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1.
J Sci Food Agric ; 100(2): 672-681, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31583701

RESUMO

BACKGROUND: Hibiscus sabdariffa is commonly used in daily life and its extract is applied widely in food and cosmetics. However, it has not been evaluated for its anti-aging effects. RESULTS: Hibiscus sabdariffa calyx aqueous extract (HSCAE) has shown potential collagenase activity suppression effects, together with tyrosinase activity inhibition, and anti-oxidation as a free radical scavenger. The current investigation demonstrated that HSCAE was not cytotoxic in skin fibroblasts, and it significantly decreased ultraviolet B (UVB)-induced reactive oxygen species (ROS) on a flow cytometry assay. Moreover, HSCAE reduced matrix metalloproteinase (MMP) expression, increased tissue inhibition of metalloproteinase (TIMP)-1 level, and enhanced collagen content by inhibiting collagenase activity. It also blocked mRNA and protein expressions of melanin production pathway key factors, including the microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and dopachrome tautomerase-2 (TRP-2). CONCLUSION: These results demonstrated, for the first time, the potential of HSCAE as a natural antioxidant with the ability to maintain collagen production and to decrease melanin syntheses under UVB radiation, for anti-aging effects. © 2019 Society of Chemical Industry.


Assuntos
Depuradores de Radicais Livres/farmacologia , Hibiscus/química , Extratos Vegetais/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Animais , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Camundongos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos
2.
Bioelectrochemistry ; 131: 107386, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31706116

RESUMO

The biocompatibility of human gingival fibroblasts (HGF) was evaluated in different concentrations of poly(vinyl alcohol) and sodium alginate (PVA/SA) nanofibres (3.5 wt% 4 wt% and 5 wt%). The PVA/SA nanofibres were deposited on the surface of an electrode microchip by using the electrospinning technique. Electrochemical impedance spectroscopy (EIS) was applied to measure the dielectric properties of each system. In order to provide a detailed analysis as well as a right physical interpretation of the EIS results, the data was fitted with an electric equivalent circuit based on the EIS and the microscopic assessments. The results registered three different time constants (TCs) of the PVA/SA scaffold which indicated different layers at different depths of the scaffold. The TCs changed their dielectric properties depending on the PVA/SA concentration. The 4 wt% system showed the highest biocompatibility properties, given that its resistance and electrochemical capacitance show the formation of a mature-stage cell interaction of HGF. The EIS data offers an exhaustive analysis of the biological activity of the cell response in real time to determine its biocompatibility features. Fluorescence analysis demonstrated a heterogeneous growth of the HGF on the PVA/SA scaffold surface.


Assuntos
Materiais Biocompatíveis , Espectroscopia Dielétrica/métodos , Gengiva/metabolismo , Tecidos Suporte , Fibroblastos/metabolismo , Gengiva/citologia , Humanos
3.
Int J Nanomedicine ; 14: 7123-7139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564869

RESUMO

Background: Poly(amidoamine) (PAMAM) dendrimers are of considerable interest when used as a carrier for topical drugs for the skin, although little is known about their possible side effects. Therefore, our study was about the impact of 2nd and 3rd generation PAMAM dendrimers on human keratinocytes and fibroblasts cells. Methods: The effect of the tested compounds on collagen biosynthesis was determined using 5[3H]-proline incorporation bioassay. Morphological changes accompanying cell growth inhibition were observed using a confocal microscope. To evaluate the percentage of apoptotic/necrotic cells and the cell growth dynamic of apoptotic features, we performed Annexin V/PI double staining assay, assessed caspase activity, and performed cell cycle analysis by flow cytometry. The flow cytometry method was also used to determine the effect of dendrimers on pro-inflammatory cytokines (IL-6, IL-8 IL-1ß). Results: The obtained results showed that as the concentration and the generation of dendrimers increased, collagen biosynthesis decreased. We also observed abnormalities in cell differentiation, which may have caused disturbed secretion of pro-inflammatory cytokines. We found that dendrimers cause chronic inflammation which may cause adverse changes in the skin, ultimately- leading to apoptosis in the case of dendrimers in lower concentrations or necrosis at higher concentrations (especially 3rd generation dendrimers). In addition, the inflammatory path induced by the tested compounds was caused by damage in the mitochondria, which we observed as a significant decrease in the mitochondrial membrane potential. Conclusion: The results of our study showed that PAMAM dendrimers can cause disorders of cell proliferation and differentiation and may be the cause of cell cycle deregulation and chronic adverse inflammation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Dendrímeros/farmacologia , Fibroblastos/metabolismo , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno/biossíntese , Dendrímeros/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fluorescência , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Necrose
4.
Int J Nanomedicine ; 14: 7237-7247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564876

