RESUMEN
In individuals with Alzheimer's disease, the brain exhibits elevated levels of IL-1ß and oxygenated cholesterol molecules (oxysterols). This study aimed to investigate the effects of side-chain oxysterols on IL-1ß expression using HMC3 microglial cells and ApoE-deficient mice. Treatment of HMC3 cells with 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol) led to increased IL-1ß expression at the transcript and protein levels. Additionally, these oxysterols upregulated the surface expression of MHC II, a marker of activated microglia. Immunohistochemistry performed on the mice showed increased microglial expression of IL-1ß and MHC II when fed a high-cholesterol diet. However, cholesterol and 24s-hydroxycholesterol did not increase IL-1ß transcript levels or MHC II expression. The extent of IL-1ß increase induced by 25OHChol and 27OHChol was comparable to that caused by oligomeric ß-amyloid, and the IL-1ß expression induced by the oxysterols was not impaired by polymyxin B, which inhibited lipopolysaccharide-induced IL-1ß expression. Both oxysterols enhanced the phosphorylation of Akt, ERK, and Src, and inhibition of these kinase pathways with pharmacological inhibitors suppressed the expression of IL-1ß and MHC II. The pharmacological agents chlorpromazine and cyclosporin A also impaired the oxysterol-induced expression of IL-1ß and upregulation of MHC II. Overall, these findings suggest that dysregulated cholesterol metabolism leading to elevated levels of side-chain oxysterols, such as 25OHChol and 27OHChol, can activate microglia to secrete IL-1ß through a mechanism amenable to pharmacologic intervention. The activation of microglia and subsequent neuroinflammation elicited by the immune oxysterols can contribute to the development of neurodegenerative diseases.
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Microglía , Oxiesteroles , Animales , Ratones , Microglía/metabolismo , Oxiesteroles/metabolismo , Enfermedades Neuroinflamatorias , Macrófagos/metabolismo , Encéfalo/metabolismoRESUMEN
Tg2576 transgenic mice for Alzheimer's disease (AD) exhibited significant phenotypes for neuropathological constipation, but no research has been conducted on the association of the fecal microbiota with dysbiosis. The correlation between fecal microbiota composition and neuropathological constipation in Tg2576 mice was investigated by examining the profile of fecal microbiota and fecal microbiota transplantation (FMT) in 9-10-month-old Tg2576 mice with the AD phenotypes and constipation. Several constipation phenotypes, including stool parameters, colon length, and histopathological structures, were observed prominently in Tg2576 mice compared to the wild-type (WT) mice. The fecal microbiota of Tg2576 mice showed decreases in Bacteroidetes and increases in the Firmicutes and Proteobacteria populations at the phylum level. The FMT study showed that stool parameters, including weight, water content, and morphology, decreased remarkably in the FMT group transplanted with a fecal suspension of Tg2576 mice (TgFMT) compared to the FMT group transplanted with a fecal suspension of WT mice (WFMT). The distribution of myenteric neurons and the interstitial cells of Cajal (ICC), as well as the enteric nervous system (ENS) function, remained lower in the TgFMT group. These results suggest that the neuropathological constipation phenotypes of Tg2576 mice may be tightly linked to the dysbiosis of the fecal microbiota.
Asunto(s)
Enfermedad de Alzheimer , Animales , Ratones , Disbiosis/microbiología , Trasplante de Microbiota Fecal/métodos , Heces/microbiología , Estreñimiento/terapia , Ratones TransgénicosRESUMEN
The recently discovered interleukin (IL)- 32 isoform IL-32θ exerts anti-metastatic effects in the breast tumor microenvironment. However, the involvement of IL-32θ in breast cancer cell proliferation is not yet fully understood; therefore, the current study aimed to determine how IL-32θ affects cancer cell growth and evaluated the responses of IL-32θ-expressing cells to other cancer therapy. We compared the functions of IL-32θ in triple-negative breast cancer MDA-MB-231 cells that stably express IL-32θ, with MDA-MB-231 cells transfected with a mock vector. Slower growth was observed in cells expressing IL-32θ than in control cells, and changes were noted in nuclear morphology, mitotic division, and nucleolar size between the two groups of cells. Interleukin-32θ significantly reduced the colony-forming ability of MDA-MB-231 cells and induced permanent cell cycle arrest at the G1 phase. Long-term IL-32θ accumulation triggered permanent senescence and chromosomal instability in MDA-MB-231 cells. Genotoxic drug doxorubicin (DR) reduced the viability of MDA-MB-231 cells not expressing IL-32θ more than in cells expressing IL-32θ. Overall, these findings suggest that IL-32θ exerts antiproliferative effects in breast cancer cells and initiates senescence, which may cause DR resistance. Therefore, targeting IL-32θ in combination with DR treatment may not be suitable for treating metastatic breast cancer.
