RESUMEN
Objective: This study aims to investigate the role of decorin in the adhesion process of Treponema pallidum subspecies pallidum (T. pallidum) to human brain microvascular endothelial cells. Methods: The study involved an in vitro experimental design. Western blot analysis was conducted to determine the protein expression level of decorin in the cells. The cells were divided into four groups: Tp group, inactivated Tp group, LPS group, and negative control group. The adhesion of T. pallidum to the cells was analyzed using darkfield microscopy counting and quantitative polymerase chain reaction (qPCR). The cells were divided into four groups based on different preprocessing treatments: control group, decorin group, DCN-siRNA group, and DCN-siRNA+decorin group. Changes in the F-actin of the cells were explored using confocal laser scanning microscopy. The cells were divided into the Tp group, Tp+decorin group, and control group. Results: Western blot analysis showed high expression of decorin in the Tp group and LPS group. Darkfield microscopy counting revealed a significantly higher number of T. pallidum adhered to a single cell in the decorin group compared to the control group. Conversely, the number of adhered T. pallidum was significantly lower in the DCN-siRNA group compared to the control group. qPCR results indicated a considerably higher T. pallidum load in the decorin group compared to the control group. In the Tp group, T. pallidum treatment induced the reorganization of F-actin, while the distribution of F-actin in the Tp+decorin group was comparable to that of the control group. Conclusions: Decorin enhances the adhesion of T. pallidum to human brain microvascular endothelial cells, suggesting that decorin may act as one of the receptors regulating the adhesion of T. pallidum to cells. Furthermore, T. pallidum treatment triggers the rearrangement of F-actin in cells, and decorin plays a protective role in this process.
Asunto(s)
Células Endoteliales , Treponema pallidum , Humanos , Treponema pallidum/genética , Treponema pallidum/metabolismo , Decorina/genética , Decorina/metabolismo , Células Endoteliales/metabolismo , Actinas/metabolismo , Globo Pálido/metabolismo , LipopolisacáridosRESUMEN
OBJECTIVES: To evaluate the effect of L-carnitine (LC) in combination with leucine supplementation on muscle strength and muscle hypertrophy in aged women participating in a resistance exercise training (RET) program. DESIGN/SETTING/PARTICIPANTS: Thirty-seven out of sixty (38.3% dropout) healthy women aged 60-75 years (mean 67.6 ± 0.7 years) completed the intervention in one of three groups. One of the supplemented groups received 1 g of L-carnitine-L-tartrate in combination with 3 g of L-leucine per day (LC+L group; n = 12), and the second supplemented group received 4 g of L-leucine per day (L group; n = 13). The control group (CON group; n = 12) received no supplementation. INTERVENTION: All three groups completed the same RET protocol involving exercise sessions twice per week for 24 weeks. MEASUREMENTS: Before and after the experiment, participants performed isometric and isokinetic muscle strength testing on the Biodex dynamometer. The cross-sectional areas of the major knee extensors and total thigh muscles were assessed using magnetic resonance imaging. Fasting serum levels of insulin-like growth factor-1 (IGF-1), myostatin and decorin, and plasma levels of total carnitine (TC) and trimethylamine-N-oxide (TMAO) levels were measured. RESULTS: The 24-week RET significantly increased muscle strength and muscle volume, but the group and time interactions were not significant for the muscle variables analyzed. Plasma total carnitine increased only in the LC+L group (p = 0.009). LC supplementation also caused a significant increase in plasma TMAO, which was higher after the intervention in the LC+L group than in the L (p < 0.001), and CON (p = 0.005) groups. The intervention did not change plasma TMAO concentration in the L (p = 0.959) and CON (p = 0.866) groups. After the intervention serum decorin level was higher than before in both supplemented groups combined (p = 0.012), still not significantly different to post intervention CON (p = 0.231). No changes in serum IGF-1 and myostatin concentrations and no links between the changes in blood markers and muscle function or muscle volume were observed. CONCLUSIONS: LC combined with leucine or leucine alone does not appear to improve the effectiveness of RET.
