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1.
Biochimie ; 189: 144-157, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34217820

RESUMO

Because of health-promoting effects, the adaptation of skeletal muscles to exercise is considered a therapeutic strategy for metabolic complications and musculoskeletal disabilities. Myokines display many beneficial effects of different exercise modalities. Among them, irisin is known as a systemic effector that positively influences several organs. There are a few studies about the effects of irisin on skeletal muscles, and irisin prosperities need to be well-defined for being an exercise mimetic. To aim this purpose, we assessed the proteome profile of mouse skeletal muscle after eight weeks of irisin injection comparing to resistance and endurance exercise treated groups. In the current study, two-dimensional gel electrophoresis was used to evaluate the protein content of the quadriceps muscle. The results were analyzed with Image Master 2D Platinum V6 software. Differentially expressed proteins were characterized by mass spectrometry (MALDI TOF/TOF) and interpreted using protein data banks and co-expression network. Irisin increases cellular ATP content by driving its overproduction through glycolysis and oxidative phosphorylation similar to two exercise protocols and as a specific property, decreases ATP consumption through creatine kinase downregulation. It also improves the microstructural properties of quadriceps muscle by increasing fiber proteins and might induce cellular proliferation and differentiation. Network analysis of differentially expressed proteins also revealed the co-expression of Irisin precursor with structural and metabolic-related proteins. The protein alterations after irisin administration display the potential of this myokine to mimic some molecular effects of exercise, suggesting it a promising candidate to improve muscle metabolism and structure.


Assuntos
Fibronectinas/farmacologia , Proteínas Musculares/metabolismo , Condicionamento Físico Animal , Proteoma/metabolismo , Músculo Quadríceps/metabolismo , Animais , Masculino , Camundongos
2.
J Complement Integr Med ; 18(2): 347-354, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34187125

RESUMO

OBJECTIVE: To evaluate the influence of irisin on the experimental paradigm of non-alcoholic fatty liver (NAFL) as a part of MetS cluster. METHODS: Forty male albino rats were divided into four groups; normal control, standard diet + irisin, high carbohydrate and fat diet (HCHF), and HCHF + irisin. After the experimental period, levels of fasting blood sugar (FBS), insulin, lipid profile, kidney functions, salusin-alpha (Sal-α), adropin, and retinol-binding protein-4 (RBP-4) were evaluated. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α) expression in skeletal muscle was evaluated by quantitative real-time PCR. Aorta, liver, pancreas, and skeletal muscle tissue samples were prepared for histopathological examination. RESULTS: Rats administrated HCHF showed elevated levels of FBS, lipid profile, kidney functions, RBP-4, and downregulation of PGC-1α expression along with a decline in levels of insulin, Sal-α, and adropin while administration of irisin significantly attenuated these levels. CONCLUSIONS: Irisin as based therapy could emerge as a new line of treatment against MetS and its related diseases.


Assuntos
Dieta Hiperlipídica , Fibronectinas/farmacologia , Síndrome Metabólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Masculino , Ratos
3.
Exp Cell Res ; 404(2): 112593, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33961841

RESUMO

AIMS: Acute lung injury (ALI) is a leading cause of mortality as a result of inflammatory cytokine overexpression and increased rates of apoptosis. Therapies for ALI are yet to be thoroughly investigated. Recent evidence has shown that irisin exerts protective effects against many types of pathologies. The present study aimed to determine the function of irisin in an ALI mouse model induced by lipopolysaccharide (LPS) and the corresponding underlying mechanisms at the tissue, cellular, and molecular levels. MAIN METHODS: We assessed irisin function in A549 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. The cell apoptosis was evaluated by flow cytometry. Western blotting and RT-PCR were used to test expression level. Animal models of ALI was established. KEY FINDINGS: We found that irisin treatment maintained lung weight, significantly reduced inflammatory cytokine expression, and alleviated lung injury by downregulating miR-199a. In LPS-stimulated cells, forced miR-199a expression downregulated Rad23b expression by targeting its 3' untranslated region, indicating that Rad23b is a direct target of miR-199a. SIGNIFICANCE: These findings reveal that irisin can alleviate ALI by inhibiting miR-199a and upregulating Rad23b expression, suggesting that irisin has clinical potential for the treatment of ALI.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibronectinas/farmacologia , MicroRNAs/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Modelos Animais de Doenças , Fibronectinas/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
4.
Biomed Res Int ; 2021: 5570229, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33997010