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Senescencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Interleucinas/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Forma del Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Inestabilidad Genómica , Humanos , Fenotipo , PloidiasRESUMEN
This study investigated the laxative effects of phlorotannins (Pt) derived from Ecklonia cava (E. cave) on chronic constipation by evaluating alterations in stool parameters, gastrointestinal motility, histopathological structure, mucin secretion, gastrointestinal hormones, muscarinic cholinergic regulation, and fecal microbiota in SD rats with loperamide (Lop)-induced constipation subjected to Pt treatment. Stool-related parameters (including stool number, weight, and water contents), gastrointestinal motility, and length of intestine were significantly enhanced in the Lop+Pt-treated group as compared to the Lop+Vehicle-treated group. A similar recovery was detected in the histopathological and cytological structure of the mid-colon of Lop+Pt-treated rats, although the level of mucin secretion remained constant. Moreover, rats with Lop-induced constipation subjected to Pt treatment showed significant improvements in water channel expression, gastrointestinal hormone secretions, and expression of muscarinic acetylcholine receptors M2/M3 (mAChRs M2/M3) and their mediators of muscarinic cholinergic regulation. Furthermore, the Lop+Pt-treated group showed a significant recovery of Bifidobacteriaceae, Muribaculaceae, Clostridiaceae, and Eubacteriaceae families in fecal microbiota. Taken together, these results provide the first evidence that exposure of SD rats with Lop-induced constipation to Pt improves the constipation phenotype through the regulation of membrane water channel expression, GI hormones, the mAChR signaling pathway, and fecal microbiota.
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Estreñimiento/tratamiento farmacológico , Laxativos/uso terapéutico , Phaeophyceae/química , Taninos/uso terapéutico , Animales , Estreñimiento/inducido químicamente , Laxativos/química , Loperamida , Masculino , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Ratas Sprague-Dawley , Taninos/químicaRESUMEN
Epimagnolin A is a lignan obtained from the flower buds of Magnolia fargesii, which is traditionally used in Asian medicine for treating headache and nasal congestion. A herbal compound fargesin obtained from M. fargesii, has exerted anti-inflammatory effects in human monocytic THP-1 cells in the previous study. The anti-inflammatory effects of epimagnolin A, however, have been not elucidated yet. In this study, it was demonstrated that epimagnolin A reduced phorbol-12-myristate-13-acetate (PMA)-induced IL-6 promoter activity and IL-6 production in human monocytic THP-1 cells. Furthermore, it was investigated the modulating effects of epimagnolin A on mitogen-activated protein kinase, nuclear factor-kappa B (NF-κB), and activator protein 1 (AP-1) activities. Phosphorylation of p38 and nuclear translocation of p50 and c-Jun were down-regulated by epimagnolin A in the PMA-stimulated THP-1 cell. The results revealed that epimagnolin A attenuated the binding affinity of NF-κB and AP-1 transcription factors to IL-6 promoter and IL-6 production through p38/NF-kB and AP-1 signaling pathways in the PMA-stimulated THP-1 cells. These results suggest that epimagnolin A can be a useful drug for the treatment of inflammatory diseases.