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Carnitina , Leucina , Entrenamiento de Fuerza , Femenino , Humanos , Carnitina/farmacología , Decorina/metabolismo , Suplementos Dietéticos , Factor I del Crecimiento Similar a la Insulina , Leucina/farmacología , Fuerza Muscular/fisiología , Músculo Esquelético , Miostatina/metabolismo , Tartratos/farmacología , Persona de Mediana Edad , AncianoRESUMEN
This study aimed to investigate the effect of bile duct-targeting lecithins- (PC-) coupled decorin (DCN) (PC-DCN) nanoliposomes against liver fibrosis in vitro and in vivo. We prepared PC-DCN nanoliposomes by using rat astrocytes, HSC-T6, to verify the antifibrosis effect of PC-DCN in vitro. First, we established a rat model of carbon tetrachloride-induced fibrosis. PC-DCN nanoliposomes were then injected into fibrotic rats via the portal vein or bile duct. The EdU assay was performed to analyze cell proliferation. Immunofluorescence staining was used to detect α-smooth muscle actin (α-SMA) expression. Western blot was performed to examine the expression of α-SMA, collagen type I alpha 1 (COL1A1), and transforming growth factor-ß (TGF-ß) protein. The levels of aspartate transaminase (AST), alanine transaminase (ALT), and total bilirubin (TBIL) were examined by enzyme-linked immunosorbent assay (ELISA) analysis. Hematoxylin and eosin (H&E) staining and Masson trichrome staining were used to determine liver tissue lesions and liver fibrosis. Compared with TGF-ß group, PC-DCN treatment could significantly reduce cell proliferation. Western blot analysis indicated that the expression of α-SMA, COL1A1, and TGF-ß was downregulated after treatment with PC-DCN in vitro and in vivo. Immunofluorescence staining confirmed that α-SMA expression was reduced by PC-DCN. Furthermore, H&E staining and Masson trichrome staining showed that the administration of PC-DCN nanoliposomes via the bile duct could reduce the extent of liver fibrosis. PCR analysis showed that PC-DCN administration could reduce proinflammatory cytokines IL-6, TNF-α, and IL-1ß expression via the bile duct. The administration of PC-DCN nanoliposomes also significantly downregulated liver function indicators ALT, AST, and TBIL. The results of our study indicated that PC-DCN could effectively reduce the extent of liver fibrosis.
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Decorina/metabolismo , Lecitinas/farmacología , Cirrosis Hepática/patología , Nanopartículas/química , Animales , Conductos Biliares/efectos de los fármacos , Conductos Biliares/patología , Tetracloruro de Carbono , Línea Celular , Proliferación Celular/efectos de los fármacos , Liposomas , Masculino , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy of uncertain etiology. Our study sought to identify a correlation between small proteoglycans decorin and biglycan expression and Kashin-Beck Disease. Immunohistochemistry was used to assess the decorin and biglycan levels in cartilage specimens from both child KBD patients, and rats fed with T-2 toxin under a selenium-deficient condition. Real-time PCR and Western blot were used to assess mRNA and protein levels of decorin and biglycan in rat cartilages, as well as in C28/I2 chondrocytes stimulated by T-2 toxin and selenium in vitro. The result showed that decorin was reduced in all zones of KBD articular cartilage, while the expression of biglycan was prominently increased in KBD cartilage samples. Increased expression of biglycan and reduced expression of decorin were observed at mRNA and protein levels in the cartilage of rats fed with T-2 toxin and selenium- deficiency plus T-2 toxin diet, when compared with the normal diet group. Moreover, In vitro stimulation of C28/I2 cells with T-2 toxin resulted in an upregulation of biglycan and downregulation of decorin, T-2 toxin induction of biglycan and decorin levels were partly rescued by selenium supplement. This study highlights the focal nature of the degenerative changes that occur in KBD cartilage and may suggest that the altered expression pattern of decorin and biglycan have an important role in the onset and pathogenesis of KBD.
