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Current cancer immunotherapeutic strategies mainly focus on remodeling the tumor microenvironment (TME) to make it favorable for antitumor immunity. Increasing attention has been paid to developing innovative immunomodulatory adjuvants that can restore weakened antitumor immunity by conferring immunogenicity to inflamed tumor tissues. Here, a galactan-enriched nanocomposite (Gal-NC) is developed from native carbohydrate structures through an optimized enzymatic transformation for effective, stable, and biosafe innate immunomodulation. Gal-NC is characterized as a carbohydrate nanoadjuvant with a macrophage-targeting feature. It is composed of repeating galactan glycopatterns derived from heteropolysaccharide structures of plant origin. The galactan repeats of Gal-NC function as multivalent pattern-recognition sites for Toll-like receptor 4 (TLR4). Functionally, Gal-NC-mediated TLR activation induces the repolarization of tumor-associated macrophages (TAMs) toward immunostimulatory/tumoricidal M1-like phenotypes. Gal-NC increases the intratumoral population of cytotoxic T cells, the main effector cells of antitumor immunity, via re-educated TAMs. These TME alterations synergistically enhance the T-cell-mediated antitumor response induced by αPD-1 administration, suggesting that Gal-NC has potential value as an adjuvant for immune checkpoint blockade combination therapies. Thus, the Gal-NC model established herein suggests a glycoengineering strategy to design a carbohydrate-based nanocomposite for advanced cancer immunotherapies.
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Neoplasias , Microambiente Tumoral , Humanos , Neoplasias/tratamento farmacológico , Imunoterapia , Imunomodulação , Macrófagos , Adjuvantes Imunológicos/farmacologiaRESUMO
Complex pectin, originating from terrestrial plant cell walls has been attracting research attention as a promising source of a new innate immune modulator. Numerous bioactive polysaccharides associated with pectin are newly reported every year, but the general mechanism of their immunological action remains unclear owing to the complexity and heterogeneity of pectin. Herein, we systematically investigated the interactions in pattern-recognition for common glycostructures of pectic heteropolysaccharides (HPSs) by Toll-like receptors (TLRs). The compositional similarity of glycosyl residues derived from pectic HPS was confirmed by conducting systematic reviews, leading to molecular modeling of representative pectic segments. Via structural investigation, the inner concavity of leucine-rich repeats of TLR4 was predicted to act as a binding motif for carbohydrate recognition, and subsequent simulations predicted the binding modes and conformations. We experimentally demonstrated that pectic HPS exhibits the non-canonical and multivalent binding aspects for TLR4 resulting in receptor activation. Furthermore, we showed that pectic HPSs were selectively clustered with TLR4 during endocytosis, inducing downstream signals to cause phenotypic activation of macrophages. Overall, we have presented a better explanation for the pattern recognition of pectic HPS and further proposed an approach to understand the interaction between complex carbohydrates and proteins.
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Pectinas , Receptor 4 Toll-Like , Conformação Molecular , Pectinas/química , Receptores Toll-Like , Animais , CamundongosRESUMO
This study aimed to investigate the anticancer activity of dried-pericarp water extract of fermented C. japonicus (CJ). The dried-pericarp water extracts of CJ were fermented using Aspergillus oryzae and Saccharomyces cerevisiae at 30 °C and 35 °C. The anticancer activities of both water extracts fermented at 30 °C and 35 °C using A. oryzae against FaDu cells were remarkably changed compared with unfermented dried-pericarp water extract of CJ, which has no anticancer activity. Cleaved-PARP, caspase 3, and apoptotic cells stained with annexin V/PI were significantly increased by treatment with A. oryzae extracts fermented at 30 °C. The insulin-like growth factor-binding protein 2 (IGFBP-2) protein level and mTOR phosphorylation by A. oryzae fermented extracts (AOFE) were dramatically reduced, and the expression levels of IGFBP-2 and phosphorylated mTOR were significantly increased depending on the glucose concentrations in FaDu cells. These results suggested that the cell viabilities in AOFE were restored as the glucose concentrations increased. Furthermore, it was confirmed LC/MS/MS that the content of gallic acid was increased by fermentation of Aspergillus oryzae (5.596 ± 0.1746 µg/mg) compared to the unfermented extract (1.620 ± 0.0432 µg/mg). Based on these results, the anticancer effect of AOFE was achieved through inhibition of the IGFBP-2/mTOR signaling pathway. These results suggest that AOFE may be a potential treatment for head and neck cancer.
