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1.
Biol Pharm Bull ; 46(12): 1769-1777, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37899248

RESUMO

Hepatocyte tight junctions (TJ) constituted blood-biliary barrier is the most important hepatic barrier for separating bile from the bloodstream, disruption or dysfunction of TJ barrier is involved in hepatobiliary manifestations of colitis, but the underlying mechanism is still not clear. This study aims to investigate the effect and underlying mechanism of tumor necrosis factor alpha (TNF-α) on hepatic TJ protein expression in blood-biliary barrier and identify its role in the pathogenesis of acute colitis-related cholestasis. Acute colitis rat model was induced by trinitrobenzene sulfonic acid (TNBS) intra-colonic administration. TJs expression of blood-biliary barrier was tested in colitis rats, the serum TNF-α level was also determined in order to elucidate the correlation of TNF-α and TJs. HepaRG cells were used to investigate the effect of TNF-α on TJs, and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway were also evaluated in rats and TNF-α treated HepaRG cells. Acute colitis was induced in rats at 5 d post TNBS, which is accompanied with cholestasis-like alteration. Serum TNF-α level was increased in colitis rats and positively correlated with the alteration of total bile acids and bilirubin, marked decrease in TJs was found in TNF-α treated HepaRG cells and the rats, down-regulated PI3K/AKT signaling pathway were also identified in TNF-α treated HepaRG cells and the rats. The study concluded that serum TNF-α mediated the down-regulation of PI3K/AKT signaling pathway, which contributed to the reduction of TJ protein expression in acute colitis-related intrahepatic cholestasis. These findings suggest that TNF-α plays an important role in the pathogenesis of intrahepatic cholestasis of colitis.


Assuntos
Colestase Intra-Hepática , Colestase , Colite , Ratos , Animais , Fator de Necrose Tumoral alfa , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Junções Íntimas/metabolismo , Colite/patologia , Transdução de Sinais , Proteínas de Junções Íntimas/metabolismo
2.
Radiol Med ; 128(2): 171-183, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36680710

RESUMO

PURPOSE: To identify molecular basis of four parameters obtained from dynamic contrast-enhanced magnetic resonance imaging, including functional tumor volume (FTV), longest diameter (LD), sphericity, and contralateral background parenchymal enhancement (BPE). MATERIAL AND METHODS: Pretreatment-available gene expression profiling and different treatment timepoints MRI features were integrated for Spearman correlation analysis. MRI feature-related genes were submitted to hypergeometric distribution-based gene functional enrichment analysis to identify related Kyoto Encyclopedia of Genes and Genomes annotation. Gene set variation analysis was utilized to assess the infiltration of distinct immune cells, which were used to determine relationships between immune phenotypes and medical imaging phenotypes. The clinical significance of MRI and relevant molecular features were analyzed to identify their prediction performance of neoadjuvant chemotherapy (NAC) and prognostic impact. RESULTS: Three hundred and eighty-three patients were included for integrative analysis of MRI features and molecular information. FTV, LD, and sphericity measurements were most positively significantly correlated with proliferation-, signal transmission-, and immune-related pathways, respectively. However, BPE did not show marked correlation relationships with gene expression alteration status. FTV, LD and sphericity all showed significant positively or negatively correlated with some immune-related processes and immune cell infiltration levels. Sphericity decreased at 3 cycles after treatment initiation was also markedly negatively related to baseline sphericity measurements and immune signatures. Its decreased status could act as a predictor for prediction of response to NAC. CONCLUSION: Different MRI features capture different tumor molecular characteristics that could explain their corresponding clinical significance.


