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BACKGROUND: A species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, we prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo, in vivo, and in vitro antiplatelet and antithrombotic effects. METHODS: Ex vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation, serotonin release, thromboxane A2 (TXA2) production and its associated activities of enzymes [cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS)], arachidonic acid (AA) release and its associated phosphorylation of phospholipase Cß3, phospholipase Cγ2 or cytosolic phospholipase A2, mitogen-activated protein kinase (MAPK) [p38 MAPK, extracellular signal-regulated kinase (ERK)], and blood coagulation time in rats were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. In vitro effects of WIB-801CE on cytotoxicity, and fibrin clot retraction in human platelets, and nitric oxide (NO) production in RAW264.7 cells or free radical scavenging activity were studied. RESULTS: Cordycepin-enriched WIB-801CE inhibited ex vivo platelet aggregation, TXA2 production, AA release, TXAS activity, serotonin release, and p38 MAPK and ERK2 phosphorylation in collagen- and ADP-activated rat platelets without affecting blood coagulation. Furthermore, WIB-801CE manifested in vivo inhibitory effect on collagen plus epinephrine-induced pulmonary thromboembolism mice model. WIB-801CE inhibited in vitro NO production and fibrin clot retraction, but elevated free radical scavenging activity without affecting cytotoxicity against human platelets. CONCLUSION: WIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. These were resulted from down-regulation of TXA2 production and its related AA release and TXAS activity, and p38MAPK and ERK2 activation. These results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.
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Cordyceps/química , Fibrinolíticos/farmacología , Extractos Vegetales/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Animales , Ácido Araquidónico/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Calcio/metabolismo , Evaluación Preclínica de Medicamentos , Masculino , Ratones Endogámicos ICR , Óxido Nítrico/metabolismo , Fosforilación , Ratas Sprague-Dawley , Serotonina/metabolismo , Tromboxano A2/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Excessive and persistent insults during endoplasmic reticulum (ER) stress lead to apoptotic cell death that is implicated in a range of chronic inflammatory diseases and cancers. Macrophage inhibitory cytokine 1 (MIC-1), a member of the transforming growth factor-ß superfamily, is diversely linked to the pathogenesis of cancer. To investigate the precise molecular mechanisms of MIC-1 gene regulation, ER stress and its related signals were studied in human colon cancer cells. Functionally, MIC-1 played pivotal roles in ER stress-linked apoptotic death, which was also influenced by C/EBP homologous protein, a well known apoptotic mediator of ER stress. ER stress enhanced MIC-1 mRNA stability instead of transcriptional activation, and there were two mechanistic translocations critical for mRNA stabilization. First, C/EBP homologous protein triggered protein kinase C-linked cytosolic translocation of the HuR/ELAVL1 (Elav-like RNA-binding protein 1) RNA-binding protein, which bound to and stabilized MIC-1 transcript. As the second critical in-and-out regulation, ER stress-activated ERK1/2 signals contributed to enhanced stabilization of MIC-1 transcript by controlling the extended holding of the nucleated mRNA in the stress granules fusing with the mRNA-decaying processing body. We propose that these two sequential in-and-out modulations can account for stabilized transcription and subsequent translation of pro-apoptotic MIC-1 gene in human cancer cells under ER stress.
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Apoptosis/fisiología , Estrés del Retículo Endoplásmico/fisiología , Regulación de la Expresión Génica/fisiología , Factor 15 de Diferenciación de Crecimiento/biosíntesis , Estabilidad del ARN/fisiología , ARN Mensajero/biosíntesis , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Línea Celular Tumoral , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Proteína 1 Similar a ELAV , Factor 15 de Diferenciación de Crecimiento/genética , Humanos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transporte de Proteínas/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Surface engineered iron oxide nanoparticles (IONPs) with catecholic ligands have been investigated as alternative T1 contrast agents. However, complex oxidative chemistry of catechol during IONP ligand exchange causes surface etching, heterogeneous hydrodynamic size distribution, and low colloidal stability because of Fe3+ mediated ligand oxidation. Herein, we report highly stable and compact (â¼10 nm) Fe3+ rich ultrasmall IONPs functionalized with a multidentate catechol-based polyethylene glycol polymer ligand through amine-assisted catecholic nanocoating. The IONPs exhibit excellent stability over a broad range of pHs and low nonspecific binding in vitro. We also demonstrate that the resultant NPs have a long circulation time (â¼80 min), enabling high resolution T1 magnetic resonance angiography in vivo. These results suggest that the amine assisted catechol-based nanocoating opens a new potential of metal oxide NPs to take a step forward in exquisite bio-application fields.
