RESUMEN
OBJECTIVE: This study investigated the relationship between the MTRA2576G polymorphism of the key enzyme in homocysteine metabolism and deep vein thrombosis (DVT), chronic venous insufficiency (CVI) and arteriosclerotic occlusion (ASO) of the lower extremities. METHODS: Genomic DNA was extracted from the peripheral blood of patients with lower-extremity vascular diseases, including 125 cases of DVT, 125 cases of CVI and 125 cases of ASO. DNA samples extracted from 197 healthy individuals were used as control samples. PCR-RFLP was used to investigate the polymorphisms of MTR in these subjects. RESULTS: The frequency of the G allele in MTR was 6.8%, 6.1% and 12.8% for the DVT group, CVI group and ASO group, respectively (p = 0.003). The frequency of the GG allele was 13.6%, 12.2% and 22.4% for the DVT group, CVI group and ASO group, respectively (p = 0.014). Only the allele frequency of GG in the ASO group was higher than that in the control group, and the disease risk was also 1.3 times higher than that in the control group (OR = 1.299, 95% CI = 1.025 â¼ 2.575). CONCLUSION: Patients with the G allele in MTR have a high risk for ASO, and the GG allele is a risk gene for ASO.
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Enfermedades Vasculares , Humanos , Alelos , Genotipo , Estudios de Casos y Controles , Factores de Riesgo , Enfermedades Vasculares/genética , Extremidad Inferior , Homocisteína , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genéticaRESUMEN
To prepare the intrauterine slow release silicone rubber bar made of Panax notoginseng and Rubia cordifolia, and finish its preliminary evaluation of in vitro releasing properties. The open mill method was used for plasticating of silicone rubber. The process parameters of the silicone rubber and drugs mixing were optimized by orthogonal test. The parameters of silicone rubber vulcanization was optimized by single factor test. The preliminary evaluation of in vitro release performance of the silicone rubber bar was conducted with ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, purpurin and rubimaillin as the indexes. The results showed that optimum technologic parameters for silicone rubber and drugs mixingï¼the roller spacing 2 mm; speed ratio 1â¶1.2; front roller temperature 55-60 â; rear roll temperature 50-55 â; and mixing time 20 min. The optimum parameters for silicone rubber vulcanizationï¼temperature 90 â, and time 60 min. The studies on release process in vitro revealed that the release process of silicone rubber bar was in line with the Higuchi equations. After 90 days, the cumulative release of ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1 was 46.7%, and the cumulative release of purpurin and rubimaillin was 51.9%. The preparation method can be applied to the preparation of silicone rubber bar, with slow release characteristics.
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Liberación de Fármacos , Medicamentos Herbarios Chinos/química , Ginsenósidos/química , Panax notoginseng/química , Rubia/química , Elastómeros de Silicona/químicaRESUMEN
BACKGROUND: A decreased autophagic capacity of bone marrow mesenchymal stromal cells (BMSCs) has been suggested to be an important cause of decreased osteogenic differentiation. A pharmacological increase in autophagy of BMSCs is a potential therapeutic option to increase osteoblast viability and ameliorate osteoporosis. AIM: To explore the effects of sinomenine (SIN) on the osteogenic differentiation of BMSCs and the underlying mechanisms. METHODS: For in vitro experiments, BMSCs were extracted from sham-treated mice and ovariectomized mice, and the levels of autophagy markers and osteogenic differentiation were examined after treatment with the appropriate concentrations of SIN and the autophagy inhibitor 3-methyladenine. In vivo, the therapeutic effect of SIN was verified by establishing an ovariectomy-induced mouse model and by morphological and histological assays of the mouse femur. RESULTS: SIN reduced the levels of AKT and mammalian target of the rapamycin (mTOR) phosphorylation in the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway, inhibited mTOR activity, and increased autophagy ability of BMSCs, thereby promoting the osteogenic differentiation of BMSCs and effectively alleviating bone loss in ovariectomized mice in vivo. CONCLUSION: The Chinese medicine SIN has potential for the treatment of various types of osteoporosis, bone homeostasis disorders, and autophagy-related diseases.