Assuntos
Imageamento por Ressonância Magnética Multiparamétrica , Terapia Neoadjuvante/métodos , Imageamento por Ressonância Magnética/métodos , Prognóstico , Estudos Retrospectivos , Meios de Contraste , Resultado do Tratamento
3.
Ther Drug Monit ; 42(4): 578-587, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32049891

RESUMO

BACKGROUND: Critically ill patients show several pathophysiological alterations that can complicate antibiotic dosing. Hence, there is a strong rationale to individualize anti-infective dosing in these patients by using therapeutic drug monitoring (TDM). The current study aimed to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound plasma concentrations of 3 commonly used antibiotics (meropenem, imipenem/cilastatin, and cefoperazone/sulbactam) in the treatment of infections in critically ill patients in China, which could be suitable for routine TDM in hospital laboratories. METHODS: The unbound drug was separated from the bound drug by ultrafiltration. Simple protein precipitation was used for sample preparation. Meropenem, imipenem/cilastatin, cefoperazone/sulbactam, and their corresponding internal standards were then resolved using the Waters CORTECS C18 column. All the compounds were detected using electrospray ionization in the positive/negative ion-switching mode. RESULTS: The calibration curves were linear for all compounds, with correlation coefficients (R) above 0.99 for total concentrations in human plasma and unbound concentrations in the plasma ultrafiltrate. For both total and unbound drugs, the relative errors and intra-assay/interassay relative standard deviations were below 15%. The limit of quantification was 0.05 mcg/mL for both total plasma concentrations and plasma ultrafiltrate concentrations of all compounds. CONCLUSIONS: The method was simple, rapid, and reliable and is currently being used to provide a TDM service to enhance the efficacious use of the 3 antibiotics.


Assuntos
Cefoperazona/sangue , Combinação Imipenem e Cilastatina/sangue , Cilastatina/sangue , Imipenem/sangue , Meropeném/sangue , Sulbactam/sangue , Cromatografia Líquida de Alta Pressão/métodos , Estado Terminal , Monitoramento de Medicamentos/métodos , Humanos , Plasma/química , Espectrometria de Massas em Tandem/métodos
4.
Biomed Chromatogr ; 32(2)2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28755504

RESUMO

Yin-zhi-huang (YZH) injection is an injectable multiherbal prescription derived from the ancient Chinese medicine formula of Yin-chen-hao-tang, which is widely used in the clinic for the treatment of jaundice and chronic liver diseases. To date, the systematic study of the components in this multiherbal prescription still lacks suitable analytical methods that are able to simultaneously detect a broad array of components at low concentrations. In this study, a new liquid chromatography-tandem mass spectrometry method using dynamic multiple reaction monitoring mode was developed to determine multiple peaks in traditional Chinese medicine preparation YZH injection. This simple, selective and sensitive method enabled the quantification of 22 components with standard materials with a lower limit of quantification of 1.46-12.5 ng/mL in cell lysates. This method was successfully applied to celluar uptake and binding investigation of components in YZH injection. The results indicated that this strategy might be a useful approach for rapidly screening of the potential bioactive candidates from YZH injection, and the discovered candidates could be used to investigate the pharmacodynamics in further studies.


Assuntos
Cromatografia Líquida/métodos , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Medicamentos de Ervas Chinesas/química , Humanos , Modelos Lineares , Compostos Orgânicos/análise , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
5.
J Hepatol ; 67(4): 770-779, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28596109