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Interleucina-6/metabolismo , Lignanos/farmacología , FN-kappa B/metabolismo , Ésteres del Forbol/farmacología , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antiinflamatorios/farmacología , Benzodioxoles/farmacología , Regulación hacia Abajo/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Células THP-1RESUMEN
Interleukin (IL)-32 is a recently discovered cytokine that appears to play an important role in human colon cancer growth. We investigated that IL-32γ in combination with TNF-α remarkably inhibited cell growth of human colon cancer cells (HCT116 and SW620) and tumor growth in xenograft-bearing nude mice. The transient enforced overexpression of IL-32γ potentiated the inhibitory effect of TNF-α on DNA synthesis, cell number and protein content, and enhanced apoptosis in colon cancer cells. We also found that knockdown of IL-32γ by siRNA showed the abolishment of cell growth inhibitory effect of TNF-α. The IL-32γ-overexpressing colon cancer cells further increased TNF-α-mediated expression of p38 MAPK as well as that of Bax, cleaved caspase-3 and -9, but decreased that of antiapoptotic proteins such as Bcl-2, cellular inhibitor of apoptosis protein (IAP) and X chromosome IAP. In xenograft model, the lipopolysaccharide (LPS)-injected (1.25 mg/kg) mice inoculated with IL-32γ-transfected HCT116 colon cancer cells were more decrease tumor volume and weight than inoculated with vector. Tumor tissues isolated from LPS-injected mice inoculated with IL-32γ-overexpressing colon cancer cells potentiated the expression levels of pro-apoptotic proteins such as cleaved caspase-3, 9 and Bax, but decreased that of Bcl-2. Furthermore, the mice increased IL-10 production, but decreased IL-6 levels in serum. In conclusion, our results suggest that IL-32γ may potentiate TNF-α-induced cell growth inhibition through activation of p38 MAPK pathways.
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Apoptosis/efectos de los fármacos , Neoplasias del Colon/patología , Citocinas/metabolismo , Interleucinas/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Interferente Pequeño/genética , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing. Maillard reaction products (MRPs) have antioxidant, antimutagenic, and antibacterial effects though 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242), a fructose-tyrosine MRP, appears to inhibit proliferation of cancer cells, its mechanism of action has not been studied in detail. The purpose of this study was to investigate the anti-proliferative effects of 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242) on two oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, through regulation of specificity protein 1 (Sp1). RESULTS: HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies. Sp1 was significantly inhibited by HPB242 in a dose-dependent manner. Furthermore, cell cycle regulating proteins and anti-apoptotic proteins, which are known as Sp1 target genes, were altered at the molecular levels. The key important regulators in the cell cycle such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, myeloid leukemia sequence 1 (Mcl-1) and survivin were significantly decreased by HPB242 or suppressed Sp1 levels, however pro-apoptotic proteins caspase3 and PARP were cleaved in HN22 and HSC4. CONCLUSIONS: HPB242 may be useful as a chemotherapeutic agent for OSCC for the purpose of treatment and prevention of oral cancer and for the improvement of clinical outcomes.
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Aldehídos/administración & dosificación , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias de la Boca/tratamiento farmacológico , Fenoles/administración & dosificación , Factor de Transcripción Sp1/genética , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Reacción de Maillard , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Factor de Transcripción Sp1/antagonistas & inhibidoresRESUMEN
The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 µM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5-2 µM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway.
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Aorta/efectos de los fármacos , Camptotecina/farmacología , Proliferación Celular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Proto-Oncogénicas c-sis/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Aorta/citología , Aorta/metabolismo , Becaplermina , Camptotecina/química , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal/fisiologíaRESUMEN
IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCε inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCε, and this interaction was totally inhibited by the PKCε inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCε and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCε and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Interleucina-6/biosíntesis , Interleucinas/biosíntesis , Monocitos/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Factor de Transcripción STAT3/metabolismo , Carcinógenos/farmacología , Línea Celular Tumoral , Activadores de Enzimas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/genética , Interleucinas/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Monocitos/citología , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Regiones Promotoras Genéticas/fisiología , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteína Quinasa C-epsilon/genética , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this study, the inhibitory effects of 1 were investigated on human colon cancer cell growth and tumor formation of xenograft mice. HCT-116 and SW-620 cells derived from human colon cancers were found to be similarly susceptible to 1, with IC50 values of 4.5 and 4.4 µM, respectively. Compound 1 induced S-phase arrest in the cell cycle progression and decreased the expression levels of cyclin A, cyclin E, and Cdk2. Conversely, 1 increased the expression of p21, a Cdk inhibitor. Compound 1 decreased Bcl-2 expression and increased the expression of Bax, and cleaved caspase-9 and -3. The effect of 1 on tumor formation when administered orally was evaluated in male BALB/c-nude mice implanted intradermally separately with HCT-116 and SW-620 cells. Tumor volumes and tumor weights in the mice treated with 1 (100 mg/kg) were decreased in both cases. These results suggest that the suppression of tumor formation by compound 1 in human colon cancer may occur through cell cycle arrest and apoptosis.