RESUMEN
A residue analytical method for the determination of bromoxynil in soil and maize was developed using high performance liquid chromatography with diode-array detector (HPLC-DAD) and high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The residual level and dissipation rate of bromoxynil in the soil and maize crops were determined using the established method. The mean half-life of bromoxynil in soil was 4.12 days with a dissipation rate of 91.25% over 21 days. The mean half-life in maize seedling was 1.14 days with a dissipation rate of 97.79% over 7 days. The final residues of bromoxynil were undetectable in maize grain, maize stem and soil at the harvest time, suggesting that residual levels of bromoxynil in maize plants are safe to humans and animals as well as to the environment.
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Herbicidas/análisis , Nitrilos/análisis , Residuos de Plaguicidas/análisis , Contaminantes del Suelo/análisis , Zea mays/química , Animales , Cromatografía Líquida de Alta Presión , Semivida , Herbicidas/química , Nitrilos/química , Residuos de Plaguicidas/química , Tallos de la Planta/química , Suelo/química , Microbiología del Suelo , Contaminantes del Suelo/química , Espectrometría de Masas en TándemRESUMEN
NF-κB (nuclear factor κB) is a regulator of hepatocellular cancer (HCC)-related inflammation and enhances HCC cells' resistance to antitumor therapies by promoting cell survival and anti-apoptosis processes. In the present work, we demonstrate that A20, a dominant-negative regulator of NF-κB, forms a complex with HSP90 (heat-shock protein 90) and causes the disassociation of the A20/HSP90 complex via downregulation of HSP90. This process restores the antitumor activation of A20. In clinical specimens, the expression level of A20 did not relate with the outcome in patients receiving sorafenib; however, high levels of HSP90 were associated with poor outcomes in these patients. A20 interacted with and formed complexes with HSP90. Knockdown of HSP90 and treatment with an HSP90 inhibitor disassociated the A20/HSP90 complex. Overexpression of A20 alone did not affect HCC cells. Downregulation of HSP90 combined with A20 overexpression restored the effect of A20. Overexpression of A20 repressed the expression of pro-survival and anti-apoptosis-related factors and enhanced HCC cells' sensitivity to sorafenib. These results suggest that interactions with HSP90 could be potential mechanisms of A20 inactivation and disassociation of the A20/HSP90 complex and could serve as a novel strategy for HCC treatment.
RESUMEN
OBJECTIVE: The aim of this study was to investigate the inhibitory effect of geniposide on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and interleukin-8 (IL-8) production in human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: Primary HUVECs were used. The mRNA/protein levels of IL-6 and IL-8 was determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). LPS-induced HUVEC migration and adhesion of monocytes to HUVECs were studied by monolayer wound healing experiments and monocytic cell adhesion assay, respectively. Expression of nuclear factor kappaB (NF-kappaB), inhibitory factor kappaB-alpha (IkappaB-alpha), p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were determined by Western blot analysis. RESULTS: Geniposide effectively inhibited LPS-induced expression of IL-6 and IL-8 in HUVECs at the transcription and translation levels. Additionally, geniposide suppressed LPS-induced HUVEC migration and U937 monocyte adhesion to HUVECs. Signal transduction studies indicate that geniposide blocked the activation of NF-kappaB, degradation of IkappaB-alpha, and phosphorylation of p38 MAPK and ERK1/2 in HUVECs challenged by LPS. CONCLUSION: The results show that geniposide can inhibit LPS-induced IL-6 and IL-8 production in HUVECs by blocking p38 MAPK and ERK1/2 signaling pathways.