RESUMO

BACKGROUND & AIMS: Macrophages play vital roles in chronic liver injury, and have been tested as a tool for cytotherapy in liver fibrosis. However, macrophages possess ontogenic and functional heterogeneities. Some subsets are pro-fibrotic, whereas others are anti-fibrotic. This study aimed to clarify which macrophage subset is efficient for cytotherapy in liver fibrosis and to elucidate the underlying mechanisms. METHODS: Liver fibrosis was induced in mice by carbon tetrachloride injection or bile duct ligation. Bone-marrow-derived macrophages (BMDMs) were polarized into M0, M1, or M2 macrophages, respectively. BMDMs were infused into mice through the tail vein at different stages of fibrogenesis. Fibrosis progression, hepatic cell populations, and related molecular changes were evaluated. RESULTS: Both M0 and M1 BMDMs significantly ameliorated liver fibrosis, but M1 exhibited stronger therapeutic effects than M0. M2 macrophages were not effective on liver fibrosis. M1 macrophages reduced the number and activation of hepatic stellate cells (HSCs), which could be attributed at least partly to increased HSC apoptosis. M1 macrophages enhanced the recruitment of endogenous macrophages into fibrotic liver, which displayed the phenotype of Ly6Clo restorative macrophages and produced matrix metalloproteinases (MMPs) and hepatic growth factor (HGF) to enhance collagen degradation and hepatocyte proliferation, respectively. M1 macrophages also increased the number of total and activated natural killer (NK) cells in the fibrotic liver, which released TNF-related apoptosis-inducing ligand (TRAIL), inducing HSC apoptosis. CONCLUSIONS: M1 macrophages, which modulate the immune microenvironment to recruit and modify the activation of endogenous macrophages and NK cells, are effective for cytotherapy in experimental liver fibrosis. Lay summary: M1 Bone marrow-derived macrophages (BMDMs) exhibit a stronger therapeutic effect by modulating the hepatic microenvironment to recruit and modify the activation of endogenous macrophages and natural killer (NK) cells, which likely lead to hepatic stellate cells (HSCs) apoptosis and hampered fibrogenesis.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Cirrose Hepática/terapia , Macrófagos/imunologia , Animais , Antígenos Ly/metabolismo , Apoptose , Tetracloreto de Carbono/toxicidade , Microambiente Celular/imunologia , Modelos Animais de Doenças , Células Estreladas do Fígado/patologia , Células Matadoras Naturais/imunologia , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Ativação de Macrófagos , Macrófagos/classificação , Macrófagos/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
6.
Hepatology ; 61(1): 303-14, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25145286

RESUMO

UNLABELLED: Macrophages play multidimensional roles in hepatic fibrosis, but their control has not been fully understood. The Notch pathway mediated by recombination signal binding protein Jκ (RBP-J), the transcription factor transactivated by signals from four mammalian Notch receptors, is implicated in macrophage activation and plasticity. In this study, by using mouse hepatic fibrosis models, we show that myeloid-specific disruption of RBP-J resulted in attenuated fibrosis. The activation of hepatic stellate cells and production of profibrotic factors including platelet-derived growth factor (PDGF)-B and transforming growth factor beta1 (TGF-ß1) reduced significantly in myeloid-specific RBP-J deficient mice. The infiltration of inflammatory cells and production of proinflammatory factors were reduced in liver of myeloid-specific RBP-J-deficient mice during fibrosis. In RBP-J-deficient macrophages, the nuclear factor kappa B (NF-κB) activation was remarkably attenuated as compared with the control. This could be attributed to the up-regulation of cylindromatosis (CYLD), a negative regulator of NF-κB, in Notch signal-compromised macrophages, because the knockdown of CYLD in RBP-J-deficient macrophages or overexpression of p65 in RBP-J knockdown cells both restored NF-κB activation and the production of proinflammatory and/or profibrotic factors by macrophages. In human hepatic fibrosis biopsies, stronger Notch activation is correlated with more severe fibrosis, which is accompanied by a lower level of CYLD but irrespective of etiological reasons. CONCLUSION: RBP-J-mediated Notch signaling is required for macrophages to promote hepatic fibrosis by up-regulation of NF-κB activation through CYLD.


Assuntos
Cisteína Endopeptidases/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Cirrose Hepática/imunologia , Macrófagos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Enzima Desubiquitinante CYLD , Células Estreladas do Fígado/fisiologia , Hepatite/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Regulação para Cima
7.
Biopharm Drug Dispos ; 37(9): 511-521, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27662517

