RESUMEN
Focal segmental glomerulosclerosis (FSGS) is a major cause of end-stage renal disease and remains without specific treatment. To identify new events during FSGS progression, we used an experimental model of FSGS associated with nephroangiosclerosis in rats injected with L-NAME (Nω-nitro-L-arginine methyl ester). After transcriptomic analysis we focused our study on the role of Isthmin-1 (ISM1, an anti-angiogenic protein involved in endothelial cell apoptosis. We studied the renal expression of ISM1 in L-NAME rats and other models of proteinuria, particularly at the glomerular level. In the L-NAME model, withdrawal of the stimulus partially restored basal ISM1 levels, along with an improvement in renal function. In other four animal models of proteinuria, ISM1 was overexpressed and localized in podocytes while the renal function was degraded. Together these facts suggest that the glomerular expression of ISM1 correlates directly with the progression-recovery of the disease. Further in vitro experiments demonstrated that ISM1 co-localized with its receptors GRP78 and integrin αvß5 on podocytes. Treatment of human podocytes with low doses of recombinant ISM1 decreased cell viability and induced caspase activation. Stronger ISM1 stimuli in podocytes dropped mitochondrial membrane potential and induced nuclear translocation of apoptosis-inducing factor (AIF). Our results suggest that ISM1 participates in the progression of glomerular diseases and promotes podocyte apoptosis in two different complementary ways: one caspase-dependent and one caspase-independent associated with mitochondrial destabilization.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria , Podocitos , Animales , Humanos , Ratas , Inhibidores de la Angiogénesis/uso terapéutico , Caspasas/metabolismo , Modelos Animales de Enfermedad , Glomeruloesclerosis Focal y Segmentaria/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , Podocitos/metabolismo , Proteinuria/metabolismoRESUMEN
Previous work has shown that dietary L-arginine (Arg) supplementation reduced white fat mass in obese rats. The present study was conducted with cell models to define direct effects of Arg on energy-substrate oxidation in hepatocytes, skeletal muscle cells, and adipocytes. BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes, and 3T3-L1 mouse adipocytes were treated with different extracellular concentrations of Arg (0, 15, 50, 100 and 400 µM) or 400 µM Arg + 0.5 mM NG-nitro-L-arginine methyl ester (L-NAME; an NOS inhibitor) for 48 h. Increasing Arg concentrations in culture medium dose-dependently enhanced (P < 0.05) the oxidation of glucose and oleic acid to CO2 in all three cell types, lactate release from C2C12 cells, and the incorporation of oleic acid into esterified lipids in BNL CL.2 and 3T3-L1 cells. Arg at 400 µM also stimulated (P < 0.05) the phosphorylation of AMP-activated protein kinase (AMPK) in all three cell types and increased (P < 0.05) NO production in C2C12 and BNL CL.2 cells. The inhibition of NOS by L-NAME moderately reduced (P < 0.05) glucose and oleic acid oxidation, lactate release, and the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in BNL CL.2 cells, but had no effect (P > 0.05) on these variables in C2C12 or 3T3-L1 cells. Collectively, these results indicate that Arg increased AMPK activity and energy-substrate oxidation in BNL CL.2, C2C12, and 3T3-L1 cells through both NO-dependent and NO-independent mechanisms.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Ácido Oléico , Ratas , Ratones , Animales , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Fosforilación , NG-Nitroarginina Metil Éster/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Ácido Oléico/farmacología , Fibras Musculares Esqueléticas/metabolismo , Adipocitos/metabolismo , Células 3T3-L1 , Glucosa/metabolismo , Hepatocitos/metabolismo , Arginina/metabolismo , Lactatos/metabolismo , Lactatos/farmacología , Músculo Esquelético/metabolismoRESUMEN
BACKGROUND: Although hypothyroidism during pregnancy may develop grave outcomes for both mothers and offspring, management of which is still a challenge due to the insufficient understanding of this disease. The close correlation between hypothyroidism and preeclampsia is well documented, suggesting that preeclampsia is a potential risk factor for the development of maternal hypothyroidism. However, the exact role of preeclampsia in gestational hypothyroidism is still obscure. OBJECTIVE: In this study, we explored the possible mechanisms of the effect of preeclampsia on thyroid function of maternal rats. METHODS: Thirty pregnant rats were randomly divided into normal pregnancy control (NOP), preeclampsia (PE), and preeclampsia supplemented with amlodipine besylate (PEAml). NG-Nitro-L-arginine-methyl ester was used to induce preeclamptic symptoms. On gestational day 21, rats were sacrificed, and then, the ultrastructure of the thyroid gland, type 1 iodothyronine deiodinase (Dio1) expression, and serum-free thyroxine (FT4), free triiodothyronine (FT3), and thyroid stimulation hormones (TSH) were assessed. RESULTS: Compared to NOP rats, results of PE rats showed that thyroid follicular cells' ultrastructure was damaged; both hepatic Dio1 mRNA and protein levels were decreased. Interestingly, these changes were ameliorated in PEAml rats. Additionally, FT4, FT3, and TSH levels have no significant differences among groups. CONCLUSION: These findings indicated that preeclampsia could disrupt synthesis, secretion, and metabolism function of thyroid hormones by damaging thyroid follicular cells and interfering Dio1 expression.
