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1.
J Agric Food Chem ; 67(35): 9789-9795, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31373816

RESUMO

Pulmonary fibrosis is a chronic lung disease characterized by abnormal accumulation of the extracellular matrix (ECM). Chronic damage of the alveolar epithelium leads to a process called "epithelial-mesenchymal transition" (EMT) and increases synthesis and deposition of ECM proteins. Therefore, inhibition of EMT might be a promising therapeutic approach for the treatment of pulmonary fibrosis. ß-Sitosterol is one of the most abundant phytosterols in the plant kingdom and the major constituent in corn silk, which is derived from the stigma and style of maize (Zea mays). In this study, we elucidated that ß-sitosterol inhibited transforming growth factor-ß1 (TGF-ß1)-induced EMT and consequently had an antifibrotic effect. ß-Sitosterol (1-10 µg/mL) significantly downregulated the TGF-ß1-induced fibrotic proteins, such as collagen, fibronectin, and α-smooth muscle actin in human alveolar epithelial cells (p < 0.01). After 24 h, relative wound density (RWD) was increased in TGF-ß1 treated group (82.16 ± 5.70) compare to the control group (64.63 ± 2.21), but RWD was decreased in ß-sitosterol cotreated group (10 µg/mL: 71.54 ± 7.39; 20 µg/mL: 65.69 ± 6.42). In addition, the changes of the TGF-ß1-induced morphological shape and protein expression of EMT markers, N-cadherin, vimentin, and E-cadherin, were significantly blocked by ß-sitosterol treatment (p < 0.01). The effects of ß-sitosterol on EMT were found to be associated with the TGF-ß1/Snail pathway, which is regulated by Smad and non-Smad signaling pathways. Taken together, these findings suggest that ß-sitosterol can be used to attenuate pulmonary fibrosis through suppression of EMT by inhibiting the TGF-ß1/Snail pathway.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , Fibrose Pulmonar/fisiopatologia , Sitosteroides/farmacologia , Zea mays/química , Actinas/genética , Actinas/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Extratos Vegetais/química , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/fisiopatologia , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
2.
Biochemistry (Mosc) ; 84(4): 358-369, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228927

RESUMO

Cytoplasmic actin structures are essential components of the eukaryotic cytoskeleton. According to the classic concepts, actin structures perform contractile and motor functions, ensuring the possibility of cell shape changes during cell spreading, polarization, and movement both in vitro and in vivo, from the early embryogenesis stages and throughout the life of a multicellular organism. Intracellular organization of actin structures, their biochemical composition, and dynamic properties play a key role in the realization of specific cellular and tissue functions and vary in different cell types. This paper is a review of recent studies on the organization and properties of actin structures in endotheliocytes, interaction of these structures with other cytoskeletal components and elements involved in cell adhesion, as well as their role in the functional activity of endothelial cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Caderinas/química , Caderinas/metabolismo , Citosol/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo
3.
Nat Commun ; 10(1): 2856, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253774

RESUMO

Microfilaments (actin) and microtubules represent the extremes in eukaryotic cytoskeleton cross-sectional dimensions, raising the question of whether filament architectures are limited by protein fold. Here, we report the cryoelectron microscopy structure of a complex filament formed from 15 protofilaments of an actin-like protein. This actin-like ParM is encoded on the large pCBH Clostridium botulinum plasmid. In cross-section, the ~26 nm diameter filament comprises a central helical protofilament surrounded by intermediate and outer layers of six and eight twisted protofilaments, respectively. Alternating polarity of the layers allows for similar lateral contacts between each layer. This filament design is stiffer than the actin filament, and has likely been selected for during evolution to move large cargos. The comparable sizes of microtubule and pCBH ParM filaments indicate that larger filament architectures are not limited by the protomer fold. Instead, function appears to have been the evolutionary driving force to produce broad, complex filaments.