Asunto(s)
Biglicano/genética , Cartílago Articular/metabolismo , Decorina/genética , Enfermedad de Kashin-Beck/genética , Animales , Cartílago Articular/patología , Niño , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/genética , Humanos , Enfermedad de Kashin-Beck/inducido químicamente , Enfermedad de Kashin-Beck/metabolismo , Enfermedad de Kashin-Beck/patología , Masculino , Ratas , Selenio/deficiencia , Selenio/metabolismo , Toxina T-2/toxicidadRESUMEN
Dentine matrix has proposed roles for directing mineralised tissue repair in dentine and bone; however, the range of bioactive components in dentine and specific biological effects on bone-derived mesenchymal stem cells (MSCs) in humans are less well understood. The aims of this study were to further elucidate the biological response of MSCs to demineralised dentine matrix (DDM) in enhancing wound repair responses and ascertain key contributing components. Dentine was obtained from human teeth and DDM proteins solubilised with ethylenediaminetetraacetic acid (EDTA). Bone marrow derived MSCs were commercially obtained. Cells with a more immature phenotype were then selected by preferential fibronectin adhesion (FN-BMMSCs) for use in subsequent in vitro assays. DDM at 10 µg/mL reduced cell expansion, attenuated apoptosis and was the minimal concentration capable of inducing osteoblastic differentiation. Enzyme-linked immunosorbent assay (ELISA) quantification of growth factors indicated physiological levels produced the above responses; transforming growth factor ß (TGF-ß1) was predominant (15.6 ng/mg DDM), with relatively lower concentrations of BMP-2, FGF, VEGF and PDGF (6.2-4.7 ng/mg DDM). Fractionation of growth factors from other DDM components by heparin affinity chromatography diminished osteogenic responses. Depletion of biglycan from DDM also attenuated osteogenic potency, which was partially rescued by the isolated biglycan. Decorin depletion from DDM had no influence on osteogenic potency. Collectively, these results demonstrate the potential of DDM for the delivery of physiological levels of growth factors for bone repair processes, and substantiate a role for biglycan as an additional adjuvant for driving osteogenic pathways.
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Materiales Biocompatibles/farmacología , Matriz Ósea/metabolismo , Regeneración Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Dentina/metabolismo , Células Madre Mesenquimatosas/citología , Biglicano/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Matriz Ósea/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Separación Celular , Cromatografía de Afinidad , Decorina/metabolismo , Fibronectinas/farmacología , Heparina/química , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismoRESUMEN
Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFß3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFß3 and bFGF2 + TGFß3 + LLLT. Indeed, the supplement of bFGF2 and TGFß3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFß3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Caballos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Traumatismos de los Tendones/veterinaria , Tendones/citología , Animales , Proliferación Celular , Células Cultivadas , Decorina/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Terapia por Luz de Baja Intensidad , Traumatismos de los Tendones/terapiaRESUMEN
BACKGROUND/AIMS: Multipotent mesenchymal stem cells affect homeostasis of adipose and joint tissues. Factors influencing their differentiation fate are of interest for both obesity and joint problems. We studied the impact of a mixture of glycosaminoglycans (GAGs) (hyaluronic acid: dermatan sulfate 1:0.25, w/w) used in an oral supplement for joint discomfort (Oralvisc™) on the differentiation fate of multipotent cells. METHODS: Primary mouse embryo fibroblasts (MEFs) were used as a model system. Post-confluent monolayer MEF cultures non-stimulated or hormonally stimulated to adipogenesis were chronically exposed to the GAGs mixture, its individual components or vehicle. The appearance of lipid laden cells, lipid accumulation and expression of selected genes at the mRNA and protein level was assessed. RESULTS: Exposure to the GAGs mixture synergistically suppressed spontaneous adipogenesis and induced the expression of cartilage extracellular matrix proteins, aggrecan core protein, decorin and cartilage oligomeric matrix protein. Hormonally-induced adipogenesis in the presence of the GAGs mixture resulted in decreased adipogenic differentiation, down-regulation of adipogenic/lipogenic factors and genes for insulin resistance-related adipokines (resistin and retinol binding protein 4), and up-regulation of oxidative metabolism-related genes. Adipogenesis in the presence of dermatan sulfate, the minor component of the mixture, was not impaired but resulted in smaller lipid droplets and the induction of a more complete brown adipocyte-related transcriptional program in the cells in the adipose state. CONCLUSIONS: The Oralvisc™ GAGs mixture can tip the adipogenic/chondrogenic fate balance of multipotent cells away from adipogenesis while favoring chondrocyte related gene expression. The mixture and its dermatan sulfate component also have modulatory effects of interest on hormonally-induced adipogenesis and on metabolic and secretory capabilities of adipose cells.