RESUMO

Background: The health hazards of silica nanoparticle (SiNP) are raising worldwide concern as SiNPs has become the second largest manufactured nanomaterial in global markets. However, insufficient data for the adverse health effects and safety evaluation of SiNPs are remaining a big question. Purpose: We evaluated the effects and related mechanism of SiNPs on pulmonary inflammation and collagen production through repeated intravenous administration in mice in a 45-day observation period. Methods: Morphological and ultrastructural change, ultradistribution of SiNPs in lungs were observed in ICR mice through intravenous administration. Oxidative damage, pro-inflammatory cytokines, hydroxyproline content, the marker of fibroblasts and epithelial-mesenchymal transition (EMT), and JAK2/STAT3 and TGF-ß1/Smad3 signaling pathways were detected to explore the lung injuries and related mechanism. Results: The results showed repeated intravenous exposure of SiNPs increased the weight of lung tissues and destroyed pulmonary histomorphological structure. The increased MDA content, depletion of SOD and GSH-Px in lungs were observed in SiNP-treated mice. The protein expressions of JAK2/STAT3 pathway were upregulated in lungs, and the levels of inflammatory cytokines TNF-α, IL-1ß, and IL-6 in serum and lungs were also elevated in SiNPs treated group. The increased hydroxyproline content indicated collagen accumulation in lungs of SiNP-treated mice. Meanwhile, the protein expressions of the marker of myofibroblast (a-SMA), the regulators in connective tissue remodeling (CTGF), TGF-ß, and p-Smad3 were all upregulated in lungs. In addition, we found intravenous administration of SiNPs-induced ultrastructural changes in type II alveolar epithelial cells but without downregulation of the protein expression of the key markers of epithelial cells (E-Cadherin). Conclusion: Our results revealed that oxidative stress and inflammation contributed to the collagen accumulation through activation of JAK2/STAT3 and TGF-ß/Smad3 pathways. It suggests that pulmonary aberrant inflammation and collagen accumulation induced by nanoparticles should be seriously considered for the safety application in diagnostics or therapeutics.


Assuntos
Colágeno/metabolismo , Janus Quinase 2/metabolismo , Nanopartículas/administração & dosagem , Pneumonia/patologia , Transdução de Sinais , Dióxido de Silício/administração & dosagem , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Administração Intravenosa , Animais , Caderinas/metabolismo , Citocinas/metabolismo , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Pulmão/patologia , Pulmão/ultraestrutura , Lesão Pulmonar/patologia , Masculino , Camundongos Endogâmicos ICR , Modelos Biológicos , Miofibroblastos/metabolismo , Nanopartículas/ultraestrutura , Estresse Oxidativo , Fator de Transcrição STAT3/metabolismo
5.
Arch Virol ; 164(12): 2995-3006, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31576460

RESUMO

Reticuloendotheliosis virus (REV) is an important representative avian retrovirus. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of REV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect changes in protein levels in chicken embryo fibroblast cells (CEFs) that were infected with REV or mock infected. In total, 605 cellular proteins were differentially expressed, among which 196, 345, and 286 were differentially expressed in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and immune system processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding, catalytic activity, and enzyme regulator activity. Pathway analysis showed that a total of 143, 167, and 179 pathways, including protein digestion and absorption, focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, Toll-like receptors, and JAK-STAT signaling, were enriched in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. In conclusion, this study is the first to analyze the protein profile of REV-infected CEFs using an iTRAQ approach. The results of this study provide valuable information for better understanding the host response to REV infection.


