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1.
Environ Health Prev Med ; 25(1): 56, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-32979924

RESUMO

BACKGROUND: We previously demonstrated that continuous exposure to nitrous acid gas (HONO) for 4 weeks, at a concentration of 3.6 parts per million (ppm), induced pulmonary emphysema-like alterations in guinea pigs. In addition, we found that HONO affected asthma symptoms, based on the measurement of respiratory function in rats exposed to 5.8 ppm HONO. This study aimed to investigate the dose-response effects of HONO exposure on the histopathological alterations in the respiratory tract of guinea pigs to determine the lowest observed adverse effect level (LOAEL) of HONO. METHODS: We continuously exposed male Hartley guinea pigs (n = 5) to four different concentrations of HONO (0.0, 0.1, 0.4, and 1.7 ppm) for 4 weeks (24 h/day). We performed histopathological analysis by observing lung tissue samples. We examined samples from three guinea pigs in each group under a light microscope and measured the alveolar mean linear intercept (Lm) and the thickness of the bronchial smooth muscle layer. We further examined samples from two guinea pigs in each group under a scanning electron microscope (SEM) and a transmission electron microscope (TEM). RESULTS: We observed the following dose-dependent changes: pulmonary emphysema-like alterations in the centriacinar regions of alveolar ducts, significant increase in Lm in the 1.7 ppm HONO-exposure group, tendency for hyperplasia and pseudostratification of bronchial epithelial cells, and extension of the bronchial epithelial cells and smooth muscle cells in the alveolar duct regions. CONCLUSIONS: These histopathological findings suggest that the LOAEL of HONO is < 0.1 ppm.


Assuntos
Enfisema/induzido quimicamente , Hiperplasia/induzido quimicamente , Exposição por Inalação/efeitos adversos , Pulmão/patologia , Ácido Nitroso/toxicidade , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Brônquios/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Cobaias , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Miócitos de Músculo Liso/efeitos dos fármacos
2.
Biomed Environ Sci ; 33(8): 583-592, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32933610

RESUMO

Objective: To screen the differentially expressed proteins (DEPs) in human bronchial epithelial cells (HBE) treated with atmospheric fine particulate matter (PM 2.5). Methods: HBE cells were treated with PM 2.5 samples from Shenzhen and Taiyuan for 24 h. To detect overall protein expression, the Q Exactive mass spectrometer was used. Gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and Perseus software were used to screen DEPs. Results: Overall, 67 DEPs were screened in the Shenzhen sample-treated group, of which 46 were upregulated and 21 were downregulated. In total, 252 DEPs were screened in the Taiyuan sample-treated group, of which 134 were upregulated and 118 were downregulated. KEGG analysis demonstrated that DEPs were mainly enriched in ubiquitin-mediated proteolysis and HIF-1 signal pathways in Shenzhen PM 2.5 samples-treated group. The GO analysis demonstrated that Shenzhen sample-induced DEPs were mainly involved in the biological process for absorption of various metal ions and cell components. The Taiyuan PM 2.5-induced DEPs were mainly involved in biological processes of protein aggregation regulation and molecular function of oxidase activity. Additionally, three important DEPs, including ANXA2, DIABLO, and AIMP1, were screened. Conclusion: Our findings provide a valuable basis for further evaluation of PM 2.5-associated carcinogenesis.


Assuntos
Poluentes Atmosféricos/análise , Brônquios/metabolismo , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Material Particulado/análise , Brônquios/efeitos dos fármacos , Biologia Computacional , Células Epiteliais/efeitos dos fármacos , Humanos , Espectrometria de Massas , Tamanho da Partícula , Proteômica
3.
Nat Commun ; 11(1): 4659, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938936

RESUMO

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.