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Adipogénesis/efectos de los fármacos , Glicosaminoglicanos/farmacología , Adipocitos/citología , Adipocitos/metabolismo , Adipoquinas/genética , Adipoquinas/metabolismo , Agrecanos/genética , Agrecanos/metabolismo , Animales , Proteína Morfogenética Ósea 2/farmacología , Células Cultivadas , Condrocitos/metabolismo , Decorina/genética , Decorina/metabolismo , Dermatán Sulfato/farmacología , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ácido Hialurónico/farmacología , Ratones , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Intense pulsed light (IPL) devices have been shown to be highly effective for the skin rejuvenation. In our study, we try to elucidate effects of IPL in fibroblast proliferation, in gene expression, and in extracellular matrix protein production. 1BR3G human skin fibroblasts were used to test the effects of an IPL device (MiniSilk FT, Deka®). Fibroblasts were divided into three groups: group 1 was irradiated with filter 800-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm) twice; group 2 was irradiated with filter 550-1200 nm (double pulse 5 ms + 5 ms, delay 10 ms, fluence 13 J/cm2) twice; and group 3 was irradiated with filter 550-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm2) twice. To determine changes in gene expression, messenger RNA (mRNA) levels for collagen types I and III and metalloproteinase 1 (MMP-1) were performed 48 h after irradiation. To determine changes in hyaluronic acid, versican, and decorin, mRNA and ELISA tests were performed after 48 h of treatment. In addition to this, a Picro-Sirius red staining for collagen was made. The study showed an increase of mRNA and hyaluronic acid, decorin, and versican production. With RT-PCR assays, an increase mRNA for collagen type I, type III, and MMP-1 was observed. Collagen and hyaluronic synthesis was increased in all groups with no differences among them, while decorin and versican synthesis was higher in those groups irradiated with 550-1200-nm filters with no dependence of type pulse or total energy dose. IPL applied in vitro cultured cells increases fibroblasts activity. Synthesis of extracellular proteins seems to be produced more specifically in determined wavelengths, which could demonstrate a biochemical mechanism light depending.
Asunto(s)
Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Rayos Láser , Metaloproteinasa 1 de la Matriz/metabolismo , Activación Transcripcional/efectos de la radiación , Línea Celular , Proliferación Celular , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Decorina/biosíntesis , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Expresión Génica , Humanos , Ácido Hialurónico/biosíntesis , Tratamiento de Luz Pulsada Intensa , Metaloproteinasa 1 de la Matriz/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/citología , Versicanos/biosíntesisRESUMEN
Jerusalem artichoke (JA) has the potential to attenuate lipid disturbances and insulin resistance (IR), but the underlying mechanisms are not well understood. In the present study, we elucidated the physiological responses and mechanisms of JA intervention with a comprehensive transcriptome analysis. Wistar rats were fed a control diet, a 60 % fructose-enriched diet (FRU), or a FRU with 10 % JA (n 6-7) for 4 weeks. An oral glucose tolerance test was carried out on day 21. Liver samples were collected for biochemical and global gene expression analyses (GeneChip® Rat Genome 230 2.0 Array, Affymetrix). Fructose feeding resulted in IR and hepatic TAG accumulation; dietary JA supplementation significantly improved these changes. Transcriptomic profiling revealed that the expression of malic enzyme 1 (Me1), associated with fatty acid synthesis; decorin (Dcn), related to fibrosis; and cytochrome P450, family 1, subfamily a, polypeptide 2 (Cyp1a2) and nicotinamide phosphoribosyltransferase (Nampt), associated with inflammation, was differentially altered by the FRU, whereas dietary JA supplementation significantly improved the expression of these genes. We established for the first time the molecular mechanisms driving the beneficial effects of JA in the prevention of type 2 diabetes and non-alcoholic fatty liver disease. We propose that 10 % JA supplementation may be beneficial for the prevention of the onset of these diseases.