Assuntos
Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium botulinum/metabolismo , Citoesqueleto/fisiologia , Citoesqueleto de Actina , Actinas/genética , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica/fisiologia , Modelos Moleculares , Conformação Proteica
4.
Parasitol Res ; 118(7): 2183-2191, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31076871

RESUMO

Cryptosporidiosis is an emergent zoonotic disease caused by the globally distributed protozoa Cryptosporidium spp. Although several Cryptosporidium studies related to humans and many animal species have been published, there are still limited studies on the epidemiology of Cryptosporidium infection in bats. The aim of this study was to determine the occurrence of Cryptosporidium spp. and to perform the molecular characterization of Cryptosporidium species and genotypes in fecal samples from bats in an urban area of the municipality of Araçatuba, state of São Paulo, Brazil. Nested PCR targeting the 18S rRNA, actin, and HSP-70 genes was performed to screen 141 fecal samples from bats and detected Cryptosporidium spp. in 16.3% (23/141) of the samples. Bidirectional sequencing identified three novel Cryptosporidium bat genotypes (XVI, XVII, and XVIII) and a new genotype (18SH) genetically similar to Cryptosporidium avium in six species of bats. This is the first report on the occurrence and molecular characterization of Cryptosporidium spp. in Brazilian bats. Zoonotic Cryptosporidium species were not found in fecal samples from bats living in an urban area in the municipality of Araçatuba, state of São Paulo, Brazil.


Assuntos
Quirópteros/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/classificação , Cryptosporidium/genética , Actinas/genética , Animais , Brasil/epidemiologia , Cryptosporidium/isolamento & purificação , Fezes/parasitologia , Genótipo , Proteínas de Choque Térmico HSP70/genética , Humanos , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Zoonoses/parasitologia
5.
Nat Cell Biol ; 21(5): 592-602, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962575

RESUMO

Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot-Marie-Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain. Interestingly, purified INF2 is not autoinhibited, suggesting the existence of other cellular inhibitors. Here, we purified an INF2 inhibitor from mouse brain tissue, and identified it as a complex of lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP-KAc-actin is dependent on the INF2 diaphanous inhibitory domain (DID). Treatment of CAP-KAc-actin-inhibited INF2 with histone deacetylase 6 releases INF2 inhibition, whereas inhibitors of histone deacetylase 6 block the activation of cellular INF2. Disease-associated INF2 mutants are poorly inhibited by CAP-KAc-actin, suggesting that focal segmental glomerulosclerosis and Charcot-Marie-Tooth disease result from reduced CAP-KAc-actin binding. These findings reveal a role for KAc-actin in the regulation of an actin assembly factor by a mechanism that we call facilitated autoinhibition.


Assuntos
Actinas/genética , Proteínas de Transporte/genética , Doença de Charcot-Marie-Tooth/genética , Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos/genética , Actinas/química , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/química , Doença de Charcot-Marie-Tooth/patologia , Glomerulosclerose Segmentar e Focal/patologia , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Humanos , Lisina/química , Camundongos , Proteínas dos Microfilamentos/química , Mutação , Ligação Proteica , Domínios Proteicos/genética
6.
Nat Cell Biol ; 21(5): 603-613, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988424

RESUMO

Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction.


Assuntos
Actinas/genética , Citoesqueleto/ultraestrutura , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Miosina Tipo II/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Células COS , Cercopithecus aethiops , Constrição , Citoesqueleto/metabolismo , Ionomicina/farmacologia , Mitocôndrias/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miosina Tipo II/química , Miosina Tipo II/metabolismo
7.
Int J Med Mushrooms ; 21(3): 301-309, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31002613

RESUMO

Real-time quantitative polymerase chain reaction (qRT-PCR) has emerged as a powerful and popular tool for quantitating differences in transcriptional gene expression levels between samples. Validation of the stability of reference genes is a fundamental step before initiating qRT-PCR assays. Sparassis latifolia is an edible and medicinal fungus containing a remarkably high concentration of ß-glucan, which has many biological and pharmacologic activities. S. latifolia may be a model species for studying fungal photobiology because its fruiting body formation requires more light than other fungi. However, suitable reference genes for qRT-PCR have not yet been determined. In the present study, 10 candidate reference genes in S. latifolia were evaluated and validated under different developmental stages and light conditions. To evaluate the suitability of candidate reference genes, three popular software programs (geNorm, NormFinder, and BestKeeper), along with the delta Ct method, were used to analyze these genes; the final ranking was determined using RefFinder. According to our results, Actin and GAPDH were expressed at the most stable levels under different developmental stages and light conditions.