Assuntos
Butiratos/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Integrinas/antagonistas & inibidores , Naftiridinas/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Administração por Inalação , Animais , Antígenos de Neoplasias/metabolismo , Bleomicina/toxicidade , Butiratos/administração & dosagem , Butiratos/metabolismo , Butiratos/farmacocinética , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/patologia , Integrinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Naftiridinas/administração & dosagem , Naftiridinas/metabolismo , Naftiridinas/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirrolidinas/administração & dosagem , Pirrolidinas/metabolismo , Pirrolidinas/farmacocinética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tomografia Computadorizada de Emissão de Fóton Único , Fator de Crescimento Transformador beta/metabolismo , Pesquisa Médica Translacional
4.
Int J Nanomedicine ; 15: 5043-5060, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764935

RESUMO

Background: Hydroxyapatite (HAP) is a common component of most idiopathic calcium oxalate (CaOx) stones and is often used as a nidus to induce the formation of CaOx kidney stones. Methods: This work comparatively studies the cytotoxicity of four kinds of HAP crystals with different sizes (40 nm to 2 µm), namely, HAP-40 nm, HAP-70 nm, HAP-1 µm, and HAP-2 µm, on human renal proximal tubular epithelial cells (HK-2). Results: HAP crystals reduce the viability and membrane integrity of HK-2 cells in a concentration-dependent manner and consequently cause cytoskeleton damage, cell swelling, increased intracellular reactive oxygen species level, decreased mitochondrial membrane potential, increased intracellular calcium concentration, blocked cell cycle and stagnation in G0/G1 phase, and increased cell necrosis rate. HAP toxicity to HK-2 cells increases with a decrease in crystal size. Conclusion: Cell damage caused by HAP crystals increases the risk of kidney stone formation.


Assuntos
Citotoxinas/química , Citotoxinas/toxicidade , Durapatita/química , Durapatita/toxicidade , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Oxalato de Cálcio/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
5.
PLoS Pathog ; 16(8): e1008760, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790753

RESUMO

Influenza A viruses (IAVs) remain a significant global health burden. Activation of the innate immune response is important for controlling early virus replication and spread. It is unclear how early IAV replication events contribute to immune detection. Additionally, while many cell types in the lung can be infected, it is not known if all cell types contribute equally to establish the antiviral state in the host. Here, we use single-cycle influenza A viruses (scIAVs) to characterize the early immune response to IAV in vitro and in vivo. We found that the magnitude of virus replication contributes to antiviral gene expression within infected cells prior to the induction of a global response. We also developed a scIAV that is only capable of undergoing primary transcription, the earliest stage of virus replication. Using this tool, we uncovered replication stage-specific responses in vitro and in vivo. Using several innate immune receptor knockout cell lines, we identify RIG-I as the predominant antiviral detector of primary virus transcription and amplified replication in vitro. Through a Cre-inducible reporter mouse, we used scIAVs expressing Cre-recombinase to characterize cell type-specific responses in vivo. Individual cell types upregulate unique sets of antiviral genes in response to both primary virus transcription and amplified replication. We also identified antiviral genes that are only upregulated in response to direct infection. Altogether, these data offer insight into the early mechanisms of antiviral gene activation during influenza A infection.


Assuntos
Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Replicação Viral , Células A549 , Animais , Antivirais/farmacologia , Proteína DEAD-box 58/metabolismo , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Células HEK293 , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia
6.
J Toxicol Sci ; 45(8): 423-434, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32741895

RESUMO

Paraquat (PQ) as a non-selective heterocyclic herbicide, has been applied worldwide for over a few decades. But PQ is very harmful to humans and rodents. The lung is the main target organ of PQ poisoning. It is an important event that lung epithelial cells are injured during PQ-induced acute lung injury and pulmonary fibrosis. As a regulator of mRNA expression, microRNA (miRNA) may play an important role in the progress. Our study was to investigate the mechanisms of PQ-induced injury of pulmonary epithelial cells through analyzing the profiling of miRNAs and their target genes. As a result, 11 differentially expressed miRNAs were screened, including 1 upregulated miRNA and 10 downregulated miRNAs in PQ-treated murine lung alveolar epithelial cells (MLE-12 cells). The bioinformatic analyses suggested that the target genes of these miRNAs were involved in mitochondrial apoptosis pathway and DNA methylation, and participated in the regulation of PI3K-Akt, mTOR, RAS, TNF, MAPK and other signal pathways which related to oxidative stress and apoptosis. This indicated that miRNAs were an important regulator of oxidative stress and apoptosis during PQ-induced injury of murine lung alveolar epithelial cells. The findings would deepen our understanding of the mechanisms of PQ-induced pulmonary injury and might provide new treatment targets for this disease.


Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Apoptose/genética , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Expressão Gênica , Herbicidas/toxicidade , MicroRNAs/genética , MicroRNAs/metabolismo , Estresse Oxidativo/genética , Paraquat/toxicidade , Alvéolos Pulmonares/citologia , Lesão Pulmonar Aguda/patologia , Animais , Células Cultivadas , Metilação de DNA/genética , Camundongos , MicroRNAs/fisiologia , Mitocôndrias/patologia
7.
Am J Physiol Renal Physiol ; 319(2): F202-F214, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32628541

RESUMO

Kidney stone disease is a crystal concretion formed in the kidneys that has been associated with an increased risk of chronic kidney disease. MicroRNAs are functionally involved in kidney injury. Data mining using a microRNA array database suggested that miR-21 may be associated with calcium oxalate monohydrate (COM)-induced renal tubular cell injury. Here, we confirmed that COM exposure significantly upregulated miR-21 expression, inhibited proliferation, promoted apoptosis, and caused lipid accumulation in an immortalized renal tubular cell line (HK-2). Moreover, inhibition of miR-21 enhanced proliferation and decreased apoptosis and lipid accumulation in HK-2 cells upon COM exposure. In a glyoxylate-induced mouse model of renal calcium oxalate deposition, increased miR-21 expression, lipid accumulation, and kidney injury were also observed. In silico analysis and subsequent experimental validation confirmed the peroxisome proliferator-activated receptor (PPAR)-α gene (PPARA) a key gene in fatty acid oxidation, as a direct miR-21 target. Suppression of miR-21 by miRNA antagomiR or activation of PPAR-α by its selective agonist fenofibrate significantly reduced renal lipid accumulation and protected against renal injury in vivo. In addition, miR-21 was significantly increased in urine samples from patients with calcium oxalate renal stones compared with healthy volunteers. In situ hybridization of biopsy samples from patients with nephrocalcinosis revealed that miR-21 was also significantly upregulated compared with normal kidney tissues from patients with renal cell carcinoma who underwent radical nephrectomy. These results suggested that miR-21 promoted calcium oxalate-induced renal tubular cell injury by targeting PPARA, indicating that miR-21 could be a potential therapeutic target and biomarker for nephrolithiasis.


Assuntos
Oxalato de Cálcio/farmacologia , Rim/lesões , MicroRNAs/farmacologia , PPAR alfa/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores/metabolismo , Oxalato de Cálcio/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Rim/metabolismo , Cálculos Renais/patologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , MicroRNAs/genética , Nefrocalcinose/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Rev Med Virol ; 30(5): e2140, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32686248

RESUMO

A knowledge-based cybernetic framework model representing the dynamics of SARS-CoV-2 inside the human body has been studied analytically and in silico to explore the pathophysiologic regulations. The following modeling methodology was developed as a platform to introduce a predictive tool supporting a therapeutic approach to Covid-19 disease. A time-dependent nonlinear system of ordinary differential equations model was constructed involving type-I cells, type-II cells, SARS-CoV-2 virus, inflammatory mediators, interleukins along with host pulmonary gas exchange rate, thermostat control, and mean pressure difference. This formalism introduced about 17 unknown parameters. Estimating these unknown parameters requires a mathematical association with the in vivo sparse data and the dynamic sensitivities of the model. The cybernetic model can simulate a dynamic response to the reduced pulmonary alveolar gas exchange rate, thermostat control, and mean pressure difference under a very critical condition based on equilibrium (steady state) values of the inflammatory mediators and system parameters. In silico analysis of the current cybernetical approach with system dynamical modeling can provide an intellectual framework to help experimentalists identify more active therapeutic approaches.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Pulmão/imunologia , Dinâmica não Linear , Pneumonia Viral/imunologia , Proteínas da Fase Aguda/antagonistas & inibidores , Proteínas da Fase Aguda/genética , Proteínas da Fase Aguda/imunologia , Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Temperatura Corporal , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Citocinas/antagonistas & inibidores , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Pulmão/efeitos dos fármacos , Pulmão/virologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Troca Gasosa Pulmonar/efeitos dos fármacos , Troca Gasosa Pulmonar/imunologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
9.
Chem Biol Interact ; 328: 109212, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32721430