Asunto(s)
Diabetes Mellitus Tipo 2/prevención & control , Dieta , Hígado Graso/prevención & control , Fructosa/administración & dosificación , Helianthus , Extractos Vegetales/administración & dosificación , Animales , Citocromo P-450 CYP1A2/genética , Decorina/genética , Ácido Graso Sintasas/metabolismo , Fructanos/administración & dosificación , Expresión Génica , Perfilación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Resistencia a la Insulina , Hígado/química , Hígado/patología , Malato Deshidrogenasa/genética , Masculino , Nicotinamida Fosforribosiltransferasa/genética , Enfermedad del Hígado Graso no Alcohólico , Fitoterapia , Raíces de Plantas/química , Ratas , Ratas Wistar , Solubilidad , Triglicéridos/metabolismoRESUMEN
Severe muscle fibrosis is the endpoint of many chronic myopathies. Identification of factors that regulate fibrosis is important for understanding its pathogenesis and for developing anti-fibrotic treatments that prevent muscle destruction. We have developed an in vitro model for screening potential anti-fibrotic agents. The model consists of three-dimensional clusters (nodules) of fibroblasts derived from Duchenne muscular dystrophy (DMD) muscle. The primary fibroblasts spontaneously and quickly form nodules resembling fibrotic foci (cells plus extracellular matrix) when grown on a solid substrate. We tested the anti-fibrotic action of suramin, decorin, and spironolactone (all with established anti-fibrotic activity) on the model. All three agents significantly reduced nodule number, and spironolactone and suramin significantly reduced nodule diameter. Nodule secretion of soluble collagen was also significantly reduced by decorin and spironolactone treatment, whereas suramin had no significant effect. Collagen I and fibronectin protein expression was significantly reduced in the culture medium of control and DMD fibroblasts by spironolactone treatment, but not by decorin and suramin treatment. Finally, in DMD fibroblast monolayers, collagen deposition was significantly reduced by all three agents. Spironolactone significantly reduced collagen I and fibronectin transcript levels, whereas decorin reduced only fibronectin. Our in vitro model of fibrogenesis has thus revealed differing anti-fibrotic effects in the three anti-fibrotic agents tested. It therefore appears as a useful and sensitive system for the testing of anti-fibrotic drugs and could be adapted for the high-throughput screening of new anti-fibrotic molecules.
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Bioensayo/métodos , Evaluación Preclínica de Medicamentos , Fibroblastos/patología , Fibrosis/tratamiento farmacológico , Distrofia Muscular de Duchenne/patología , Western Blotting , Colágeno/genética , Colágeno/metabolismo , Decorina/farmacología , Decorina/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espironolactona/farmacología , Espironolactona/uso terapéutico , Suramina/farmacología , Suramina/uso terapéuticoRESUMEN
PURPOSE: To describe new affected individuals of Franceschetti's original pedigree of hereditary recurrent erosion and to classify a unique entity called Franceschetti corneal dystrophy. DESIGN: Observational case series. METHODS: Slit-lamp examination of 10 affected individuals was conducted. Biomicroscopic examinations were supplemented by peripheral corneal biopsy in 1 affected patient with corneal haze. Tissue was processed for light and electron microscopy and immunohistochemistry was performed. DNA analysis was carried out in 12 affected and 3 nonaffected family members. RESULTS: All affected individuals suffered from severe ocular pain in the first decade of life, attributable to recurrent corneal erosions. Six adult patients developed bilateral diffuse subepithelial opacifications in the central and paracentral cornea. The remaining 4 affected individuals had clear corneas in the pain-free stage of the disorder. Histologic and immunohistochemical examination of the peripheral cornea in a single patient showed a subepithelial, avascular pannus. There was negative staining with Congo red. DNA analysis excluded mutations in the transforming growth factor beta-induced (TGFBI) gene and in the tumor-associated calcium signal transducer 2 (TACSTD2) gene. CONCLUSION: We have extended the pedigree of Franceschetti corneal dystrophy and elaborated its natural history on the basis of clinical examinations. A distinctive feature is the appearance of subepithelial opacities in adult life, accompanied by a decreased frequency of recurrent erosion attacks. Its clinical features appear to distinguish it from most other forms of dominantly inherited recurrent corneal erosion reported in the literature.