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Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Monoterpenos/farmacología , Fase S/efectos de los fármacos , Tropolona/análogos & derivados , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Genes bcl-2/efectos de los fármacos , Genes bcl-2/genética , Células HCT116 , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Monoterpenos/química , Tropolona/química , Tropolona/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP rho/efectos de los fármacosRESUMEN
BACKGROUND: Moisturisers prevent and treat dry skin. They can also protect sensitive skin, improve skin tone and texture, and mask imperfections. Herbal medicines or their extracts have been available as topical formulations and cosmetics. Arctium lappa L. (Asteraceae) has been used to treat inflammatory disorders and various skin problems. It could be a candidate herbal medicine for treating dry skin condition.This study aims to establish the efficacy and safety of a proposed herbal moisturising cream containing Arctium lappa L. seed extract, which has been approved by the Korean Ministry of Food and Drug Safety for use in cosmetics. METHODS/DESIGNS: This study is a randomised, double-blind, placebo-controlled study with two parallel groups (proposed herbal moisturising cream vs. placebo cream). We will recruit 66 healthy male and female participants, aged 20 to 65 years, who have been diagnosed with dry skin conditions. Participants will be randomly allocated to receive either the proposed herbal moisturising cream or a placebo cream for four weeks. Each participant will be examined for signs and symptoms before and after using the cream. Skin hydration, sebum (oily secretion) levels and transepidermal water loss (TEWL; constitutive loss of water from the skin surface) will be assessed. Participants will also be asked to fill out a health-related quality of life questionnaire. Safety will be assessed using blood tests, urine analysis, a pregnancy test, and the assessment of vital signs. DISCUSSION: This trial will utilise high-quality methodologies in accordance with both consolidated standards for reporting trials guidelines and the guidelines for clinical trials of cosmetics products that are aimed at expressions and advertisement approval in Korea. It will evaluate the clinical efficacy and safety of a proposed herbal moisturising cream containing Arctium lappa L. seed extract to treat dry skin conditions and provide itch relief. Moreover, we will also employ health-related quality of life questionnaires to assess changes in the quality of life. The results of this study will be used to present the evidence needed to request advertising/display allowances, in compliance with the recently amended Cosmetics Act for advertisement in Korea. TRIAL REGISTRATION: Current Controlled Trials ISRCTN46216631.
Asunto(s)
Arctium/química , Extractos Vegetales/administración & dosificación , Adulto , Anciano , Análisis de Varianza , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Extractos Vegetales/efectos adversos , Prurito/tratamiento farmacológico , Crema para la Piel/administración & dosificación , Crema para la Piel/efectos adversos , Enfermedades de la Piel/tratamiento farmacológico , Encuestas y Cuestionarios , Resultado del TratamientoRESUMEN
A series of novel 4-O-methylhonokiol analogs were synthesized in light of revealing structure-activity relationship for inhibitory effect of COX-2 enzyme. The key strategy of the molecular design was oriented towards modification of the potential metabolic soft spots (e.g., phenol and olefin) or by altering the polar surface area via incorporating heterocycles such as isoxazole and triazole. Most of all exhibited the inhibitory effects on COX-2 and PGF(1) production but not macrophage NO production. Especially, aryl carbamates 10 and 11 exhibited more potent inhibitory activity against COX-2 and PGF(1) production.
