de Células , Células Cultivadas , Electrorretinografía , Etiquetado Corte-Fin in Situ , Células Fotorreceptoras de Vertebrados/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Ratas Mutantes , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatologíaRESUMEN
Alzheimer's disease (AD) is a growing health concern worldwide. MicroRNAs (miRNAs) have been extensively studied in many diseases, including AD. To identify differentially expressed miRNAs (DEmiRNAs) and genes specific to AD, we used bioinformatic analyses to investigate candidate miRNA-mRNA pairs involved in the pathogenesis of AD. We focused on differentially expressed genes (DEGs) that are targets of DEmiRNAs. The GEO2R tool and the HISAT2-DESeq2 software were used to identify DEmiRNAs and DEGs. Bioinformatic tools available online, such as TAM and the Database for Annotation, Visualization and Integrated Discovery (DAVID), were used to perform functional annotation and enrichment analysis. Targets of miRNAs were predicted using the miRTarBase. The Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape, which are available online, were utilized to construct protein-protein interaction (PPI) networks and identify hub genes. Furthermore, transcription factors (TFs) encoded by the DEGs were predicted using the TransmiR database and TF-miRNA-mRNA networks were constructed. Finally, the expression profile of a hub gene in peripheral blood mononuclear cells was compared between healthy individuals and AD patients. We identified 26 correlated miRNA-mRNA pairs. In the parietal lobe, miRNA-mRNA pairs involved in protein folding were enriched, and in the frontal lobe, miRNA-mRNA pairs involved in synaptic transmission, abnormal protein degradation, and apoptosis were enriched. In addition, HSP90AB1 in peripheral blood mononuclear cells was found to be significantly downregulated in AD patients, and this was consistent with its expression profile in the parietal lobe of AD patients. Our results provide brain region-specific changes in miRNA-mRNA associations in AD patients, further our understanding of potential underlying molecular mechanisms of AD, and reveal promising diagnostic and therapeutic targets for AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Lóbulo Frontal/metabolismo , MicroARNs/metabolismo , Lóbulo Parietal/metabolismo , ARN Mensajero/metabolismo , Enfermedad de Alzheimer/genética , Biología Computacional , Bases de Datos Genéticas , Regulación hacia Abajo , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , MicroARNs/genética , Mapas de Interacción de Proteínas , ARN Mensajero/genética , Regulación hacia ArribaRESUMEN
The increasing applications of single-layer molybdenum disulfide (SLMoS2) pose great potential risks associated with environmental exposure. This study found that metallic-phase SLMoS2 with nanoscale (N-1T-SLMoS2, â¼400 nm) and microscale (M-1T-SLMoS2, â¼3.6 µm) diameters at 10-25 mg/L induced significant algal growth inhibition (maximum 72.7 and 74.6%, respectively), plasmolysis, and oxidative damage, but these alterations were recoverable. Nevertheless, membrane permeability, chloroplast damage, and chlorophyll biosynthesis reduction were persistent. By contrast, the growth inhibition (maximum 55.3%) and adverse effects of nano-sized semiconductive-phase SLMoS2 (N-2H-SLMoS2, â¼400 nm) were weak and easily alleviated after 96 h of recovery. N-1T-SLMoS2 (0.011 µg/h) and N-2H-SLMoS2 (0.008 µg/h) were quickly biodegraded to soluble Mo compared with M-1T-SLMoS2 (0.004 µg/h) and excreted by algae. Incomplete biodegradation of SLMoS2 (26.8-43.9%) did not significantly mitigate its toxicity. Proteomics and metabolomics indicated that the downregulation of proteins (50.7-99.2%) related to antioxidants and photosynthesis and inhibition of carbon fixation and carbohydrate metabolism contributed to the persistent phytotoxicity. These findings highlight the roles and mechanisms of the size and phase in the persistent phytotoxicity of SLMoS2, which has potential implications for risk assessment and environmental applications of nanomaterials.
Asunto(s)
Molibdeno , Nanoestructuras , Disulfuros/toxicidad , Molibdeno/toxicidad , FotosíntesisRESUMEN
Manure, soil, and vegetable samples were collected from different-sized livestock farms in Xinxiang, China. The residues of sulfadiazine, sulfamonomethoxine, and sulfamethoxazole were analyzed by high-performance liquid chromatography. The results indicated that the concentration ranges of the three total sulfonamides in manure, soil, and vegetables were 10.13-566.23 µg kg-1, 7.60-176.26 µg kg-1, and 0-32.70 µg kg-1, respectively. The mean concentrations were 219.71 µg kg-1, 70.73 µg kg-1, and 7.08 µg kg-1 for manure, soil, and vegetables, respectively. The mean concentrations in soil were lower than the ecotoxic effect trigger value (100 µg kg-1), indicating a low risk for organisms in soil. The concentrations of the three sulfonamides varied significantly in different types of vegetables and all were lower than the acceptable daily intake value (50 µg (kg day)-1). However, the potential ecotoxicity and danger to human and animal health via accumulation of the antibiotic in the food chain cannot be ignored.
Asunto(s)
Antibacterianos/análisis , Ganado , Estiércol/análisis , Contaminantes del Suelo/análisis , Suelo/química , Sulfonamidas/análisis , Verduras/química , Animales , China , Cromatografía Líquida de Alta Presión , Medición de RiesgoRESUMEN
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.