RESUMO

Hydroxychloroquine (HCQ) is frequently used medications for many auto-immunity diseases. However, HCQ induced retinal toxicity, which might result in irreversible retinopathy, is one of the most important complications of HCQ. However, the molecular mechanism underlying the HCQ retinal toxicity is still not well known. Retinal pigment epithelium, in which HCQ is highly enriched due to the tissue-specific affinity of HCQ, is considered to play important role in HCQ retinopathy. Herein, we used a metabolomics approach based on liquid chromatography-mass spectrometry to investigate the metabolic changes in retinal pigment epithelial cells (ARPE-19) with HCQ exposure at 6 h and 24 h. ARPE-19 cells were treated with HCQ at sub-lethal concentration 20 (IC 20), which was determined with MTT assay. Untargeted metabolic profiling revealed 9 and 15 metabolites that were significantly different between control group and HCQ exposure group at 6 h and 24 h, respectively. Enrichment and pathway analysis highlighted ascorbate and aldarate metabolism, d-Glutamine and d-glutamate metabolism and C5-Branched dibasic acid metabolism were disturbed after HCQ exposure. These findings increased our knowledge about the metabolic perturbation induced by HCQ exposure and indicated that metabolic profiling in the ARPE-19 cells might be helpful in understanding the mechanism of HCQ retinal toxicity and exploring potential biomarker.


Assuntos
Células Epiteliais/metabolismo , Hidroxicloroquina/toxicidade , Redes e Vias Metabólicas , Metabolômica , Epitélio Pigmentado da Retina/metabolismo , Espectrometria de Massas em Tandem , Adulto , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Análise Discriminante , Células Epiteliais/efeitos dos fármacos , Humanos , Análise dos Mínimos Quadrados , Metaboloma/efeitos dos fármacos , Análise Multivariada , Análise de Componente Principal
10.
Ecotoxicol Environ Saf ; 203: 110956, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32678753

RESUMO

BACKGROUND: Atmospheric pollutants could induced over-expression of Muc5ac, which is a major pathological feature in acute exacerbation of Chronic Obstructive Pulmonary Disease (COPD) and fatal asthma. Notch signaling pathway could promote mucus cell proliferation and mucus secretion. However, the effects of Notch signaling pathway on the airway mucus secretion induced by PM2.5 remain unknown. In this study, we investigated the role of the Notch signaling pathway on Muc5ac by atmospheric PM2.5 in Beas-2B cell. METHODS: The mRNA and protein levels of the Notch1-4, downstream target gene Hes1 and Muc5ac in the Notch signaling pathway were detected by qPCR and western after Beas-2B cells were exposed to PM2.5 of different concentrations for 12h, 24h, and 48h. RESULTS: The longer the exposure time and the higher the concentration of PM2.5, the lower the survival rate of Beas-2B cells. The expressions of Hes1 and Muc5ac in mRNA and protein were significantly increased after PM2.5 exposure. Correlation analysis indicated that there was a positive correlation between the expression of Muc5ac and Hes1 in mRNA and protein. CONCLUSION: Atmospheric PM2.5 can induce the express of Muc5ac, the Notch signaling pathway may be involved in the regulation of Muc5ac by Hes1.


Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais/efeitos dos fármacos , Mucina-5AC/biossíntese , Material Particulado/toxicidade , Receptores Notch/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais
11.
Gene ; 759: 144999, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32717305