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Antineoplásicos/farmacologia , Aspergillus oryzae/química , Camellia/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Extratos Vegetais/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Fermentação , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas , Água/químicaRESUMO
Bromochlorophene (BCP) has shown good properties in sterilization and antibacterial activity and is widely used as a household chemical. We evaluated the genotoxicity, single and repeated-dose 28-day oral toxicity, and dermal application of a BCP suspension in Sprague-Dawley (SD) rats. For the single-dose toxicity study, a dose of 25-1,000 mg per kg of bodyweight (mg/kg b.w.) of BCP was given once orally to SD rats. Mortality and clinical signs were observed and recorded for the first 30 min after treatment, at 4 h post-administration, and then at least once daily for 14 days after administration. For the repeated-dose 28-day toxicity study, the high dose was set at 1,000 mg/kg b.w. and the middle, middle-low, and low dose were set to 500, 250, and 125 mg/kg, respectively. Hematology and biochemistry parameters were examined. Gross pathologic and histopathologic examinations were performed on selected tissues from all animals. A bacterial reverse mutation assay, in vitro chromosomal aberration assay, and in vivo micronucleus assay were performed to assess genotoxicity-dermal application exposure assessment of BCP in rats. A high oral approximate lethal dose (ALD) of 1,000 mg/kg was observed in the single-dose toxicity test. During the repeated-dose 28-day time period, most animal deaths after administration occurred during the first 3 weeks. The 1,000 mg/kg b.w. oral dose caused the death of six male rats (6/7) and four female rats (4/7). At 500 mg/kg b.w., the female rats showed mortality (1/7). For the biochemistry assays, cholesterol was increased significantly compared to vehicle in both sexes in the 250 and 500 mg/kg groups. Histopathological changes with treatment-related findings were observed in the pancreas in female rats treated with a high dose of BCP compared with the vehicle group. BCP showed no genotoxic effect. These data suggested that the ALD of BCP, estimated as a non-genotoxic substance, was over 1,000 mg/kg b.w. in the single-dose toxicity study, and the NOAEL of BCP was considered to be 250 mg/kg b.w. for male and female rats after repeated oral administration for 28 days under the present study conditions.
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In this study, we developed a simple screening procedure for the determination of 18 anthelmintics (including benzimidazoles, macrocyclic lactones, salicylanilides, substituted phenols, tetrahydropyrimidines, and imidazothiazoles) in five animal-derived food matrices (chicken muscle, pork, beef, milk, and egg) using liquid chromatography-tandem mass spectrometry. Analytes were extracted using acetonitrile/1% acetic acid (milk and egg) and acetonitrile/1% acetic acid with 0.5 mL of distilled water (chicken muscle, pork, and beef), and purified using saturated n-hexane/acetonitrile. A reversed-phase analytical column and a mobile phase consisting of (A) 10 mM ammonium formate in distilled water and (B) methanol were used to achieve optimal chromatographic separation. Matrix-matched standard calibration curves (R 2 ≥0.9752) were obtained for concentration equivalent to ×1/2, ×1, ×2, ×3, ×4, and ×5 fold the maximum residue limit (MRL) stipulated by the Korean Ministry of Food and Drug Safety. Recoveries of 61.2-118.4%, with relative standard deviations (RSDs) of ≤19.9% (intraday and interday), were obtained for each sample at three spiking concentrations (×1/2, ×1, and ×2 the MRL values). Limits of detection, limits of quantification, and matrix effects were 0.02-5.5 µg/kg, 0.06-10 µg/kg, and -98.8 to 13.9% (at 20 µg/kg), respectively. In five samples of each food matrix (chicken muscle, pork, beef, milk, and egg) purchased from large retailers in Seoul that were tested, none of the target analytes were detected. It has therefore been shown that this protocol is adaptable, accurate, and precise for the quantification of anthelmintic residues in foods of animal origin.
