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1.
Mil Med Res ; 8(1): 18, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33685528

RESUMO

BACKGROUND: Vacuum sealing drainage (VSD) and epidermal growth factor (EGF) both play an important role in the treatment of wounds. This study aims to explore the effects of the combination of VSD and EGF on wound healing and the optimal concentration and time of EGF. METHODS: We tested the proliferation and migration capacity of HaCaT and L929 cells at different EGF concentrations (0, 1, 5, 10, and 100 ng/ml) and different EGF action times (2, 10, and 30 min). A full-thickness skin defect model was established using male, 30-week-old Bama pigs. The experiment included groups as follows: routine dressing change after covering with sterile auxiliary material (Control), continuous negative pressure drainage of the wound (VSD), continuous negative pressure drainage of the wound and injection of EGF 10 min followed by removal by continuous lavage (V + E 10 min), and continuous negative pressure drainage of the wound and injection of EGF 30 min followed by removal by continuous lavage (V + E 30 min). The wound healing rate, histological repair effect and collagen deposition were compared among the four groups. RESULTS: An EGF concentration of 10 ng/ml and an action time of 10 min had optimal effects on the proliferation and migration capacities of HaCaT and L929 cells. The drug dispersion effect was better than drug infusion after bolus injection effect, and the contact surface was wider. Compared with other groups, the V + E 10 min group promoted wound healing to the greatest extent and obtained the best histological score. CONCLUSIONS: A recombinant human epidermal growth factor (rhEGF) concentration of 10 ng/ml can promote the proliferation and migration of epithelial cells and fibroblasts to the greatest extent in vitro. VSD combined with rhEGF kept in place for 10 min and then washed, can promote wound healing better than the other treatments in vivo.


Assuntos
Fator de Crescimento Epidérmico/uso terapêutico , Hormônio do Crescimento Humano/uso terapêutico , Tratamento de Ferimentos com Pressão Negativa/normas , Cicatrização/efeitos dos fármacos , Animais , Fator de Crescimento Epidérmico/farmacologia , Hormônio do Crescimento Humano/farmacologia , Tratamento de Ferimentos com Pressão Negativa/métodos , Suínos
2.
Nat Commun ; 12(1): 613, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504774

RESUMO

Induction of intrinsic liver regeneration is an unmet need that can be achieved by temporally activating key hepatocyte regenerative pathways. Here, we establish an efficient, safe, non-integrative method to transiently express hepatocyte-growth-factor (HGF) and epidermal-growth-factor (EGF) in hepatocytes via nucleoside-modified, lipid-nanoparticle-encapsulated mRNA (mRNA-LNP) delivery in mice. We confirm specific hepatotropism of mRNA-LNP via intravenous injection of firefly luciferase encoding mRNA-LNP, with protein expression lasting about 3 days. In the liver, virtually all hepatocytes are transfected along with a subpopulation of endothelial and Kupffer cells. In homeostasis, HGF mRNA-LNP efficiently induce hepatocyte proliferation. In a chronic liver injury mouse model recapitulating non-alcoholic fatty liver disease, injections of both HGF and EGF mRNA-LNP sharply reverse steatosis and accelerate restoration of liver function. Likewise, HGF and EGF mRNA-LNP accelerate liver regeneration after acetaminophen-induced acute liver injury with rapid return to baseline ALT levels. This study introduces mRNA-LNP as a potentially translatable safe therapeutic intervention to harness liver regeneration via controlled expression of endogenous mitogens in vivo.