Asunto(s)
Hipotiroidismo/metabolismo , Yoduro Peroxidasa/metabolismo , Hígado/enzimología , Preeclampsia/metabolismo , Glándula Tiroides/ultraestructura , Hormonas Tiroideas/sangre , Animales , Femenino , Hipotiroidismo/complicaciones , NG-Nitroarginina Metil Éster/metabolismo , Embarazo , Preñez , Ratas , Ratas Sprague-Dawley , Factores de Riesgo , Tirotropina/sangre , Tiroxina/sangre , Factores de Tiempo , Triyodotironina/sangreRESUMEN
Anti-oxidant and anti-inflammatory properties of caffeic acid (CA) have been reported recently. In this study, the therapeutic effects of CA on ethanol-induced ulcer and the roles of nitric oxide and cholinergic pathways in these effects were investigated. Ulcer was induced by ethanol via oral gavage. Ulcer induced rats were treated with either vehicle (ulcer group) or CA (100, 250 or 500 mg/kg, per oral gavage). Macroscopic evaluation showed that 250 mg/kg CA was the effective dose. To elucidate the action mechanism of CA, 10 mg/kg l-NAME or 1 mg/kg atropine sulfate was administered to 250 mg/kg CA treated groups. All rats were decapitated 1 h after ulcer induction and gastric samples were scored macroscopically and microscopically, and analyzed for myeloperoxidase (MPO), malondialdehyde (MDA), and glutathione (GSH) levels. ANOVA test was used for statistical analyses. Macroscopic and microscopic damage scores, MDA levels and MPO activity were increased while GSH levels were decreased in ulcer group. Treatment with 250 mg/kg and 500 mg/kg CA reduced macroscopic and microscopic damage scores, decreased MPO activity and MDA levels, and preserved the depleted glutathione significantly. l-NAME administration before CA treatment elevated MDA levels, MPO activity and depleted glutathione. However, atropine sulfate had no effect on biochemical parameters. We conclude that CA ameliorates ethanol-induced gastric mucosal damage, and NO pathway contributes to this effect. On the other hand, there is a lack of evidence for the contribution of the muscarinic cholinergic system.
Asunto(s)
Ácidos Cafeicos/farmacología , Etanol/farmacología , Mucosa Gástrica/diagnóstico por imagen , Óxido Nítrico/metabolismo , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Antiulcerosos/farmacología , Antioxidantes/metabolismo , Colinérgicos/farmacología , Modelos Animales de Enfermedad , Mucosa Gástrica/metabolismo , Glutatión/metabolismo , Masculino , Malondialdehído/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , Peroxidasa/metabolismo , Fitoterapia/métodos , Extractos Vegetales/farmacología , Ratas , Ratas Sprague-Dawley , Úlcera Gástrica/metabolismoRESUMEN
BACKGROUND: We aimed to analyze whether a diet supplemented with a standard dose of copper (Cu) in the form of nanoparticles, as an alternative to carbonate, exerts beneficial effects within the vasculature and improves the blood antioxidant status. METHODS: Male Wistar rats were fed for 8 weeks with a diet supplemented with Cu (6.5 mg Cu/kg in the diet) either as nanoparticles (40 nm diameter) or carbonate - the control group. Moreover, a negative control was not supplemented with Cu. At 12 weeks of age, blood samples, internal organs and thoracic aorta were taken for further analysis. Blood antioxidant mechanism was measured together with Cu and Zn. RESULTS: Diet with Cu as nanoparticles resulted in an elevated catalase activity and ferric reducing ability of plasma, however decreased Cu (plasma), and ceruloplasmin (Cp) compared to carbonate. The participation of vasoconstrictor prostanoid was increased, as indomethacin did not modify the acetylcholine (ACh)-induced response. Arteries from Cu nanoparticle and carbonate rats exhibited a reduced maximal contraction to potassium chloride and an increased response to noradrenaline. The endothelium-dependent vasodilation to ACh was enhanced while exogenous NO donor, sodium nitroprusside, did not modify the vascular response. Down-regulation of BKCa channels influenced hyperpolarizing mechanism. The superoxide dismutase and HDL-cholesterol were decreased opposite to an increased lipid hydroperoxides, malondialdehyde, Cu (plasma and liver) and Cp. CONCLUSION: Despite the increased antioxidant capacity in blood of Cu nanoparticle fed rats, vasoconstrictor prostanoids and NO are involved in vascular regulation.