RESUMO

The reduction in estrogen levels results in a decrease in bone density at menopause. Irisin is a myokine that modulates the benefits of exercise, which may include bone health. This study was planned to examine irisin's impact in preventing osteoporosis after ovariectomy. 4 groups of female albino rats (10 rats/group): control, sham-operated, ovariectomized (OVX-control), and OVX-irisin-treated. Serum levels of bone markers [osteocalcin (OC), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), calcium (Ca++), phosphorus (P)], glucose, and insulin were being measured. Body mass index, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), dry and ash femur weight, and bone contents of Ca++ and P were investigated. The femur was examined histopathologically. The OVX-control group showed an increase in serum levels of OC, BALP, TRAP, calcium, phosphorus, BMI, glucose, insulin, and HOMA-IR (P < 0.05) and a reduction in dry and ash weight of the femur, the concentration of calcium and phosphorus content in bone ash (P < 0.05). The OVX-irisin-treated group exhibited a decrease in serum levels of OC, BALP and TRAP, calcium, phosphorus, BMI, glucose, insulin, HOMA-IR (P < 0.05), and a rise in dry and ash weight of the femur, the concentration of calcium and phosphorus in bone ash (P < 0.05). Histological examination of the distal femur diaphysis of the OVX-irisin-treated group exhibited proper bone architecture and density compared with that of the OVX-control group. It is concluded that irisin treatment in the OVX rats safeguarded the regular bone architecture and normal levels of serum bone biomarkers. Irisin may be a possible novel target in the prohibition of postmenopausal osteoporosis.


Assuntos
Fibronectinas/farmacologia , Osteoporose , Ovariectomia , Substâncias Protetoras/farmacologia , Animais , Modelos Animais de Doenças , Feminino , Fêmur/química , Fêmur/efeitos dos fármacos , Osteoporose/etiologia , Osteoporose/metabolismo , Ratos
5.
Exp Cell Res ; 403(2): 112599, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33848551

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational research. Depending on the maturity of cardiac cultures and the intended applications, obtaining hiPSC-CMs as a single-cell, monolayer or three-dimensional clusters can be challenging. Here, we defined strategies to replate hiPSC-CMs on early days (D15-30) or later more mature (D60-150) differentiation cultures. After generation of hiPSCs and derivation of cardiomyocytes, four dissociation reagents Collagenase A/B, Collagenase II, TrypLE, EDTA and five different extracellular matrix materials Laminin, iMatrix-511, Fibronectin, Matrigel, and Geltrex were comparatively evaluated by imaging, cell viability, and contraction analysis. For early cardiac differentiation cultures mimicking mostly the embryonic stage, the highest adhesion, cell viability, and beating frequencies were achieved by treatment with the TrypLE enzyme. Video-based contraction analysis demonstrated higher beating rates after replating compared to before treatment. For later differentiation days of more mature cardiac cultures, dissociation with EDTA and replating cells on Geltrex or Laminin-derivatives yielded better recovery. Cardiac clusters at various sizes were detected in several groups treated with collagenases. Collectively, our findings revealed the selection criteria of the dissociation approach and coating matrix for replating iPSC-CMs based on the maturity and the requirements of further downstream applications.


Assuntos
Técnicas de Cultura de Células , Meios de Cultura/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Adulto , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Colágeno/farmacologia , Colagenases/farmacologia , Meios de Cultura/química , Combinação de Medicamentos , Feminino , Fibronectinas/farmacologia , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/análogos & derivados , Insulina/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Laminina/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Proteoglicanas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
6.
Exp Biol Med (Maywood) ; 246(14): 1597-1606, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33882700

RESUMO

This study aimed to track and evaluate the effect of low-dose irisin on the browning of white adipose tissue (WAT) in mice using magnetic resonance imaging (MRI) noninvasively in vivo. Mature white adipocytes extracted from mice were cultured, induced and characterized before being treated by irisin. The volume and fat fraction of WAT were quantified using MRI in normal chow diet and high fat mice after injection of irisin. The browning of cultured white adipocytes and WAT in mice were validated by immunohistochemistry and western blotting for uncoupling protein 1 (UCP1) and deiodinase type II (DIO2). The serum indexes were examined with high fat diet after irisin intervention. UCP1 and DIO2 in adipocytes showed increases responding to the irisin treatment. The size of white adipocytes in mice receiving irisin intervention was reduced. MRI measured volumes and fat fraction of WAT were significantly lower after Irisin treatment. Blood glucose and cholesterol levels were reduced in high fat diet mice after irisin treatment. Irisin intervention exerted browning of WAT, resulting reduction of volume and fat fraction of WAT as measured by MRI. Furthermore, it improved the condition of mice with diet-induced obesity and related metabolic disorders.


