RESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive respiratory disorder characterized by poor prognosis, often presenting with acute exacerbation. The primary cause of death associated with IPF is acute exacerbation of IPF (AE-IPF). However, the pathophysiology of acute exacerbation has not been clearly elucidated yet. This study aims to investigate the underlying pathophysiological molecular mechanism in a mouse AE-PF model. C57BL/6J mice were intratracheally administered bleomycin (BLM, 5 mg/kg) to induce pulmonary fibrosis. After 14 days, lipopolysaccharide (LPS, 2 mg/kg) was injected via the trachea route. Histological assessments, including H&E and Masson staining, as well as inflammatory indicators, were included to evaluate the induction of AE-PF by BLM and LPS in mice. Transcriptomic profiling of pulmonary tissues identified CSF3 as one of the top 10 upregulated DEGs in AE-PF mice. Indeed, administration of exogenous CSF3 protein exacerbated AE-PF in mice. Mechanistically, CSF3 disrupted alveolar epithelial barrier integrity and permeability by regulating specialized cell adhesion complexes such as tight junctions (TJs) and adherens junctions (AJs) via PI3K/p-Akt/Snail pathway, contributing to the aggravation of AE-PF in mice. Moreover, the discovery of elevated sera CSF3 indicated a notable increase in IPF patients during the exacerbation of the disease. Pearson correlation analysis in IPF patients revealed significant positive associations between CSF3 levels and KL-6 levels, LDH levels, CRP levels, respectively. These results provide mechanistic insights into the role of CSF3 in exacerbating of lung fibrotic disease and indicate monitoring CSF3 levels may aid in early clinical decisions for alternative therapy in the management of rapidly progressing IPF.
Assuntos
Bleomicina , Fibrose Pulmonar Idiopática , Camundongos Endogâmicos C57BL , Animais , Humanos , Camundongos , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/induzido quimicamente , Masculino , Modelos Animais de Doenças , Progressão da Doença , Feminino , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Transdução de Sinais , Pessoa de Meia-Idade , Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição da Família Snail/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Transforming growth factor ß (TGF-ß) signaling plays critical roles in both physiological and pathological conditions. In the tumor microenvironment, TGF-ß are well demonstrated as a tumor inducer, which also promote tumor growth and metastasis. SMAD family is an important TGF-ß signalling transducer, which consists of receptor-regulated SMADs (R-SMADs), common-mediator SMADs (co-SMADs), and inhibitory SMADs (I-SMADs). Smad7 is one of the I-SMADs which has been proved to block TGF-ß signalling transduction in both tumor cells and immune cells. Accumulated evidence has suggested SMAD7 acted as a tumor suppressor in various cancer types, such as colorectal cancer, pancreatic cancer and skin melanoma, etc. However, the role of SMAD7 in melanoma lung metastasis has not been well studied. Here, we first investigated the role of SMAD7 on tumor cell viability by overexpressing SMAD7 in murine melanoma cell line B16-F10. Our results showed that SMAD7 overexpression slightly impaired B16-F10 cells growth, promoted cell apoptosis and arrested the cell cycle at S phase. In vivo study showed that SMAD7 overexpression inhibited B16-F10 lung metastasis. Further mechanism study suggested that SMAD7 promoted T cells activation by decreasing regulatory T cells (Tregs) infiltrating into the tumor microenvironment. In summary, our results proved that tumor cell derived SMAD7 inhibited melanoma lung metastasis by impairing the migration capacity of Tregs.
RESUMO
Delayed encephalopathy after acute carbon monoxide (CO) poisoning (DEACMP) is the most severe and clinically intractable complication that occurs following acute CO poisoning. Unfortunately, the mechanism of DEACMP is still vague. Growing evidence indicates that delayed cerebral damage after CO poisoning is related to oxidative stress, abnormal neuro-inflammation, apoptosis and immune-mediated injury. Our recent report indicated that methylene blue (MB) may be a promising therapeutic agent in the prevention of neuronal cell death and cognitive deficits after transient global cerebral ischaemia (GCI). In this study, we aimed to investigate the potential of MB therapy to ameliorate the signs and symptoms of DEACMP. Rats were exposed to 1000 ppm CO for 40 min. in the first step; CO was then increased to 3000 ppm, which was maintained for another 20 min. The rats were implanted with 7-day release Alzet osmotic mini-pumps subcutaneously under the back skin, which provided MB at a dose of 0.5 mg/kg/day 1 hr after CO exposure. The results showed that MB significantly suppressed oxidative damage and expression of pro-inflammatory factors, including tumour necrosis factor-α and interleukin (IL)-1ß. MB treatment also suitably modulated mitochondrial fission and fusion, which is helpful in the preservation of mitochondrial function. Furthermore, MB dramatically attenuated apoptosis and neuronal death. Lastly, behavioural studies revealed that MB treatment preserved spatial learning and memory in the Barnes maze test. Our findings indicated that MB may have protective effects against DEACMP.