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Células Endoteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Interleucina-6/antagonistas & inhibidores , Interleucina-6/biosíntesis , Interleucina-8/antagonistas & inhibidores , Interleucina-8/biosíntesis , Iridoides/farmacología , Lipopolisacáridos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Western Blotting , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas I-kappa B/antagonistas & inhibidores , Indicadores y Reactivos , Monocitos/efectos de los fármacos , FN-kappa B/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Células U937 , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of bis(12)-hupyridone in rat blood has been developed and validated. Chromatographic separation was carried out in an Agilent Extend C(18) 5microm column (length, 250mm; inner diameter, 4.6mm) using a mixture of water-acetonitrile-trifluoroacetic acid (81:19:0.04, v/v/v) as the mobile phase at a flow rate of 1mL/min, with detection at 229nm. The method used for the bis(12)-hupyridone quantification showed linearity for concentration range of 0.1-7.5microg/mL with r(2)=0.9991. The limit of detection and quantification of this method were 0.05microg/mL and 0.1microg/mL, respectively. The intra- and inter-day variations of the analysis were less than 4.22% with standard errors less than 13.3%. The developed method was successfully applied to the pharmacokinetic study of bis(12)-hupyridone after intravenous administration of 5mg/kg and intraperitoneal administration of 10 and 20mg/kg in rats.
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Inhibidores de la Colinesterasa/sangre , Inhibidores de la Colinesterasa/farmacocinética , Quinolonas/sangre , Quinolonas/farmacocinética , Acetonitrilos/química , Animales , Área Bajo la Curva , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Congelación , Semivida , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Estructura Molecular , Control de Calidad , Quinolonas/química , Quinolonas/farmacología , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrofotometría Ultravioleta/métodos , Ácido Trifluoroacético/química , Agua/químicaRESUMEN
We have recently reported that bis(7)-tacrine could prevent glutamate-induced neuronal apoptosis through NMDA receptors. In this study, we demonstrated that in cultured rat cortical neurons, bis(7)-tacrine (IC(50), 0.02 microM) prevented glutamate-induced excitotoxicity more substantially than memantine (IC(50), 0.7 microM). In addition, bis(7)-tacrine was more efficient than memantine in buffering the intracellular Ca(2+) triggered by glutamate. In cultured rat hippocampal neurons, bis(7)-tacrine inhibited 50 microM NMDA-activated current in a concentration-dependent manner with an IC(50) of 0.68+/-0.07 microM, which is five times more potent than that produced by memantine (IC(50), 3.41+/-0.36 microM; p<0.05). By contrast, bis(7)-tacrine, up to 5 microM, did not significantly affect the current activated by 50 microM AMPA or 50 microM kainate. These results suggest that bis(7)-tacrine is more potent than memantine against glutamate-induced neurotoxicity by selectively inhibiting NMDA-activated current.
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Corteza Cerebral/efectos de los fármacos , Citoprotección , Antagonistas de Aminoácidos Excitadores/farmacología , Memantina/farmacología , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tacrina/análogos & derivados , Animales , Calcio/análisis , Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Agonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/toxicidad , Ácido Kaínico/farmacología , Ratas , Ratas Sprague-Dawley , Tacrina/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
The present study aims to investigate the preclinical intestinal absorption of bis(7)-tacrine (B7T) using different absorption models. In addition, potential intestinal and liver first-pass metabolism was evaluated by in vitro incubation of B7T with rat intestine and liver microsome. Results showed that the permeability of B7T across artificial membrane was pH dependent with rapid diffusion achieved at both pH 6.8 and 7.4. However, the absorptive permeability of B7T in Caco-2 cell model was substantially lower than that in the artificial membrane accompanied with over 56% of B7T being trapped within Caco-2 cells. In the rat in situ intestinal perfusion model, B7T was subject to an extensive intestinal extraction (>90%) with extremely low concentration of B7T detected in mesenteric blood, which was further found to be associated with the high tissue binding (99.9%) of B7T. In vitro incubation of B7T with rat liver and intestinal microsomes revealed that hydroxylation of B7T might mainly occur in rat liver rather than intestine. In conclusion, B7T is expected to have a low oral bioavailability in vivo, which may be due to its poor intestinal permeability, significant tissue binding and hepatic hydroxylation metabolism.