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Lateral flow assays (LFA) enable development of portable and rapid diagnostic kits; however, their capacity to detect low levels of disease markers remains poor. Here, we report a highly sensitive pregnancy test kit as a proof of concept, by combining brush-type ligand-coated quantum beads (B-type QBs) and nanobody, which can control the antibody orientation and enhance sensitivity. The brush-type ligand provided excellent dispersion stability and high-binding capacity toward antibody. Fc-binding nanobody increased the antigen-binding capacity of conjugated antibodies on the B-type QBs. To facilitate convenient acquisition of the LFA results, we developed a smartphone-based reader with a 3D-printed optical imaging module, and validated the diagnostic performance of the sensing platform. The pregnancy test kit achieved a 5.1 pg mL-1 limit of detection, corresponding to the levels for early-stage detection of heart disease and malaria. Our LFA application can potentially be expanded to diagnosis other diseases by simply changing the antibody pair in the kit.
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Técnicas Biosensibles , Pruebas de Embarazo , Anticuerpos , Técnicas Biosensibles/métodos , Femenino , Humanos , Ligandos , EmbarazoRESUMEN
The surface charge of iron oxide nanoparticles (IONPs) plays a critical role in the interactions between nanoparticles and biological components, which significantly affects their toxicity in vitro and in vivo. In this study, we synthesized three differently charged IONPs (negative, neutral, and positive) based on catechol-derived dopamine, polyethylene glycol, carboxylic acid, and amine groups, via reversible addition-fragmentation chain transfer-mediated polymerization (RAFT polymerization) and ligand exchange. The zeta potentials of the negative, neutral, and positive IONPs were -39, -0.6, and +32 mV, respectively, and all three IONPs showed long-term colloidal stability for three months in an aqueous solution without agglomeration. The cytotoxicity of the IONPs was studied by analyzing cell viability and morphological alteration in three human cell lines, A549, Huh-7, and SH-SY5Y. Neither IONP caused significant cellular damage in any of the three cell lines. Furthermore, the IONPs showed no acute toxicity in BALB/c mice, in hematological and histological analyses. These results indicate that our charged IONPs, having high colloidal stability and biocompatibility, are viable for bio-applications.
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Silica aerogels have attracted much attention owing to their excellent thermal insulation properties. However, the conventional synthesis of silica aerogels involves the use of expensive and toxic alkoxide precursors and surface modifiers such as trimethylchlorosilane. In this study, cost-effective water-glass silica aerogels were synthesized using an eco-friendly catechol derivative surface modifier instead of trimethylchlorosilane. Polydopamine was introduced to increase adhesion to the SiO2 surface. The addition of 4-tert-butyl catechol and hexylamine imparted hydrophobicity to the surface and suppressed the polymerization of the polydopamine. After an ambient pressure drying process, catechol-modified aerogel exhibited a specific surface area of 377 m2/g and an average pore diameter of approximately 21 nm. To investigate their thermal conductivities, glass wool sheets were impregnated with catechol-modified aerogel. The thermal conductivity was 40.4 mWm-1K-1, which is lower than that of xerogel at 48.7 mWm-1K-1. Thus, by precisely controlling the catechol coating in the mesoporous framework, an eco-friendly synthetic method for aerogel preparation is proposed.
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Chemical characterizations of biochemically functionalized single nanoparticles are necessary to optimize the nanoparticle surface functionality in recently advanced nanobiological applications but have not yet been fully explored because of technical difficulties. Exploiting the photoinduced force exerted on a light-illuminated nanoscale tip, nanoscale mid-infrared hyperspectral images with a 10 nm spatial resolution of a monolayer ligand-functionalized single gold nanoparticle under ambient and environmental conditions are presented. We extend our study to the diagnosis of nanoscale heterogeneous chemical contaminants which come from a particle functionalization process but are undetectable in conventional ensemble-averaged imaging technique. High sensitivity and high spatial resolution are achieved via the strongly localized tip-enhanced force at the junction between the gold-coated tip and the functionalized nanoparticle in photoinduced force microscopy, which far exceeds the capability of the conventional methods. The present study paves a new way to directly detect heterogeneous nanochemicals at the single-component level, which is necessary to evaluate nanomaterial safety in biomedical applications.