Assuntos
Hepatócitos/patologia , Lipídeos/química , Regeneração Hepática/fisiologia , Fígado/patologia , Nanopartículas/química , Nucleosídeos/metabolismo , RNA Mensageiro/metabolismo , Acetaminofen , Animais , Proliferação de Células/efeitos dos fármacos , Doença Crônica , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/farmacologia , Feminino , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Injeções , Fígado/efeitos dos fármacos , Fígado/lesões , Fígado/fisiopatologia , Testes de Função Hepática , Regeneração Hepática/efeitos dos fármacos , Camundongos Endogâmicos C57BL
3.
Nat Commun ; 12(1): 71, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397984

RESUMO

Signaling complexes are often organized in a spatiotemporal manner and on a minute timescale. Proximity labeling based on engineered ascorbate peroxidase APEX2 pioneered in situ capture of spatiotemporal membrane protein complexes in living cells, but its application to cytosolic proteins remains limited due to the high labeling background. Here, we develop proximity labeling probes with increased labeling selectivity. These probes, in combination with label-free quantitative proteomics, allow exploring cytosolic protein assemblies such as phosphotyrosine-mediated protein complexes formed in response to minute-scale EGF stimulation. As proof-of-concept, we systematically profile the spatiotemporal interactome of the EGFR signaling component STS1. For STS1 core complexes, our proximity proteomics approach shows comparable performance to affinity purification-mass spectrometry-based temporal interactome profiling, while also capturing additional-especially endosomally-located-protein complexes. In summary, we provide a generic approach for exploring the interactome of mobile cytosolic proteins in living cells at a temporal resolution of minutes.


Assuntos
Citosol/metabolismo , Proteômica , Transdução de Sinais , Biotina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Humanos , Fenóis , Mapeamento de Interação de Proteínas , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de Tempo
4.
PLoS One ; 15(8): e0238155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841278

RESUMO

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.


Assuntos
Antineoplásicos/farmacologia , Sinalização do Cálcio , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Gefitinibe/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Estrenos/farmacologia , Humanos , Fatores de Transcrição NFATC/biossíntese , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores
5.
Rev. habanera cienc. méd ; 19(4): e3048, ilus
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1139171

RESUMO

RESUMEN Introducción: la Insuficiencia renal crónica es un problema de salud pública, por lo que surge la necesidad de incrementar las acciones dirigidas a su prevención, diagnóstico e intervenciones terapéuticas eficaces; estudios recientes emplean el Factor de crecimiento epidérmico para evaluar su efecto reno-protector en variables funcionales; sin embargo se carece de estudios relacionados con los efectos de este producto sobre las características histológicas de riñones insuficientes. Objetivo: determinar los posibles efectos protectores y/o reparadores del Factor de crecimiento epidérmico humano recombinante sobre las características histológicas de riñones con insuficiencia renal crónica. Material y métodos: se trabajó con tres series, formadas por un grupo control y uno experimental cada una, de cinco animales, a los grupos experimentales se les realizó ablación quirúrgica de 5/6 de la masa renal. La serie A se conformó por animales controles, la B por los tratados con Factor de crecimiento epidérmico 24 horas antes del procedimiento y la C por los tratados con el bioproducto 24 horas después. Pasados 56 días del acto operatorio a los animales se les practicó la eutanasia y se procedió al estudio histológico del riñón. Resultados: en los animales de la serie A se observaron alteraciones histológicas en los corpúsculos y túbulos renales, en la serie B se observó que la mayor parte del parénquima renal presentó características normales y los de la serie C mostraron un daño renal incrementado. Conclusiones: El Factor de crecimiento epidérmico humano recombinante posee efecto reno-protector, sin embargo, no ofrece efecto reno-reparador(AU)


ABSTRACT Introduction: Chronic Kidney Failure is a public health problem; therefore, the need for actions aimed at its prevention, diagnosis and effective therapeutic interventions is a most. Recent studies use the Epidermal Growth Factor to evaluate its reno-protective effect on functional variables; however, there are no studies related to the effects of this product on histological features of kidney in chronic failure. Objective: To determine the possible protective and / or repair effects of recombinant human epidermal growth factor on the histological features of kidneys in chronic renal failure. Material and methods: We worked with three series, each consisting of a control group and an experimental group of five animals. The experimental groups underwent 5/6 surgical ablation of the renal mass. Series A consisted of control animals, series B included those animals treated with Epidermal Growth Factor 24 hours before the procedure and series C was made up of those animals treated with the bioproduct 24 hours after the procedure. Fifty-six days after surgical act, euthanasia was practiced on the animals and the kidneys were histologically studied. Results: Histological alterations were observed in the renal corpuscles and tubules in the animals included in series A; in series B it was observed that most of the renal parenchyma presented normal characteristics and those in series C showed increased kidney damage. Conclusions: Recombinant human epidermal growth factor has a reno-protective effect; however, it does not offer a repair effect of acute kidney(AU)