Asunto(s)
Inhibidores de la Colinesterasa/farmacocinética , Absorción Intestinal/fisiología , Nootrópicos/farmacocinética , Tacrina/análogos & derivados , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Biotransformación , Células CACO-2 , Inhibidores de la Colinesterasa/metabolismo , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , Epitelio/metabolismo , Glucurónidos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Masculino , Nootrópicos/metabolismo , Permeabilidad , Ratas , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta , Tacrina/metabolismo , Tacrina/farmacocinéticaRESUMEN
The lipophilicity and solubility profiles of bis(12)-hupyridone (B12H) and bis(7)-tacrine (B7T), two novel acetylcholinesterase inhibitors dimerized from huperzine A fragments and tacrine, respectively, were investigated over a broad pH range. Lipophilicity was assessed by both shake flask method with 1-octanol-water system and a reverse-phase HPLC system with methanol-water as mobile phase. The former method was used for determining the lipophilicities of the ionized forms (log D) of the dimers while the latter method was used for that of the neutral forms (log P). The log P values for B12H and B7T were found to be 5.4 and 8.2, respectively, indicating that the two dimers are highly lipophilic. The solubilities of both dimers were found to be affected by pH. The solubility of B12H was >1.41 mg/ml when the pH was <7, but <0.06 mg/ml when the pH was >8. The solubility of B7T was >0.26 mg/ml when the pH was <9, but <0.005 mg/ml when the pH was >12. The ionic strength of a solution could affect the solubilities considerably (11.16 mg/ml for B12H and 12.71 mg/ml for B7T in water; 2.07 mg/ml for B12H and 0.36 mg/ml for B7T in saline). The ionization constants (pK(a)) of the two dimers were determined by UV spectrophotometry. Both dimers were found to have two pK(a) values: 7.5+/-0.1 (pK(a1)) and 10.0+/-0.2 (pK(a2)) for B12H; and 8.7+/-0.1 (pK(a1)) and 10.7+/-0.4 (pK(a2)) for B7T. Furthermore, an in vivo pharmacological assay conducted in mice showed that a maximum AChE inhibition occurred 15 min after the single-dose and intraperitoneal administration of either dimer. This indicates that the two dimers may easily cross the blood-brain barrier. In summary, these physiochemical characteristics suggest that the two dimers may be promising candidates for the development of better drugs for Alzheimer's disease.
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Química Física/métodos , Inhibidores de la Colinesterasa/química , Quinolonas/química , Sesquiterpenos/química , Tacrina/análogos & derivados , Tacrina/química , Administración Oral , Algoritmos , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Química Física/normas , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/farmacología , Cromatografía Líquida de Alta Presión/métodos , Dimerización , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Quinolonas/farmacología , Sesquiterpenos/administración & dosificación , Sesquiterpenos/farmacología , Solubilidad , Espectrofotometría Ultravioleta/métodos , Tacrina/administración & dosificación , Tacrina/farmacologíaRESUMEN
An analytical method using on-line high performance liquid chromatography-tandem mass spectrometry with electrospray ionization was developed and applied for the quantification of bis(7)-tacrine (B7T) in rat blood. B7T and pimozide (internal standard, IS) were extracted in a single step from 100 microl of alkalized blood with ethyl acetate. Analytes were separated using an Extend C-18 column at 25 degrees C. The elution was achieved isocratically with a mobile phase composed of 0.05% aqueous formic acid and acetonitrile (60:40, v/v) at a flow rate of 0.35 ml/min. Quantification was achieved by monitoring the selected ions at m/z 247 for B7T and m/z 462-->m/z 328 for pimozide. Retention times were 1.45 and 2.23 min for B7T and IS, respectively. Calibration curves were linear in the range from 86.4 to 2160.0 ng/ml. The established method is rapid, selective and sensitive for the identification and quantification of B7T in biological samples. The assay is accurate (bias <10%) and reproducible (intra- and inter-day variation <10%), with detection and quantification limit of 3.6 and 42.3 ng/ml, respectively. Furthermore, it was successfully applied for the pharmacokinetic measurement of B7T in rat with a single intravenous administration at 0.3mg/kg.