Assuntos
Basidiomycota/genética , Genes Fúngicos/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Actinas/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética
8.
In Vitro Cell Dev Biol Anim ; 55(5): 387-394, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30993556

RESUMO

This study aimed at investigating the expression of osteoblast and chondrocyte-related genes in mesenchymal stem cells (MSCs), derived from rabbit adipose tissue, under mechanical vibration. The cells were placed securely on a vibrator's platform and subjected to 300 Hz of sinusoidal vibration, with a maximum amplitude of 10 µm, for 45 min per day, and for 14 consequent days, in the absence of biochemical reagents. The negative control group was placed in the conventional culture medium with no mechanical loading. The expression of osteoblast and chondrocyte-related genes was investigated using real-time polymerase chain reaction (real-time PCR). In addition, F-actin fiber structure and alignment with the help of actin filament fluorescence staining were evaluated, and the level of metabolic activity of MSCs was determined by the methyl thiazolyl tetrazolium assay. The real-time PCR study showed a significant increase of bone gene expression in differentiated cells, compared with MSCs (P < 0.05). On the other hand, the level of chondrocyte gene expression was not remarkable. Applying mechanical vibration enhanced F-actin fiber structure and made them aligned in a specific direction. It was also found that during the differentiation process, the metabolic activity of the cells increased (P < 0.05). The results of this work are in agreement with the well-accepted fact that the MSCs, in the absence of growth factors, are sensitive to low-amplitude, high-frequency vibration. Outcomes of this work can be applied in cell therapy and tissue engineering, when regulation of stem cells is required.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Estresse Mecânico , Vibração/uso terapêutico , Actinas/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiologia , Animais , Células da Medula Óssea/fisiologia , Diferenciação Celular/genética , Condrócitos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Coelhos , Engenharia Tecidual
9.
Mol Phylogenet Evol ; 135: 31-44, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30844445

RESUMO

Phylogenetic relationships and phylogeography of six species of Caucasian barbels, the genus Barbus s. str., were studied based on extended geographic coverage and using mtDNA and nDNA markers. Based on 27 species studied, matrilineal phylogeny of the genus Barbus is composed of two clades - (a) West European clade, (b) Central and East European clade. The latter comprises two subclades: (b1) Balkanian subclade, and (b2) Ponto-Caspian one that includes 11 lineages mainly from Black and Caspian Sea drainages. Caucasian barbels are not monophyletic and subdivided for two groups. The Black Sea group encompasses species from tributaries of Black Sea including re-erected B. rionicus and excluding B. kubanicus. The Caspian group includes B. ciscaucasicus, B. cyri (with B. goktschaicus that might be synonymized with B. cyri), B. lacerta from the Tigris-Euphrates basin and B. kubanicus from the Kuban basin. Genetic structure of Black Sea barbels was influenced by glaciation-deglaciation periods accompanying by freshwater phases, periods of migration and colonization of Black Sea tributaries. Intra- and intergeneric hybridization among Caucasian barbines was revealed. In the present study, we report about finding of B. tauricus in the Kuban basin, where only B. kubanicus was thought to inhabit. Hybrids between these species were detected based on both mtDNA and nDNA markers. Remarkably, Kuban population of B. tauricus is distant to closely located conspecific populations and we consider it as relic. We highlight revealing the intergeneric hybridization between evolutionary tetraploid (2n = 100) B. goktschaicus and evolutionary hexaploid (2n = 150) Capoeta sevangi in Lake Sevan.


Assuntos
Cyprinidae/classificação , Cyprinidae/genética , Hibridização Genética , Filogenia , Filogeografia , Actinas/genética , Animais , Sequência de Bases , Teorema de Bayes , Mar Negro , DNA Mitocondrial/genética , Variação Genética , Haplótipos/genética , Íntrons/genética , Fatores de Tempo
10.
Nat Commun ; 10(1): 1128, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850660

RESUMO

Scleroderma is an autoimmune rheumatic disorder accompanied by severe fibrosis in skin and other internal organs. During scleroderma progression, resident fibroblasts undergo activation and convert to α-smooth muscle actin (α-SMA) expressing myofibroblasts (MFBs) with increased capacity to synthesize collagens and fibrogenic components. Accordingly, MFBs are a major therapeutic target for fibrosis in scleroderma and treatment with blocking MFBs could produce anti-fibrotic effects. TLY012 is an engineered human TNF-related apoptosis-inducing ligand (TRAIL) which induces selective apoptosis in transformed cells expressing its cognate death receptors (DRs). Here we report that TLY012 selectively blocks activation of dermal fibroblasts and induces DR-mediated apoptosis in α-SMA+ MFBs through upregulated DR5 during its activation. In vivo, TLY012 reverses established skin fibrosis to near-normal skin architecture in mouse models of scleroderma. Thus, the TRAIL pathway plays a critical role in tissue remodeling and targeting upregulated DR5 in α-SMA+ MFBs is a viable therapy for fibrosis in scleroderma.