Assuntos
Animais , Ratos , Fator de Crescimento Epidérmico/farmacologia , Insuficiência Renal Crônica/tratamento farmacológico , Rim/efeitos dos fármacos
6.
Sci Rep ; 10(1): 7620, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376896

RESUMO

Epithelial-mesenchymal transition (EMT) is a major cellular process in which epithelial cells lose cell polarity and cell-cell adhesion and become motility and invasiveness by transforming into mesenchymal cells. Catechol is one of the natural compounds present in fruits and vegetables and has various pharmacological and physiological activities including anti-carcinogenic effects. However, the effects of catechol on EMT has not been reported. Epidermal growth factor (EGF) is one of the growth factors and is known to play a role in inducing EMT. The present study showed that catechol suppressed not only the morphological changes to the mesenchymal phenotype of epithelial HCC cells, but also the reduction of E-cadherin and the increment of Vimentin, which are typical hallmark of EMT. In addition, catechol suppressed EMT-related steps such as migration, invasion, anoikis resistance acquisition, and stem cell-like characterization through the EGFR-AKT-ERK signaling pathway during liver cancer metastasis. Therefore, these results suggest that catechol may be able to regulate the early metastasis of liver cancer in vitro.


Assuntos
Carcinoma Hepatocelular/patologia , Catecóis/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail/metabolismo
7.
Res Vet Sci ; 131: 232-243, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32417693

RESUMO

Cyadox, a new antibacterial agent as the quinoxaline-1, 4-dioxides, has a good antibacterial and growth-promoting effect, and has the advantages of lower toxicity, adequate safety and faster absorption. Seven differential expressed genes (DEGs) induced by cyadox were screened in swine liver tissues, including Insulin-like Growth Factor-1 (IGF-1), Epidermal Growth Factor (EGF), Poly ADP-ribose polymerase (PARP), the Defender Against Apoptotic Death 1 (DAD1), Complement Component 3 (C3), Transketolase (TK) and cyadox-related novel gene (CRNG). To elucidate the signal mechanism that cyadox altered these genes expression, the time-effect relationship and signaling pathways related to 7 DEGs induced by cyadox were determined in Porcine Kidney-15 (PK-15) cells by RT-qPCR and the application of various signal pathway inhibitors. The phosphorylation levels of signal factors in PK-15 cells were detected by Western blot. The analyses demonstrated that, the mRNA expressions of 7 DEGs were significantly enhanced by cyadox mainly through the phosphoinositide 3-kinase (PI3K) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) signaling pathways in PK-15 cells. Furthermore, EGF might be the early response gene of cyadox to activate downstream signaling pathways and regulates the expression of other related genes or directly exerting biological effects. In brief, cyadox mainly regulates the expression of these 7 genes by PI3K and NF-кB signaling pathways to exert it's antibacterial and growth-promoting activity in PK-15 cells.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transcrição Genética/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , NF-kappa B/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Quinoxalinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Suínos
8.
Nat Commun ; 11(1): 1711, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32249764

RESUMO

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk.