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Enfermedad de Alzheimer/tratamiento farmacológico , Cromatografía Líquida de Alta Presión/métodos , Tacrina/análogos & derivados , Espectrometría de Masas en Tándem/métodos , Animales , Área Bajo la Curva , Estabilidad de Medicamentos , Congelación , Semivida , Inyecciones Intravenosas , Masculino , Tasa de Depuración Metabólica , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tacrina/administración & dosificación , Tacrina/sangre , Tacrina/química , Tacrina/farmacocinética , Tacrina/uso terapéutico , Factores de TiempoRESUMEN
Methylene blue (MB) can ameliorate behavioral, neurochemical, and neuropathological impairments in animal models of acute and chronic neurodegenerative disorders, but the underlying mechanism remains unclear. Myocyte enhancer factor 2 (MEF2D) is known to promote neuronal survival in several models, and several survival and death signals converge on MEF2D and regulate its activity. Here, we investigated the role of MEF2D in the neuroprotective effect of MB against glutamate-induced toxicity in HT22 neuronal cells. Our results showed that MB, event at less than 100 nM, improved the viability of HT22 cells exposed to 2 mM glutamate. MB attenuated the mitochondrial impairment and quenches the reactive oxygen species (ROS) induced by glutamate. Surprisingly, MB at 50-200 nM did not affect the Nrf2/HO-1 pathway, an important endogenous anti-oxidative system. Further study showed that MB increased the transcription and translation of MEF2D. In addition, MB upregulated the expression of mitochondrial NADH dehydrogenase 6 (ND6) in a MEF2D-dependent manner. Knockdown of MEF2D abolished both MB-medicated increase of ND6 and MB-induced neuroprotection against glutamate-induced toxicity. Moreover, we showed that MB promoted Akt function activity, suppressed GSK-3ß activity, and increased MEF2D level in hippocampus of mice and HT22 cells. These findings for the first time demonstrate that MB protects HT22 neuronal cells against glutamate-induced cell death partially via the regulation of MEF2D-associated survival pathway.
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Hipocampo/efectos de los fármacos , Azul de Metileno/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Hipocampo/metabolismo , Factores de Transcripción MEF2/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The benzodiazepine diazepam impairs memory and long-term potentiation (LTP) in the hippocampus. Here, we investigate the effect of diazepam on LTP of C-fiber evoked field potentials in spinal dorsal horn, which is relevant to pathological pain. LTP of C-fiber evoked field potentials was recorded in the superficial layers of spinal dorsal horn in urethane-anesthetized Sprague--Dawley rats. Diazepam was applied locally at the recording spinal segments before and after LTP induction by tetanic stimulation. We found (1) Diazepam completely blocked LTP induction. (2) Diazepam and midazolam reversed spinal LTP, when applied at 30 min after LTP induction and depressed but could not reverse spinal LTP, when applied at 3 h after LTP induction. (3) Pretreatment with benzodiazepine receptor antagonist flumazenil or GABA(A) receptor antagonist bicuculline completely blocked the inhibitory effects of diazepam on spinal LTP. In contrast, when the inhibitory effect of diazepam was fully established, neither of these antagonists was capable of reversing the inhibition by diazepam. (4) Spinal application of the GABA(A) receptor agonist 3-amino-1-propanesulfonic acid (3-APSA) at a dose of 50 microg, produced a transient inhibition of spinal LTP. These results suggest that diazepam might prevent and depress spinal plastic change produced by noxious stimulation via activation of the GABA(A) -benzodiazepine receptor complex.