Asunto(s)
Antioxidantes/metabolismo , Cobre/administración & dosificación , Nanopartículas/administración & dosificación , Óxido Nítrico/metabolismo , Prostaglandinas/metabolismo , Acetilcolina/metabolismo , Animales , Ceruloplasmina/metabolismo , Suplementos Dietéticos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , Nitroprusiato/metabolismo , Cloruro de Potasio/metabolismo , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Previous studies have investigated the cardiovascular activity of Gynura procumbens Merr. single-solvent extracts. The objective of this study was to evaluate the in vitro vasorelaxant properties and the underlying pharmacological mechanisms of serial extracts and fractions of Gynura procumbens (GP). The leaves of GP were serially extracted with petroleum ether, chloroform, methanol and water using the maceration method. Suspended aortic ring preparations were pre-contracted with phenylephrine (PE 1 µM), followed by cumulative addition of GP extracts (0.25-3 mg/mL). The petroleum ether extract (GPPE) was the most potent among the four extracts. Pre-incubation of endothelium-intact aorta with atropine (1 µM), indomethacin (10 µM), methylene blue (10 µM), propranolol (1 µM) and potassium channel blockers such as TEA (1 µM), glibenclamide (10 µM), 4-aminopyridine (1 µM) and barium chloride (10 mM) had no effect on GPPE-induced vasorelaxation. The vasorelaxant effect of GPPE was partly diminished by pretreatment of aortic rings preparations with L-NAME (10 µM) and even more so in endothelium-denuded aortic rings, indicating a minimal involvement of endothelium-dependent pathway in GPPE-induced vasorelaxation. The calcium-induced vasocontractions were antagonized significantly and concentration-dependently by GPPE in calcium free and high potassium medium. These results illustrate that Ca2+ antagonizing actions of GPPE in rat isolated aorta are comparable to that of verapamil and may be mainly responsible for its vasodilation effect. The antioxidant activity of GPPE supports its vasorelaxant effect by attenuating the production of deleterious free radicals and reactive oxygen species in the vasculature.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Asteraceae/química , Extractos Vegetales/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Aorta Torácica/metabolismo , Calcio/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Masculino , NG-Nitroarginina Metil Éster/metabolismo , Fitoterapia/métodos , Hojas de la Planta/química , Ratas , Ratas Sprague-DawleyRESUMEN
AIM: This study aims to investigate the effect of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Ex) on placental tissue and angiogenesis in rats with preeclampsia (PE). METHOD: The expression of MSC surface markers were identified by flow cytometry. Alizarin red staining and oil red O staining were used to examine osteogenic and adipogenic differentiation of hucMSCs. Western blotting was used to determine expressions of CD63 and CD81 in hucMSC-Ex. PE rat models were established using endothelial nitric oxide synthase, eNOS)NG-Nitro-l-arginine Methyl Ester, which were then treated with exosome (Exo) of low dosage (L-Exo), Exo of medium dosage (M-Exo) and Exo of high dosage (H-Exo). The blood pressure at the 15d, 17d and 19d of pregnancy and 24-h urinary protein were measured. TUNEL staining and immunohistochemistry were applied to detect the cell apoptosis and micro-vascular density (MVD) in placental tissues, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to examine serum levels of vascular endothelial growth factor (VEGF) and soluble fms-like tyrosine kinase receptor-1 (sFlt1). RESULTS: In vitro cultured hucMSCs showed expression of MSC surface markers (CD29, CD90 and CD105), and no expression of CD34 and CD45. Besides, the isolated exosomes expressed the exosome markers (CD63 and CD81). In response to the treatment of L-Exo, M-Exo and H-Exo, the blood pressure of PE rat models on the 17 d and the 19 d as well as the 24-h urinary protein were substantially decreased. Moreover, at the 21 d, PE rat models treated with L-Exo, M-Exo and H-Exo exhibited an increase in the number and quality of fetuses, placenta quality, MVD and VEGF expression, but substantial decreased cell apoptosis and expression of sFlt1. The influence of Exos was exerted in a dosage dependent manner. CONCLUSION: hucMSC-Ex, in a dose-dependent manner, can improve the morphology of placental tissue in rats with PE, by inhibiting cell apoptosis and promoting angiogenesis in placental tissue.