RESUMO

Drug interactions are one of the commonest causes of side effects, particularly in long-term therapy. The aim of the current study was to investigate the possible effects of metoprolol on the pharmacokinetics of metformin in rats and to clarify the mechanism of drug interaction. In this study, rats were treated with metformin alone or in combination with metoprolol. Plasma, urine and tissue concentrations of metformin were determined by HPLC. Western blotting and real-time qPCR were used to evaluate the expression of rOCTs and rMATE1. The results showed that, after single or 7-day repeated administration, the plasma concentrations of metformin in the co-administration group were significantly decreased compared with that in the metformin group. However, the parameter V/F of metformin in the co-administration group was markedly increased compared with that in the metformin group. The hepatic, renal and muscular Kp of metformin were markedly elevated after co-administration with metoprolol. Consistently, metformin uptake in rat kidney slices was significantly induced by metoprolol. In addition, multiple administrations of metoprolol significantly reduced the expression of rMATE1 in rat kidney as well as the urinary excretion of metformin. Importantly, after long-term administration, lactic acid and uric acid levels in the co-administration group were increased by 25% and 26%, respectively, compared with that in the metformin group. These results indicate that metoprolol can decrease the plasma concentration of metformin via the induction of hepatic, renal and muscular uptake, and long-term co-administration of metformin and metoprolol can cause elevated lactic acid and uric acid levels. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Rim/metabolismo , Fígado/metabolismo , Metformina/sangue , Metoprolol/metabolismo , Músculo Esquelético/metabolismo , Antagonistas de Receptores Adrenérgicos beta 1/sangue , Antagonistas de Receptores Adrenérgicos beta 1/metabolismo , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Interações Medicamentosas/fisiologia , Hipoglicemiantes/sangue , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Metformina/farmacologia , Metoprolol/sangue , Metoprolol/farmacologia , Músculo Esquelético/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Distribuição Aleatória , Ratos , Ratos Wistar
8.
Can J Physiol Pharmacol ; 93(8): 633-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26053941

RESUMO

Hepatic transporters and drug metabolizing enzymes (DMEs) play important roles in the pharmacological effects and (or) side-effects of many drugs, and are regulated by several mediators, including neurotransmitters. This work aimed to investigate whether serum levels of 5-hydroxytryptamine (5-HT) affected the expression of hepatic transporters or DMEs. The expression of hepatic transporters was assessed using the Western-blot technique in a 2,4,6-trinitrobenzenesulfonic-acid-induced rat model of post-infectious irritable bowel syndrome (PI-IBS), in which serum levels of 5-HT were significantly elevated. To further clarify the underlying mechanism, the 5-HT precursor 5-hydroxytryptophan (5-HTP) and the 5-HT depleting agent parachlorophenylalanine (pCPA) were applied to adjust serum levels of 5-HT. Serum levels of 5-HT were measured using LC-MS/MS; the expression of hepatic transporters, DMEs, and nuclear receptors were examined by Western-blot technique. Our results showed that in PI-IBS rats the expression of multidrug resistance protein 2 (Mrp2) was significantly decreased, while colonic enterochromaffin cell density and serum levels of 5-HT were all significantly increased. Moreover, 5-HTP treatment significantly increased serum levels of 5-HT and decreased the expression of Mrp2 and glycoprotein P (P-gp), whereas treatment with pCPA markedly decreased serum levels of 5-HT and increased the expression of Mrp2 and P-gp. Our results indicated that serum 5-HT regulates the expression of Mrp2 and P-gp, and the underlying mechanism may be related to the altered expression of the nuclear receptor constitutive androstane receptor (CAR).


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Síndrome do Intestino Irritável/sangue , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Serotonina/sangue , 5-Hidroxitriptofano/administração & dosagem , Animais , Colo/efeitos dos fármacos , Colo/metabolismo , Receptor Constitutivo de Androstano , Modelos Animais de Doenças , Células Enterocromafins/efeitos dos fármacos , Células Enterocromafins/metabolismo , Fenclonina/administração & dosagem , Síndrome do Intestino Irritável/induzido quimicamente , Síndrome do Intestino Irritável/fisiopatologia , Fígado/efeitos dos fármacos , Masculino , Ratos Wistar , Transdução de Sinais , Ácido Trinitrobenzenossulfônico
9.
Biomed Chromatogr ; 29(9): 1453-60, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25753317