Assuntos
Técnicas de Cultura de Células/métodos , Linhagem da Célula , Glândulas Mamárias Humanas/citologia , Organoides/citologia , Organoides/metabolismo , Células-Tronco/citologia , Adulto , Proteína BRCA1/genética , Neoplasias da Mama , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Feminino , Humanos , Glândulas Mamárias Humanas/química , Glândulas Mamárias Humanas/metabolismo , Pessoa de Meia-Idade , Organoides/química , Análise de Célula Única , Células-Tronco/química , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
9.
Sci Rep ; 10(1): 6596, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313130

RESUMO

Enhancement of endogenous neurogenesis after ischemic stroke may improve functional recovery. We previously demonstrated that medium B, which is a combination with epidermal growth factor (EGF) and fibronectin, can promote neural stem/progenitor cell (NSPC) proliferation and migration. Here, we showed that medium B promoted proliferation and migration of cultured NSPCs onto various 3-dimentional structures. When rat cortical neurons with oxygen glucose deprivation (OGD) were co-cultured with NSPCs, medium B treatment increased neuronal viability and reduced cell apoptosis. In a rat model with transient middle cerebral artery occlusion (MCAO), post-insult intraventricular medium B treatment enhanced proliferation, migration, and neuronal differentiation of NSPCs and diminished cell apoptosis in the infarct brain. In cultured post-OGD neuronal cells and the infarct brain from MCAO rats, medium B treatment increased protein levels of Bcl-xL, Bcl-2, phospho-Akt, phospho-GSK-3ß, and ß-catenin and decreased the cleaved caspase-3 level, which may be associated with the effects of anti-apoptosis. Notably, intraventricular medium B treatment increased neuronal density, improved motor function and reduced infarct size in MCAO rats. In summary, medium B treatment results in less neuronal death and better functional outcome in both cellular and rodent models of ischemic stroke, probably via promotion of neurogenesis and reduction of apoptosis.


Assuntos
Apoptose , Isquemia Encefálica/tratamento farmacológico , Ventrículos Cerebrais/patologia , Fator de Crescimento Epidérmico/uso terapêutico , Fibronectinas/uso terapêutico , Neurogênese , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Isquemia Encefálica/complicações , Isquemia Encefálica/fisiopatologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ventrículos Cerebrais/fisiopatologia , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/farmacologia , Fibronectinas/farmacologia , Glucose/deficiência , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Ventrículos Laterais/patologia , Ventrículos Laterais/fisiopatologia , Masculino , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/ultraestrutura , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oxigênio , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/fisiopatologia
10.
PLoS One ; 15(4): e0231250, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275673

RESUMO

Single-cell expression analysis is an effective tool for studying the dynamics of cell population profiles. However, the majority of statistical methods are applied to individual profiles and the methods for comparing multiple profiles simultaneously are limited. In this study, we propose a nonparametric statistical method, called Decomposition into Extended Exponential Family (DEEF), that embeds a set of single-cell expression profiles of several markers into a low-dimensional space and identifies the principal distributions that describe their heterogeneity. We demonstrate that DEEF can appropriately decompose and embed sets of theoretical probability distributions. We then apply DEEF to a cytometry dataset to examine the effects of epidermal growth factor stimulation on an adult human mammary gland. It is shown that DEEF can describe the complex dynamics of cell population profiles using two parameters and visualize them as a trajectory. The two parameters identified the principal patterns of the cell population profile without prior biological assumptions. As a further application, we perform a dimensionality reduction and a time series reconstruction. DEEF can reconstruct the distributions based on the top coordinates, which enables the creation of an artificial dataset based on an actual single-cell expression dataset. Using the coordinate system assigned by DEEF, it is possible to analyze the relationship between the attributes of the distribution sample and the features or shape of the distribution using conventional data mining methods.


Assuntos
Biomarcadores/metabolismo , Análise de Célula Única , Estatísticas não Paramétricas , Adulto , Simulação por Computador , Bases de Dados como Assunto , Fator de Crescimento Epidérmico/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Tempo
11.
Tissue Cell ; 63: 101319, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32223947