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Distrofias Hereditarias de la Córnea/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/genética , Biomarcadores/metabolismo , Biopsia , Cadherinas/metabolismo , Moléculas de Adhesión Celular/genética , Niño , Condroitín/metabolismo , Claudinas/metabolismo , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Opacidad de la Córnea/etiología , Análisis Mutacional de ADN , Decorina/metabolismo , Dermatán Sulfato/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Dolor Ocular/etiología , Femenino , Humanos , Inmunohistoquímica , Masculino , Linaje , Recurrencia , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: To explore whether Radix Dipsaci (RD) exhibits beneficial effects on tendon healing. METHODS: An attempt was made to explore the in vitro effects of a hot water extract of RD on gene expression of procollagen Type I (COL1A1), procollagen Type III (COL3A1) and decorin in cultured tendon fibroblasts, and its in vivo effects in a well-established rat model of patellar tendon donor site injury. RESULTS: It was found that gene expression of COL3A1 and decorin in cultured tendon fibroblasts was significantly increased by RD, but that COL1A1 was not affected. In vivo studies showed that RD increased blood vessels in the wound but did not significantly affect the expression of COL1A1, COL3A1 and decorin at day 14 post-injury. The ultimate tensile stress of the healing tendon was not significantly improved by either local injection or oral administration of hot water extract of RD (P > 0.05). CONCLUSION: The present findings imply that RD per se does not significantly improve tendon healing. Further investigation of RD in a herbal formula may be necessary to test its efficacy in tendon injuries.
Asunto(s)
Dipsacaceae , Medicamentos Herbarios Chinos/uso terapéutico , Fibroblastos/efectos de los fármacos , Ligamento Rotuliano/lesiones , Fitoterapia , Traumatismos de los Tendones/tratamiento farmacológico , Cicatrización de Heridas/efectos de los fármacos , Animales , Células Cultivadas , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/metabolismo , Decorina/metabolismo , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/farmacología , Fibroblastos/metabolismo , Masculino , Ligamento Rotuliano/efectos de los fármacos , Ligamento Rotuliano/metabolismo , Ligamento Rotuliano/trasplante , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Traumatismos de los Tendones/etiología , Traumatismos de los Tendones/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Achilles tendon problems are commonly encountered in sports medicine and low-level laser therapy (LLLT) is widely used in rehabilitative applications to decrease pain, reduce inflammatory processes, and promote tissue healing. This study examined the effects on the proliferation of porcine Achilles tendon fibroblasts and gene expression, using different doses of low-level laser irradiation (LLLI). Four groups of identically cultured fibroblasts were exposed to LLLI and harvested after 24 h. The control group (Group 1) was subjected to no LLLI. Other groups received 1 J/cm2 (Group 2), 2 J/cm2 (Group 3), and 3 J/cm2 (Group 4), respectively. Cell proliferation and mRNA expressions of type I collagen and decorin were then measured. When compared to the control group, the cell proliferation of irradiated Achilles tendon fibroblasts in the other three groups increased significantly by 13% +/- 0.8% (Group 2), 30% +/- 0.4% (Group 3), and 12% +/- 0.6% (Group 4) respectively. But progressively higher laser intensity did not achieve a correspondingly higher cell proliferation effect in Achilles tendon fibroblasts. The mRNA expressions of decorin and type I collagen in fibroblasts with LLLI were significantly higher (p < 0.05). Therefore, suitable dosages of LLLI may result in more effective tissue healing by promoting type I collagen and decorin synthesis. However, these positive effects of LLLI on the repair of the Achilles tendon in humans should be further investigated in clinic.