RESUMO

Clostridium perfringens beta2 (CPB2), a key virulence factor, is produced by C. perfringens type C that is the main pathogenic microorganism causing diarrhea in piglets. However, little is known concerning the toxic damage effect of CPB2 on intestinal cells of piglets. In present study, CPB2 toxin obtained by genetic recombination technology was evaluated for its cytotoxicity property using the intestinal porcine epithelial (IPEC-J2) cells, which aims to attempt to understand and explain its mechanism of action in porcine small intestinal epithelial cells. IPEC-J2 cells were treated with different concentrations of CPB2 toxin (5, 10, 20, 30, 40, and 50 µg/mL), and MTT assay results showed that the cell viability of CPB2-treated IPEC-J2 cells decreased in a dose-dependent manner. Lactate dehydrogenase (LDH) assay results revealed that CPB2 significantly increased the LDH release, relative to the control. The expression of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8) gradually increased, while the expression of interleukin 10 (IL-10) gradually decreased in IPEC-J2 cells with increasing concentration of CPB2 (10-30 µg/mL), as analyzed by quantitative real-time PCR (RT-qPCR). Also, CPB2 increased the content of intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) of IPEC-J2 cells. Western blot and immunofluorescence results demonstrate that CPB2 decreased the expression of zonula occludens (ZO-1), claudin12 (CLDN12) and occludin (OCLN) in IPEC-J2 cells. In addition, CPB2 increased Bax expression, and inhibited Bcl-2 and Bcl-xL expression, as measured by Western blot. Considering all of these findings, it was concluded that CPB2 toxin shows significant cytotoxicity, cell growth inhibition and increase in cell permeability in IPEC-J2 cells in a concentration-dependent manner, thus leading to abnormal cell apoptosis and functions in porcine small intestinal epithelial cells.


Assuntos
Toxinas Bacterianas/toxicidade , Células Epiteliais/efeitos dos fármacos , Estresse Oxidativo , Animais , Apoptose , Linhagem Celular , Claudinas/genética , Claudinas/metabolismo , Células Epiteliais/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Potencial da Membrana Mitocondrial , Ocludina/genética , Ocludina/metabolismo , Suínos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Vis Exp ; (159)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32478724

RESUMO

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.


Assuntos
Ar , Brônquios/patologia , Células Epiteliais/patologia , Exposição por Inalação/análise , Modelos Biológicos , Material Particulado/toxicidade , Testes de Toxicidade , Automação , Técnicas de Cultura de Células , Linhagem Celular , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Nanoestruturas/toxicidade
14.
Nat Chem Biol ; 16(7): 766-775, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483376

RESUMO

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first 'delete' step to remove heterogeneous N-glycoforms of a certain subclass and a second 'insert' step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.


Assuntos
Membrana Celular/química , Células Epiteliais/química , Glicoconjugados/química , Oligossacarídeos/química , Receptores Opioides delta/química , Animais , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetulus , Encefalina Leucina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Glicoconjugados/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Oligossacarídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Transgenes
15.
Nucleic Acids Res ; 48(13): 7454-7467, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32520327

RESUMO

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Processamento de RNA , Aminofenóis/farmacologia , Brônquios/citologia , Linhagem Celular Tumoral , Células Cultivadas , Agonistas dos Canais de Cloreto/farmacologia , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Mutação , Quinolonas/farmacologia
16.
Am J Physiol Renal Physiol ; 319(1): F93-F105, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32475133

RESUMO

The long noncoding RNA nuclear enriched abundant transcript 1 (NEAT1) has been reported to promote liver fibrosis progression. However, its molecular mechanism in renal fibrosis was not elucidated. In the present study, an in vitro model of renal fibrosis was established with HK-2 and HKC-8 cells treated with transforming growth factor-ß1. C57BL/6 mice were used for the in vivo model with unilateral ureteral obstruction. Our results indicated that NEAT1 and collagen type I levels were significantly upregulated, whereas miR-129 was obviously downregulated, in the progression of renal fibrosis. Meanwhile, NEAT1 knockdown or miR-129 overexpression inhibited collagen type I deposition, the epithelial-mesenchymal transition process, and the inflammation response to suppress renal fibrosis. NEAT1 directly targeted miR-129, and miR-129 directly bound to collagen type I. Downregulation of miR-129 reversed inhibition of renal fibrosis induced by NEAT1 silencing, and upregulation of collagen type I also reversed inhibition of renal fibrosis caused by miR-129 overexpression. NEAT1 knockdown alleviated renal fibrosis in mice subjected to unilateral ureteral obstruction. In conclusion, NEAT1 sponged miR-129 to modulate the epithelial-mesenchymal transition process and inflammation response of renal fibrosis by regulation of collagen type I. Our study indicates a novel role in the regulation of renal fibrosis and provides a new potential treatment target for renal fibrosis.