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Receptor activator of NF-κB ligand (RANKL) induces generation of intracellular reactive oxygen species (ROS), which act as second messengers in RANKL-mediated osteoclastogenesis. Dual oxidase maturation factor 1 (Duoxa1) has been associated with the maturation of ROS-generating enzymes including dual oxidases (Duox1 and Duox2). In the progression of osteoclast differentiation, we identified that only Duoxa1 showed an effective change upon RANKL stimulation, but not Duox1, Duox2, and Duoxa2. Therefore, we hypothesized that Duoxa1 could independently act as a second messenger for RANKL stimulation and regulate ROS production during osteoclastogenesis. Duoxa1 gradually increased during RANKL-induced osteoclastogenesis. Using siRNA or retrovirus transduction, we found that Duoxa1 regulated RANKL-stimulated osteoclast formation and bone resorption positively. Furthermore, knockdown of Duoxa1 decreased the RANKL-induced ROS production. During Duoxa1-related control of osteoclastogenesis, activation of tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated early signaling molecules including MAPKs, Akt, IκB, Btk, Src and PLCγ2 was affected, which sequentially modified the mRNA or protein expression levels of key transcription factors in osteoclast differentiation, such as c-Fos and NFATc1, as well as mRNA expression of osteoclast-specific markers. Overall, our data indicate that Duoxa1 plays a crucial role in osteoclastogenesis via regulating RANKL-induced intracellular ROS production and activating TRAF6-mediated signaling.
Assuntos
Oxidases Duais/metabolismo , Regulação da Expressão Gênica , Osteoclastos/citologia , Osteogênese , Ligante RANK/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Diferenciação Celular , Oxidases Duais/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Osteoclastos/metabolismo , Ligante RANK/genética , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
Securinine (Sec) is a naturally derived compound separated from the roots of Securinega suffruticosa, which has long been used as a herbal medicine. Sec is widely known as a GABA receptor antagonist, it is also known as an innate immune cell agonist and has been reported to increase macrophage activity and promote monocyte maturation. On the basis of these studies, we investigated the effect of Sec on osteoclast differentiation and bone resorbing function. We have found that Sec inhibits RANKL-induced osteoclast differentiation, fusion, actin ring formation, and bone resorbing function by the inhibition of gene expression associated with each stage. Moreover, Sec significantly suppressed osteoclastogenesis by decreasing the phosphorylation of p38, Akt, JNK, IκB, and PLCγ2, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos and NFATc1. Finally, Sec effectively protected bone loss induced by the excessive inflammatory responses and activity of osteoclasts in vivo by a micro-CT and histological analysis. In conclusion, our findings suggest that Sec may be a promising drug for bone metabolic diseases such as osteoporosis, which is associated with the excessive activity of osteoclasts.
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Azepinas/uso terapêutico , Doenças Ósseas Metabólicas/tratamento farmacológico , Medicina Herbária/métodos , Compostos Heterocíclicos de Anel em Ponte/uso terapêutico , Lactonas/uso terapêutico , Osteogênese/efeitos dos fármacos , Piperidinas/uso terapêutico , Animais , Azepinas/farmacologia , Doenças Ósseas Metabólicas/patologia , Diferenciação Celular , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Humanos , Lactonas/farmacologia , Camundongos , Piperidinas/farmacologiaRESUMO
The increase of bone-resorbing osteoclast activity in bone remodeling is the major characteristic of various bone diseases. Thus, inhibiting osteoclastogenesis and bone-resorbing function may be an effective therapeutic target for bone diseases. Betulinic acid (BA), a natural plant-derived pentacyclic triterpenoid compound, is known to possess numerous pharmacological and biochemical properties including anti-inflammatory, anticancer, and antiadipogenic activity. However, the effect of BA on osteoclast differentiation and function in bone metabolism has not been demonstrated so far. In this study, we investigated whether BA could suppress RANKL-induced osteoclastogenesis and bone resorption. Interestingly, BA significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt and IκB, as well as PLCγ2-Ca2+ signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos and NFATc1. The inhibition of these pathways by BA was once more confirmed by retrovirus infection of constitutively active (CA)-Akt and CA-Ikkß retrovirus and measurement of Ca2+ influx. BA also significantly inhibited the expression of osteoclastogenesis-specific marker genes. Moreover, we found that BA administration restored the bone loss induced through acute lipopolysaccharide injection in mice by a micro-CT and histological analysis. Our findings suggest that BA is a potential therapeutic candidate for bone diseases involving osteoclasts.