RESUMO

A simple, specific and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), α-hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC-C18 column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, and post-column splitting (1:4) was used to give optimal interface flow rates (0.2 mL/min) for MS detection; the total run time was 8.5 min. Mass spectrometric detection was achieved using a triple-quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42-7000 ng/mL for MET, 2.05-4200 ng/mL for HMT and 1.95-4000 ng/mL for DMT. The analytical method was successfully applied to herb-drug interaction study of MET and breviscapine after administration of breviscapine (12.5 mg/kg) and MET (40 mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/análise , Flavonoides/sangue , Metoprolol/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Medicamentos de Ervas Chinesas/farmacocinética , Flavonoides/farmacocinética , Interações Ervas-Drogas , Masculino , Metoprolol/análogos & derivados , Metoprolol/farmacocinética , Ratos , Ratos Wistar
10.
Yao Xue Xue Bao ; 50(12): 1603-6, 2015 Dec.
Artigo em Zh | MEDLINE | ID: mdl-27169283

RESUMO

This study aims to investigate the change of plasma concentration of digoxin (DIG) in rats with ovariectomy. Twelve female SD rats were randomly assigned into ovariectomized group and sham group (n = 6). All rats plasma was collected after a single dose of 2 mg x kg(-1) DIG administrated orally, serum DIG concentration was determined by LC-MS/MS. The level of P-gp in the intestinal was analyzed by Western blotting. Pharmacokinetic calculations were performed on each individual using DAS 2.0 practical pharmacokinetic software. Compared with the sham group, C(max) of ovariectomized group decreased significantly (P < 0.01). There was no significant difference of AUC(0-t), and the level of P-gp was elevated in ovariectomized group. It was found that C(max) of DIG was significantly reduced after ovariectomy, and the change was associated with the decreased level of estrogen, which contributes to the increased level of P-gp.


Assuntos
Digoxina/sangue , Digoxina/farmacocinética , Ovariectomia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Western Blotting , Cromatografia Líquida , Modelos Animais de Doenças , Estrogênios/sangue , Feminino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem
11.
Yi Chuan ; 37(6): 599-604, 2015 06.
Artigo em Zh | MEDLINE | ID: mdl-26351057

RESUMO

Genetics is one of the most important courses for undergraduate students majoring in life science. In recent years, new knowledge and technologies are continually updated with deeper understanding of life science. However, the teaching model of genetics is still based on theoretical instruction, which makes the abstract principles hard to understand by students and directly affects the teaching effect. Thus, exploring a new teaching model is necessary. We have carried out a new teaching model, literature-based learning, in the course on Microbial Genetics for undergraduate students majoring in biotechnology since 2010. Here we comprehensively analyzed the implementation and application value of this model including pre-course knowledge, how to choose professional literature, how to organize teaching process and the significance of developing this new teaching model for students and teachers. Our literature-based learning model reflects the combination of "cutting-edge" and "classic" and makes book knowledge easy to understand, which improves students' learning effect, stimulates their interests, expands their perspectives and develops their ability. This practice provides novel insight into exploring new teaching model of genetics and cultivating medical talents capable of doing both basic and clinical research in the "precision medicine" era.


Assuntos
Genética/educação , Aprendizagem , Ensino
12.
BMC Cancer ; 14: 463, 2014 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-24952875