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Diazepam/farmacología , Hipnóticos y Sedantes/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Células del Asta Posterior/efectos de los fármacos , Animales , Electrofisiología , Potenciales Evocados/efectos de los fármacos , Flumazenil/farmacología , Moduladores del GABA/farmacología , Masculino , Fibras Nerviosas Amielínicas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Taurina/análogos & derivados , Taurina/farmacologíaRESUMEN
Lipids metabolism was involved in the process of many types of tumor and alkylglycerone phosphate synthase (AGPS) was considered implicated in tumor process. Benzyl isothiocyanate (BITC) showed the inhibitory effect of tumor and AGPS activity, therefore, we screened a group of small molecular compound based on BITC by computer-aid design targeting AGPS and the results showed that the derivants could suppress the proliferation, the expression of tumor related genes such as survivin and Bcl-2, and the level of ether lipids such as lysophosphatidic acid ether (LPAe) and platelet activating factor ether (PAFe); however, the activity of caspase-3/8 was improved in glioma U87MG and hepatic carcinoma HepG2 cells in vitro.
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Transferasas Alquil y Aril/metabolismo , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Isotiocianatos/farmacología , Neoplasias/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Diseño Asistido por Computadora , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Glioma/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Interleukin-8 (IL-8) expression by melanoma cells may influence their metastatic capabilities. Tetramethylpyrazine (TMP) from Ligusticum wallichil Franch. possesses anti-inflammatory and antitumor activities. It has recently been suggested that autocrine IL-8 may play a role in tumor cell survival, invasion and migration. The role of TMP in association with IL-8 in the tumor cell migratory process remains unclear. The purpose of the present study was to determine whether TMP influences the migratory ability of a human ovarian carcinoma cell line (SKOV3) via regulation of IL-8 expression in vitro. Cell counts showed that treatment of SKOV3 with TMP (25-100 µg/ml) for 24 h did not decrease cell numbers, while an effect of TMP on the down-regulation of the expression of IL-8 was observed. In addition, migration of SKOV3 cells was suppressed after treatment with TMP (25-100 µg/ml) for 24 h. Therefore, expression of IL-8 by SKOV3 cells correlates with their metastatic potential. Western blot analysis revealed that ERK1/2 and p38 phosphorylation was blocked by TMP. Furthermore, IL-8 mRNA expression was inhibited significantly after co-incubation with PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor), respectively. Notably, these changes were the results of activator protein-1 (AP-1) activity suppression rather than that of NF-κB. Our data suggest that TMP may inhibit tumor cell invasion and migration, at least in part, through its down-regulation of IL-8 expression. Our results provide evidence that anti-inflammation plays an important role in integrative cancer therapies.
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Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Interleucina-8/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias Ováricas/patología , Pirazinas/farmacología , Factor de Transcripción AP-1/metabolismo , Adhesión Celular , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Cocultivo , Femenino , Genes Reporteros , Humanos , Interleucina-8/metabolismo , Luciferasas/biosíntesis , Luciferasas/genética , Monocitos/citología , Monocitos/efectos de los fármacos , Fosforilación , Transducción de Señal , Transcripción GenéticaRESUMEN
Cisplatin (CDDP) is a major chemotherapeutic drug used in the treatment of human ovarian cancer. Tubeimoside I (TBMS1) has also shown potent antitumor and antitumor-promoting effects, and may offer a promising new approach in the effective treatment of CDDP-resistant human ovarian cancers. This study aimed to investigate the effect of TBMS1 in sensitizing CDDP in CDDP-resistant human ovarian cancer cells (A2780/DDP). A variety of methods were employed to measure cell apoptosis, p38, ERK1/2 and glutathione S-transferase (GST)-π expressions. It was found that TBMS1 combined with CDDP promoted cell apoptosis, decreased proliferation activity and increased cytosolic Ca2+ levels. Bcl-2 protein expression was down-regulated but Bax was up-regulated. Moreover, GST-π mRNA and protein expression were decreased. TBMS1 reduced the resistance of the cells to CDDP-induced cytotoxicity. Both the p38 inhibitor (SB203580) and the ERK1/2 inhibitor (PD98059) effectively blocked this effect. These results suggest that TBMS1 can effectively sensitize CDDP in CDDP-resistant human ovarian cancer cells through the down-regulation of the ERK1/2 and the up-regulation of the p38 signaling pathways.