Asunto(s)
Exosomas/metabolismo , Células Madre Mesenquimatosas/citología , Neovascularización Patológica/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Sustancias Protectoras/metabolismo , Cordón Umbilical/citología , Animales , Apoptosis/fisiología , Presión Sanguínea/fisiología , Diferenciación Celular/fisiología , Femenino , Humanos , Etiquetado Corte-Fin in Situ/métodos , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Embarazo , Ratas , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO) synthase type III (NOS-3) overexpression induces cell death in hepatoblastoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. The first-generation adenoviruses were designed to overexpress NOS-3 or green fluorescent protein, and luciferase complementary DNA under the regulation of murine alpha-fetoprotein (AFP) and Rous Sarcoma Virus (RSV) promoters, respectively. Both adenovirus and Hepa 1-6 cells were used for in vitro and in vivo experiments. Adenoviruses were administered through the tail vein 2 weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8, -9 and -3 activities in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by Nω-nitro-l-arginine methyl ester hydrochloride, p53 and CD95 small interfering RNA. AFP-NOS-3/RSV-luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.
Asunto(s)
Carcinoma Hepatocelular/terapia , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Neoplasias Hepáticas/terapia , Óxido Nítrico Sintasa de Tipo III/genética , Adenoviridae/genética , Animales , Carcinoma Hepatocelular/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular Tumoral , Proliferación Celular , Daño del ADN , ADN Complementario/genética , ADN Complementario/metabolismo , Modelos Animales de Enfermedad , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Vectores Genéticos , Hígado/citología , Hígado/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/terapia , Neoplasias Hepáticas/genética , Ratones , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Virus del Sarcoma de Rous/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMEN
Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 µmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 µmol·L(-1)), significantly blocked SCU (10-1 000 µmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 µmol·L(-1)), did not significantly change the effects of SCU (10-1 000 µmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 µmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 µmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.
Asunto(s)
Apigenina/farmacología , Hipoxia de la Célula , Vasos Coronarios/efectos de los fármacos , Glucuronatos/farmacología , Daño por Reperfusión/fisiopatología , Vasodilatación/efectos de los fármacos , Animales , Moléculas de Adhesión Celular/efectos de los fármacos , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico , Proteínas de Microfilamentos/efectos de los fármacos , NG-Nitroarginina Metil Éster/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Fosfoproteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/complicaciones , Transducción de Señal/efectos de los fármacos , Tionucleótidos/metabolismo , Tionucleótidos/farmacología , Vasodilatación/fisiologíaRESUMEN
OBJECTIVE: The aim of this study was to elucidate the mechanism(s) responsible for the vasorelaxant effect of Nigella sativa (N. sativa). METHODS: The activity of different concentrations of N. sativa extract was evaluated on contractile responses of isolated aorta to KCl and phenylephrine (PE). RESULTS: The extract (2-14 mg/mL) induced a concentration dependent relaxation both in endothelium-intact and endothelium-denuded aortic rings precontracted by PE (10(-6) M) and KCl (6 × 10(-2) M). Extract reduced PE- and KCl-induced contractions in presence of cumulative concentrations of calcium (10(-5)-10(-2) M) significantly. L-NAME and indomethacin had no effect on vasorelaxation effect of extract in PE-induced contraction. Diltiazem and heparin reduced significantly this vasorelaxation at a concentration of 14 mg/mL of extract; however, N. sativa-induced relaxation was not affected by ruthenium red. Tetraethylammonium chloride reduced the extract-induced relaxation in concentrations of 2-6 mg/mL of extract significantly but glibenclamide reduced this relaxative effect in all concentrations of extract. CONCLUSIONS: The inhibitory effect of N. sativa seed extract on the contraction induced by PE and KCl was endothelium-independent. This relaxation was mediated mainly through the inhibition of Ca(2+) and KATP channels and also intracellular calcium release.