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Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Inhibidores de la Ciclooxigenasa 2/síntesis química , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/química , Diseño de Fármacos , Lignanos/química , Lignanos/farmacología , Prostaglandinas F/metabolismo , Animales , Compuestos de Bifenilo/síntesis química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/química , Activación Enzimática/efectos de los fármacos , Lignanos/síntesis química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Óxido Nítrico/metabolismo , Relación Estructura-ActividadRESUMEN
Sphingomyelin is the most abundant sphingolipid in mammalian cells and is mostly present in the plasma membrane. A new analytical method using high-performance liquid chromatography (HPLC) was developed to quantify sphingomyelin in mouse plasma and tissues, 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells. Sphingomyelin and dihydrosphingomyelin, an internal standard, were separated by high-performance thin-layer chromatography and simultaneously hydrolyzed with sphingolipid ceramide N-deacylase and sphingomyelinase to release sphingosine and dihydrosphingosine, respectively. Sphingomyelin content was measured by HPLC following o-phthalaldehyde derivatization. Sphingomyelin concentrations in 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells were 60.10 ± 0.24, 62.69 ± 0.08, and 58.38 ± 0.37 pmol/µg protein, respectively, whereas those in brain, kidney, and liver of ICR mice were 55.60 ± 0.43, 43.75 ± 0.21, and 22.26 ± 0.14 pmol/µg protein. The sphingomyelin concentration in mouse plasma was 407.40 ± 0.31 µM. The limits of detection and quantification for sphingomyelin were 5 and 20 pmol, respectively, in the HPLC analysis with fluorescence detection. This sensitivity was sufficient for analyzing sphingomyelin in biological samples. In conclusion, this analytical method is a sensitive and specific technique for quantifying sphingomyelin and was successfully applied to diverse biological samples with excellent reproducibility.
RESUMEN
High-risk variants of human papillomavirus (HPV) induce cervical cancer by persistent infection, and are regarded as the principal aetiological factor in this malignancy. The pro-inflammatory cytokine interleukin-32 (IL-32) is present at substantial levels in cervical cancer tissues and in HPV-positive cervical cancer cells. In this study, we identified the mechanism by which the high-risk HPV-16 E7 oncogene induces IL-32 expression in cervical cancer cells. We used antisense transfection, over-expression, or knock-down of IL-32 to assess the effects of the HPV-16 E7 oncogene on IL-32 expression in cervical cancer cells. Cyclo-oxygenase 2 (COX-2) inhibitor treatment was conducted, and the expression levels, as well as the promoter activities, of IL-32 and COX-2 were evaluated in human HPV-positive cervical cancer cell lines. E7 antisense treatment reduced the expression levels and promoter activities of COX-2, which is constitutively expressed in HPV-infected cells. Constitutively expressed IL-32 was also inhibited by E7 antisense treatment. Moreover, IL-32 expression was blocked by the application of the selective COX-2 inhibitor, NS398, whereas COX-2 over-expression resulted in increased IL-32 levels. These results show that the high-risk variant of HPV induces IL-32 expression via E7-mediated COX-2 stimulation. However, E7 and COX-2 were down-regulated in the IL-32γ over-expressing cells and recovered by IL-32 small interfering RNA, indicating that E7 and COX-2 were feedback-inhibited by IL-32γ in cervical cancer cells.
Asunto(s)
Regulación Viral de la Expresión Génica/inmunología , Interleucinas/metabolismo , Proteínas E7 de Papillomavirus/biosíntesis , Infecciones por Papillomavirus/inmunología , Transducción de Señal/inmunología , Neoplasias del Cuello Uterino/virología , Western Blotting , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , Ciclooxigenasa 2/inmunología , Ciclooxigenasa 2/metabolismo , Femenino , Expresión Génica , Papillomavirus Humano 16/inmunología , Humanos , Inmunohistoquímica , Inflamación/inmunología , Inflamación/metabolismo , Interleucinas/inmunología , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/metabolismo , Displasia del Cuello del Útero/inmunología , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/virologíaRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are the transcriptional factor that regulate glucose and lipid homeostasis and widely well-known as molecular targets for improvement of metabolic disorder. Because major transcriptional activity of PPARs depends on their proper ligands, the studies for PPAR ligands have been continuously developed. We previously reported the simple enzyme-linked immunosorbent assay (ELISA) systems to screen PPAR ligands and a chemical library including flavonoid derivatives have applied to these systems. In this study, we introduce two compounds (KU16476 and KU28843) identified as PPARγ partial agonists by a screening ELISA for PPARγ ligand. KU16476 and KU28843 significantly increased binding between PPARγ and SRC-1 in a simple ELISA system. Co-activator recruiting-induced abilities of two compounds were less than that of indomethacin, a well-known PPARγ agonist. To determine whether these compounds would be PPARγ partial agonists, each candidate with indomethacin were applied to a simple ELISA based on binding between PPARγ and SRC-1. Cotreatment with indomethacin significantly increased binding between PPARγ and SRC-1 than treatment of indomethacin or candidate alone. Two compounds had no considerable cytotoxicities, induced partial adipogenesis, and accumulated lipid droplets in 3T3-L1 fibroblast. Also, these two compounds enhanced expression of PPARγ-mediated genes such as aP2 and UCP-2. By docking study, we confirmed that two compounds bound well to the active site of PPARγ with hydrophobic interactions. We suggest that two compounds identified by a simple ELISA system can be PPARγ partial agonists. These PPARγ partial agonists and these studies to find out novel PPARγ agonists may contribute to drug development against metabolic disorders.