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Nanopartículas de Magnetita/química , Oro/química , Ligandos , Microscopía de Fuerza Atómica/métodos , Polietilenglicoles/química , Propiedades de SuperficieRESUMEN
In this article, the facile synthesis of sea urchin-shaped LiFePO4 nanoparticles by thermal decomposition of metal-surfactant complexes and application of these nanoparticles as a cathode in lithium ion secondary batteries is demonstrated. The advantages of this work are a facile method to synthesize interesting LiFePO4 nanostructures and its synthetic mechanism. Accordingly, the morphology of LiFePO4 particles could be regulated by the injection of oleylamine, with other surfactants and phosphoric acid. This injection step was critical to tailor the morphology of LiFePO4 particles, converting them from nanosphere shapes to diverse types of urchin-shaped nanoparticles. Electron microscopy analysis showed that the overall dimension of the urchin-shaped LiFePO4 particles varied from 300 nm to 2 µm. A closer observation revealed that numerous thin nanorods ranging from 5 to 20 nm in diameter were attached to the nanoparticles. The hierarchical nanostructure of these urchin-shaped LiFePO4 particles mitigated the low tap density problem. In addition, the nanorods less than 20 nm attached to the edge of urchin-shaped nanoparticles significantly increased the pathways for electronic transport.
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Conversion reaction materials (transition metal oxides, sulfides, phosphides, etc.) are attractive in the field of lithium-ion batteries because of their high theoretical capacity and low cost. However, the realization of these materials in lithium-ion batteries is impeded by large voltage hysteresis, high polarization, inferior cycle stability, rate capability, irreversible capacity loss in first cycling, and dramatic volume change during redox reactions. One method to overcome these problems is the introduction of amorphous materials. This work introduces a facile method to synthesize amorphous and crystalline dinickel phosphide (Ni2P) nanoparticle clusters with identical morphology and presents a direct comparison of the two materials as anode materials for rechargeable lithium-ion batteries. To assess the effect of crystallinity and hierarchical structure of nanomaterials, it is crucial to conserve other factors including size, morphology, and ligand of nanoparticles. Although it is rarely studied about synthetic methods of well-controlled Ni2P nanomaterials to meet the above criteria, we synthesized amorphous, crystalline Ni2P, and self-assembled Ni2P nanoparticle clusters via thermal decomposition of nickel-surfactant complex. Interestingly, simple modulation of the quantity of nickel acetylacetonate produced amorphous, crystalline, and self-assembled Ni2P nanoparticles. A 0.357 M nickel-trioctylphosphine (TOP) solution leads to a reaction temperature limitation (â¼315 °C) by the nickel precursor, and crystalline Ni2P (c-Ni2P) nanoparticles clusters are generated. On the contrary, a lower concentration (0.1 M) does not accompany a temperature limitation and hence high reaction temperature (330 °C) can be exploited for the self-assembly of Ni2P (s-Ni2P) nanoparticle clusters. Amorphous Ni2P (a-Ni2P) nanoparticle clusters are generated with a high concentration (0.714 M) of nickel-TOP solution and a temperature limitation (â¼290 °C). The a-Ni2P nanoparticle cluster electrode exhibits higher capacities and Coulombic efficiency than the electrode based on c-Ni2P nanoparticle clusters. In addition, the amorphous structure of Ni2P can reduce irreversible capacity and voltage hysteresis upon cycling. The amorphous morphology of Ni2P also improves the rate capability, resulting in superior performance to those of c-Ni2P nanoparticle clusters in terms of electrode performance.