Assuntos
Adipócitos/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Fibronectinas/farmacologia , Adipócitos/metabolismo , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Branco/diagnóstico por imagem , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Fibronectinas/metabolismo , Iodeto Peroxidase/metabolismo , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Desacopladora 1/metabolismo
7.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809872

RESUMO

The epigenetic mechanisms controlling germ cell development and differentiation are still not well understood. Sirtuin-1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD)-dependent histone deacetylase and belongs to the sirtuin family of deacetylases. It catalyzes the removal of acetyl groups from a number of protein substrates. Some studies reported a role of SIRT1 in the central and peripheral regulation of reproduction in various non-primate species. However, testicular SIRT1 expression and its possible role in the testis have not been analyzed in primates. Here, we document expression of SIRT1 in testes of different primates and some non-primate species. SIRT1 is expressed mainly in the cells of seminiferous tubules, particularly in germ cells. The majority of SIRT1-positive germ cells were in the meiotic and postmeiotic phase of differentiation. However, SIRT1 expression was also observed in selected premeiotic germ cells, i.e., spermatogonia. SIRT1 co-localized in spermatogonia with irisin, an endocrine factor specifically expressed in primate spermatogonia. In marmoset testicular explant cultures, SIRT1 transcript levels are upregulated by the addition of irisin as compared to untreated controls explants. Rhesus macaques are seasonal breeders with high testicular activity in winter and low testicular activity in summer. Of note, SIRT1 mRNA and SIRT1 protein expression are changed between nonbreeding (low spermatogenesis) and breeding (high spermatogenesis) season. Our data suggest that SIRT1 is a relevant factor for the regulation of spermatogenesis in primates. Further mechanistic studies are required to better understand the role of SIRT1 during spermatogenesis.


Assuntos
Regulação da Expressão Gênica , Sirtuína 1/genética , Testículo/metabolismo , Animais , Callithrix , Fibronectinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Macaca mulatta , Masculino , Primatas , Estações do Ano , Sirtuína 1/metabolismo , Transcrição Genética
8.
Reprod Biol Endocrinol ; 19(1): 18, 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536035

RESUMO

OBJECTIVE: The aim of this study is to investigate the effect of irisin on leukemia inhibitory factor (LIF) and integrin αvß3 in implantation failure uterus. METHODS: Early pregnant rats were randomly divided into normal group (N), mifepristone treated group (M), irisin group (I) and progestin group (P). The implantation failure model was established using mifepristone. Second, we evaluated the average number of embryos and detected the expression of LIF and integrin αvß3 protein and mRNA in endometrium. RESULTS: Compared with group M, the average number of embryos was significantly higher in group N, P and I, the expression of LIF and integrin αvß3 in endometrium was significantly higher in group N, P and I. CONCLUSION: Irisin could improve the poor receptive state of endometrium by promoting LIF and integrin αvß3 secretion to improve blastocyst implantation in rats of implantation failure.


Assuntos
Implantação do Embrião/efeitos dos fármacos , Fibronectinas/farmacologia , Integrina alfaVbeta3/genética , Fator Inibidor de Leucemia/genética , Animais , Implantação do Embrião/genética , Perda do Embrião/induzido quimicamente , Perda do Embrião/genética , Perda do Embrião/metabolismo , Perda do Embrião/patologia , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Fibronectinas/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intramusculares , Integrina alfaVbeta3/metabolismo , Fator Inibidor de Leucemia/metabolismo , Mifepristona/farmacologia , Gravidez , Progestinas/administração & dosagem , Progestinas/farmacologia , Ratos , Ratos Wistar
9.
J Vis Exp ; (167)2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33522507

RESUMO

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Neovascularização Fisiológica , Engenharia Tecidual/métodos , Tecidos Suporte/química , Actinas/metabolismo , Biomarcadores/metabolismo , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Fibronectinas/farmacologia , Imunofluorescência , Humanos , Impressão Tridimensional , Imagem com Lapso de Tempo
10.
Arch Med Res ; 52(2): 182-190, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33067011