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Evaluación Preclínica de Medicamentos/métodos , PPAR gamma/agonistas , Células 3T3-L1 , Adipogénesis , Animales , Muerte Celular , Células HEK293 , Humanos , Ligandos , Ratones , Modelos Moleculares , Peso Molecular , Coactivador 1 de Receptor Nuclear/metabolismo , PPAR gamma/genética , Unión Proteica , Activación TranscripcionalRESUMEN
Data from clinical studies indicate that inhibitors of Class I and Class II histone deacetylase (HDAC) enzymes show great promise for the treatment of cancer. Zolinza (SAHA, Zolinza) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma. As a part of our ongoing effort to identify novel small molecules to target these important enzymes, we have prepared two series of benzothiazole-containing analogues of SAHA. It was found that several compounds with 6C-bridge linking benzothiazole moiety and hydroxamic functional groups showed good inhibition against HDAC3 and 4 at as low as 1 µg/ml and exhibited potent cytotoxicity against five cancer cell lines with average IC(50) values of as low as 0.81 µg/ml, almost equipotent to SAHA.
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Antineoplásicos/química , Benzotiazoles/química , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Ácidos Hidroxámicos/química , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Estructura Terciaria de Proteína , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , VorinostatRESUMEN
Adipocyte differentiation has been a target in anti-obesity strategies and is known to be closely related to lipid metabolism. Ceramide, a major sphingolipid metabolite, has been implicated in differentiation. In this study, we investigated whether ceramide biosynthesis is related to adipogenesis in 3T3-L1 cells. Preadipocytes can be differentiated synchronously by a mixture of adipogenic inducers including 3-isobutyl-1-methylxanthine, dexamethasone and insulin. The number of lipid droplets and the triglyceride content, which are differentiation biomarkers, gradually increased during adipogenesis. Interestingly, ceramide and sphingosine contents in the differentiated cells were decreased compared to those in preadipocytes. When the preadipocytes were treated with an 3-isobutyl-1-methylxanthine- or dexamethasone- or insulin-deficient mixture of inducers, the cellular ceramide levels were significantly increased compared with those in cells treated with the complete set of inducers. When preadipocytes were treated with 0, 0.1 or 1 µg/ml insulin along with 3-isobutyl-1-methylxanthine and dexamethasone, the ceramide levels were decreased and the triglyceride content was increased in a concentration-dependent manner. When the cells were treated with epigallocatechin gallate, an adipocyte differentiation inhibitor, during adipogenesis, the ceramide levels of adipocytes were increased and the fat content was decreased. In conclusion, our findings demonstrate that cellular ceramide levels are inversely correlated with adipocyte differentiation.