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Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , NF-kappa B/antagonistas & inibidores , Osteogênese/efeitos dos fármacos , Triterpenos Pentacíclicos/farmacologia , Fosfolipase C gama/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/química , Transdução de Sinais/efeitos dos fármacos , Animais , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Osteoclastos/efeitos dos fármacos , Triterpenos Pentacíclicos/química , Fosfolipase C gama/química , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/química , Ligante RANK/metabolismo , Ácido BetulínicoRESUMO
Chronic and extensive exposure of ultraviolet (UV)-irradiation causes human skin sunburn, inflammation, or photoaging, which is associated with downregulated collagen synthesis. This study investigated the effects of fermented blackberry (Rubus fruticosus B., FBB) by Lactobacillus plantarum JBMI F5 (LP) on UVB-induced photoaging in human foreskin fibroblast (Hs68) as well as in SKH-1 hairless mice. FBB pretreatment inhibited UVB-mediated type-1 procollagen degradation, matrix metalloproteinase (MMP)-1 and MMP-2 protein expression, and suppressed nuclear factor-κB (NF-κB) activation as well as mitogen-activated protein kinase (MAPK) phosphorylation in Hs68. In addition, FBB administration diminished the wrinkle formation in dorsal skin and epidermal thickening in UVB-irradiated hairless mice. Moreover, UVB-induced Type-1 procollagen reduction and antioxidant enzyme inactivation were reversed by FBB administration. These results suggest that FBB may have antiphotoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of reactive oxygen species and related MAPK and NF-κB signaling. Therefore, FBB can be a potential candidate for protecting skin aging against UV irradiation.
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Fibroblastos/efeitos dos fármacos , Lactobacillus plantarum/metabolismo , Extratos Vegetais/farmacologia , Rubus/química , Envelhecimento da Pele/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Fermentação , Fibroblastos/efeitos da radiação , Prepúcio do Pênis/citologia , Frutas/química , Masculino , Camundongos , Camundongos Pelados , Extratos Vegetais/química , Envelhecimento da Pele/efeitos da radiaçãoRESUMO
BACKGROUND: This study aimed to analyze the association of low vitamin D status with thyroid autoimmunity and dysfunction in the Korean population according to sex and menopausal status in women. METHODS: This study was based on the data acquired from the 6th Korea National Health and Nutrition Examination Survey. We enrolled 4,356 subjects who had data of thyroid function, antithyroid peroxidase antibody (TPOAb), and serum 25-hydroxyvitamin D (25[OH]D) levels. We excluded subjects who were pregnant and who had a history of thyroid disease or thyroid cancer, and those with transient thyroid dysfunction who tested negative for TPOAb (TPOAb[-]). RESULTS: TPOAb positivity (TPOAb[+]) with thyroid dysfunction (subclinical and overt hypothyroidism) was more prevalent in the vitamin D deficient group than in the vitamin D insufficient and sufficient groups including premenopausal (P=0.046) and postmenopausal women (P=0.032), although no significant differences were observed in men. The mean serum 25(OH)D level was significantly lower in the TPOAb(+) with thyroid dysfunction group than in the TPOAb(+) with euthyroidism and TPOAb(-) groups of premenopausal women (P=0.001), although no significant differences were observed in men and postmenopausal women. Multivariate binary logistic regression analysis, adjusted for age, body mass index, and current smoking status, showed that vitamin D insufficiency and deficiency were significantly associated with TPOAb(+) with thyroid dysfunction in premenopausal women (P<0.001), although no significant associations were observed in men and postmenopausal women. CONCLUSION: Low vitamin D status was significantly associated with thyroid autoimmunity and dysfunction in the Korean population, especially in premenopausal women.
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Samul-Tang (SMT), consisting of four medicinal herbs, is a well-known herbal prescription treating hematological disorders related symptoms. Our previous study demonstrated that SMT attenuated inflammation of vascular endothelial cells. In condition of retained vascular dysfunction, vascular inflammation is initiated and results in activation of smooth muscle cells (SMCs). Activated SMCs lose control of cell cycle regulation and migrate into intima, resulting in formation of atheroma. Here, we further investigated whether SMT suppresses proliferation and migration of SMCs. SMT showed antiproliferative effects on SMCs by suppressing [3H]-thymidine incorporation against TNF-α stimulation. Underlying mechanisms of antiproliferative effects were found to be resulting from cell cycle regulation. SMT downregulated expression of cyclin D1-CDK4 and cyclin E-CDK2 complexes and upregulated p21waf1/cip1 and p27kip1. SMT also suppressed migration of SMCs against TNF-α stimulation. This is thought to have resulted from suppressing MMP2 and MMP9 expressions and ROS production. In summary, SMT attenuates abnormal migration of vascular smooth muscle cells via regulating cell cycle and suppressing MMPs expression and ROS production. Our study suggests that SMT, a traditionally used herbal formula, protects vascular smooth muscle cells and might be used as an antiatherosclerotic drug.