RESUMO

BACKGROUND: Irisin has been considered a prognostic factor in several cardiovascular diseases. Nevertheless, no data are available on the role of irisin in cardiac remodeling. AIM OF THE STUDY: This study aimed to determine the potential role of irisin in cardiac remodeling and explore potential mechanisms. METHODS: A total of 40 rats that underwent transverse abdominal aortic constriction (TAC) surgery or sham operation were divided into four groups: sham + saline (NS), sham + irisin, TAC + NS, and TAC + irisin. After 6 weeks of treatment, echocardiography was performed to assess in vivo cardiac morphology. The left ventricular myocardium was prepared and observed by pathological examination. The effect of irisin on cardiomyocyte apoptosis and the expression of oxidative stress and cardiac hypertrophy markers were observed. Then, the effect of irisin on the Akt signaling system was also detected. RESULTS: The rats in the TAC group displayed obvious signs of cardiac dysfunction and cardiac hypertrophy, and irisin treatment could reverse these changes. Irisin could inhibit the expression of nicotinamide adenine dinucleotide phosphate oxidase 2 and xanthine oxidase in TAC rats and increase the expression of antioxidant enzymes. Furthermore, the expression of phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), and phosphorylated glycogen synthase kinase 3ß (p-GSK3ß) was much higher in the cardiac remodeling groups (p <0.05 vs. sham rats). Irisin could relieve the inhibition effect and reduce the expression level of these three proteins. CONCLUSIONS: Irisin treatment could significantly improve cardiac remodeling by inhibiting oxidative stress via attenuating the Akt signaling activation.


Assuntos
Apoptose/efeitos dos fármacos , Fibronectinas/uso terapêutico , Coração/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Fibronectinas/farmacologia , Humanos , Masculino , Ratos
11.
Gene ; 769: 145209, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33038421

RESUMO

As an important complication of diabetes mellitus, diabetic cardiomyopathy (DCM) is thought to arise as a result of insulin resistance (IR) in cardiomyocytes. Improving IR in cardiomyocytes may therefore be a way to treat DCM. A recently discovered myokine, irisin, has been shown to be significantly associated with increased insulin sensitivity both in clinical and pre-clinical studies of diabetes mellitus. Based on previously research, we hypothesized that irisin may be a potential candidate for increasing the insulin sensitivity of cardiomyocytes. The aim of the present study was to examine the ability of irisin to affect IR induced by treatment of rat cardiomyocyte H9c2 cells with palmitic acid (PA) and to explore its underlying mechanism. Differentiated H9c2 cells were treated with 500 µM PA, 200 ng/mL irisin, and 500 µM PA + 200 ng/mL irisin with or without 100 nM rapamycin (RAP) for 24 h. We found that coincubation with 200 ng/mL irisin for 24 h significantly increased insulin-stimulated glucose consumption compared to the 500 µM PA group alone. Additionally, coincubation with irisin significantly alleviated the degree of autophagy compared to the 500 µM PA group alone as evidenced by monodansylcadaverine (MDC) fluorescence, the LC3II/LC3I protein levels ratio, and the protein levels of Atg5 and Atg7. Coincubation with irisin increased the levels of PI3Kp110α, pAkt and Akt compared to the 500 µM PA group alone. All these effects of irisin were reversed by RAP. Our results indicate that irisin improves IR in H9c2 cells, possibly in part by inhibiting autophagy through activating the PI3K/Akt pathway.


Assuntos
Autofagia/efeitos dos fármacos , Fibronectinas/farmacologia , Resistência à Insulina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Recombinantes de Fusão/farmacologia
12.
Methods Mol Biol ; 2217: 17-25, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215373

RESUMO

Integrin activation is a crucial event for multiple biological functions. Therefore, methods to detect integrin activation are vital. Since the main cellular function of integrins is adhesion, we and others utilize this feature to measure integrin activation. Here, we describe how to detect the activity of the fibronectin-binding integrin α5ß1 using a fusion of glutathione S-transferase (GST) to the 9th, 10th, and 11th type III repeats on fibronectin (GST-FNIII9-11). Moreover, we detail how to measure αvß3 integrin activity using the ligand-mimetic WOW-1 antibody that selectively binds unoccupied αvß3 integrins. Finally, we describe methods of testing ligation of fibronectin-binding integrins utilizing monoclonal antibodies against ligand-induced binding sites (LIBS).