RESUMO

Salivary epidermal growth factor (EGF) plays an important role in the maintenance of the oral and gastro-esophageal mucosa. Sialoadenectomy delays healing of oral wounds and affects lingual papillae. In this work, we aimed to determine the effect of EGF deficiency induced by sialoadenectomy and evaluate the effect of exogenous EGF administration on the lingual papillae and taste buds in rats. Thirty male adult Wistar albino rats were equally divided into 3 groups; sham-operated control group, sialoadenectomy group and group of sialoadenectomy + EGF. EGF was given 8 weeks after sialoadenectomy in a dose of 1 µg /ml/day in drinking water for 2 weeks. The anterior two-thirds of the tongue was dissected and cut longitudinally into two halves; one half for light microscope and the other for electron microscope examinations. Saliva and blood were collected to determine salivary and plasma EGF. Our results revealed that sialoadenectomy significantly reduced plasma and saliva levels of EGF which resulted in severe disruption of the architecture of lingual papillae. These changes were effectively improved by the exogenous EGF administration. In conclusion, EGF supplementation reversed the effects of sialoadenectomy and restored almost normal architecture of lingual papillae and taste buds.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Glândulas Salivares/metabolismo , Papilas Gustativas/metabolismo , Língua/metabolismo , Animais , Fator de Crescimento Epidérmico/deficiência , Fator de Crescimento Epidérmico/farmacologia , Mucosa Esofágica/efeitos dos fármacos , Mucosa Esofágica/metabolismo , Humanos , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Ratos , Saliva/efeitos dos fármacos , Saliva/metabolismo , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/cirurgia , Papilas Gustativas/efeitos dos fármacos , Papilas Gustativas/cirurgia , Língua/efeitos dos fármacos , Língua/patologia , Língua/cirurgia
12.
Int J Nanomedicine ; 15: 1421-1435, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184596

RESUMO

Purpose: In this study, we aim to explore the effects of graphene oxide (GO), a derivative of graphene, nanoparticles of four different sizes on the cellular fate of mouse neural stem cells (mNSCs). Methods: GO NPs were characterized with transmission electron microscopy (TEM), scanning electron micrography (SEM), atomic force microscopy (AFM) and Raman Spectra analysis. The cytotoxic effects of the GO NPs of different sizes on the mNSCs were determined using CCK-8 assay, Annexin V-APC/ 7-AAD staining and EdU staining assays. We investigated the biological and the mechanisms of GO NPs on cells using immunofluorescence analysis and quantitative real-time PCR (qPCR). Results: The average hydrodynamic sizes of the GO NPs were 417 nm, 663 nm, 1047 nm, and 4651 nm, with a thickness of approximately 22.5 nm, 17.7 nm, 22.4 nm, and 13.4 nm, respectively. GO NPs of all sizes showed low cytotoxicity at a concentration of 20 µg/mL on the mNSCs. Immunostaining demonstrated that treatment with GO NPs, especially the 663 nm ones, enhanced the self-renewal ability of mNSCs in the absence of EGF and bFGF. Under differentiation medium conditions that are free of mitogenic factors, all the GO NPs, particularly the 4651 nm ones, increased the expression level of Tuj1 and GFAP. With regards to the migration ability, we found that 417 nm GO-NP-treated mNSCs migrated over a longer distance than the control group obviously. In addition, higher expression of Rap1, Vinculin and Paxillin was observed in the GO NP-treated groups compared to the control group. mRNA-Sequence analysis and Western blotting results suggested that the 4651 nm GO NPs triggered positive neuronal differentiation through phosphorylation of ERK1/2 by the downregulating of TRPC2. Conclusion: GO NPs play an important role in the applications of inducing self-renewal and differentiation of mNSC, and are promising in the future for further studies.