Assuntos
Actinas/genética , Derme/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Escleroderma Sistêmico/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Actinas/metabolismo , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular , Colágeno/genética , Colágeno/metabolismo , Derme/metabolismo , Derme/patologia , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Engenharia de Proteínas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Transdução de Sinais
11.
Forensic Sci Int ; 298: 58-63, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30878465

RESUMO

The first appearance of ribonucleic acid (RNA) in forensic science research was in 1984 in the study of post-mortem tissues. Since then, many studies have explored the role of gene expression and its potential applications in forensic science. The two main RNA molecules that have been subject to increasing interest in the forensic science community are messenger RNA (mRNA) and microRNA (miRNA). Identification of body fluid type and estimating the time since deposition can be of immense value to criminal investigations. Determining the time since deposition or age of a biological stain can help to indicate either when a crime happened, or whether the biological evidence was deposited before/after a known crime event, in order for samples to be excluded. The research presented here has used reverse transcription quantitative PCR to examine the relative expression ratio (RER) in two types of body fluid-specific markers (saliva and semen), to develop a method to estimate the age of biological stains. mRNA and miRNA markers specific to saliva and semen, along with three reference genes were selected. Biological samples from 20 participants were stored in a dark dry place at room temperature to simulate natural ageing. A series of desired ageing points were set and total RNA was extracted when samples reached each desired point. The degradation behaviour of each RNA marker was analysed, showing that they exhibited unique degradation profiles across a one-year storage interval for saliva and semen samples, where miRNAs and the U6 reference gene were shown to have high stability. The RERs exhibit a non-linear relationship with body fluid stain age and can be considered as a potential method for body fluid stain age estimation, hence the time since deposition.


Assuntos
Marcadores Genéticos , MicroRNAs/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Saliva/química , Sêmen/química , Actinas/genética , Actinas/metabolismo , Genética Forense , Humanos , Masculino , Protaminas/genética , Protaminas/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Nuclear Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Fatores de Tempo
12.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837367

RESUMO

Atrial fibrillation (AF) is the most frequently diagnosed cardiac arrhythmia worldwide. Patients with permanent atrial fibrillation are at an increased risk of developing valvular heart disease. Atrial fibrosis occurs in this pathophysiological setting. LIM kinase 1 (LIMK1) is a serine/threonine kinase that regulates microtubule stability and actin polymerization in fibroblasts. LIMK1 has been implicated in the pathogenesis of atrial fibrillation. Clinical data and biopsies of the right atrial appendage were collected from 50 patients with valvular heart disease who underwent heart valve replacement surgery. Data from patients with permanent atrial fibrillation (AF) and patients with sinus rhythm (SR) were compared. We found that AF patients had upregulated expression of LIMK1 as well as higher fibrosis. Transforming growth factor-ß (TGF-ß) stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts as well as upregulated expression of LIMK1. Downregulation of LIMK1 by siRNA inhibited TGF-ß induced fibroblast-myofibroblast transition, as evidenced by the downregulation of the expression of several differentiation markers, namely alpha-smooth muscle actin and type I and III collagen. Our findings revealed that increased LIMK1 protein levels may contribute to atrial fibrosis, and suggested that LIMK1 might be involved in AF development by promoting fibrogenesis associated with TGF-ß.


Assuntos
Fibrilação Atrial/genética , Fibrose/genética , Doenças das Valvas Cardíacas/genética , Quinases Lim/genética , Actinas/genética , Animais , Fibrilação Atrial/complicações , Fibrilação Atrial/fisiopatologia , Biópsia , Colágeno Tipo I/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose/fisiopatologia , Regulação da Expressão Gênica/efeitos dos fármacos , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Doenças das Valvas Cardíacas/complicações , Doenças das Valvas Cardíacas/fisiopatologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , RNA Interferente Pequeno/farmacologia , Fator de Crescimento Transformador beta/farmacologia
13.
Acta Biochim Biophys Sin (Shanghai) ; 51(4): 402-410, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30877755