Assuntos
Anticorpos/metabolismo , Fibronectinas/genética , Immunoblotting/métodos , Integrina alfa5beta1/genética , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Anticorpos/química , Sítios de Ligação , Adesão Celular , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica/métodos , Integrina alfa5beta1/agonistas , Integrina alfa5beta1/metabolismo , Mimetismo Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sequências Repetitivas de Aminoácidos
13.
Life Sci ; 267: 118954, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359670

RESUMO

The scientific interest in irisin, a myokine discovered in 2012, has grown exponentially in recent years. Irisin, which is mainly produced in skeletal muscle, influences the browning process of adipose tissue and lipid and energy metabolism. Recent discoveries highlight that the potential of this hormone may have been underestimated. In the first part of this review, reports on irisin structure and molecules involved in its metabolic pathway are shown. Furthermore, data related to unclear aspects are also reported: distribution, different gene expression of its precursors in different tissues, physiological levels of circulating irisin, and pharmacokinetic and pharmacodynamic profile. The second part of this work focuses on exogenous stimuli and pharmacological agents which regulate the metabolic pathway of irisin and its serum concentration. In addition to physical exercise and exposure to low temperatures, which were early recognized as exogenous stimuli able to promote the production of this myokine, preclinical and clinical evidence demonstrates the ability of natural and synthetic molecules to interfere with this metabolic pathway. Current experimental data on irisin cannot dissolve all doubts related to this interesting molecule, but they certainly underline its potential for therapeutic purposes. Thus, identification of new pharmacological tools able to act on the irisin pathway is a challenging issue for biomedical research.


Assuntos
Fibronectinas/metabolismo , Fibronectinas/farmacologia , Fibronectinas/fisiologia , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético/fisiologia , Exercício Físico/fisiologia , Humanos , Músculo Esquelético/metabolismo , Fatores de Transcrição/metabolismo
14.
J Vis Exp ; (165)2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33283783

RESUMO

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 Ω x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.


Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/citologia , Criopreservação , Células Endoteliais/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Biomarcadores/metabolismo , Barreira Hematoencefálica/citologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Colágeno Tipo IV/farmacologia , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibronectinas/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos
15.
ACS Appl Mater Interfaces ; 12(51): 56908-56923, 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33314916

RESUMO

Encapsulation devices are an emerging barrier technology designed to prevent the immunorejection of replacement cells in regenerative therapies for intractable diseases. However, traditional polymers used in current devices are poor substrates for cell attachment and induce fibrosis upon implantation, impacting long-term therapeutic cell viability. Bioactivation of polymer surfaces improves local host responses to materials, and here we make the first step toward demonstrating the utility of this approach to improve cell survival within encapsulation implants. Using therapeutic islet cells as an exemplar cell therapy, we show that internal surface coatings improve islet cell attachment and viability, while distinct external coatings modulate local foreign body responses. Using plasma surface functionalization (plasma immersion ion implantation (PIII)), we employ hollow fiber semiporous poly(ether sulfone) (PES) encapsulation membranes and coat the internal surfaces with the extracellular matrix protein fibronectin (FN) to enhance islet cell attachment. Separately, the external fiber surface is coated with the anti-inflammatory cytokine interleukin-4 (IL-4) to polarize local macrophages to an M2 (anti-inflammatory) phenotype, muting the fibrotic response. To demonstrate the power of our approach, bioluminescent murine islet cells were loaded into dual FN/IL-4-coated fibers and evaluated in a mouse back model for 14 days. Dual FN/IL-4 fibers showed striking reductions in immune cell accumulation and elevated levels of the M2 macrophage phenotype, consistent with the suppression of fibrotic encapsulation and enhanced angiogenesis. These changes led to markedly enhanced islet cell survival and importantly to functional integration of the implant with the host vasculature. Dual FN/IL-4 surface coatings drive multifaceted improvements in islet cell survival and function, with significant implications for improving clinical translation of therapeutic cell-containing macroencapsulation implants.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Fibrose/prevenção & controle , Ilhotas Pancreáticas/metabolismo , Polímeros/química , Sulfonas/química , Animais , Adesão Celular/efeitos dos fármacos , Fibronectinas/química , Fibronectinas/farmacologia , Luciferina de Vaga-Lumes/farmacologia , Interleucina-4/química , Interleucina-4/farmacologia , Ilhotas Pancreáticas/diagnóstico por imagem , Ilhotas Pancreáticas/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas/instrumentação , Transplante das Ilhotas Pancreáticas/métodos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica/efeitos dos fármacos , Imagem Óptica , Próteses e Implantes , Células RAW 264.7
16.
Bull Exp Biol Med ; 170(1): 158-163, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33231802