Assuntos
Antídotos/farmacologia , Encefalopatias/prevenção & controle , Região CA1 Hipocampal/efeitos dos fármacos , Intoxicação por Monóxido de Carbono/tratamento farmacológico , Azul de Metileno/farmacologia , Neurônios/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encefalopatias/metabolismo , Encefalopatias/patologia , Encefalopatias/fisiopatologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Intoxicação por Monóxido de Carbono/metabolismo , Intoxicação por Monóxido de Carbono/fisiopatologia , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Aprendizagem Espacial/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The pro-inflammatory activity of interleukin 17, which is produced by the IL-23/IL-17 axis, has been associated with the pathogenesis of traumatic brain injury (TBI). The study investigated the potential role of IL-17 in secondary brain injury of TBI in a rat model. Our data showed that the levels of IL-17 increased from 6 h to 7 days and peaked at 3 days, in both the CNS and serum, which were consistent with the severity of secondary brain injury. The IL-23 inhibitor suberoylanilide hydroxamic acid (SAHA) treatment markedly decreased the expressions of IL-17 and apoptosis-associated proteins cleaved caspase-3 and increased the protein ratio of Bcl-2 (B cell lymphoma/leukemia-2)/Bax (Bcl-2-associated X protein). Meanwhile, neuronal apoptosis was reduced, and neural function was improved after SAHA treatment. This study suggests that IL-17 is involved in secondary brain injury after TBI. Administering an IL-23 inhibitor and thereby blocking the IL-23/IL-17 axis may be beneficial in the treatment of TBI.
Assuntos
Dano Encefálico Crônico/fisiopatologia , Lesões Encefálicas Traumáticas/fisiopatologia , Interleucina-17/fisiologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Dano Encefálico Crônico/etiologia , Dano Encefálico Crônico/metabolismo , Dano Encefálico Crônico/prevenção & controle , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Inflamação , Interleucina-17/sangue , Interleucina-17/líquido cefalorraquidiano , Interleucina-17/genética , Interleucina-23/antagonistas & inibidores , Interleucina-23/fisiologia , Masculino , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , VorinostatAssuntos
Antibióticos Antineoplásicos/química , Doxorrubicina/química , Portadores de Fármacos , Micelas , Polímeros/química , Animais , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Dextranos/química , Ditiotreitol/química , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Composição de Medicamentos , Cinética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Oxirredução , Tamanho da Partícula , Poliésteres/química , Solubilidade , Tecnologia Farmacêutica/métodosRESUMO
Reduction-responsive biodegradable micelles were developed from disulfide-linked dextran-b-poly(epsilon-caprolactone) diblock copolymer (Dex-SS-PCL) and applied for triggered release of doxorubicin (DOX) in vitro and inside cells. Dex-SS-PCL was readily synthesized by thiol-disulfide exchange reaction between dextran orthopyridyl disulfide (Dex-SS-py, 6000 Da) and mercapto PCL (PCL-SH, 3100 Da). Dynamic light scattering (DLS) measurements showed that Dex-SS-PCL yielded micelles with an average size of about 60 nm and a low polydispersity index (PDI 0.1-0.2) in PB (50 mM, pH 7.4). Interestingly, these micelles formed large aggregates rapidly in response to 10 mM dithiothreitol (DTT), most likely due to shedding of the dextran shells through reductive cleavage of the intermediate disulfide bonds. DOX could be efficiently loaded into the micelles with a drug loading efficiency of about 70%. Notably, the in vitro release studies revealed that Dex-SS-PCL micelles released DOX quantitatively in 10 h under a reductive environment, mimicking that of the intracellular compartments such as cytosol and the cell nucleus, whereas only about 27% DOX was released from reduction insensitive Dex-PCL micelles in 11.5 h under otherwise the same conditions and about 20% DOX released from Dex-SS-PCL micelles in 20 h under the nonreductive conditions. The cell experiments using fluorescence microscopy and confocal laser scanning microscopy (CLSM) showed clearly that DOX was rapidly released to the cytoplasm as well as to the cell nucleus. MTT studies revealed a markedly enhanced drug efficacy of DOX-loaded Dex-SS-PCL micelles as compared to DOX-loaded reduction-insensitive Dex-PCL micelles. These reduction-responsive biodegradable micelles have appeared highly promising for the targeted intracellular delivery of hydrophobic chemotherapeutics in cancer therapy.