Asunto(s)
Proteína 1 Similar a Quitinasa-3/metabolismo , MicroARNs/fisiología , Retina/metabolismo , Degeneración Retiniana/prevención & control , Epitelio Pigmentado de la Retina/metabolismo , Animales , Autofagia , Western Blotting , Línea Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Electrorretinografía , Etiquetado Corte-Fin in Situ , Masculino , Microscopía Electrónica de Transmisión , Ratas , Ratas Mutantes , Ratas Sprague-Dawley , Retina/fisiopatología , Degeneración Retiniana/enzimología , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/fisiopatología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Pueblo Asiatico/genética , Mutación , Retinitis Pigmentosa/genética , Rodopsina/genética , Adulto , China/epidemiología , Codón sin Sentido , Análisis Mutacional de ADN , Electrorretinografía , Femenino , Mutación del Sistema de Lectura , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Mutación Missense , Linaje , Retina/fisiopatología , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/fisiopatología , Trastornos de la Visión/fisiopatología , Campos Visuales/fisiología , Adulto JovenRESUMEN
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.
Asunto(s)
Barrera Hematorretinal/efectos de los fármacos , Retinopatía Diabética/tratamiento farmacológico , Eritropoyetina/administración & dosificación , Ocludina/metabolismo , Vasos Retinianos/fisiopatología , Regulación hacia Arriba , Proteína de la Zonula Occludens-1/metabolismo , Animales , Western Blotting , Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Retinopatía Diabética/fisiopatología , Inyecciones Intravítreas , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Vasos Retinianos/efectos de los fármacosRESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Transición Epitelial-Mesenquimal/fisiología , Epitelio Pigmentado de la Retina/citología , Animales , Biomarcadores/metabolismo , Western Blotting , Desdiferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Reacción en Cadena de la Polimerasa , Epitelio Pigmentado de la Retina/metabolismo , PorcinosRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/fisiopatología , MicroARNs/fisiología , Estrés Oxidativo/fisiología , Retina/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Análisis de Varianza , Animales , Far-Western Blotting , Células Cultivadas , Electrorretinografía , Células Ependimogliales/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Macrófagos/microbiología , Fenotipo , Infecciones por Pseudomonas/microbiología , Cicatrización de Heridas/fisiología , Animales , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/microbiología , Expresión Génica/fisiología , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
UNLABELLED: Tissue-specific stem/progenitor cells are essential to mediate organogenesis and tissue homeostasis. In addition, these cells have attracted significant interest for their therapeutic potential. However, it remains challenging to expand most types of these cells in vitro. In this study we devised a screening strategy aimed at identifying growth factors and small molecules that can sustain self-renewal of mouse hepatoblasts. This approach began with a defined basal condition, on top of which collections of growth factors and bioactive small molecules were screened for maintaining self-renewal of primary hepatoblasts. The initially identified proteins and small molecules were then combined in the basal media for subsequent screening to identify additional molecules that can synergistically promote hepatoblast self-renewal. This strategy was performed iteratively to eventually define a small molecule and growth factor cocktail, including epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor ß receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate, which was sufficient to sustain long-term self-renewal of the murine hepatoblasts under chemically defined conditions. These expanded hepatoblasts retain the ability to respond to liver developmental cues and produce functional hepatocytes and form bile duct-like structures. CONCLUSION: Our work established a chemically defined condition that allows long-term expansion of hepatoblasts, improved our understanding of hepatoblast self-renewal, and highlights the power of phenotypic screening to enable self-renewal of somatic stem/progenitor cells.
Asunto(s)
Técnicas de Cultivo de Célula , Hepatocitos/citología , Hígado/citología , Células Madre/citología , Animales , Benzamidas , Medios de Cultivo , Dioxoles , Factor de Crecimiento Epidérmico , Femenino , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Ratones Endogámicos , Embarazo , Pirazoles , Piridinas , Pirimidinas , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Células Madre/efectos de los fármacosRESUMEN
In this study, the roles of hMMS2 (human methyl methanesulfonate sensitive mutant 2) gene encoding the human ubiquitin-conjugating enzyme E2 variant 2 in the drug resistance in human colon carcinoma were investigated by using a well-differentiated human colorectal carcinoma L-OHP-resistant cell line, THC8307/L-OHP. THC8307/L-OHP cells were transfected via liposome along with plasmid pcDNA6.2-GW/EmGFP-miR-MMS2 expressing both miRNA against hMMS2 and GFP, followed by real-time fluorescent quantitative PCR and immunofluorescence to select stable transfectants with significantly reduced hMMS2 expression. Compared with untransfected or pcDNA6.2-GW/EmGFP vector-transfected cells, the hMMS2-depleted cells displayed significantly (P<0.05) reduced half inhibition concentration(IC50) resistance index (RI) and colony-forming efficiency (CFE) upon treatment with oxaliplatin (L-OHP), while its relative reverse efficiency(RRE) was significantly higher (P<0.05) than the control cells, indicating compromised ability of cell proliferation. Indeed, Rho-damine 123 staining and flow cytometry analyses revealed an increased rate of apoptosis in hMMS2-depleted cells while no difference in cell proliferation or apoptosis was observed between the two control cell lines. The above observations collec-tively indicate that suppression of hMMS2 reverses L-OHP tolerance in differentiated human colorectal carcinoma cells by promoting apoptosis.