RESUMO

BACKGROUND: Aberrantly activated Notch signaling has been found in more than 50% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Current strategies that employ γ-secretase inhibitors (GSIs) to target Notch activation have not been successful. Many limitations, such as non-Notch specificity, dose-limiting gastrointestinal toxicity and GSI resistance, have prompted an urgent need for more effective Notch signaling inhibitors for T-ALL treatment. Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) has been demonstrated to suppress Notch activation in vitro, suggesting that FHL1C may be new candidate target in T-ALL therapy. However, the role of FHL1C in T-ALL cells remained unclear. METHODS: Using RT-PCR, we amplified full-length human FHL1C, and constructed full-length and various truncated forms of FHL1C. Using cell transfection, flow cytometry, transmission electron microscope, real-time RT-PCR, and Western blotting, we found that overexpression of FHL1C induced apoptosis of Jurkat cells. By using a reporter assay and Annexin-V staining, the minimal functional sequence of FHL1C inhibiting RBP-J-mediated Notch transactivation and inducing cell apoptosis was identified. Using real-time PCR and Western blotting, we explored the possible molecular mechanism of FHL1C-induced apoptosis. All data were statistically analyzed with the SPSS version 12.0 software. RESULTS: In Jurkat cells derived from a Notch1-associated T-ALL cell line insensitive to GSI treatment, we observed that overexpression of FHL1C, which is down-regulated in T-ALL patients, strongly induced apoptosis. Furthermore, we verified that FHL1C-induced apoptosis depended on the RBP-J-binding motif at the C-terminus of FHL1C. Using various truncated forms of FHL1C, we found that the RBP-J-binding motif of FHL1C had almost the same effect as full-length FHL1C on the induction of apoptosis, suggesting that the minimal functional sequence in the RBP-J-binding motif of FHL1C might be a new drug candidate for T-ALL treatment. We also explored the molecular mechanism of FHL1C overexpression-induced apoptosis, which suppressed downstream target genes such as Hes1 and c-Myc and key signaling pathways such as PI3K/AKT and NF-κB of Notch signaling involved in T-ALL progression. CONCLUSIONS: Our study has revealed that FHL1C overexpression induces Jurkat cell apoptosis. This finding may provide new insights in designing new Notch inhibitors based on FHL1C to treat T-ALL.


Assuntos
Apoptose/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas Musculares/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/metabolismo , Motivos de Aminoácidos , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Progressão da Doença , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais
13.
Front Immunol ; 15: 1379853, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38650937

RESUMO

Introduction: Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. Methods: HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. Results: In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. Discussion: iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.


Assuntos
Fenótipo , Animais , Camundongos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos/imunologia , Proliferação de Células , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Apoptose , Fagocitose , Movimento Celular/imunologia
14.
Mol Biol Rep ; 40(2): 1531-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23138187

RESUMO

Dendritic cells (DCs) are professional antigen presenting cells that activate and modulate immune responses, but the mechanisms underlying DC activation have not been fully understood. In this study, we investigated the role of Notch signaling in DC activation by using murine bone marrow-derived DCs. Triggering of Toll-like receptors (TLRs) of DCs led to upregulated expression of Notch ligands. Disruption of Notch signaling by the deletion of RBP-J, the critical transcription factor mediating the canonical signaling from all Notch receptors, resulted in a reduced capacity of DCs in activating T cells. Moreover, RBP-J deficiency altered the polarization of T cell activation, as manifested by downregulated interferon-γ and upregulated interleukin-4 and -10 expressions after LPS or Poly(I:C) stimulation. Furthermore, we found that RBP-J(-/-) DCs had reduced intracellular calcium after TLR-triggering. Immunofluorescent staining showed that RBP-J deficient DCs exhibited attenuated cytoskeleton reorganization when contacting T cells. In summary, our results suggested that the canonical Notch signaling promotes the cytoskeleton reorganization and the TLR-mediated DC activation.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Comunicação Celular , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Deleção de Genes , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais , Linfócitos T/fisiologia , Regulação para Cima
15.
Mol Biol Rep ; 40(3): 2097-105, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23224519

RESUMO

The transcription factor recombination signal binding protein-Jκ (RBP-J) is the critical transcription factor downstream to all four mammalian Notch receptors. Although it has been reported that Notch signaling pathway is involved in bone remodeling, the importance of RBP-J in osteoclastogenesis has not been fully explored. To investigate the role of RBP-J in osteoclastogenesis, we conditionally deleted RBP-J systemically in bone marrow (BM) or specifically in macrophages. We found that disruption of RBP-J in BM resulted in an obvious decrease in trabecular bone mass associated with an increase in osteoclasts, leading to osteopenia. Disruption of RBP-J in macrophages phenocopied the phenotypes of RBP-J deletion in BM with respect to osteoclastogenesis, suggesting that the osteopenia in RBP-J deficient mice is essentially resulted from increased osteoclastogenesis. Furthermore, we found that RBP-J deletion in osteoclasts resulted in a dramatic increase in tartrate-resistant acid phosphatase expression. These findings demonstrate a negatively role of RBP-J in the differentiation of osteoclasts and suggest that Notch pathway may be a new therapeutic target for bone diseases related to increased osteoclastogenesis.