RESUMO

Epicardial progenitor cells (EpiCs) which are derived from the proepicardium have the potential to differentiate into coronary vascular smooth muscle cells during development. Whether sphingosine 1-phosphate (S1P), a highly hydrophobic zwitterionic lysophospholipid in signal transduction, induces the differentiation of EpiCs is unknown. In the present study, we demonstrated that S1P significantly induced the expression of smooth muscle cell specific markers α-smooth muscle actin and myosin heavy chain 11 in the EpiCs. And the smooth muscle cells differentiated from the EpiCs stimulated by S1P were further evaluated by gel contraction assay. To further confirm the major subtype of sphingosine 1-phosphate receptors (S1PRs) involved in the differentiation of EpiCs, we used the agonists and antagonists of different S1PRs. The results showed that the S1P1/S1P3 antagonist VPC23019 and the S1P2 antagonist JTE013 significantly attenuated EpiCs differentiation, while the S1P1 agonist SEW2871 and antagonist W146 did not affect EpiCs differentiation. These results collectively suggested that S1P, principally through its receptor S1P3, increases EpiCs differentiation into VSMCs and thus indicated the importance of S1P signaling in the embryonic coronary vasculature, while S1P2 plays a secondary role.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Células-Tronco Embrionárias Murinas/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Pericárdio/citologia , Esfingosina/análogos & derivados , Actinas/genética , Actinas/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Pericárdio/embriologia , Fosfosserina/análogos & derivados , Fosfosserina/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Esfingosina/farmacologia
14.
Mol Med Rep ; 19(5): 3548-3554, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864692

RESUMO

Previously, we demonstrated that Rhizoma Paridis saponins (RPS), the major active component of Rhizoma Paridis, may exhibit hepatoprotective effects. The present study aimed to identify the potential mechanism of RPS on hepatic injury and improvement in hepatic fibrosis (HF). A HF model was created in Sprague­Dawley rats by administration of carbon tetrachloride. RPS was administered for treatment following creation of the HF model. The protein and mRNA expression of vascular endothelial growth factor (VEGF), platelet­derived growth factor (PDGF), extracellular signal­regulated kinase (ERK)1/2 and α­smooth muscle actin (SMA) was detected by reverse transcription quantitative polymerase chain reaction and western blot analysis. RPS was demonstrated to improve hepatic inflammation and decrease HF severity according to hematoxylin and eosin and Masson trichrome staining. Following RPS treatment, the level of alanine aminotransferase, aspartate aminotransferase and malondialdehyde, and expression levels of the mRNA and protein of VEGF, ERK1/2, PDGF and α­SMA in the model group was decreased. By contrast, the content of glutathione­PX and superoxide dismutase was increased. These data suggest that RPS may treat HF primarily through downregulation of the expression levels of the mRNA and phosphorylated VEGF, ERK1/2, PDGF and α­SMA proteins.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Rizoma/química , Saponinas/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores , Biópsia , Cromatografia Líquida de Alta Pressão , Imuno-Histoquímica , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Saponinas/química , Saponinas/farmacocinética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Mol Med Rep ; 19(4): 3305-3313, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816527

RESUMO

Progressive renal fibrosis is a common complication of chronic kidney disease that results in end­stage renal disorder. It is well established that several microRNAs (miRs) function as critical regulators implicated in fibrotic diseases. However, the role of miR­181 in the development and progression of renal fibrosis remains unclear, and the precise mechanism has not yet been fully defined. The present study identified the functional implications of miR­181 expression during renal fibrosis. miR­181 exhibited significantly reduced expression in the serum of renal fibrosis patients and in the kidneys of mice with unilateral ureteral obstruction (UUO). In addition, miR­181 downregulated the expression of human α­smooth muscle actin (α­SMA) in response to angiotensin II stimulation. Transfection with miR­181 mimics significantly suppressed the expression levels of α­SMA, connective tissue growth factor, collagen type I α1 (COL1A1) and collagen type III α1 (COL3A1) in NRK49F cells. Notably, early growth response factor­1 (Egr1) was identified as a direct target gene of miR­181. Furthermore, in vivo experiments revealed that treatment with miR­181 agonist strongly rescued kidney impairment induced by UUO, as supported by Masson's trichrome staining of kidney tissues and reverse transcription­quantitative polymerase chain reaction analysis of COL1A1 and COL3A1 mRNA levels. Therefore, miR­181 may be regarded as an important mediator in the control of profibrotic markers during renal fibrosis via binding to Egr1, and may be a promising new target in the diagnosis and therapy of renal fibrosis.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Nefropatias/genética , MicroRNAs/genética , Interferência de RNA , Actinas/genética , Actinas/metabolismo , Adulto , Idoso , Animais , Biomarcadores , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Fibrose , Humanos , Imuno-Histoquímica , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Ratos
16.
Mol Cells ; 42(4): 356-362, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30841028

