RESUMO
BACKGROUND: Psoriasis is a common chronic inflammatory skin disorder that causes patches of thick red skin and silvery scales and affects 1-3% of the population, which reduces patient's quality of life. Understanding the pathogenesis of psoriasis is crucial for developing novel therapeutic strategies. METHODS: HaCaT and NHEK cells were treated with TNF-α in vitro. A mouse model of psoriasis was established by topical imiquimod application on back skin. LncRNA MEG3 was cloned into the pcDNA3.1 vector and transfected in TNF-α-treated HaCaT and NHEK cells to overexpress its expression. Liposome-encapsulated pcDNA3.1-MEG3 was injected into imiquimod-treated mice via tail vein. RT-qPCR and western blot assays were used to examine the expression of lncRNA MEG3, IL-6, IL-8, IFN-γ, IL-1ß, LC3, Beclin 1, p62, p-p65, p65, NLRP3, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR respectively. The secretion of IL-6, IL-8, IFN-γ and IL-1ß was determined using ELISA assay. Immunofluorescence and immunohistochemistry methods were performed for analyzing the expression of LC3 and NLRP3 in cells and skin tissues respectively. LY294002 was used to block the PI3K/AKT/mTOR signalling. MTT assay was applied to test the toxicity of LY294002 to HaCaT and NHEK cells. RESULTS: LncRNA MEG3 expression levels were downregulated in TNF-α-treated HaCaT and NHEK cells and skin tissues of psoriatic mice model. TNF-α treatment enhanced inflammation and suppressed autophagy in HaCaT and NHEK cells, which were largely reversed by overexpression of lncRNA MEG3. Autophagy puncta and NLRP3 inflammasome assembly showed the same patterns with the expression of inflammation and autophagy markers in TNF-α-treated HaCaT and NHEK cells with or without lncRNA MEG3 overexpression. TNF-α-induced activation of the PI3K/AKT/mTOR signalling was abolished by lncRNA MEG3 overexpression in HaCaT and NHEK cells. Blocking the PI3K/AKT/mTOR signalling inhibited TNF-α-induced inflammation and restored autophagy level in TNF-α-treated HaCaT and NHEK cells. Overexpression of lncRNA MEG3 suppressed inflammation, promoted autophagy and inhibited the activation of the PI3K/AKT/mTOR signalling in a mouse model of psoriasis. CONCLUSION: LncRNA MEG3 facilitates autophagy and suppresses inflammation in TNF-α-treated keratinocytes and psoriatic mice, which is dependent on the PI3K/AKT/mTOR signalling pathway. Our study enhances the understanding of psoriasis and provides potential therapeutic targets for psoriasis.
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Autofagia/genética , Inflamação/genética , Queratinócitos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Psoríase/genética , RNA Longo não Codificante/metabolismo , Animais , Autofagia/efeitos dos fármacos , Cromonas/farmacologia , Feminino , Células HaCaT , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos Endogâmicos BALB C , Morfolinas/farmacologia , Psoríase/patologia , RNA Longo não Codificante/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfaRESUMO
INTRODUCTION: Water is a critical nutrient, and it is important for the maintenance of the physiological function of the human body [
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Afeto/efeitos dos fármacos , Cognição/efeitos dos fármacos , Comportamento de Ingestão de Líquido , Estudantes/psicologia , Água/farmacologia , Adolescente , China , Dieta Saudável/psicologia , Método Duplo-Cego , Ingestão de Líquidos , Feminino , Comportamentos Relacionados com a Saúde , Humanos , Masculino , Concentração Osmolar , Universidades , Adulto JovemRESUMO
In this study, we report the cloning and expression of a functional glucoside 3-dehydrogenase (G3DH) gene from Sphingobacterium faecium ZJF-D6. This gene is 1686 bp in length and encodes a peptide of 562 amino acids. The G3DH gene was successfully expressed in E. coli, and the recombinant enzyme could oxidize glucosides, galactosides and analogues at C-3 position. The sequence and multiple alignment analysis showed that the enzyme has highest identity with G3DHs from Paraglaciecola polaris LMG 21857, Aliiglaciecola lipolytica E3 and Halomonas sp. alpha-15. The recombinant G3DH was purified on Ni-NTA column and exhibited the highest activity at pH 7.6 and 30 °C. It was sensitive to acid and alkali, and showed well thermostability. The SfG3DH could oxidize a wild range of sugars. When recombinant E. coli BL21 cells were used as catalyst, a high rate of conversion to N-p-nitrophenyl-3-ketovalidamine was achieved, and no p-nitroaniline was detected. This process offers a promising approach to fulfill substrate of 3-ketovalidoxylamine A C-N lyase production.