Assuntos
Antibióticos Antineoplásicos/química , Doxorrubicina/química , Portadores de Fármacos/química , Poliésteres/química , Polímeros/química , Portadores de Fármacos/síntese química , Espectroscopia de Ressonância Magnética , Micelas , Polímeros/síntese química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Recombinant human myxovirus resistance protein A (MxA) was successfully expressed by an Escherichia coli expression system. After immunization and cell fusion, a mouse hybridoma (3C2) producing MAbs to MxA was established. Hybridoma 3C2 was further characterized using indirect ELISA, Western blot analysis, immunofluorescent staining, and immunoprecipitation. The ELISA results showed that the titer of 3C2 was between 1:6400 and 1:12800 in ascitic fluids. The isotype of the monoclonal antibody was tested to be IgG1kappa. 3C2 can also specifically recognize human MxA protein in various formats by Western blot analysis, immunofluorescent staining, and immunoprecipitation assay. We further demonstrated that 3C2 could be used to detected MxA expression induce by type I interferon in A549 cell line and human peripheral blood mononuclear cells by Western blot in a dose-dependent manner.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Ligação ao GTP/imunologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Antivirais/farmacologia , Líquido Ascítico , Western Blotting , Cães , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Proteínas de Ligação ao GTP/genética , Humanos , Hibridomas/metabolismo , Imunização , Imunoprecipitação , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Resistência a Myxovirus , Células NIH 3T3/efeitos dos fármacos , Células NIH 3T3/imunologia , Células NIH 3T3/metabolismo , Plasmídeos , Proteínas Recombinantes/genéticaRESUMO
Hepatocellular carcinoma (HCC) is the fifth most common malignancy caused by environmental and genetic factors. MicroRNAs (miRNAs) are a class of short non-coding RNAs with posttranscriptional regulatory functions. They participate in diverse biological pathways and function as gene regulators. Genetic polymorphisms in 3' untranslated regions (3' UTRs) targeted by miRNAs alter the strength of miRNA binding, with consequences on regulation of target genes thereby affecting the individual's cancer risk. We have previously predicted polymorphisms falling in miRNA-binding regions of cancer genes. We selected an insertion/deletion (Indel) polymorphism (rs3783553) in the 3' UTR of interleukin (IL)-1alpha (IL1A) for a case-control study in a Chinese population. With samples from 403 HCC patients and 434 healthy control individuals, strong evidence of association was observed for the variant homozygote. This association was validated in a second independent case-control study with 1074 HCC patients and 1239 healthy control individuals (odds ratio = 0.62; 95% confidence interval = 0.49-0.78). We further show that the 'TTCA' insertion allele for rs3783553 disrupts a binding site for miR-122 and miR-378, thereby increasing transcription of IL-1alpha in vitro and in vivo. These findings suggest that functional polymorphism rs3783553 in IL1A could contribute to HCC susceptibility. Considering IL-1alpha affects not only various phases of the malignant process, such as carcinogenesis, tumor growth and invasiveness, but also patterns of interactions between malignant cells and the host's immune system, our results indicated that IL-1alpha may be a promising target for immunotherapy, early diagnosis and intervention of HCC.
Assuntos
Regiões 3' não Traduzidas , Carcinoma Hepatocelular/genética , Deleção de Genes , Interleucina-1alfa/metabolismo , Neoplasias Hepáticas/genética , MicroRNAs/genética , Mutagênese Insercional , Polimorfismo Genético , Adulto , Idoso , Sítios de Ligação , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Processamento Pós-Transcricional do RNA , RiscoRESUMO
Biodegradable micelles with sheddable poly(ethylene glycol) shells were developed based on disulfide-linked poly(ethylene glycol)-b-poly(epsilon-caprolactone) (PEG-SS-PCL) diblock copolymer and applied for rapid intracellular release of doxorubicin (DOX). PEG-SS-PCL was prepared with controlled block lengths via exchange reaction between PEG orthopyridyl disulfide and mercapto PCL. The micelles formed from PEG-SS-PCL, though sufficiently stable in water, were prone to fast aggregation in the presence of 10mm dithiothreitol (DTT), due to shedding of the PEG shells through reductive cleavage of the intermediate disulfide bonds. Interestingly, the in vitro release studies revealed that these shell-sheddable micelles released DOX quantitatively within 12h under a reductive environment analogous to that of the intracellular compartments such as cytosol and the cell nucleus. In contrast, minimal drug release (<20%) was observed within 24h for the reduction insensitive PEG-PCL micelles under the same conditions as well as for PEG-SS-PCL micelles under the non-reductive conditions. Remarkably, cell experiments showed that these shell-sheddable micelles accomplished much faster release of DOX inside cells and higher anticancer efficacy as compared to the reduction insensitive control. These shell-sheddable biodegradable micelles are highly promising for the efficient intracellular delivery of various lipophilic anticancer drugs to achieve improved cancer therapy.