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Cisplatino/farmacología , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias Ováricas/enzimología , Saponinas/farmacología , Triterpenos/farmacología , Regulación hacia Arriba/efectos de los fármacos , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/uso terapéutico , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Gutatión-S-Transferasa pi/genética , Gutatión-S-Transferasa pi/metabolismo , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saponinas/uso terapéutico , Triterpenos/uso terapéutico , Proteína X Asociada a bcl-2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIM: To investigate the effect of puerarin on lipopolysaccharide (LPS)-activated N9 microglia cells. METHODS: MTT was used to detect the cytotoxic effect of puerarin and LPS on N9 microglia cells. Flow cytometry was used to determine the effect of puerarin on cellular production of nitric oxide (NO) and reactive oxygen species (ROS), apoptosis and cell cycle in LPS-activated N9 microglia cells. The amount of NO released into the supernatant was detected by Griess assay. The HE staining was used to show the morphological response of LPS-activated N9 microglia cells to puerarin. RESULTS: Morphologically, amoeba-like LPS-activated N9 cells turned to be round in quiescence after treated with puerarin (100-200 micromol/L). Puerarin (50-200 micromol/L) caused less amount of NO released into the supernatant, with 12.43+/-0.11 micromol/L of NO at a dose of 200 micromol/L puerarin compared with 23.45+/-0.19 micromol/L in untreated control (P<0.01). Likewise, puerarin (200 micromol/L) completely abolished the ROS-producing ability of LPS-activated N9 cells, with a basal ROS level comparable to the non-LPS-treated control. Puerarin also exerted an anti-apoptotic effect on the LPS-activated N9 cells and rescued the cells from G(0)/G(1) arrest. CONCLUSION: Puerarin antagonizes activation effect of LPS on N9 microglia cells and might be a useful drug for the treatment of neurodegenerative disorders.
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Isoflavonas/farmacología , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Ratones , Microglía/citología , Microglía/metabolismo , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
AIM OF THE STUDY: To determine the inhibitory effect of tetramethylpyrazine (TMP) on lipopolysaccharide (LPS)-induced over-production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in N9 microglial cells. MATERIALS AND METHODS: N9 cells were pretreated with vehicle or TMP and then exposed to LPS for the time indicated. Cell viability was determined by methylthiazoyltetrazolium (MTT) assay. Nitrite assay was performed by Griess reaction. Expression of iNOS mRNA was examined by RT-PCR. Protein levels of iNOS, p38 mitogen-activated protein kinase (MAPK), ERK1/2, JNK, phosphatidylinositol 3-kinase (PI3K) and Akt were determined by western blot analysis. Formation of reactive oxygen species (ROS) was evaluated by fluorescence image system. RESULTS: TMP inhibited LPS-induced over-production of NO and iNOS in N9 cells. TMP also inhibited the NF-kappaB translocation from cytoplasm into nucleus of N9 cells. In addition, TMP showed blocking effect on the phosphorylation of p38 MAPK, ERK1/2, JNK and Akt, but not PI3K. Further, TMP suppressed the formation of intracellular ROS in LPS-induced N9 cells. CONCLUSIONS: TMP inhibited production of NO and iNOS in LPS-induced N9 cells through blocking MAPK and PI3K/Akt activation and suppressing ROS production.