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Clonagem Molecular/métodos , Glucose Desidrogenase/genética , Glucose Desidrogenase/metabolismo , Nitrofenóis/metabolismo , Sphingobacterium/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosídeos/metabolismo , Glucosídeos/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sphingobacterium/genética , Especificidade por Substrato , TemperaturaRESUMO
OBJECTIVE: To study the chemical constituents from the rhizome of Valeriana jatamansi. METHODS: The chemical constituents were separated and purified by silica gel, medium pressure column chromatography, and preparative HPLC. Their structures were determined by physicochemical properties and spectral data. RESULTS: Six compounds were isolated from the dibromochloromethane extract in the rhizome of Valeriana jatamansi, and identified as decursidin (1), decursitin B (2), decursitin A (3), 3'(S)-acetoxy-4'(R)-angeloyloxy-3',4'-dihydroxanthyletin (4), 8-acetoxyl-pathchouli alcohol (5) and dibutyl phthalate (6). CONCLUSION: Compounds 1-4 are coumarins which are isolated from this genus for the first time,and compound 6 is isolated from this genus for the first time.
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Compostos Fitoquímicos/análise , Rizoma/química , Valeriana/química , Cromatografia Líquida de Alta Pressão , Cumarínicos/análise , Nardostachys/químicaRESUMO
PURPOSE: Microplastics have been widely detected in the environment and marine organisms. However, few studies have investigated the presence of microplastics in humans. This preliminary study identified and quantified the microplastic content in human feces. METHODS: A total of 26 young male students aged 18-25 years were recruited from Beijing, China. A self-administered 7-day 24-h fluid intake record was used to document fluid intake, and food intake was recorded for 3 days. Feces were collected by participants using a sterile fecal collector. Microplastics in the remaining fecal residues were measured and identified using fourier transform infrared micro-spectroscopy. RESULTS: Eventually, twenty-four participants completed the study. The fecal samples of 23 (95.8%) participants tested positive for microplastics. In these 23 samples, the abundance of microplastics varied from 1 particle/g to 36 particles/g (size 20 to 800 µm). The summed mass of all microplastic particles per participant ranged from 0.01 to 14.6 mg. Qualitative analysis of the microplastics indicated the presence of one to eight types of microplastics in each sample, with polypropylene (PP) being the most abundant; it was found in 95.8% of fecal samples. We examined associations between water intake habits and the abundance of microplastics in their feces. A moderate correlation was observed between packaged water and beverage intake and microplastic abundance in feces (r = 0.445, P = 0.029). CONCLUSION: Various types of microplastics were detected in human feces, with PP being found in the highest proportion. There may be an association between water intake habits and microplastic abundance in feces.
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Microplásticos , Poluentes Químicos da Água , Adolescente , Adulto , Pequim , China , Monitoramento Ambiental , Fezes/química , Humanos , Masculino , Plásticos , Poluentes Químicos da Água/análise , Adulto JovemRESUMO
BACKGROUND: It was reported that microRNA-21(miR-21) was differentially expressed in the keratinocytes of psoriasis patients, and it may influence the apoptosis and proliferation of cells. The role of lncRNA maternally expressed gene3 (MEG3), a competing endogenous RNAs of miR-21, in the progression of psoriasis remains unclear. We aimed to unfold the influence of MEG3 and miR-21 on the proliferation and apoptosis of psoriasis epidermal cells. METHODS: 50µg/L TNF-α was used to treat HaCaTs and NHEKs cells for 24 h, and then different experiments were conducted. qRT-PCR were applied for measuring the mRNA level of MEG3, miR-2, and caspase-8, and the protein expression of caspase-8 was measured with western blotting. Flow cytometry was used for assessing apoptosis. Cell proliferation was detected using MTT and colony formation assays. Dual luciferase reporter assay was applied for confirming the binding site between MEG3 and miR-21, miR-21 and Caspase-8. RESULTS: A cell model for in vitro studying the role of MEG3 in psoriasis pathophysiology was established using HaCaT and HHEKs. MEG3 was significantly down-regulated in HaCaT, HHEKs, and psoriatic skin samples. MEG3 inhibits proliferation and promotes apoptosis of Activated-HaCaT (Act-HaCaT) and Activated-HHEKs (Act- HHEK) by regulating miR-21, and the binding site between MEG3 and miR-21 was identified. We also found that miR-21 could inhibit the level of caspase-8 and identified the binding site between caspase-8 and miR-21. Some down-stream proteins of caspase-8, Cleaved caspase-8, cytc, and apaf-1 were regulated by miR-21 and MEG3. CONCLUSION: MEG3/miR-21 axis may regulate the expression of caspase-8, and further influence the proliferation and apoptosis of psoriasis keratinocyte, Act-HaCaT and Act- HHEK. Therefore, our findings may provide a new thought for the study of pathogenesis and treatment of psoriasis.