Asunto(s)
Aorta/efectos de los fármacos , Canales de Calcio/metabolismo , Endotelio Vascular/efectos de los fármacos , Nigella sativa/química , Extractos Vegetales/farmacología , Canales de Potasio/metabolismo , Semillas/química , Vasodilatación/efectos de los fármacos , Animales , Aorta/metabolismo , Calcio/metabolismo , Endotelio Vascular/metabolismo , Contracción Muscular/efectos de los fármacos , NG-Nitroarginina Metil Éster/metabolismo , Extractos Vegetales/química , Ratas , Ratas Wistar , Vasodilatadores/farmacocinéticaRESUMEN
Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca(2+) and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Glutamatos/farmacología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico/biosíntesis , Extractos Vegetales/farmacología , Apoptosis/efectos de los fármacos , Calcio/análisis , Calcio/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Flavonoides/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Morfolinas/farmacología , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ornitina/análogos & derivados , Ornitina/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Té/química , Vasodilatación/efectos de los fármacosRESUMEN
The mechanisms underlying the regenerative capacity of endothelial progenitor cells (EPCs) are not fully understood. We hypothesized that biosynthesis of tetrahydrobiopterin is an important mechanism responsible for the stimulatory effects of peroxisome proliferator-activated receptor-δ (PPARδ) activation on regenerative function of human EPCs. Treatment of human EPCs with a selective PPARδ agonist GW501516 for 24 hours increased the levels of mRNA, protein, and enzymatic activity of GTP cyclohydrolase I (GTPCH I), as well as the production of tetrahydrobiopterin. The effects of GW501516 were mediated by suppression of PTEN expression, thereby increasing phosphorylation of AKT. The AKT signaling also mediated GW501516-induced phosphorylation of endothelial NO synthase. In addition, activation of PPARδ significantly enhanced proliferation of EPCs. This effect was abolished by the GTPCH I inhibitor, 2,4-diamino-6-hydroxypyrimidine, or genetic inactivation of GTPCH I with small interfering RNA but not by inhibition of endothelial NO synthase with N(G)-nitro-l-arginine methyl ester. Supplementation of NO did not reverse 2,4-diamino-6-hydroxypyrimidine-inhibited 5-bromodeoxyuridine incorporation. Furthermore, transplantation of human EPCs stimulated re-endothelialization in a mouse model of carotid artery injury. Pretreatment of EPCs with GW501516 significantly enhanced the ability of transplanted EPCs to repair denuded endothelium. GTPCH I-small interfering RNA transfection significantly inhibited in vivo regenerative capacity of EPCs stimulated with GW501516. Thus, in human EPCs, activation of PPARδ stimulates expression and activity of GTPCH I and biosynthesis of tetrahydrobiopterin via PTEN-AKT signaling pathway. This effect enhances the regenerative function of EPCs.
Asunto(s)
Biopterinas/análogos & derivados , Células Endoteliales/fisiología , PPAR delta/metabolismo , Regeneración/fisiología , Células Madre/fisiología , Animales , Biopterinas/biosíntesis , Proliferación Celular , GTP Ciclohidrolasa/metabolismo , Humanos , Ratones , NG-Nitroarginina Metil Éster/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Spontaneously hypertensive rat (SHR) offspring from L-arginine- and antioxidant-supplemented SHR dams had persistent lower blood pressure in adulthood. We investigated the influence of vascular mechanism in this effect. We analyzed response to acetylcholine and phenylephrine in aorta and superior mesenteric arteries from Wistar-Kyoto (WKY), SHR, and SHR perinatally supplemented with L-arginine and 4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl (TEMPOL; SHR-suppl). Supplements reduced blood pressure persistently in SHR. Relaxation to acetylcholine was greater in WKY than SHR and remained unmodified in SHR-suppl compared with SHR. Acute TEMPOL did not alter relaxation to acetylcholine in WKY but increased it similarly in SHR and SHR-suppl. Phenylephrine contraction was increased in SHR compared to WKY. In SHR-suppl, this response was similar to SHR. Endothelium removal or N-nitro-L-arginine methyl ester (L-NAME) increased contraction to phenylephrine more in WKY than SHR. In SHR-suppl, this was similar to SHR. In both SHR and SHR-suppl, TEMPOL similarly reduced phenylephrine response. This effect was prevented by L-NAME. Results exposed reinforce the concept that oxidative stress during perinatal period is a contributing factor to the development of hypertension in SHR. Results also reveal that the beneficial effect of this supplementation does not appear to be related to improved endothelial function, suggesting that other regulatory mechanisms of blood pressure may be involved.