Assuntos
Fibroblastos/metabolismo , Doenças das Aves Domésticas/genética , Proteoma/genética , Vírus da Reticuloendoteliose/fisiologia , Infecções por Retroviridae/veterinária , Animais , Embrião de Galinha , Galinhas , Fibroblastos/química , Fibroblastos/virologia , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologia , Proteoma/química , Proteoma/metabolismo , Proteômica , Vírus da Reticuloendoteliose/genética , Infecções por Retroviridae/genética , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/virologia , Espectrometria de Massas em Tandem
6.
Medicine (Baltimore) ; 98(40): e17126, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31577702

RESUMO

BACKGROUND: The aim of this study was to investigate the role of n-acetyl cysteine (NAC) in the lipopolysaccharide (LPS)-mediated induction of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) synthesis by human periodontal ligament fibroblast cells (hPDLFs). In addition, we aimed to determine the involvement of the nuclear factor-kappa B (NF-κB) pathway in any changes in IL-1ß and TNF-α expression observed in response to LPS and NAC. METHODS: HPDLFs were obtained by primary culture. The culture medium used in this experiment was Dulbecco's Modified Eagle Medium (DMEM low-glucose). Cells were stimulated with various concentrations of NAC or LPS. Cell proliferation was measured at various time-points with the cell Counting Kit 8 (CCK-8) assay. mRNA levels of IL-1ß and TNF-α were determined by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Protein levels of IL-1ß and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). Protein and mRNA expression levels of NF-κB were measured by western blot and RT-qPCR. RESULTS: The results showed that LPS treatment in hPDLFs induced mRNA and protein expression of IL-1ß, TNF-α, and NF-κB. However, these effects were eliminated by pretreatment with NAC. Pretreatment with both NAC (1 mmol/L) and BAY11-7082 (10 µmol/L) significantly inhibited the NF-κB activity induced by LPS. CONCLUSION: NAC inhibits the LPS-mediated synthesis of tumor TNF-α and IL-1ß in hPDLFs, through the NF-κB pathway.


Assuntos
Acetilcisteína/farmacologia , Fibroblastos/efeitos dos fármacos , Interleucina-1beta/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Humanos , Interleucina-1beta/biossíntese , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Ligamento Periodontal , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
7.
Nature ; 574(7779): 553-558, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645721

RESUMO

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.


Assuntos
Envelhecimento/metabolismo , Reprogramação Celular , Senescência Celular/fisiologia , Fibroblastos/metabolismo , Cicatrização , Animais , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/metabolismo , Judeus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Processos Estocásticos , Fatores de Tempo , Cicatrização/efeitos dos fármacos
8.
Braz J Med Biol Res ; 52(9): e8551, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482977

RESUMO

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Western Blotting , Fibroblastos/metabolismo , Imunofluorescência , Camundongos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
9.
Adv Gerontol ; 32(3): 357-363, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31512421

RESUMO

The aim of this work was to examine the content of Piezo1 in fibroblasts and blood vessels of human dermis from the development until deep aging (from 20 weeks of pregnancy until 85 years old), and defining of a role of Piezo1 in age-dependent changes in the number of fibroblasts and blood vessels in the dermis. Piezo1, proliferating cells nuclear antigen (PCNA), endothelial cells marker CD31 were detected with indirect immunohistochemical technique. Results showed that a portion of fibroblasts with positive staining for Piezo1 in the dermis is decreased from 20 weeks of pregnancy to 40 years old. Percent of Piezo1 positive fibroblasts in dermis is increased sufficiently since 41 years old until 60-85 years old group. The content of Piezo1 in blood vessels in the human dermis is decreased sufficiently from 20 weeks of pregnancy until 40 years old. Age-related changes in the content of Piezo1 in fibroblasts and blood vessels is not associated with an age-related decrease in total number and percent of PCNA positive fibroblasts, the number of blood vessels in the dermis.