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Caspase 8/metabolismo , Queratinócitos/metabolismo , MicroRNAs/metabolismo , Psoríase , RNA Longo não Codificante/metabolismo , Adulto , Apoptose , Linhagem Celular , Proliferação de Células , Feminino , Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Psoríase/metabolismo , RNA Longo não Codificante/genética , Transdução de SinaisRESUMO
A spectrophotometric screening method for avermectin oxidizing microbes by determination of 4â³-oxo-avermectin was established based on the reaction between 4â³-oxo-avermectin and 2,4-dinitrophenylhydrazine. Combined with a gradient HPLC assay, microorganisms capable of regioselectively oxidizing avermectin to 4â³-oxo-avermectin were successfully obtained by this method.
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Ivermectina/análogos & derivados , Oxidantes , Espectrofotometria/métodos , Bactérias/genética , Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , DNA Bacteriano , Enzimas , Ivermectina/análise , Ivermectina/metabolismo , Oxirredução , Fenil-Hidrazinas/metabolismo , RNA Ribossômico 16S/genética , Streptomyces/genética , Streptomyces/metabolismoRESUMO
A soluble glucoside 3-dehydrogenase (G3DH) was purified from a newly isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251. The enzyme was purified to 35.71-fold with a yield of 41.91 % and was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 62 kDa. The sequences of two peptides of the enzyme were all contained in a GMC family oxidoreductase (EFK55866) by mass spectrometry analysis. The optimal pH of the enzyme was around 6.2. The enzyme was stable within a pH range of 5.0-6.6 and was sensitive to heat. G3DH from S. faecium exhibited extremely broad substrate specificity and well regioselectivity to validoxylamine A. The enzyme was completely inhibited by Hg2Cl2 and partly inhibited by Cu(2+), Fe(2+), Ca(2+), and Cd(2+). The apparent K m values for D-glucose, sucrose, and validoxylamine were calculated to be 1.1, 1.7, and 2.1 mM, respectively. With this purified enzyme, 3-keto sucrose was prepared at pH 5.0, 30 °C for 10 h with a yield of 28.7 %.
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Glucose Desidrogenase/biossíntese , Glucose Desidrogenase/metabolismo , Sphingobacterium/metabolismo , Biotecnologia , Glucose Desidrogenase/genética , Glucose Desidrogenase/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Transporte Proteico , Sphingobacterium/isolamento & purificação , Especificidade por Substrato , Sacarose/química , Sacarose/metabolismo , TemperaturaRESUMO
A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH 7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0-10.5. The optimal temperature was found to be near 40 degrees C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca2+. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K (m) value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.
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Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/isolamento & purificação , Stenotrophomonas/enzimologia , Proteínas de Bactérias/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Ponto Isoelétrico , Cinética , Peso Molecular , Stenotrophomonas/química , Especificidade por SubstratoRESUMO
3-Ketovalidoxylamine A C-N lyase is one of three key enzymes in the production of valienamine, which is a potent glucosidase inhibitor from validamycin A. N-p-Nitrophenyl-3-ketovalidamine, used as the substrate of 3-ketovalidoxylamine A C-N lyase, was prepared from N-p-nitrophenylvalidamine with free cells of Stenotrophomonas maltrophilia CCTCC M 204024. The yield and selectivity of N-p-nitrophenyl-3-ketovalidamine from cells were improved by treatment with 10 mM ethylenediaminetetraacetic acid. The optimal pH and temperature for N-p-nitrophenyl-3-ketovalidamine formation was pH 6.0 and 30 degrees C, respectively. N-p-Nitrophenyl-3-ketovalidamine was formed with a yield of 0.68 in the first batch.
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Carbono-Nitrogênio Liases/metabolismo , Nitrofenóis/metabolismo , Stenotrophomonas/metabolismo , Cicloexenos/química , Cicloexenos/metabolismo , Ácido Edético/farmacologia , Hexosaminas/química , Hexosaminas/metabolismo , Concentração de Íons de Hidrogênio , Inositol/análogos & derivados , Inositol/química , Inositol/metabolismo , Cinética , Modelos Químicos , Estrutura Molecular , Nitrofenóis/química , Stenotrophomonas/efeitos dos fármacos , Especificidade por Substrato , TemperaturaRESUMO
A soluble glucoside 3-dehydrogenase (G3DH) from Stenotrophomonas maltrophilia CCTCC M 204024, recently isolated from wheat soil in our laboratory, was purified to 37.4-fold with a yield of 24.7% and was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 66 kDa. 2,6-Dichlorophenolindophenol (DCPIP) and ferricyanide were able to act as artificial electron acceptors for the enzyme. The optimal pH of G3DH was in the range of 6.0-7.0 in the presence of DCPIP. The enzyme was stable in the pH range of 4.4-10.6 and was sensitive to heat. G3DH exhibited extremely broad substrate specificity by converting many sugars to their corresponding 3-ketoglucosides. They produced a characteristic spectrum by alkaline treatment with a peak at 340 nm. The apparent Km values for validoxylamine A and D: -glucose were 8.3 and 1.1 mM, respectively. Cu2+, Ag2+, and Hg2Cl2 inhibited the activity of G3DH.