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[This corrects the article on p. 105 in vol. 33, PMID: 28747975.].
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BACKGROUND: Ligustilide is a bioactive phthalide derivative isolated from Cnidii Rhizoma (Cnidium officinale, rhizome) and Angelicae Gigantis Radix (Angelica gigas Nakai, root) which are both medicinal herbs used to treat circulatory disorders. Vascular endothelium is a central spot in developing cardiovascular diseases and chronic vascular inflammation might result in atherosclerosis development. PURPOSE: We previously found out that a traditional herbal formula, Samul-Tang (Si-Wu-Tang, containing Cnidii Rhizoma and Angelicae Gigantis Radix), attenuated vascular inflammation in human umbilical vein endothelial cells (HUVECs). However, which compound was responsible for vascular protective action remained unclear. Here, we investigated vascular protective potential of an isolated single compound, (Z)-ligustilide. METHODS: MTT assay, western blotting, immunofluorescence, electrophoretic mobility shift assay was performed. BCECF-AM, CM-H2DCFDA, DAF-FM diacetate were used as a fluorescent indicator. RESULTS: Ligustilide suppressed HL-60 monocyte adhesion and CAMs (ICAM-1, VCAM-1, E-selectin) expression in HUVECs. Ligustilide significantly inhibited TNF-α-increased production of ROS and activated NF-κB signaling pathway. Also, ligustilide treated HUVECs exhibited significant HO-1 induction via Nrf2 nuclear translocation and endothelial NO synthesis. CONCLUSION: Present study demonstrates that ligustilde attenuates vascular inflammation and activate defense system of endothelial cell. Ligustilide is a bioactive compound which might prevent cardiovascular complications such as thrombosis or atherosclerosis.
Assuntos
4-Butirolactona/análogos & derivados , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Vasculite/tratamento farmacológico , 4-Butirolactona/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Selectina E/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Células HL-60 , Células Endoteliais da Veia Umbilical Humana , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Vasculite/metabolismoRESUMO
Brassinin, a phytoalexin firstly identified as a constituent of Chinese cabbage, has been demonstrated to exhibit antiproliferative effects on various cancer cell lines, by reducing reactive oxygen species (ROS) production via regulation of the antioxidant pathway. The present study aimed to explore the protective effects of brassinin in TNFαinduced vascular inflammation in human umbilical vein endothelial cells (HUVECs). Pretreatment with brassinin significantly inhibited adhesion of U937 cells to TNFαinduced HUVECs in a dosedependent manner. Brassinin treatment decreased the expression levels of cell adhesion molecules, including intracellular adhesion molecule1 (ICAM1), vascular cell adhesion molecule1 (VCAM1), and endothelialselectin (Eselectin) following stimulation with TNFα in HUVECs. In addition, pretreatment with brassinin decreased the protein expression levels of nuclear factor (NF)κB p65 in the nucleus, suggesting that brassinin inhibited NFκB p65 nuclear translocation. Brassinin treatment also markedly decreased the mRNA expression levels of interleukin8 in a dosedependent manner. Finally, brassinin pretreatment significantly decreased TNFαinduced intracellular reactive oxygen species (ROS) production in HUVECs compared with control. The present results therefore suggest that brassinin may serve as a potential therapeutic agent for atherosclerosis.