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While incorporation of nanoparticles in a polymer matrix generally enhances the physical properties, effective control of the nanoparticle/polymer interface is often challenging. Here, we report a dramatic enhancement of the mechanical properties of polymer nanocomposites (PNCs) using a simple physical grafting method. The PNC consists of low molecular weight poly(ethylene glycol) (PEG) and silica nanoparticles whose surfaces are modified with dopamine-modified PEG (DOPA-mPEG) brush polymers. With DOPA-mPEG grafting, the nanoparticle surface can be readily altered, and the shear modulus of the PNC is increased by a factor of 105 at an appropriate surface grafting density. The detailed microstructure and mechanical properties are examined with small-angle X-ray scattering (SAXS) and oscillatory rheometry experiments. The attractive interactions between particles induced by DOPA-mPEG grafting dramatically improve the mechanical properties of PNCs even in an unentangled polymer matrix, which shows a much higher shear modulus than that of a highly entangled polymer matrix.
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In this study, we investigated the effects of cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha on collagen-stimulated platelet aggregation. CE-WIB801C dose dependently inhibited collagen-induced platelet aggregation, and had a synergistic effect together with cordycepin (W-cordycepin) from CE-WIB801C on the inhibition of collagen-induced platelet aggregation. CE-WIB801C and cordycepin stimulated the phosphorylation of VASP (Ser(157)) and the dephosphorylation of PI3K and Akt, and inhibited the binding of fibrinogen to glycoprotein IIb/IIIa (αIIb/ß3) and the release of ATP and serotonin in collagen-induced platelet aggregation. A-kinase inhibitor Rp-8-Br-cAMPS reduced CE-WIB801C-, and cordycepin-increased VASP (Ser(157)) phosphorylation, and increased CE-WIB801C-, and cordycepin-inhibited the fibrinogen binding to αIIb/ß3. Therefore, we demonstrate that CE-WIB801C-, and cordycepin-inhibited fibrinogen binding to αIIb/ß3 are due to stimulation of cAMP-dependent phosphorylation of VASP (Ser(157)), and inhibition of PI3K/Akt phosphorylation. These results strongly indicate that CE-WIB801C and cordycepin may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.
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In this study, we investigated the effect of cordycepin-enriched (CE)-WIB801C from Cordyceps militaris on ADP (20 µM)-stimulated platelet aggregation. CE-WIB801C dose-dependently inhibited ADP-induced platelet aggregation, and its IC50 value was 18.5 µg/mL. CE-WIB801C decreased TXA2 production, but did not inhibit the activities of COX-1 and thromboxane synthase (TXAS) in ADP-activated platelets, which suggests that the inhibition of TXA2 production by CE-WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. CE-WIB801C inhibited ATP release and [Ca(2+)]i mobilization, and increased cAMP level and IP3RI (Ser(1756)) phosphorylation in ADP-activated platelets. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased CE-WIB801C-inhibited [Ca(2+)]i mobilization, and strongly inhibited CE-WIB801C-increased IP3RI (Ser(1756)) phosphorylation. CE-WIB801C elevated the phosphorylation of VASP (Ser(157)), an A-kinase substrate, but inhibited fibrinogen binding to αIIb/ß3. These results suggest that CE-WIB801C-elevated cAMP involved in IP3RI (Ser(1756)) phosphorylation to inhibit [Ca(2+)]i mobilization and, VASP (Ser(157)) phosphorylation to inhibit αIIb/ß3 activation. Therefore, in this study, we demonstrate that CE-WIB801C may have a preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.
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Adenosina Difosfato/farmacología , Señalización del Calcio/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Desoxiadenosinas/farmacología , Fibrinógeno/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Extractos Vegetales/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenosina Trifosfato/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Calcio/metabolismo , Cordyceps/química , AMP Cíclico/metabolismo , Ciclooxigenasa 1/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones de Hierba-Droga , Humanos , Fosforilación/efectos de los fármacos , Extractos Vegetales/química , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Tionucleótidos/farmacología , Tromboxano A2/metabolismo , Tromboxano-A Sintasa/metabolismoRESUMEN
AIM: In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca(2ï¼)-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A2 (TXA2)-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 µg/mL)-stimulated platelet aggregation. METHODS: Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37â in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 µg/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca(2ï¼) ([Ca(2ï¼)]i), TXA2, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation. RESULTS: CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA2, an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca(2ï¼)]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca(2ï¼)-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca(2ï¼) release from internal stores (i.e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca(2ï¼)]i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca(2ï¼)]i mobilization and TXA2 production via the inhibition of the COX-1 activity. CONCLUSIONS: These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.