RESUMO

In this work, an optimal protocol was developed for obtaining adhesion culture of neural stem/progenitor cells (NSPC) of rat olfactory mucosa. During the development of the protocol, the conditions for cell culturing on adhesion substrates fibronectin and laminin in DMEM/F-12 and neurobasal media with the same culture additives were compared. Cell proliferation was maximum during culturing on both substrates in the neurobasal medium. Using the immunofluorescence method, we found that culturing on fibronectin in the neurobasal medium ensured maximum (52.22%) content of nestin-positive cells in comparison with other culturing conditions. The highest percentage of ßIII-tubulin-positive cells was detected in cultures growing on fibronectin in the neurobasal medium and in DMEM/F-12 (79.11 and 83.52%, respectively). Culturing in adhesion cultures in the neurobasal medium on fibronectin allowed obtaining cultures enriched with NSPC and neurons differentiating from them in a quantity sufficient for further transplantation. The developed protocol can be recommended for obtaining NPSC from human olfactory mucosa for the treatment of spinal cord injuries.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Meios de Cultura/farmacologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Mucosa Olfatória/citologia , Animais , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Fibronectinas/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Laminina/farmacologia , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Cultura Primária de Células , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/terapia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
17.
Endocrinol Metab (Seoul) ; 35(3): 647-655, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32981307

RESUMO

BACKGROUND: Hepatic stellate cells (HSCs) are known to play a fundamental role in the progression of liver fibrosis. Once HSCs are activated, they are involved in proliferation, migration, and contractility which are characteristics of liver fibrogenesis. Recent studies have shown that irisin, a myokine secreted during physical exercise, has a protective effect in various metabolic diseases, especially in renal fibrosis. However, whether irisin is involved in HSC activation and other processes associated with liver fibrosis has not yet been investigated. In this study, we reveal the role of irisin in HSC activation as well as in proliferation, migration, and contractile properties of HSCs in vitro. METHODS: LX-2 cells, immortalized human HSCs, were treated with transforming growth factor beta 1 (TGF-ß1), a core regulator of HSC fibrosis, with or without irisin, and markers of the aforementioned processes were analyzed. Further, an inflammatory response was stimulated with TGF-ß1 and lipopolysaccharide (LPS) in combination with irisin and the expression of cytokines was measured. RESULTS: Recombinant irisin significantly suppressed the expression of TGF-ß1-stimulated fibrosis markers including alpha-smooth muscle actin and collagen type 1 alpha 1 and prevented the TGF-ß1-induced proliferation, migration, and contractility of LX-2 cells. Additionally, irisin ameliorated the production of interleukin-6 (IL-6) and IL-1ß induced by TGF-ß1 and LPS treatments. CONCLUSION: These findings suggested that irisin potently improved the progression of hepatic fibrosis by regulating HSC activation, proliferation, migration, contractility, and HSC-mediated production of inflammatory cytokine.


Assuntos
Fibronectinas/farmacologia , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/patologia , Fator de Crescimento Transformador beta1/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Proteínas Recombinantes/farmacologia
18.
Biomed Res Int ; 2020: 6537371, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32934963

RESUMO

As a common disorder, acute kidney injury (AKI) is characterized by high mortality and morbidity, and current therapeutic options for AKI remain limited. Irisin, a muscle factor, plays an important role in metabolic disorders. However, the role of irisin in AKI is still unclear. To assess the effect of irisin on the course of AKI, we used an ischemia/reperfusion (I/R) C57BL/6 mouse model. Supplementation with irisin attenuated kidney injury induced by I/R, as shown by decreases in the levels of serum creatinine and blood urea nitrogen. Animal model studies also showed that irisin pretreatment upregulates the expression of uncoupling protein 2 (UCP2) and protects against the renal cell apoptosis and oxidative stress caused by I/R. In vitro, hypoxia/recovery (H/R) treatment was applied to induce tubular cell apoptosis. Irisin pretreatment ameliorated the cell apoptosis induced by H/R, while transfection of UCP2 siRNA significantly reduced the protective effect of irisin in cells after H/R. In addition, AMPK signaling may be involved in irisin-mediated upregulation of UCP2 in a renal proximal tubular epithelial cell (PTEC) model. Thus, the renoprotective effect of irisin on AKI may be mediated through increasing the expression of UCP2 in kidneys after I/R.