Assuntos
Vasos Sanguíneos , Derme , Fibroblastos , Canais Iônicos , Envelhecimento da Pele , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Vasos Sanguíneos/fisiologia , Criança , Pré-Escolar , Derme/irrigação sanguínea , Derme/citologia , Derme/embriologia , Derme/crescimento & desenvolvimento , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Lactente , Canais Iônicos/metabolismo , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Antígeno Nuclear de Célula em Proliferação/metabolismo , Envelhecimento da Pele/fisiologia
10.
Biol Res ; 52(1): 52, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31540582

RESUMO

BACKGROUND: Long noncoding RNAs (lncRNAs) have been reported to be associated with dermis process during burn wound healing. This study aimed to investigate the role of lncRNA X-inactive specific transcript (XIST) in human skin fibroblasts (HSF) and extracellular matrix (ECM) as well as the regulatory network of XIST/microRNA-29b-3p (miR-29b-3p)/collagen 1 alpha 1 (COL1A1). METHODS: The wound samples were collected from 25 patients with deep partial thickness burn at day 5 after burn. The thermal injured model was established using HSF cells. The expressions of XIST, miR-29b-3p and COL1A1 were measured by quantitative real-time polymerase chain reaction and western blot. ECM synthesis, cell proliferation and migration were detected by western blot, cell counting kit-8 and trans-well assays, respectively. The interaction between miR-29b-3p and XIST or COL1A1 was explored by bioinformatics analysis and luciferase reporter assay. RESULTS: The expressions of XIST and COL1A1 were enhanced but miR-29b-3p expression was decreased after thermal injury. XIST overexpression promoted ECM synthesis, cell proliferation and migration in thermal injured HSF cells. However, XIST knockdown played an opposite effect. miR-29b-3p overexpression inhibited ECM synthesis, cell proliferation and migration, which was reversed by XIST. COL1A1 silence suppressed ECM synthesis, cell proliferation and migration by miR-29b-3p targeting. Moreover, COL1A1 up-regulation weakened the effect of XIST silence on ECM synthesis and HSF cell function. CONCLUSION: XIST promoted ECM synthesis, cell proliferation and migration by sponging miR-29b-3p and targeting COL1A1 in HSF cells after thermal injury, indicating the promoting role of XIST in wound healing.


Assuntos
Queimaduras/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , RNA Longo não Codificante/metabolismo , Western Blotting , Queimaduras/genética , Movimento Celular , Proliferação de Células , Matriz Extracelular/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Reação em Cadeia da Polimerase em Tempo Real
11.
Cell Mol Life Sci ; 76(21): 4179-4201, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31563998

RESUMO

Uncontrolled scarring, or fibrosis, can interfere with the normal function of virtually all tissues of the body, ultimately leading to organ failure and death. Fibrotic diseases represent a major cause of death in industrialized countries. Unfortunately, no curative treatments for these conditions are yet available, highlighting the critical need for a better fundamental understanding of molecular mechanisms that may be therapeutically tractable. The ultimate indispensable effector cells responsible for deposition of extracellular matrix proteins that comprise scars are mesenchymal cells, namely fibroblasts and myofibroblasts. In this review, we focus on the biology of these cells and the molecular mechanisms that regulate their pertinent functions. We discuss key pro-fibrotic mediators, signaling pathways, and transcription factors that dictate their activation and persistence. Because of their possible clinical and therapeutic relevance, we also consider potential brakes on mesenchymal cell activation and cellular processes that may facilitate myofibroblast clearance from fibrotic tissue-topics that have in general been understudied.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Humanos , Células-Tronco Mesenquimais/citologia , Miofibroblastos/citologia , Miofibroblastos/metabolismo , RNA não Traduzido/metabolismo , Transdução de Sinais
12.
Life Sci ; 233: 116714, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31376370

RESUMO

Increased levels of particulate matter (PM) air pollutants in East Asia have resulted in detrimental health impacts increasing morbidity and mortality. Epidemiological studies suggest a possible relation between the cutaneous exposure of PM and increased oxidative stress and inflammation which lead to skin lesions. The present study utilizes an integrated cell culture model of keratinocytes and fibroblasts to mimic viable skin layers and investigate the possible effects of PM exposure after penetration through corneocytes. The skin perfection is upheld by homeostatic functionality of epidermal cells and the integrity of connective tissues. Exposure to xenobiotics could alter the skin cell homeostasis aggravating premature skin aging. Stimulation of HaCaT keratinocytes by PM collected from Beijing, China (CPM) increased the intracellular ROS levels triggering a cascade of events aggravating inflammatory responses and connective tissue degradation. In HDF fibroblasts, treatment with preconditioned keratinocyte culture media augmented inflammatory responses, cellular differentiation, and connective tissue degradation. Above events were marked by the increased intracellular ROS, inflammatory mediators, pro-inflammatory cytokines, matrix metalloproteinases (MMP)-1 and -2 levels, collagenase, and elastase activity. Fucosterol treatment of keratinocytes dose-dependently attenuated the detrimental effects both in keratinocytes and fibroblasts restoring the conditions near to physiological levels. Further evaluations could be advanced on developing fucosterol, in forms such as rejuvenating cosmeceuticals which could attenuate detrimental responses of CPM exposure.