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Plaquetas/efectos de los fármacos , Ácidos Cafeicos/farmacología , Calcio/metabolismo , AMP Cíclico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Animales , Plaquetas/metabolismo , Western Blotting , Moléculas de Adhesión Celular/metabolismo , Ciclooxigenasa 1/metabolismo , Electrofisiología , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tromboxano A2/metabolismoRESUMEN
In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its IC50 value was 175 µg/ml. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated [Ca(2+)]i mobilization and thromboxane A2 (TXA2) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated [Ca(2+)]i level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor (IP3R) phosphorylation. These results suggest that the inhibition of [Ca(2+)]i mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CE-WIB801C suppressed TXA2 production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS). These results suggest that the inhibition of TXA2 production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent Ca(2+)-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.
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In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea catechins from green tea leaves, on activities of cyclooxygenase (COX)-1 and thromboxane synthase (TXAS), thromboxane A2 (TXA2) production associated microsomal enzymes. EGCG inhibited COX-1 activity to 96.9%, and TXAS activity to 20% in platelet microsomal fraction having cytochrome c reductase (an endoplasmic reticulum marker enzyme) activity and expressing COX-1 (70 kDa) and TXAS (58 kDa) proteins. The inhibitory ratio of COX-1 to TXAS by EGCG was 4.8. These results mean that EGCG has a stronger selectivity in COX-1 inhibition than TXAS inhibition. In special, a nonsteroid anti-inflammatory drug aspirin, a COX-1 inhibitor, inhibited COX-1 activity by 11.3% at the same concentration (50 µM) as EGCG that inhibited COX-1 activity to 96.9% as compared with that of control. This suggests that EGCG has a stronger effect than that of aspirin on inhibition of COX-1 activity. Accordingly, we demonstrate that EGCG might be used as a crucial tool for a strong negative regulator of COX-1/TXA2 signaling pathway to inhibit thrombotic disease-associated platelet aggregation.
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In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with IC50 value of about 81.1 µg/mL. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free Ca(2+) ([Ca(2+)]i), one of aggregation-inducing molecules. Of two Ca(2+)-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of [Ca(2+)]i elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMPdependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-Ser(157) phosphorylation through A-kinase activation to inhibit [Ca(2+)]i mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.
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NF-κB expression and activity are strictly regulated in gut epithelia to prevent overstimulation of pro-inflammatory responses following exposure to commensal bacteria. The effects of epithelial EGR-1 on responses to bacterial NF-κB-activating lipopolysaccharide (LPS) in intestinal epithelial cells under ribosomal stress were assessed. This was done to determine the potential of EGR-1 as a modulator of epithelial NF-κB signaling. Nuclear translocation of phosphorylated p65 protein was observed in the cells exposed to LPS although chemokine expression was marginally affected. In contrast, simultaneous exposure to LPS and ribosomal insults prevented epithelial NF-κB activation while chemokine expression was enhanced. The effect of EGR-1, another pro-inflammatory signaling mediator, was monitored to determine the involvement of this factor on chemokine production in response to this co-treatment. Similar to the previously reported ribosomal stress response, EGR-1 expression was elevated by ribosomal insults alone and positively affected gene expression of pro-inflammatory chemokines in the intestinal epithelial cells. However, EGR-1 suppression led to super-induction of chemokines by simultaneous treatment with LPS and ribosomal insult, indicating that EGR-1 is a negative modulator of chemokine gene expression. Particularly, mucosal ribosomal insult-triggered EGR-1 mediated PPARγ induction, which counteracted NF-κB activation by LPS. It can be thus concluded that EGR-1 regulates pro-inflammatory NF-κB activation by LPS via PPARγ although EGR-1 is a positive mediator of chemokine expression following ribosomal insult in intestinal epithelial cells.
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Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Enterocitos/metabolismo , Ribosomas/metabolismo , Línea Celular , Quimiocinas/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Enterocitos/inmunología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Transducción de Señal , Estrés Fisiológico , Regulación hacia ArribaRESUMEN
AIM: In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation. METHODS: Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) ([Ca(2+)](i)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation. RESULTS: EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca(2+)](i) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit [Ca(2+)](i) mobilization and TXA(2) production on collagen-induced platelet aggregation. CONCLUSIONS: These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.