Asunto(s)
Antioxidantes/metabolismo , Arginina/metabolismo , Acetilcolina/química , Animales , Animales Recién Nacidos , Antioxidantes/química , Presión Sanguínea , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/farmacología , Relación Dosis-Respuesta a Droga , Hipertensión/tratamiento farmacológico , Hipertensión/patología , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico/química , Estrés Oxidativo , Fenilefrina/química , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Marcadores de SpinRESUMEN
We simultaneously assessed benefits and risks of niacin-bound chromium (NBC) intake at varying doses over a prolonged period of time (>1.2 years) in male and female Sprague-Dawley (SD) rats. We performed the study in two phases. First, we followed 60 male and 60 female SD rats, each gender divided into six groups. Through day 150 (phase 1A), all SD rats received a high sucrose diet (30% w/w) with or without different concentrations of NBC. The male/female groups were: 1] control without NBC n = 10, 2] low NBC (2.8 ppm, n = 10), 3] medium NBC (8.7 ppm, n = 20), 4] high NBC (28.0 ppm, n = 20). Based on dosing, we refer to the three treatment groups as 1X, 3X, and 10X. During days 151-312 (phase 1B), NBC was removed from diets of one half of the 3X and 10X groups. These are referred to as 3X satellite and 10X satellite. In phase 2 (days 313-460), males from groups 1X, 3X, 10X, 3X satellite, and 10X satellite received the same 3X dose of NBC (8.7 ppm). The last two groups also ingested different doses of a formulation of natural products in addition to NBC. We examined blood pressure, the renin-angiotensin system (RAS), nitric oxide (NO), and insulin systems and inflammatory parameters. Results in male and female SD rats were comparable. NBC lowered systolic blood pressure (SBP) in a dose-dependent fashion; however, after 200 days, the SBP of the low dose group (1X) began to rise and returned to baseline control. After raising the dose of NBC to 3X, the SBP in the 1X group decreased significantly once more. When half the test rats (3X and 10X) were deprived of NBC, SBP rose gradually to control levels after 2 to 3 months. However, the SBP decreased significantly once more when each satellite group returned to the 3X dose. Special testing suggests that NBC at adequate dosing increases insulin sensitivity, lowers HbA1C, decreases activity of the RAS, at least in part, through ACE inhibition, enhances NO activity, and is without signs of toxicity. The addition of a formula composed of antioxidants and immune modulators to the chromium regimen caused even faster and more profound changes in SBP than with NBC alone. We conclude that NBC at adequate dosing is effective in male and female SD rats on certain metabolic parameters over a prolonged period, effects that disappear over months after NBC is removed. When dosing is returned, the effectiveness of NBC returns. Low doses of NBC may lose their effect over time. No signs of toxicity were observed.
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Cromo , Dieta , Suplementos Dietéticos , Niacina , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Animales , Análisis Químico de la Sangre , Glucemia/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Cromo/administración & dosificación , Cromo/química , Cromo/metabolismo , Ingestión de Líquidos , Ingestión de Alimentos , Inhibidores Enzimáticos/metabolismo , Femenino , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Losartán/metabolismo , Masculino , NG-Nitroarginina Metil Éster/metabolismo , Niacina/administración & dosificación , Niacina/química , Niacina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: The initial development of pulsed electromagnetic field (PEMF) therapy and its evolution over the last century for use in clinical surgery has been slow, primarily because of lack of scientifically-derived, evidence-based knowledge of the mechanism of action. OBJECTIVE: Our objective was to review the major scientific breakthroughs and current understanding of the mechanism of action of PEMF therapy, providing clinicians with a sound basis for optimal use. METHODS: A literature review was conducted, including mechanism of action and biologic and clinical studies of PEMF. Using case illustrations, a holistic exposition on the clinical use of PEMF in plastic surgery was performed. RESULTS: PEMF therapy has been used successfully in the management of postsurgical pain and edema, the treatment of chronic wounds, and in facilitating vasodilatation and angiogenesis. Using scientific support, the authors present the currently accepted mechanism of action of PEMF therapy. CONCLUSIONS: This review shows that plastic surgeons have at hand a powerful tool with no known side effects for the adjunctive, noninvasive, nonpharmacologic management of postoperative pain and edema. Given the recent rapid advances in development of portable and economical PEMF devices, what has been of most significance to the plastic surgeon is the laboratory and clinical confirmation of decreased pain and swelling following injury or surgery.
