RESUMO
BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.
Assuntos
Citoglobina/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Cápsula de Tenon/metabolismo , Colágeno/análise , Citoglobina/farmacologia , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/análise , Humanos , RNA Mensageiro/análise , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Xishuangbanna, a border area of China, Burma and Laos, had its first major DENV-1 outbreak in 2017. This study aims to explore the genetic characterization, potential source and evolution of the viruses in outbreak. METHODS: The structural protein C/prM/E genes of viruses isolated from local residents or Burmese travelers were sequenced followed by mutation, phylogenetic, homologous recombination, molecular clock and demographic reconstruction analysis. RESULTS: Phylogenetic analysis revealed that all of the strains were classified as three cluster of DENV-1. Cluster 1, 2 and 3 were most similar to China Guangzhou 2011, China Hubei 2014 and Laos 2008 strain, respectively. Among 236 base mutations, 31 caused nonsynonymous mutations when compared with the DENV-1SS. No homologous recombination signal was discovered. The structural protein of these strains had similar three-dimensional structure. Only site 434 showed differences among five predicted protein binding sites. Molecular clock phylogenetic and demographic reconstruction analysis showed that DENV-1 became highly diversified in 1972 followed by a slightly decreased period until 2017. CONCLUSIONS: Dengue isolated strains show diversification between Burma and China. Amino acid substitution (I440T) may lead to weakened virulence of the epidemic strains. DENV-1 became highly diversified in 1972 followed by a slightly decreased period.
Assuntos
Vírus da Dengue/genética , Dengue/epidemiologia , Surtos de Doenças , Genes Virais , Substituição de Aminoácidos , China/epidemiologia , Demografia , Vírus da Dengue/isolamento & purificação , Técnicas de Genotipagem , Humanos , Laos/epidemiologia , Mianmar/epidemiologia , Filogenia , Filogeografia , Conformação Proteica , Proteínas Estruturais Virais/genéticaRESUMO
BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.
Assuntos
Humanos , Matriz Extracelular/metabolismo , Cápsula de Tenon/metabolismo , Fibroblastos/metabolismo , Citoglobina/metabolismo , RNA Mensageiro/análise , Colágeno/análise , Fibronectinas/análise , Fator A de Crescimento do Endotélio Vascular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Citoglobina/farmacologiaRESUMO
BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.
Assuntos
Autofagia/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Autofagia/fisiologia , Linhagem Celular , Citometria de Fluxo , Humanos , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.