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Adipocitos/metabolismo , Adipogénesis , Ceramidas/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Animales , Catequina/análogos & derivados , Catequina/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Terapia Molecular Dirigida , Obesidad/tratamiento farmacológico , Esfingosina/metabolismo , Triglicéridos/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is becoming one of the most common chronic liver diseases in the world. One of the features of NAFLD is hepatic fat accumulation, which further causes hepatic steatosis, fibrosis, and inflammation. Saponins, the major pharmacologically active ingredients isolated from Panax notoginseng, contain several ginsenosides, which have various pharmacological and therapeutic functions. However, the ginsenoside-specific molecular mechanism of saponins in NAFLD remains unknown. This study aimed to elucidate the effects of ginseng saponin extract and its ginsenosides on hepatic steatosis, fibrosis, and inflammation and their underlying action mechanism in NAFLD. Mice were fed a fast food diet (FFD) for 16 weeks to induce NAFLD and then treated with saponin extract (50 or 150 mg/kg) for the remaining nine weeks to determine the effects of saponin on NAFLD. Saponin extract administration significantly alleviated FFD-induced hepatic steatosis, fibrosis, and inflammation. Particularly, saponin extract, compared with conventional red ginseng, contained significantly increased amounts of ginsenosides (Rh1 (10.34-fold) and Rg2 (7.1-fold)). In vitro Rh1 and Rg2 treatments exerted an anti-steatotic effect in primary hepatocytes, an antifibrotic effect in hepatic stellate cells, and anti-inflammatory and pro-mitophagy effects in immortalized mouse Kupffer cells. Mechanistically, saponin extract alleviated lipopolysaccharide-induced NLRP3 inflammasome activation by promoting mitophagy. In conclusion, saponin extract inhibited inflammation-mediated pathological inflammasome activation in macrophages, thereby preventing NAFLD development. Thus, saponin extract administration may be an alternative method for NAFLD prevention.
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Ginsenósidos/farmacología , Inflamasomas/antagonistas & inhibidores , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Panax/química , Extractos Vegetales/farmacología , Saponinas/farmacología , Animales , Modelos Animales de Enfermedad , Comida Rápida/efectos adversos , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiologíaRESUMEN
This experiment was designed to know whether (-)-epigallocatethin-3-O-gallate (EGCG) counteracts caffeine-induced hyperactivity, and its potential mechanisms in mice. EGCG inhibited methamphetamine-induced, cocaine-induced and caffeine-induced horizontal hyperlocomotion and rearing activity. EGCG also inhibited hyperlocomotion and rearing activity induced by apomorphine, a D1/D2-like agonist. Moreover, EGCG inhibited climbing behavior, a typical stereotyped behavior induced by stimulation of dopamine receptors through the activation of those receptors by apomorphine. From this experiment, we suggest that EGCG inhibits hyperactivity induced by psychostimulants including caffeine, in part by modulating dopaminergic transmission, and these inhibitory effects of EGCG counteract the stimulant actions of caffeine in green tea.
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Cafeína/toxicidad , Catequina/análogos & derivados , Estimulantes del Sistema Nervioso Central/toxicidad , Hipercinesia/prevención & control , Animales , Catequina/farmacología , Hipercinesia/inducido químicamente , Masculino , Ratones , Ratones Endogámicos ICR , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/efectos de los fármacos , Receptores de Dopamina D2/metabolismo , Conducta Estereotipada/efectos de los fármacos , Té/químicaRESUMEN
Lidocaine is a local anesthetic agent used in the form of injection and topical cream. However, these formulation types have limitations of being either painful or slow-acting, thereby hindering effective and complete clinical performance of lidocaine. Dissolving microneedles (DMNs) are used to overcome these limitations owing to their fast onset time and minimally invasive administration methods. Using hyaluronic acid and lidocaine to produce the drug solution, a lidocaine HCl encapsulated DMN (Li-DMN) was fabricated by centrifugal lithography. The drug delivery rate and local anesthetic quality of Li-DMNs were evaluated using the pig cadaver insertion test and Von Frey behavior test. Results showed that Li-DMNs could deliver sufficient lidocaine for anesthesia that is required to be utilized for clinical level. Results from the von Frey test showed that the anesthetic effect of Li-DMNs was observed within 10 min after administration, thus confirming fast onset time. A toxicity test for appropriate clinical application standard was conducted with a microbial limit test and an animal skin irritation test, showing absence of skin irritation and irritation-related microorganisms. Overall, Li-DMN is a possible alternative drug delivery method for local anesthesia, meeting the requirements for clinical conditions and overcoming the drawbacks of other conventional lidocaine administration methods.