Assuntos
Fibroblastos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Material Particulado/efeitos adversos , Dermatopatias/tratamento farmacológico , Pele/efeitos dos fármacos , Estigmasterol/análogos & derivados , Poluentes Atmosféricos/efeitos adversos , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , NF-kappa B/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Dermatopatias/etiologia , Dermatopatias/metabolismo , Dermatopatias/patologia , Estigmasterol/farmacologia
13.
Cell Prolif ; 52(5): e12668, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31379046

RESUMO

OBJECTIVES: Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle-like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle-related cells. MATERIALS AND METHODS: Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three-dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two-dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. RESULTS: The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core-shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell-cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. CONCLUSIONS: Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.


Assuntos
Técnicas de Cultura de Células/métodos , Derme/citologia , Fibroblastos/citologia , Queratinócitos/citologia , Células Cultivadas , Derme/metabolismo , Fibroblastos/metabolismo , Humanos , Hidrogéis/química , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal
14.
Life Sci ; 234: 116779, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31430452

RESUMO

Emerging evidence has revealed that microRNAs (miRNAs) play critical roles in keloid pathogenesis. However, potential molecular mechanism of keloid formation remains unclear. In the present study, our findings showed that miR-152-3p mRNA expression level was notably up-regulated in keloid tissues and keloid fibroblasts compared with that of normal skin tissues and normal skin fibroblasts, respectively. Furthermore, miR-152-3p inhibition remarkably suppressed cell proliferation, which was increased by miR-152-3p overexpression. Cell invasion was also significantly decreased by miR-152-3p inhibition, whereas was increased by miR-152-3p overexpression. The mRNA and protein expression levels of extracellular matrix components including type I collagen, type III collagen and fibronectin were decreased by miR-152-3p inhibition, but were increased by miR-152-3p overexpression. In addition, results of dual-luciferase reporter assay indicated that FOXF1 is a direct target of miR-152-3p. FOXF1 overexpression significantly inhibits cell proliferation, invasion, and extracellular matrix in keloid fibroblasts, and the suppressive effects of miR-152-3p mimic on these functions were notably partly reversed by FOXF1 overexpression. Taken together, these findings indicated that miR-152-3p regulates cell proliferation, invasion and extracellular matrix expression through targeting FOXF1 in keloid fibroblasts, suggesting that miR-152-3p is a novel and promising molecular target for keloid treatment.


Assuntos
Matriz Extracelular/patologia , Fibroblastos/patologia , Fatores de Transcrição Forkhead/genética , Queloide/patologia , MicroRNAs/genética , Regiões 3' não Traduzidas , Adolescente , Adulto , Movimento Celular , Proliferação de Células , Células Cultivadas , Matriz Extracelular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Queloide/genética , Regulação para Cima , Adulto Jovem
15.
Nat Cell Biol ; 21(9): 1093-1101, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31451768

RESUMO

Mechanistic target of rapamycin (mTOR) kinase functions in two multiprotein complexes: lysosomal mTOR complex 1 (mTORC1) and mTORC2 at the plasma membrane. mTORC1 modulates the cell response to growth factors and nutrients by increasing protein synthesis and cell growth, and repressing the autophagy-lysosomal pathway1-4; however, dysfunction in mTORC1 is implicated in various diseases3,5,6. mTORC1 activity is regulated by phosphoinositide lipids7-10. Class I phosphatidylinositol-3-kinase (PI3K)-mediated production of phosphatidylinositol-3,4,5-trisphosphate6,11 at the plasma membrane stimulates mTORC1 signalling, while local synthesis of phosphatidylinositol-3,4-bisphosphate by starvation-induced recruitment of class II PI3K-ß (PI3KC2-ß) to lysosomes represses mTORC1 activity12. How the localization and activity of PI3KC2-ß are regulated by mitogens is unknown. We demonstrate that protein kinase N (PKN) facilitates mTORC1 signalling by repressing PI3KC2-ß-mediated phosphatidylinositol-3,4-bisphosphate synthesis downstream of mTORC2. Active PKN2 phosphorylates PI3KC2-ß to trigger PI3KC2-ß complex formation with inhibitory 14-3-3 proteins. Conversely, loss of PKN2 or inactivation of its target phosphorylation site in PI3KC2-ß represses nutrient signalling via mTORC1. These results uncover a mechanism that couples mTORC2-dependent activation of PKN2 to the regulation of mTORC1-mediated nutrient signalling by local lipid signals.