Assuntos
Grafite/farmacologia , Nanopartículas/química , Células-Tronco Neurais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Grafite/química , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Células-Tronco Neurais/fisiologia , Tamanho da Partícula , Fosforilação/efeitos dos fármacos , Análise Espectral Raman , Canais de Cátion TRPC/metabolismo , Tubulina (Proteína)/metabolismo
13.
Endocrinology ; 161(4)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32147716

RESUMO

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone that promotes intestinal growth and proliferation through downstream mediators, including epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1). EGF synergistically enhances the proliferative actions of IGF-1 in intestinal cell lines, and both of these factors are known to be essential for the trophic effects of GLP-2 in vivo. However, whether EGF and IGF-1 interact to mediate the proliferative actions of GLP-2 in vivo remains unknown. Normal and knockout (KO) mice lacking the intestinal epithelial IGF-1 receptor (IE-IGF-1R) were therefore treated chronically with EGF and/or long-acting human hGly2GLP-2, followed by determination of intestinal growth parameters. Intestines from control and IE-IGF-1R KO mice were also used to generate organoids (which lack the GLP-2 receptor) and were treated with EGF and/or IGF-1. Combination treatment with EGF and hGly2GLP-2 increased small intestinal weight and crypt-villus height in C57Bl/6 mice in an additive manner, whereas only hGly2GLP-2 treatment increased crypt cell proliferation. However, although combination treatment also increased small intestinal weight and crypt-villus height in IE-IGF-1R KO mice, the proliferative responses to hGly2GLP-2 alone or with EGF were diminished in these animals. Finally, IGF-1 treatment of organoids undergoing EGF withdrawal was not additive to the effect of EGF replacement on proliferation, but could restore normal proliferation in the absence of EGF. Together, these findings demonstrate that the intestinal proliferative effects of hGly2GLP-2 are augmented by exogenous EGF in a manner that is partially dependent upon IE-IGF-1R signaling.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Intestino Delgado/metabolismo , Camundongos , Camundongos Knockout , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos
14.
J Neurosci ; 40(13): 2618-2632, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32079647

RESUMO

Sensory hair cell losses underlie the vast majority of permanent hearing and balance deficits in humans, but many nonmammalian vertebrates can fully recover from hearing impairments and balance dysfunctions because supporting cells (SCs) in their ears retain lifelong regenerative capacities that depend on proliferation and differentiation as replacement hair cells. Most SCs in vertebrate ears stop dividing during embryogenesis; and soon after birth, vestibular SCs in mammals transition to lasting quiescence as they develop massively thickened circumferential F-actin bands at their E-cadherin-rich adherens junctions. Here, we report that treatment with EGF and a GSK3 inhibitor thinned the circumferential F-actin bands throughout the sensory epithelium of cultured utricles that were isolated from adult mice of either sex. That treatment also caused decreases in E-cadherin, ß-catenin, and YAP in the striola, and stimulated robust proliferation of mature, normally quiescent striolar SCs. The findings suggest that E-cadherin-rich junctions, which are not present in the SCs of the fish, amphibians, and birds which readily regenerate hair cells, are responsible in part for the mammalian ear's vulnerability to permanent balance and hearing deficits.SIGNIFICANCE STATEMENT Millions of people are affected by hearing and balance deficits that arise when loud sounds, ototoxic drugs, infections, and aging cause hair cell losses. Such deficits are permanent for humans and other mammals, but nonmammals can recover hearing and balance after supporting cells regenerate replacement hair cells. Mammalian supporting cells lose the capacity to proliferate around the time they develop unique, exceptionally reinforced, E-cadherin-rich intercellular junctions. Here, we report the discovery of a pharmacological treatment that thins F-actin bands, depletes E-cadherin, and stimulates proliferation in long-quiescent supporting cells within a balance epithelium from adult mice. The findings suggest that high E-cadherin in those supporting cell junctions may be responsible, in part, for the permanence of hair cell loss in mammals.