RESUMO

The binding of MS2 bacteriophage coat protein (MCP) to MS2 binding site (MBS) RNA stem-loop sequences has been widely used to label mRNA for live-cell imaging at single-molecule resolution. However, concerns have been raised recently from studies with budding yeast showing aberrant mRNA metabolism following the MS2-GFP labeling. To investigate the degradation pattern of MS2-GFP-labeled mRNA in mammalian cells and tissues, we used Northern blot analysis of ß-actin mRNA extracted from the Actb-MBS knock-in and MBS×MCP hybrid mouse models. In the immortalized mouse embryonic cell lines and various organ tissues derived from the mouse models, we found no noticeable accumulation of decay products of ß-actin mRNA compared with the wild-type mice. Our results suggest that accumulation of MBS RNA decay fragments does not always happen depending on the mRNA species and the model organisms used.


Assuntos
Actinas/química , Actinas/genética , Proteínas do Capsídeo/metabolismo , Levivirus/genética , Actinas/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Embrião de Mamíferos/citologia , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Levivirus/metabolismo , Camundongos , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Organismos Livres de Patógenos Específicos
17.
Invest Ophthalmol Vis Sci ; 60(4): 1156-1164, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30908581

RESUMO

Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown antifibrotic effects on several diseases. The aims of the present in vitro study were to investigate the antifibrotic effects of bromfenac (a kind of NSAID) on primary human pterygium fibroblasts (HPFs) and primary human conjunctival fibroblasts (HConFs), as well as to explore the possible mechanisms of these effects. Methods: The cells used in this study were primary HPFs and HConFs, and profibrotic activation was induced by transforming growth factor-beta1 (TGF-ß1). Western blot, quantitative real-time PCR, and immunofluorescence (IF) assays were used to detect the effects of TGF-ß1 and bromfenac on the synthesis of fibronectin (FN), type III collagen (COL3), and alpha-smooth muscle actin (α-SMA) in HPFs and HConFs; the changes of signaling pathways were detected by Western blot; cell migration ability was detected by wound healing assay; cell proliferation ability was detected by CCK-8 assay; and pharmaceutical inhibitions of the downstream signaling pathways of TGF-ß1 were used to assess their possible associations with the effects of bromfenac. Results: Bromfenac suppressed the TGF-ß1-induced protein expression of FN (0.59 ± 0.07 folds, P = 0.008), COL3 (0.48 ± 0.08 folds, P = 0.001), and α-SMA (0.61 ± 0.03 folds, P = 0.008) in HPFs. Bromfenac also attenuated TGF-ß1-induced cell migration (0.30 ± 0.07 folds, P < 0.001), cell proliferation (0.64 ± 0.03 folds, P = 0.002) and the expression levels of p-AKT (0.66 ± 0.08 folds, P = 0.032), p-ERK1/2 (0.69 ± 0.11 folds, P = 0.003), and p-GSK-3ß-S9 (0.65 ± 0.10 folds, P = 0.002) in HPFs. PI3K/AKT inhibitor (wortmannin) and MEK/ERK inhibitor (U0126) reduced the TGF-ß1-induced synthesis of FN, COL3, and α-SMA in HPFs. All the results were similar in HConFs. Conclusions: Bromfenac protects against TGF-ß1-induced synthesis of FN, α-SMA, and COL3 in HPFs and HConFs at least in part by inactivating the AKT and ERK pathways.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzofenonas/farmacologia , Bromobenzenos/farmacologia , Túnica Conjuntiva/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pterígio/patologia , Pterígio/prevenção & controle , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Idoso , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Túnica Conjuntiva/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose/patologia , Fibrose/prevenção & controle , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt , Pterígio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Sincalida/farmacologia , Fator de Crescimento Transformador beta1/farmacologia
18.
J Vet Med Sci ; 81(5): 646-652, 2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-30880304

RESUMO

Activin E, a secreted peptide encoded by the inhibin/activin ßE subunit gene, is a member of the transforming growth factor-ß superfamily, which is predominantly expressed in the liver. Recent reports have suggested that activin E plays a role in energy homeostasis as a hepatokine. Here, using transgenic mice overexpressing activin E under the control of the ß-actin promoter, we demonstrate that activin E controls energy metabolism through brown/beige adipocytes. The glucose tolerance test and insulin tolerance test showed that the insulin sensitivity was improved in the transgenic mice. Furthermore, the mice had a high body temperature compared with wild-type mice. The transgenic brown adipose tissue and mesenteric white adipose tissue showed upregulation of uncoupling protein 1, which enables energy dissipation as heat by uncoupling oxidative phosphorylation from ATP production. Present results indicate that activin E activates energy expenditure through brown/beige adipocyte activation, suggesting that activin E has high potential for obesity therapy.