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Medicina Basada en la Evidencia , Magnetoterapia , Procedimientos de Cirugía Plástica/instrumentación , Cicatrización de Heridas/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Enfermedad Crónica , Fracturas Óseas/terapia , Humanos , NG-Nitroarginina Metil Éster/metabolismo , Procedimientos de Cirugía Plástica/métodos , Resistencia a la Tracción/fisiologíaRESUMEN
Ascertaining the upstream regulatory mechanisms of hyperthermia-induced apoptosis is important to understand the role of hyperthermia in combined modality cancer therapy. Accordingly, we investigated whether (i) hyperthermia-induced apoptosis is mediated through the nitric oxide (NO) signaling pathway and (ii) inhibition of post-translational modification of IkappaBalpha and down regulation of NFkappaB-DNA binding activity is an intermediate step in NO-dependent apoptosis in MCF-7 breast cancer cells. For hyperthermia treatment, the cells were exposed to 43 degrees C. Intracellular NO levels measured by the fluorescent intensity of DAF-2A and iNOS expression by immunobloting revealed an increased level of iNOS dependent NO production after 43 degrees C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43 degrees C treatments. EMSA analysis showed a dose-dependent inhibition of NFkappaB-DNA binding activity. The hyperthermia-mediated inhibition of NFkappaB was persistent even after 48 h. Inhibition of NO by L-NAME rescued the NFkappaB-DNA binding activity and inhibits heat-induced apoptosis. Similarly, over-expression of NFkappaB by transient transfection inhibits heat-induced apoptosis. These results demonstrate that apoptosis upon hyperthermia exposure of MCF-7 cells is regulated by NO-mediated suppression of NFkappaB.
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Apoptosis/fisiología , Neoplasias de la Mama/terapia , Hipertermia Inducida , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Animales , Línea Celular Tumoral , Terapia Combinada , ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , Proteínas I-kappa B/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: There is a major societal concern relating to the addictive properties of analgesic drugs such as morphine with regard to alleviating pain. Because of this, alternative methods of pain relief are, and have been, actively pursued. An extremely promising method for treatment of low to moderate levels of chronic pain in humans is transcutaneous electrical nerve stimulation (TENS). MATERIAL/METHODS: All experiments utilized the invertebrate marine bivalve mollusc Mytilus edulis pedal ganglia. TENS was achieved using a stimulation apparatus developed by Professor Han of Peking University. TENS experiments employed 2 stimulation protocols: 1) low 2 Hz frequency at 5 mA current, 2) alternating low and high frequencies at 2 and 100 Hz, respectively at 5 mA current. Real-time measurements of nitric oxide (NO), using an amperometric probe, measured NO released into the tissue bath subsequent to TENS. RESULTS: Pooled M. edulis pedal ganglia exposed to TENS demonstrate that stimulation at 2 Hz and 5 mA current promotes time-dependent release of NO. In another experiment, pooled ganglia were stimulated at alternating frequencies of 2 Hz and 100 Hz and 5 mA, which also released NO in a time-dependent manner. Unstimulated control ganglia did not release significant amounts of NO. NO release was antagonized by naloxone and L-NAME exposure, demonstrating that it was receptor and nitric oxide synthase mediated, respectively. CONCLUSIONS: It would appear that TENS stimulates endogenous morphine release since NO release was blocked by naloxone and opioid peptides do not release NO. The present study is highly suggestive of the occurrence of this same mechanism in mammalian neural systems since all biochemical and signaling components are present. Furthermore, it would appear that this process has evolutionary survival value since it occurs in an animal that evolved 500 million years ago.