Assuntos
Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células Ciliadas Auditivas/efeitos dos fármacos , Sáculo e Utrículo/efeitos dos fármacos , Actinas/metabolismo , Animais , Caderinas/genética , Células Ciliadas Auditivas/metabolismo , Camundongos , Piridinas/farmacologia , Pirimidinas/farmacologia , Sáculo e Utrículo/metabolismo , beta Catenina/metabolismo
15.
Int J Mol Sci ; 21(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906564

RESUMO

Multiple isoforms of 14-3-3 proteins exist in different organisms. In mammalian cells, 14-3-3 protein has seven isoforms (α/ß, ε, η, γ, σ, θ/τ, and δ/ζ), with α and δ representing the phosphorylated versions of ß and ζ, respectively. While the existence of multiple isoforms may represent one more level of regulation in 14-3-3 signaling, our knowledge regarding the isoform-specific functions of 14-3-3 proteins is very limited. Determination of the subcellular localization of the different 14-3-3 isoforms could give us important clues of their specific functions. In this study, by using indirect immunofluorescence, subcellular fractionation, and immunoblotting, we studied the subcellular localization of the total 14-3-3 protein and each of the seven 14-3-3 isoforms; their redistribution throughout the cell cycle; and their translocation in response to EGF in Cos-7 cells. We showed that 14-3-3 proteins are broadly distributed throughout the cell and associated with many subcellular structures/organelles, including the plasma membrane (PM), mitochondria, ER, nucleus, microtubules, and actin fibers. This broad distribution underlines the multiple functions identified for 14-3-3 proteins. The different isoforms of 14-3-3 proteins have distinctive subcellular localizations, which suggest their distinctive cellular functions. Most notably, 14-3-3ƞ is almost exclusively localized to the mitochondria, 14-3-3γ is only localized to the nucleus, and 14-3-3σ strongly and specifically associated with the centrosome during mitosis. We also examined the subcellular localization of the seven 14-3-3 isoforms in other cells, including HEK-293, MDA-MB-231, and MCF-7 cells, which largely confirmed our findings with Cos-7 cells.


Assuntos
Proteínas 14-3-3/metabolismo , Ciclo Celular , Fator de Crescimento Epidérmico/farmacologia , Proteínas 14-3-3/genética , Animais , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitose , Isoformas de Proteínas/metabolismo , Transporte Proteico , Frações Subcelulares/metabolismo
16.
Mol Med Rep ; 21(1): 329-337, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31939627

RESUMO

Previous studies have demonstrated that the expression of CARD10 is closely associated with the occurrence of tumors, and its role is mainly to promote tumor progression by activating the transcription factor NF­κB. However, the signaling pathway in renal cancer remains unclear. The objective of the present study was to investigate the ability of caspase recruitment domain 10 (CARD10) to regulate the NF­κB signaling pathway and promote the progression of renal cell carcinoma (RCC). Expression of CARD10 in ACHN, 786­O and HK­2 cells was evaluated via western blot analysis, as was the epidermal growth factor (EGF)­induced activation of NF­κB signaling pathway­related proteins in cells. The expression of CARD10 was inhibited by CARD10 short hairpin RNA transfection. Cell cycle analysis and MTT assays were used to evaluate cell proliferation. Cell apoptosis was analyzed via flow cytometry. The invasion of renal cell lines was detected via Transwell cell migration and invasion assays in vitro. The results showed that CARD10 expression was significantly higher in RCC cells than in normal renal tubular epithelial cells. CARD10 silencing inhibited the proliferation, invasion and migration of RCC cells. EGF stimulation upregulated the activation of the NF­κB pathway in RCC cells. Inhibition of CARD10 expression inhibited NF­κB activation in RCC cells. Taken together, these data suggested that CARD10 promotes the progression of renal cell carcinoma by regulating the NF­κB signaling pathway. Thus, this indicated that CARD10 may be a novel therapeutic target in RCC.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Fator de Crescimento Epidérmico/farmacologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
17.
Prostate ; 80(5): 412-423, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31995655

RESUMO

BACKGROUND: Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells. METHODS: We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes. RESULTS: Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR. CONCLUSION: We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Aminoquinolinas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Células PC-3 , Pirimidinas/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteína Companheira de mTOR Insensível à Rapamicina/deficiência , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Sirolimo/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
Dermatol Ther ; 33(1): e13196, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31849151