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Tendón Calcáneo/efectos de la radiación , Proliferación Celular/efectos de la radiación , Terapia por Luz de Baja Intensidad , Cicatrización de Heridas/efectos de la radiación , Animales , Células Cultivadas , Colágeno Tipo I/biosíntesis , Decorina , Proteínas de la Matriz Extracelular/biosíntesis , Fibroblastos/efectos de la radiación , Expresión Génica/efectos de la radiación , Proteoglicanos/biosíntesis , ARN Mensajero/metabolismo , PorcinosAsunto(s)
Proteínas de la Matriz Extracelular/genética , Fototerapia , Proteoglicanos/genética , Piel/efectos de la radiación , Rayos Ultravioleta , Adulto , Anciano , Anciano de 80 o más Años , Decorina , Proteínas de la Matriz Extracelular/metabolismo , Expresión Génica/efectos de la radiación , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Proteoglicanos/metabolismo , Piel/metabolismoRESUMEN
Patients with localized scleroderma underwent an 8-week course of ultraviolet A1 phototherapy. At baseline, decorin levels of lesional skin were significantly lower than those of nonlesional skin and healthy control subjects. After ultraviolet A1 phototherapy, decorin levels of lesional skin were significantly higher than baseline. Decorin may be of significance in the pathogenesis of localized scleroderma.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Fototerapia , Proteoglicanos/metabolismo , Esclerodermia Limitada/metabolismo , Piel/metabolismo , Adulto , Decorina , Femenino , Humanos , Persona de Mediana Edad , Esclerodermia Limitada/terapiaRESUMEN
Expression of decorin using the vaccinia virus/T7 expression system resulted in secretion of two distinct glycoforms: a proteoglycan substituted with a single chondroitin sulfate chain and N-linked oligosaccharides and a core protein glycoform substituted with N-linked glycans but without a glycosaminoglycan chain. In this report, we have addressed two distinct questions. What is the rate-limiting step in glycosaminoglycan synthesis? Is glycosylation with either N-linked oligosaccharides or glycosaminoglycan required for secretion of decorin? N-terminal sequencing of the core protein glycoform, the addition of benzyl-beta-d-xyloside, and a UDP-xylose: core protein beta-d-xylosyltransferase activity assay show that xylosylation is a rate-limiting step in chondroitin sulfate biosynthesis. Decorin can be efficiently secreted with N-linked oligosaccharides alone or with a single chondroitin sulfate chain alone; however, there is severely impaired secretion of core protein devoid of any glycosylation. A decorin core protein mutant devoid of N-linked oligosaccharide attachment sites will not be secreted by Chinese hamster ovary cells deficient in xylosyltransferase or by parental Chinese hamster ovary wild type cells if the xylosyltransferase recognition sequence is disrupted. This finding suggests that quality control mechanisms sensitive to an absence of N-linked oligosaccharides can be abrogated by interaction of the core protein with the glycosaminoglycan synthetic machinery. We propose a model of regulation of decorin secretion that has several components, including appropriate substitution with N-linked oligosaccharides and factors involved in glycosaminoglycan synthesis.
Asunto(s)
Proteoglicanos/química , Animales , Northern Blotting , Células CHO , Línea Celular , Sulfatos de Condroitina/química , Cricetinae , ADN Complementario/metabolismo , Decorina , Escherichia coli/metabolismo , Proteínas de la Matriz Extracelular , Glicosaminoglicanos/química , Glicósidos/química , Glicosilación , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Mutación , Oligosacáridos/química , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteoglicanos/genética , Proteoglicanos/metabolismo , Proteínas Recombinantes/química , Temperatura , Factores de Tiempo , Tunicamicina/farmacología , Uridina Difosfato Xilosa/química , Virus Vaccinia/metabolismo , Xilosa/químicaRESUMEN
The chondroitin/dermatan sulfate proteoglycans (CS/DSPGs), biglycan, decorin, and versican play several important roles in extracellular matrix influencing matrix organization, cell proliferation, and recruitment. Moreover, they bind and regulate growth factors in the extracellular matrix. We have previously shown that cultured human lung fibroblasts treated with transforming growth factor-beta (TGF-beta) alone or in combination with epidermal growth factor and platelet-derived growth factor, increase the production of these PGs. In this report, we describe that the structure of their galactosaminoglycan side chains is altered, albeit there is no alteration of polysaccharide length. The findings showed that iduronic acid content is reduced by 50% in decorin and biglycan, whereas 4-O-sulfation is increased 2-fold in versican. To unravel the mechanism behind these changes, the activities of chondroitin C-5 epimerase and of O-sulfotransferases in cellular fractions prepared from fibroblasts were quantitated, and transcript levels of the relevant sulfotransferases were measured by real time polymerase chain reaction (RT-PCR). The C-5 epimerase activity was reduced by 25% in TGF-beta1 treated cells and 50% in fibroblasts treated with the growth factor combination. No change in activity in dermatan 4-O sulfotransferase was observed, and only a minor decrease in dermatan 4-O-sulfotransferase-1 (D4ST-1) mRNA was observed. On the other hand, chondroitin 4-O sulfotransferase activity increased 2-fold upon TGF-beta1 treatment and 3-fold upon treatment with the growth factor combination. This is in agreement with a 2-fold up-regulation of chondroitin-4-O-sulfotransferase 1 (C4ST-1) mRNA, and no changes in chondroitin-4-O-sulfotransferase 2 (C4ST-2) mRNA. Thus, cellular activity and transcript level correlated well with the changes in the structure of the dermatan/chondroitin sulfate chains.