Assuntos
Ativinas/farmacologia , Adipócitos Bege/metabolismo , Adipócitos Marrons/metabolismo , Resistência à Insulina , Actinas/genética , Actinas/metabolismo , Ativinas/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Intolerância à Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Termogênese/efeitos dos fármacos , Proteína Desacopladora 1/metabolismo
19.
Int J Mol Sci ; 20(6)2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875826

RESUMO

BACKGROUND: Interleukin-1 (IL-1)ß and IL-1 receptor antagonist (IL-1Ra) have been proposed as important mediators during chronic liver diseases. We aimed to determine whether the modulation of IL-1ß signaling with IL-1Ra impacts on liver fibrosis. METHODS: We assessed the effects of IL-1ß on human hepatic stellate cells (HSC) and in mouse models of liver fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride treatment (CCl-4). RESULTS: Human HSCs treated with IL-1ß had increased IL-1ß, IL-1Ra, and MMP-9 expressions in vitro. HSCs treated with IL-1ß had reduced α-smooth muscle actin expression. These effects were all prevented by IL-1Ra treatment. In the BDL model, liver fibrosis and Kuppfer cell numbers were increased in IL-1Ra KO mice compared to wild type mice and wild type mice treated with IL-1Ra. In contrast, after CCl-4 treatment, fibrosis, HSC and Kupffer cell numbers were decreased in IL-1Ra KO mice compared to the other groups. IL-1Ra treatment provided a modest protective effect in the BDL model and was pro-fibrotic in the CCl-4 model. CONCLUSIONS: We demonstrated bivalent effects of IL-1Ra during liver fibrosis in mice. IL-1Ra was detrimental in the CCl-4 model, whereas it was protective in the BDL model. Altogether these data suggest that blocking IL-1-mediated inflammation may be beneficial only in selective liver fibrotic disease.


Assuntos
Actinas/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-1beta/genética , Cirrose Hepática/genética , Metaloproteinase 9 da Matriz/genética , Animais , Tetracloreto de Carbono/efeitos adversos , Contagem de Células , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Macrófagos do Fígado/citologia , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos do Fígado/imunologia , Cirrose Hepática/etiologia , Cirrose Hepática/imunologia , Masculino , Camundongos , Regulação para Cima
20.
Biol Pharm Bull ; 42(3): 401-410, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828072

RESUMO

Ridaifen (RID)-B is an analog derived from tamoxifen (TAM). TAM has an antitumor effect by acting as an antagonist to estrogen receptor (ER). However, TAM is known to also induces apoptosis in cancer cells that do not have ER. We clarified that RID-B induces cell death at a lower concentration than TAM, and causes ER-independent apoptosis and autophagy. Based on the results of previous studies, we assumed that RID-B had a unique target different from ER and examined structural activity correlation to determine what kinds of structural features are related to RID-B activity. As a result, we found there was activity even without one of phenyl groups (Ar3) in RID-B and revealed that two pyrrolidine side chains peculiar to RID-B are related to the action. Furthermore, analogs with shorter alkyl side chains induced autophagy, but analogs with certain length of alkyl side chains induced apoptosis. Also, although there is no doubt that RID-B induces apoptosis by causing mitochondrial injury, our results suggested that such injury induced mitochondria-selective autophagy. We revealed that RID-B induce mitophagy and that this mitophagy is a defense mechanism against RID-B. Our results suggest that autophagy was induced against apoptosis caused by mitochondrial dysfunction in RID-B, so the combination of autophagy inhibitor and anticancer-drug can be effective for cancer treatment.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pirrolidinas/química , Pirrolidinas/farmacologia , Tamoxifeno/análogos & derivados , Actinas/genética , Actinas/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Degradação Mitocondrial , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Tamoxifeno/química , Tamoxifeno/farmacologia
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