RESUMO

After burns, protecting tissues by medicines in the zone of stasis reduces the width and depth of injury. This study's goal was to reduce burned tissue damage in the zone of stasis using epidermal growth factor (EGF). Forty-eight Wistar rats were separated into three groups. In all groups, the burn procedure was applied following the comb burn model. In Group 1, no postburn treatment was administered. In Group 2, physiological saline solution (0.3 cc) was injected intradermally and in Group 3, EGF (0.3 cc) was injected intradermally into stasis zone tissues after the burn procedure. Surviving tissue rates were 24.0% in Group 1, 25.3% in Group 2, and 70.2% in Group 3. The average numbers of cells stained with Nrf2, HO-1, and the number of apoptotic cells were 230, 150, and 17.5 in Group 1, 230, 145, and 15.0 in Group 2, and 370, 230, and 0 in Group 3, respectively. Values in Group 3 were found to be statistically significantly different than those of Groups 1 and 2; there was no difference between Groups 1 and 2. This study shows that EGF protects zone of stasis tissue from burn damage.


Assuntos
Queimaduras/tratamento farmacológico , Fator de Crescimento Epidérmico/administração & dosagem , Cicatrização/efeitos dos fármacos , Animais , Queimaduras/patologia , Modelos Animais de Doenças , Progressão da Doença , Fator de Crescimento Epidérmico/farmacologia , Feminino , Injeções Intradérmicas , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Pele/patologia , Resultado do Tratamento
19.
Theriogenology ; 142: 320-327, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711691

RESUMO

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ±â€¯1.2 vs. 100% ±â€¯0.0), presence of first polar body (65.9% ±â€¯1.2 vs. 70.5% ±â€¯1.8), nuclear status in second metaphase (62.5% ±â€¯11.6 vs. 68.4% ±â€¯4.9), cytoplasmic maturation (100.0% ±â€¯0.7 vs. 75.0% ±â€¯0.7), reactive oxygen species levels (0.5 ±â€¯0.2 vs. 0.3 ±â€¯0.1), and mitochondrial membrane potential (1.1 ±â€¯0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.


Assuntos
Adenina/análogos & derivados , Artiodáctilos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ionomicina/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Adenina/farmacologia , Animais , Artiodáctilos/embriologia , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Partenogênese/fisiologia
20.
Theriogenology ; 142: 284-290, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711701

RESUMO

The aims of this study were to investigate the effects of epidermal growth factor (EGF) and progesterone on the development, viability and the gene expression of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (∼0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5 °C, with 5% CO2 in air, for 18 days, in TCM-199+ (n = 63) alone (control medium) or supplemented with 10 ng/mL progesterone (n = 64), 10 ng/mL EGF (n = 61) or both EGF and progesterone (n = 66). The effects of these treatments on growth, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and cyclin B1 were evaluated, significantly different (p < 0.05). The results showed that there was a progressive increase in follicular diameter in all treatments, but only follicles cultured in medium supplemented with EGF had increased significantly in diameter when compared to follicles cultured in the control medium at the end of the culture period, significantly different (p < 0.05). A positive interaction between EGF and progesterone was not observed. In addition, the presence of EGF, progesterone or both in culture medium did not influence the rate of follicle survival and antrum formation. However, the presence of only progesterone in cultured medium increased the expression of mRNAs for GDF9 and cyclin B1 in oocytes. EGF also significantly increased the levels of mRNAs for cMOS and GDF9 when compared to follicles cultured in control medium. Ultrastructural analyzes showed that cultured follicles in all treatments maintained the integrity of granulosa cells. In conclusion, the EGF promotes the development of secondary follicles cultured in vitro for 18 days and increases the expression of cMOS and GDF9, while progesterone alone or in association with EGF have not a positive effect on follicular growth. However, progesterone increases the expression of GDF9 and cyclin B1 in oocytes.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Progesterona/farmacologia , Animais , Bovinos , Células Cultivadas , Feminino , Genes mos/efeitos dos fármacos , Genes mos/genética , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Fator 9 de Diferenciação de Crescimento/genética , Folículo Ovariano/fisiologia
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