Asunto(s)
Condroitín/química , Citocinas/metabolismo , Dermatán Sulfato/química , Regulación de la Expresión Génica , Factor de Crecimiento Transformador beta/metabolismo , Carbohidrato Epimerasas/química , Línea Celular , Células Cultivadas , Sulfatos de Condroitina/química , Cartilla de ADN/química , ADN Complementario/metabolismo , Decorina , Disacáridos/química , Factor de Crecimiento Epidérmico/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Fibroblastos/metabolismo , Humanos , Ácido Idurónico/química , Microsomas/metabolismo , Polímeros/química , Polisacáridos/química , Proteoglicanos/química , ARN/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfotransferasas/química , Sulfotransferasas/metabolismo , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta1 , Regulación hacia ArribaRESUMEN
ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.
Asunto(s)
Proteínas de la Matriz Extracelular , Metaloendopeptidasas/química , Proteínas ADAM , Proteína ADAMTS4 , Alanina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Proteínas Portadoras/química , Cartílago Articular/metabolismo , Dominio Catalítico , Bovinos , Línea Celular Tumoral , Membrana Celular/metabolismo , Sulfatos de Condroitina/química , Clonación Molecular , ADN Complementario/metabolismo , Decorina , Electroforesis en Gel de Poliacrilamida , Epítopos/química , Fibromodulina , Eliminación de Gen , Vectores Genéticos , Ácido Glutámico/química , Humanos , Hidrólisis , Interleucina-1/metabolismo , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Procolágeno N-Endopeptidasa , Unión Proteica , Estructura Terciaria de Proteína , Proteoglicanos/química , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Porcinos , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transferrina/químicaRESUMEN
BACKGROUND: Ultraviolet A1 (340-400 nm, UVA1) phototherapy is highly effective in sclerotic lesions of systemic sclerosis (SSc). Histological evaluation of skin specimens obtained before and after UVA1 phototherapy revealed loosening of collagen bundles and the appearance of small collagen fibers. We have previously shown that UVA1 irradiation induced collagenase in vitro study by using SSc fibroblasts. The increased levels of mRNA and protein of decorin in SSc fibroblasts were reported. In this study, we focus on the lesional expression of small dermatan sulfate proteoglycan, decorin that has a role of binding to collagen and fibrillogenesis. CASE PRESENTATION: We employed immunohistochemical analysis of decorin before and after UVA1 phototherapy. The skin specimens from three patients who were effectively treated with UVA1 phototherapy were analysed. Monoclonal antibody 6B6 as the specific reactivity to decorin was used. The increased decorin was focally accumulated in the newly synthesized collagen fibers in the sclerotic lesion of SSc. After UVA1 phototherapy, decorin was decreased in upper to middle dermis, although decorin was slightly increased in papillary dermis. CONCLUSIONS: These results suggest that decreased and normalized levels of accumulated decorin may relate to the efficacy of sclerotic lesions in UVA1 phototherapy.
Asunto(s)
Proteoglicanos/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/terapia , Terapia Ultravioleta/métodos , Anciano , Decorina , Proteínas de la Matriz Extracelular , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Proteoglicanos/análisisRESUMEN
Cancer has been attributed to 3 causes: pollution, infection, and poor nutrition. Conventional treatments include surgery, chemotherapy, and radiotherapy. The author proposes that immunotherapy also be considered. Among other environmental influences, dietary deficiencies and carcinogenic viral infections must be investigated and treated wherever possible. It has been suggested that mushrooms, in particular, have a structure that is immunomodulatory because it resembles the proteoglycan structure in the human extracellular matrix, and both are metabolically active. Inasmuch as mitochondria have a bacterial origin, proteoglycans may have a mushroom origin. The author describes a study which shows that natural killer cells can double in number with 8 wk of treatment with Coriolus versicolor. Also described is an epidemiological survey of cancer deaths among Flammulina velutipes farmers in Japan, which found that the mushroom farmers had lower rates of cancer deaths than controls who were not involved in mushroom farming.