Assuntos
Lipídeos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/metabolismo , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
16.
Anticancer Res ; 39(8): 4061-4064, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366488

RESUMO

BACKGROUND/AIM: Circulating tumor cells (CTCs) may have an important role in metastasis. CTC clusters, which contain fibroblasts, indicate poor prognosis. In the present study, we used our malignant lymphoma metastatic mouse model to compare the effect of a choline-deficient-diet (CDD) and the control diet (CD) on fibroblasts in CTCs. MATERIALS AND METHODS: We compared the number and morphology of CTCs in CDD and CD mice using color-coded imaging with fluorescent proteins. Malignant lymphoma EL4 cells expressing RFP were injected in the spleen of transgenic C57B/6-GFP mice, which were fed a CDD or CD. Two weeks later, we harvested and observed the number of CTCs and fibroblast-like cells both in heart blood and portal blood. Imaging of CTC morphology was performed with smeared glass slides and in culture. RESULTS AND CONCLUSION: There was no significant difference in the number of CTCs between CDD and CD mice. The number of fibroblast-like cells in the CTC microenvironment in CD mouse portal blood was significantly larger than in CDD mouse portal blood. These differences may be caused by deficiency in choline that leads to less metastasis in choline-deficient-diet-induced fatty liver.


Assuntos
Colina/metabolismo , Linfoma/sangue , Células Neoplásicas Circulantes/metabolismo , Células Estromais/metabolismo , Animais , Linhagem Celular Tumoral , Deficiência de Colina/sangue , Deficiência de Colina/genética , Deficiência de Colina/patologia , Dieta/efeitos adversos , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Fluorescência Verde/química , Humanos , Proteínas Luminescentes/química , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Células Estromais/patologia , Microambiente Tumoral/genética
17.
Clin Exp Rheumatol ; 37 Suppl 119(4): 69-75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31365333

RESUMO

OBJECTIVES: Relaxin is a potent anti-fibrotic hormone that has been tested to ameliorate fibrosis in systemic sclerosis (SSc), but with controversial results. The aim of the study is to sequence relaxin receptor gene RXFP1 and to assess its mRNA expression and protein levels in the skin of SSc patients and healthy subjects. METHODS: Fibroblasts were isolated from unaffected/affected skin samples of (n=16) limited-cutaneous-SSc-(LcSSc) and from affected ones of (n=4) diffuse-cutaneous-SSc-(DcSSc) patients. Fibroblasts from healthy subjects were used as controls. Sequencing of exonic target regions of interest for RXFP1 gene was performed, coupled with mRNA transcript variant analysis. RXFP1 mRNA and protein levels were assessed by quantitative-real-time-PCR-(qRT-PCR) and by immunocytochemistry-(ICC). Alpha-smooth-muscle-actin-(α-SMA) synthesis induced by transforming-growth-factor-beta-1-(TGF-ß1) stimulation was investigated in all fibroblasts with and without pre-treatment with serelaxin (a recombinant form of human relaxin-2 targeting the receptor RXFP1). RESULTS: Sequencing of RXFP1 gene showed no relevant mutations in all fibroblast populations. The analysis of mRNA transcripts revealed the presence of 13 different mRNA isoforms of RXFP1 (7 coding and 6 non-coding) upregulated in LcSSc/DcSSc-affected samples and not in LcSSc-unaffected and in healthy ones. On the contrary, ICC demonstrated the absence of RXFP1 in LcSSc/DcSSc-affected fibroblasts and the presence in LcSSc-unaffected and in healthy ones. To prove these findings, serelaxin pre-incubation was unable to counteract TGF-ß1-driven upregulation of α-SMA in LcSSc/DcSSc-affected fibroblasts only, but not in LcSSc-unaffected and healthy ones. CONCLUSIONS: The absence/altered expression of relaxin receptor RXFP1 in the affected fibroblasts of SSc patients could explain the inefficacy of relaxin-based anti-fibrotic treatments in the disease.