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Indóis/farmacologia , Inflamação/prevenção & controle , Tiocarbamatos/farmacologia , Fator de Necrose Tumoral alfa/toxicidade , Adesão Celular/efeitos dos fármacos , Selectina E/análise , Selectina E/genética , Selectina E/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana , Humanos , Indóis/química , Molécula 1 de Adesão Intercelular/análise , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-8/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiocarbamatos/química , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Células U937 , Molécula 1 de Adesão de Célula Vascular/análise , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Ginsenosides from Panax ginseng are well known for their diverse pharmacological effects including antithrombotic activity. Since adventitious roots of mountain ginseng (ARMG) also contain various ginsenosides, blood flow-improving effects of the dried powder and extract of ARMG were investigated. Rats were orally administered with dried powder (PARMG) or ethanol extract (EARMG) of ARMG (125, 250 or 500 mg/kg) or aspirin (30 mg/kg, a reference control) for 3 weeks. Forty min after the final administration, carotid arterial thrombosis was induced by applying a 70% FeCl3-soaked filter paper outside the arterial wall for 5 min, and the blood flow was monitored with a laser Doppler probe. Both PARMG and EARMG delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at high doses. In mechanism studies, a high concentration of EARMG inhibited platelet aggregation induced by collagen in vitro. In addition, EARMG improved the blood lipid profiles, decreasing triglyceride and cholesterol levels. Although additional action mechanisms remain to be clarified, it is suggested that ARMG containing high amount of ginsenosides such as Rg3 improves blood flow not only by inhibiting oxidative thrombosis, but also by modifying blood lipid profiles.
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OBJECTIVE: This study aimed to evaluate whether Hwangryunhaedoktang (HHT), a herbal compound, has an inhibitory effect on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages. METHODS: The effects of HHT were evaluated by confirming nitric oxide (NO) production and expression of inducible NO synthase (iNOS) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated RAW264.7 macrophages via the Griess assay, Western blotting, and real-time reverse transcription quantitative polymerase chain reaction. Western blot analyses and luciferase assays were used to evaluate whether HHT has an effect on the phosphorylation and translocation of nuclear factor-κB (NF-κB). The secretion and expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined via enzyme-linked immunosorbent assay and Western blot analyses. RESULTS: HHT suppressed LPS-induced NO production and expression of iNOS in a dose-dependent manner. Additionally, MAPKs activation was also attenuated via inhibition of phosphorylation of extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinase and p38 which were related to inflammatory pathway. Furthermore, HHT also effectively attenuated NF-κB activation and its translocation to the nucleus, a process that is closely linked to inflammation. LPS normally induced the expression of inflammatory cytokines such as TNF-α and IL-6, but the secretion and expression of TNF-α and IL-6 were significantly attenuated by pretreating the cells with HHT. CONCLUSION: HHT suppressed LPS-induced NO production by blocking the activation of NF-κB and MAPK signaling pathways in RAW264.7 macrophages. Furthermore, HHT may have an anti-inflammatory effect by suppressing the LPS-induced secretion of TNF-α and IL-6. Therefore, the traditional herbal formula HHT might be a useful potential therapeutic agent for inflammation.
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Anti-Inflamatórios/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Óxido Nítrico/biossíntese , Extratos Vegetais/farmacologia , Animais , Interleucina-6/metabolismo , Camundongos , NF-kappa B/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Chong-Myung-Tang (CMT) extract is widely used in Korea as a traditional herbal tonic for increasing memory capacity in high-school students and also for numerous body ailments since centuries. The use of CMT to improve the learning capacity has been attributed to various plant constituents, especially black ginseng, in it. Therefore, in this study, we have first investigated whether black ginseng-enriched CMT extracts affected spatial learning using the Morris water maze (MWM) test. Their molecular mechanism of action underlying improvement of learning and memory was examined in vitro. METHODS: We used two types of black ginseng-enriched CMT extracts, designated as CM-1 and CM-2, and evaluated their efficacy in the MWM test for spatial learning behavior and their anti-inflammatory effects in BV2 microglial cells. RESULTS: Our results show that both black ginseng-enriched CMT extracts improved the learning behavior in scopolamine-induced impairment in the water maze test. Moreover, these extracts also inhibited nitric oxide production in BV2 cells, with significant suppression of expression of proinflammatory cytokines, especially inducible nitric oxide synthase, cyclooxygenase-2, and interleukin-1ß. The protein expression of mitogen-activated protein kinase and nuclear factor-κB pathway factors was also diminished by black ginseng-enriched CMT extracts, indicating that it not only improves the memory impairment, but also acts a potent anti-inflammatory agent for neuroinflammatory diseases. CONCLUSION: Our research for the first time provides the scientific evidence that consumption of black ginseng-enriched CMT extract as a brain tonic improves memory impairment. Thus, our study results can be taken as a reference for future neurobehavioral studies.