Assuntos
Injúria Renal Aguda/tratamento farmacológico , Fibronectinas/genética , Proteínas Quinases/genética , Traumatismo por Reperfusão/genética , Proteína Desacopladora 2/genética , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Fibronectinas/farmacologia , Humanos , Rim/efeitos dos fármacos , Rim/lesões , Rim/patologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos
19.
Cell Biol Int ; 44(11): 2315-2325, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32770767

RESUMO

High glucose (HG)-induced cardiomyocytes (CMs) injury is a leading cause of diabetic cardiomyopathy with little treatment options. Irisin, a new myokine, which is cleaved from its precursor fibronectin type III domain-containing protein 5 (FNDC5), has aroused great attention as an essential cardioprotective factor and glucose metabolism regulator but little was known on diabetic cardiomyopathy yet. Here, we aim to clarify the role of irisin in the HG-induced CMs injury. Neonatal Sprague-Dawley rat CMs were cultured in a normal or HG medium for 12, 24, and 48 hr, respectively before exposing to irisin. The apoptosis level was determined by terminal-deoxynucleotidyl transferase-mediated-dUTP nick end-labeling assay. Cell viability was measured with the conventional methyl thiazolyl tetrazolium assay. Moreover, reactive oxygen species production was evaluated by dihydroethidium staining. Inflammatory factors, namely tumor necrosis factor-α, interleukin-6, interleukin-1ß were determined by enzyme-linked immunosorbent assay kits. Furthermore, protein and messenger RNA (mRNA) expressions were measured by western blot and quantitative real-time polymerase chain reaction, respectively. HG increases the apoptosis of CMs and activated the inflammatory responses and oxidative stress in CMs. Meanwhile, the mRNA and protein expressions of FNDC5 are decreased after HG exposure. Nevertheless, the increased apoptosis is alleviated by irisin treatment. Notably, irisin suppresses the inflammatory responses and oxidative stress in injured CMs. Mechanically, after the administration of Compound C, AMP-activated protein kinase (AMPK) inhibitor, these cardioprotective effects resulting from irisin are reversed. Irisin plays a significant role in antiapoptosis, anti-inflammation, antioxidative stress in HG-induced CMs via AMPK/mammalian target of the rapamycin signaling pathway.


Assuntos
Cardiomiopatias Diabéticas/fisiopatologia , Fibronectinas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cardiomiopatias Diabéticas/metabolismo , Feminino , Fibronectinas/farmacologia , Glucose/metabolismo , Masculino , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo
20.
Cerebrovasc Dis ; 49(4): 346-354, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756048

RESUMO

BACKGROUND: Inflammatory response exerts an important role in ischemia/reperfusion (I/R) injury. TLR4 and myeloid differentiation factor 88 (MyD88) are key components in inflammation and are involved in the cerebral I/R injury. Irisin is a skeletal muscle-derived myokine produced after exercise, which was found to suppress inflammation. In this study, we investigated whether irisin could protect the brain from I/R injury through the TLR4/MyD88 pathway. METHODS: Male Sprague Dawley rats (20 months, 190 ∼ 240 g) were pretreated with irisin at 10, 50, or 100 mg/kg for consecutive 3 days and then subjected to surgery of middle cerebral artery occlusion or sham operation. Infarct size and neuron loss were measured to evaluate brain damage. The mRNA and protein levels of TLR4 and MyD88 were measured by in situ hybridization and immunohistochemistry, respectively. NF-κB activation was assessed by electrophoretic mobility shift assay. Neurological function was evaluated by neurobehavior score test and passive avoidance test. RESULTS: Irisin could reduce neuronal damage and neurofunctional impairment after I/R injury. This effect was mediated by downregulating the TLR4/MyD88 and inhibiting NF-κB activation. CONCLUSION: Irisin plays a beneficial effect in I/R injury through regulating the TLR4/MyD88 pathway.


Assuntos
Encéfalo/efeitos dos fármacos , Fibronectinas/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fator 88 de Diferenciação Mieloide/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Receptor 4 Toll-Like/metabolismo , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Atividade Motora/efeitos dos fármacos , NF-kappa B/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais
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