Assuntos
Fibroblastos/metabolismo , Relaxina , Esclerodermia Difusa , Escleroderma Sistêmico , Idoso , Feminino , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes , Relaxina/metabolismo , Esclerodermia Difusa/metabolismo , Esclerodermia Difusa/patologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia
18.
Aquat Toxicol ; 213: 105229, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31255889

RESUMO

Although the global use of the 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (p,p'-DDT) has been prohibited, its persistence in the environment has caused long-lasting exposure on marine mammals. Our previous studies revealed exceedingly high residue levels of DDTs in Indo-Pacific humpback dolphins (Sousa chinensis) from the Pearl River Estuary region, China. However, the molecular mechanisms of p,p'-DDT toxicity on the dolphin are largely unknown. This study conducted the first cytotoxicity effect exploration of p,p'-DDT on the dolphin skin fibroblasts (ScSFs) to enhance the understanding of the cellular and molecular regulation impacts. ScSF cells were exposed to p,p'-DDT (28∼168 µM) for 24, 48 and 72 h. The exposure remarkably decreased viability of ScSF cells, possibly due to the synergetic effects of cell cycle arrest and apoptosis via DNA damage and mitochondria dysfunction. The DNA damage and mitochondria dysfunction were likely triggered by an increase of cellular reactive oxygen species (ROS), alteration in mitochondrial membrane potential, reduction in the cellular ATP levels, decreased expression of the genes CDK1, CDK4, cyclin B1, cyclin D1 and apoptosis regulator Bcl-2, release of cytochrome c, and activation of caspase-3, caspase-8 and caspase-9. Moreover, caspase inhibitor displayed protective activity against p,p'-DDT-induced apoptosis, indicating that caspases played a central role in p,p'-DDT-triggered apoptosis in the ScSF cells. We hypothesize apoptosis likely plays a minor role in cytocidal effects induced by p,p'-DDT exposure, but the mechanisms remain unclear. Overall, this research provides new evidence of the cytotoxic mechanisms underlying p,p'-DDT exposure on humpback dolphin skin cells, and suggests that p,p'-DDT contamination is one of key health concern issues for the protection of this marine mammal.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , DDT/toxicidade , Golfinhos/metabolismo , Exposição Ambiental , Fibroblastos/citologia , Mitocôndrias/metabolismo , Pele/citologia , Animais , Caspases/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Poluentes Químicos da Água/toxicidade
19.
Int Heart J ; 60(4): 944-957, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31257341

RESUMO

Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.


Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genética , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/patologia
20.
Nat Commun ; 10(1): 3027, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289275

RESUMO

Fibrosis accompanying wound healing can drive the failure of many different organs. Activated fibroblasts are the principal determinants of post-injury pathological fibrosis along with physiological repair, making them a difficult therapeutic target. Although activated fibroblasts are phenotypically heterogeneous, they are not recognized as distinct functional entities. Using mice that express GFP under the FSP1 or αSMA promoter, we characterized two non-overlapping fibroblast subtypes from mouse hearts after myocardial infarction. Here, we report the identification of FSP1-GFP+ cells as a non-pericyte, non-hematopoietic fibroblast subpopulation with a predominant pro-angiogenic role, characterized by in vitro phenotypic/cellular/ultrastructural studies and in vivo granulation tissue formation assays combined with transcriptomics and proteomics. This work identifies a fibroblast subtype that is functionally distinct from the pro-fibrotic αSMA-expressing myofibroblast subtype. Our study has the potential to shift our focus towards viewing fibroblasts as molecularly and functionally heterogeneous and provides a paradigm to approach treatment for organ fibrosis.


Assuntos
Fibroblastos/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Transplante de Medula Óssea , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose/etiologia , Fibrose/patologia , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/citologia , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Quimeras de Transplante
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