Assuntos
Colágeno Tipo I/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Nitrogênio da Ureia Sanguínea , Linhagem Celular , Creatinina/sangue , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/patologia , Camundongos , MicroRNAs/genética , RNA Longo não Codificante/genética , Fator de Crescimento Transformador beta1/farmacologia
17.
Nat Med ; 26(7): 1063-1069, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483361

RESUMO

The mucosal epithelium is a common target of damage by chronic bacterial infections and the accompanying toxins, and most cancers originate from this tissue. We investigated whether colibactin, a potent genotoxin1 associated with certain strains of Escherichia coli2, creates a specific DNA-damage signature in infected human colorectal cells. Notably, the genomic contexts of colibactin-induced DNA double-strand breaks were enriched for an AT-rich hexameric sequence motif, associated with distinct DNA-shape characteristics. A survey of somatic mutations at colibactin target sites of several thousand cancer genomes revealed notable enrichment of this motif in colorectal cancers. Moreover, the exact double-strand-break loci corresponded with mutational hot spots in cancer genomes, reminiscent of a trinucleotide signature previously identified in healthy colorectal epithelial cells3. The present study provides evidence for the etiological role of colibactin in human cancer.


Assuntos
Neoplasias Colorretais/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Peptídeos/farmacologia , Policetídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Mutação/efeitos dos fármacos , Motivos de Nucleotídeos/efeitos dos fármacos
18.
Clin Sci (Lond) ; 134(12): 1357-1376, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32490513

RESUMO

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-ß1 (TGF-ß1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-ß1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.


Assuntos
Células Epiteliais/enzimologia , Túbulos Renais Proximais/patologia , Doenças Metabólicas/enzimologia , Quinases Associadas a rho/antagonistas & inibidores , Adolescente , Animais , Anti-Inflamatórios/farmacologia , Criança , Pré-Escolar , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Feminino , Fibrose , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Lactente , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Doenças Metabólicas/patologia , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Células RAW 264.7 , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Obstrução Ureteral/enzimologia , Obstrução Ureteral/patologia , Quinases Associadas a rho/metabolismo
19.
PLoS One ; 15(6): e0235118, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579601

RESUMO

During diabetes, renal proximal tubular cells (PTC) are exposed to a combination of high glucose and hypoxic conditions, which plays a relevant role in the development of diabetic kidney disease (DKD). In this work, a time-series proteomic study was performed to analyse the effect of a diabetic-like microenvironment induced changes on HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O2) were compared to cells in control conditions for up to 48 h. Diabetic conditions increased the percentage of death cells after 24 and 48 h, but no differences in the protein/cell ratio were found. The relative protein quantification using dimethyl-labeling and UHPLC-MS/MS analysis allowed the identification of 317, 296 and 259 proteins at 5, 24 and 48 h, respectively. The combination of statistical and time expression profile analyses indicated an increased expression of proteins involved in glycolysis, and a decrease of cytoskeletal-related proteins. The exposure of HK-2 cells to high glucose and hypoxia reproduces some of the effects of diabetes on PTC and, with the limitations inherent to in vitro studies, propose new mechanisms and targets to be considered in the management of DKD.


Assuntos
Células Epiteliais/metabolismo , Glucose/metabolismo , Túbulos Renais Proximais/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Hipóxia Celular , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Nefropatias Diabéticas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucose/farmacologia , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo
20.
Biochem Pharmacol ; 178: 114123, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32593613

RESUMO

Commonly used drugs for treating many conditions are either natural products or derivatives. In silico modelling has identified several natural products including quercetin as potential highly effective disruptors of the initial infection process involving binding to the interface between the SARS-CoV-2 (Covid-19) Viral Spike Protein and the epithelial cell Angiotensin Converting Enzyme-2 (ACE2) protein. Here we argue that the oral route of administration of quercetin is unlikely to be effective in clinical trials owing to biotransformation during digestion, absorption and metabolism, but suggest that agents could be administered directly by alternative routes such as a nasal or throat spray.


Assuntos
Betacoronavirus/efeitos dos fármacos , Produtos Biológicos/farmacologia , Peptidil Dipeptidase A/metabolismo , Quercetina/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/metabolismo , Betacoronavirus/fisiologia , Produtos Biológicos/administração & dosagem , Produtos Biológicos/química , Ensaios Clínicos como Assunto/métodos , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Estrutura Molecular , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Quercetina/administração & dosagem , Quercetina/química , Internalização do Vírus/efeitos dos fármacos
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