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BACKGROUND: Korean ginseng, Panax ginseng Meyer, has been used as a traditional oriental medicine to treat illness and promote health for several thousand years. Ginsenosides are the main constituents for the pharmacological effects of P. ginseng. Since several ginsenosides, including ginsenoside (G)-Rg3 and G-Rp1, have reported antiplatelet activity, here we investigate the ability of G-Rp4 to modulate adenosine diphosphate (ADP)-induced platelet aggregation. The ginsenoside Rp4, a similar chemical structure of G-Rp1, was prepared from G-Rg1 by chemical modification. METHODS: To examine the effects of G-Rp4 on platelet activation, we performed several experiments, including antiplatelet ability, the modulation of intracellular calcium concentration, and P-selectin expression. In addition, we examined the activation of integrin αIIbß3 and the phosphorylation of signaling molecules using fibrinogen binding assay and immunoblotting in rat washed platelets. RESULTS: G-Rp4 inhibited ADP-induced platelet aggregation in a dose-dependent manner. We found that G-Rp4 decreased calcium mobilization and P-selectin expression in ADP-activated platelets. Moreover, fibrinogen binding to integrin αIIbß3 by ADP was attenuated in G-Rp4-treated platelets. G-Rp4 significantly attenuated phosphorylation of extracellular signal-regulated protein kinases 1 and 2, p38, and c-Jun N-terminal kinase, as well as protein kinase B, phosphatidylinositol 3-kinase, and phospholipase C-γ phosphorylations. CONCLUSION: G-Rp4 significantly inhibited ADP-induced platelet aggregation and this is mediated via modulating the intracellular signaling molecules. These results indicate that G-Rp4 could be a potential candidate as a therapeutic agent against platelet-related cardiovascular diseases.
RESUMO
15 K is 1,2, 3-triazolyl ester of ketorolac, an old pain-killer, that blocks PAK1 by its R-form and inhibits COX-2 by its S-form. Mainly due to a robust increase in cell-permeability, 15K is over 500 times more potent than ketorolac in both anti-cancer and anti-PAK1 activities in cell culture with IC50 around 24 nM. However, 15K has no anti-AKT activity. Angiogenesis requires at least the kinase PAK1, and perhaps the kinase AKT as well, and is essential for a robust growth of solid tumors. Thus, in this study, we examined the potential antiangiogenic activity of 15K both in ovo and cell culture, prior to its in vivo (xenograft) anti-cancer activity test. The IC50 of 15K against the embryonic angiogenesis in ovo in CAM (chorioallantoic membrane) assay is around 1 nmol/egg. Surprizingly, however, 15K failed to inhibit the tube formation of HUVECs (human umbilical vein endothelial cells) in cell culture even at high as 150 µM. In an attempt to solve this mystery, we tested both in ovo as well as HUVECs-based anti-angiogenic activity of a potent survivin-suppressor called YM155, which blocks PAK1, in addition to AKT. YM155 is slightly more potent than 15K in CAM assay with IC50 around 0.5 nmol/egg, and apparenty inhibits the tube formation of HUVECs with IC50 around 18 nM. According to a few previous findings with the direct PAK1-inhibitor frondoside A (FRA), the tube formation of HUVECs depends solely on PAK1. Thus, the failure of 15K to affect their tube formation is most likely due to their drug (15K)-resistance. Furthermore, unlike FRA, YM155 killed HUVECs with IC50 around 18 nM, clearly indicating that AKT is essential for survival of HUVECs, instead of their tube formation.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Imidazóis/farmacologia , Cetorolaco/farmacologia , Naftoquinonas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Survivina/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Quinases Ativadas por p21/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Ésteres , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Concentração Inibidora 50 , Survivina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Zigoto , Quinases Ativadas por p21/metabolismoRESUMO
Crowberry, Empetrum nigrum var. japonicum, is widely used in folk medicine and grows naturally in Korea. Although some constituents and biological activity of Korean crowberry have been examined, there is little detailed information available. In this study, we investigated the effects of ethanol extracts of crowberry (EECB) on the inhibition of angiogenesis, both in vitro and in vivo. The effects of EECB were tested on in vitro models of angiogenesis, that is, tube formation and proliferation of human umbilical vein endothelial cells (HUVECs). EECB exhibited significant inhibitory effects on tube formation of HUVECs in a concentration-dependent manner. In addition, crowberry significantly suppressed the proliferation of HUVECs in a concentration-dependent manner. Furthermore, strong antiangiogenic activity of EECB samples was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). These results indicate that crowberry may have potential applications in the prevention and treatment of angiogenesis-dependent human diseases.