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Depuradores de Radicales Libres/farmacología , Microglía/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Proteína Oncogénica v-akt/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pirazinas/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Animales , Western Blotting , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/aislamiento & purificación , Lipopolisacáridos/toxicidad , Ratones , Microglía/enzimología , Microglía/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Pirazinas/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Chitosan oligosaccharides (COS) have been reported to exert anti-fungal activities, antitumour activities and immuno-enhancing effects. However, the potential roles of COS in the treatment of vascular inflammations remain unknown. In the present study, we examined the effects of COS on interleukin-6 (IL-6) production in human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). Induction of HUVECs with LPS (100 ng/ml) increased the mRNA expression and protein secretion of IL-6 (versus the vehicle-treated group, p < 0.01), which were significantly reverted by the pre-treatment with COS (50-200 microg/ml) for 24 hr before LPS exposure (versus the LPS-treated group, p < 0.05 or 0.01). Signal transduction studies showed that the pre-treatment of HUVECs with COS (50-200 microg/ml) for 24 hr markedly inhibited the LPS-induced over-expression of phosphorylated p38 mitogen-activated protein kinase (MAPK), phosphorylated ERK1/2 and nuclear factor kappaB (NF-kappaB). Moreover, the LPS-induced NF-kappaB activation was suppressed by the specific ERK1/2 inhibitor PD98059 (30 microM) (versus the LPS-treated group, p < 0.01), but not by the specific p38 MAPK inhibitor SB203580 (25 microM). Additionally, both MAPK inhibitors markedly suppressed LPS-induced IL-6 mRNA expression in HUVECs (versus the LPS-treated group, p < 0.01). In conclusion, our results suggest that COS inhibit LPS-induced up-regulation of IL-6 in HUVECs, and this can be regulated by at least two parallel signalling pathways: one via p38 MAPK pathway independent of NF-kappaB activation and one via ERK1/2 pathway dependent on NF-kappaB activation.
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Quitosano/farmacología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-6/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quitosano/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Regulación de la Expresión Génica , Humanos , Interleucina-6/genética , Lipopolisacáridos/farmacología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Oligosacáridos/farmacología , Fosforilación , Venas Umbilicales/citología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Tetramethylpyrazine has been widely used in traditional Chinese medicine to treat cardiovascular diseases such as atherosclerosis and hypertension. The underlying mechanism of cardioprotective effects, however, remains to be elucidated. Here, using human umbilical vein endothelial cells (HUVECs), we have assessed the protective effect of tetramethylpyrazine on H(2)O(2)-induced oxidative damage. After pre-incubation with tetramethylpyrazine (50, 100 and 150 microg/ml) for 24 hr., viability loss in H(2)O(2)-induced HUVECs (76.5% of the control level, p < 0.05, at 400 microM of H(2)O(2) for 12 hr.) was restored in a concentration-dependent manner, and the maximal recovery (88.7% of the control level, p < 0.05) was achieved with tetramethylpyrazine at 150 microg/ml. The production of reactive oxygen species was suppressed by measuring fluorescent intensity of 2',7'-dichorofluorescein (83.1% of the H(2)O(2)-treated group, p < 0.05, at 150 microg/ml of tetramethylpyrazine). Tetramethylpyrazine also increased activities of superoxide dismutase and glutathione peroxidase (144.1% and 118.3% of the H(2)O(2)-treated group, respectively, p < 0.05, at 150 microg/ml of tetramethylpyrazine). In addition, tetramethylpyrazine reduced levels of malonaldehyde, intracellular nitric oxide and nitric oxide synthase (83.8%, 91.2% and 78.7% of the H(2)O(2)-treated group, respectively, p < 0.05, at 150 microg/ml of tetramethylpyrazine). Furthermore, pre-incubation of tetramethylpyrazine with HUVECs for 24 hr. resulted in reduction of apoptosis and removal of cell cycle arrest in the S phase (56.6% and 59.7% of the H(2)O(2)-treated group, respectively, p < 0.01, at 150 microg/ml of tetramethylpyrazine). Altogether, these results suggest that tetramethylpyrazine has a protective effect on H(2)O(2)-induced oxidative damage in HUVECs due to its antioxidant and antiapoptotic properties.