Assuntos
Doenças Ósseas Metabólicas/genética , Diferenciação Celular/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Osteoclastos/citologia , Osteoclastos/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Animais , Doenças Ósseas Metabólicas/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Receptores Notch/metabolismo , Transdução de Sinais , Fosfatase Ácida Resistente a Tartarato
16.
Front Immunol ; 14: 1193081, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37680624

RESUMO

Yolk sac-derived microglia and peripheral monocyte-derived macrophages play a key role during Parkinson's disease (PD) progression. However, the regulatory mechanism of microglia/macrophage activation and function in PD pathogenesis remains unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and activation. In this study, with an 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride-induced acute murine PD model, we found that Notch signaling was activated in amoeboid microglia accompanied by a decrease in tyrosine hydroxylase (TH)-positive neurons. Furthermore, using myeloid-specific RBP-J knockout (RBP-JcKO) mice combined with a PD model, our results showed that myeloid-specific disruption of RBP-J alleviated dopaminergic neurodegeneration and improved locomotor activity. Fluorescence-activated cell sorting (FACS) analysis showed that the number of infiltrated inflammatory macrophages and activated major histocompatibility complex (MHC) II+ microglia decreased in RBP-JcKO mice compared with control mice. Moreover, to block monocyte recruitment by using chemokine (C-C motif) receptor 2 (CCR2) knockout mice, the effect of RBP-J deficiency on dopaminergic neurodegeneration was not affected, indicating that Notch signaling might regulate neuroinflammation independent of CCR2+ monocyte infiltration. Notably, when microglia were depleted with the PLX5622 formulated diet, we found that myeloid-specific RBP-J knockout resulted in more TH+ neurons and fewer activated microglia. Ex vitro experiments demonstrated that RBP-J deficiency in microglia might reduce inflammatory factor secretion, TH+ neuron apoptosis, and p65 nuclear translocation. Collectively, our study first revealed that RBP-J-mediated Notch signaling might participate in PD progression by mainly regulating microglia activation through nuclear factor kappa-B (NF-κB) signaling.


Assuntos
NF-kappa B , Doença de Parkinson , Animais , Camundongos , Microglia , Ativação de Macrófagos , Transdução de Sinais , Dopamina
17.
Cell Immunol ; 278(1-2): 84-90, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23121979

RESUMO

Microglia participates in the regulation of many inflammation-related pathological processes in the central nervous system, but how microglial activation is regulated has not been fully understood. Here, by using a microglial cell line, we show that microglia, like other macrophages, are activated by inflammatory stimuli in a polarized manner. The LPS-polarized M1 microglia appeared to be unable to respond to a secondary IL4 stimulation, while IL4-polarized M2 microglia could respond to secondary LPS stimulation. We also show that Notch signaling is involved in microglial polarization. When Notch signaling was blocked, the M1 polarization was suppressed, while the M2 polarization was promoted. Withdraw of the Notch signal inhibitor did not permit M2 N9 cells to re-polarize to M1 upon LPS stimulation, suggesting that the effects of Notch blockade on microglial polarization could be "memorized" by cells. These results suggest complicated mechanisms including epigenetic programs in the regulation of macrophage polarization.


Assuntos
Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Receptores Notch/imunologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Linhagem Celular , Epigênese Genética , Camundongos , Microglia/citologia , Microglia/imunologia , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Transdução de Sinais
18.
Hepatology ; 54(3): 979-88, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21633967

RESUMO

UNLABELLED: Hepatic ischemia/reperfusion (I/R) injury is initiated by reactive oxygen species (ROS) accumulated during the early reperfusion phase after ischemia, but cellular mechanisms controlling ROS production and scavenging have not been fully understood. In this study, we show that blocking Notch signal by knockout of the transcription factor RBP-J or a pharmacological inhibitor led to aggravated hepatic I/R injury, as manifested by deteriorated liver function and increased apoptosis, necrosis, and inflammation, both in vitro and in vivo. Interruption of Notch signaling resulted in increased intracellular ROS in hepatocytes, and a ROS scavenger cured exacerbated hepatic I/R injury after Notch signaling blockade, suggesting that Notch signal deficiency aggravated I/R injury through increased ROS levels. Notch signal blockade resulted in down-regulation of Hes5, leading to reduced formation of the Hes5-STAT3 complex and hypophosphorylation of STAT3, which further attenuated manganese superoxide dismutase (MnSOD) expression and increased ROS and apoptosis. Indeed, overexpression of a constitutively active STAT3 rescued MnSOD expression and I/R injury-induced apoptosis in the absence of Notch signaling. Finally, forced Notch activation by ligand stimulation or Hes5 overexpression reduced intracellular ROS and protected hepatocytes from apoptosis after I/R injury through the activation of STAT3 and MnSOD expression. CONCLUSION: Notch signal protects hepatocytes from I/R injury by Hes5-dependent activation of STAT3, which activates the expression of MnSOD, leading to the scavenging of ROS.


Assuntos
Hepatócitos/metabolismo , Janus Quinase 2/fisiologia , Fígado/irrigação sanguínea , Espécies Reativas de Oxigênio/metabolismo , Receptores Notch/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose , Células Cultivadas , Citoproteção , Camundongos , Camundongos Endogâmicos C57BL , Superóxido Dismutase/fisiologia
19.
Protein Expr Purif ; 85(1): 109-16, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22813925

RESUMO

Signal regulatory protein (SIRP) α, a transmembrane protein belonging to the immunoglobulin superfamily, is a receptor for CD47. The interaction between SIRPα and CD47 plays an important role in regulating the phagocytosis of leukemia cells and leukemia stem cells (LSCs) by macrophages. Blocking antibodies against CD47 have been shown to promote phagocytosis of LSCs by macrophages. Here, we consider an alternative way to interrupt the interaction between CD47 and SIRPα. We expressed the extracellular domains of the human SIRPα (hSIRP(ext)) and the human CD47 (hCD47(ext)) in Escherichia coli as Trx fusion proteins, and purified them by using affinity chromatography. We show that the purified fusion protein Trx-SIRP(ext) could interact in vitro with Trx-hCD47(ext). Moreover, Trx-SIRP(ext) could effectively bind to Jurkat T-ALL cells, which expressed CD47 at a high level. CD47(ext), on the other hand, bound to human macrophages. In vitro phagocytosis assay showed that these fusion proteins could enhance the phagocytosis of Jurkat cells by macrophage, with Trx-hSIRP(ext) showed a higher efficiency than Trx-CD47(ext). These results indicated that the soluble Trx-hSIRP(ext) and Trx-CD47(ext) polypeptides could be alternative molecules to interrupt CD47-SIRPα interaction between leukemia cells and macrophages, and might be potentially useful for the targeted therapy of leukemia.


Assuntos
Antígenos de Diferenciação/genética , Antígenos de Diferenciação/farmacologia , Antígeno CD47/genética , Antígeno CD47/farmacologia , Escherichia coli/genética , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Receptores Imunológicos/genética , Antígenos de Diferenciação/química , Antígenos de Diferenciação/imunologia , Antígeno CD47/química , Antígeno CD47/imunologia , Humanos , Células Jurkat , Leucemia de Células T/tratamento farmacológico , Leucemia de Células T/imunologia , Macrófagos/imunologia , Redobramento de Proteína , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia
20.
Mol Biol Rep ; 39(5): 5643-50, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22203480

RESUMO

Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP-MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfocinas/metabolismo , Macrófagos/enzimologia , Proteínas de Membrana/metabolismo , Óxido Nítrico/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Linfocinas/genética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
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