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Neuronas/metabolismo , Óxido Nítrico/metabolismo , Péptidos Opioides/metabolismo , Estimulación Eléctrica Transcutánea del Nervio/métodos , Animales , Calibración , Ganglios/metabolismo , Modelos Biológicos , Morfina/farmacología , Mytilus edulis/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , Naloxona/farmacología , Sistema Nervioso/metabolismo , Dolor/tratamiento farmacológico , Transducción de SeñalRESUMEN
Se estudiaron con herramientas morfológicas, los efectos de diferentes dosis de Losartan sobre el remodelamiento cardiovascular, en ratas deficientes en óxido nítrico. 30 ratas Wistar, con 15 semanas de edad, fueron separadas en 6 grupos. control (C), L-NAME (LN), y 4 grupos en que administró LN junto con Losartan, en diferentes dosis (1, 5, 20 y 40 mg/kg/dia). El L-NAME fue administrado durante 9 semanas y la administración de Losartan se inició en la segunda semana de experimentación. Se estudiaron el corazón, la parte torácica de la aorta y la arteria mesentérica craneal, con microscopía de luz y estereología. La presión arterial (PA) aumentó desde la primera semana de administración de L-NAME. El tratamiento con Losartan, en las dosis de 20 y 40 mg/kg/día, fue eficiente para reducir la PA después de la séptima semana de tratamiento. El remodelamiento cardiaco adverso en el grupo LN se caracterizó por intensa fibrosis intersticial, disminución de la microvascularización miocárdica e hipertrofia y consecuente pérdida de cardiomiocitos. La estructura de la pared de la aorta (densidad por área de núcleos de células musculares lisas y densidad de superficie de lamelas), y la relación media/luz de la arteria mesentérica craneal, también fueron muy alteradas por la administración de L-NAME. Sólo en una dosis igual o mayor que 20 mg/kg/día, el Losartan tuvo efecto benéfico tratando estas alteraciones. En conclusión, tanto el corazón como la pared arterial de ratas deficientes en óxido nítrico, presentan un proceso de remodelamiento acentuado, y éste es eficientemente tratado con Losartan en diferentes dosis. La eficiencia del tratamiento con Losartan en el modelo de bloqueo de la síntesis de óxido nítrico se correlaciona con el efecto hipotensor de la droga, principalmente en las dosis más elevadas.
We studied with morphological tools the effects of different doses of Losartan upon the cardiovascular remodeling in nitric oxide deficient rats. At 15 weeks of age, thirty Wistar rats were separated in six groups: control (C), L-NAME (LN), and four groups were LN was given plus Losartan at different doses (1, 5, 20 and 40 mg/kg/day). The L-NAME was given for 9 weeks, the Losartan administration starting on the 2nd week of experiment. We studied the heart, thoracic aorta and superior mesenteric artery with light microscopy and stereology. The blood pressure (BP) increased since the first week of L-NAME administration, the Losartan treatment at doses of 20 and 40 mg/kg/day was efficient to reduce BP after the 7th week of treatment. The cardiac adverse remodeling in the LN group was characterized by intense interstitial fibrosis, impairment of the myocardial microvascularization, cardiomyocyte hypertrophy and consequent loss of cardiomyocytes. The aortic wall structure (density per area of smooth muscle cell nuclei and surface density of lamellae), and the superior mesenteric artery media/lumen ratio were also strongly affected by L-NAME administration. Only in the dose equal or higher than 20 mg/kg/day Losartan showed beneficial effects treating these alterations. In conclusion, both the heart and the arterial wall of NO deficient rats suffer a marked adverse remodeling process that is efficiently treated by a dose-dependent Losartan administration. The efficiency of Losartan treatment in this model of NO synthesis blockade correlates with the hypotensor effect of the drug mainly in the high dose treatment.
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Animales , Recién Nacido , NG-Nitroarginina Metil Éster/administración & dosificación , NG-Nitroarginina Metil Éster/metabolismo , NG-Nitroarginina Metil Éster/uso terapéutico , Sistema Renina-Angiotensina , Sistema Renina-Angiotensina/fisiología , Fenómenos Fisiológicos Cardiovasculares , Ratas Wistar/anatomía & histología , Ratas Wistar/crecimiento & desarrolloRESUMEN
Electroacupuncture (EA) is widely used to treat disorders of the nervous system, such as stroke. The aim of the present study was to investigate the effect of EA on cerebral blood flow (CBF) in cerebral ischemic rats. We developed an animal model of cerebral ischemia (CI) by occluding the blood flow of both common carotid arteries in Sprague-Dawley (SD) rats; 2 or 15 Hz EA was applied to both Zusanli acupoints. The levels of nitric oxide (NO) in the peripheral blood and amounts of calcitonin gene-related peptide (CGRP) in the cerebral cortex and thalamus were measured. In addition, L-N (G)-nitro arginine methyl ester (L-NAME) was used to measure the changes in CBF induced by EA in rats with and without CI. The results indicated that both 2 and 15 Hz EA increase the mean CBF in rats with and without CI. However, neither 2 nor 15 Hz EA induced changes in levels of NO in peripheral blood or changes in CGRP levels in cerebral cortex and thalamus. In addition, L-NAME did not change the increase in CBF. We concluded that both 2 and 15 Hz EA at both Zusanli acupoints induced the increase of CBF in rats with and without CI. Whether the effect of EA is related to NO or CGRP will be investigated in a future study.
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Isquemia Encefálica/fisiopatología , Circulación Cerebrovascular , Electroacupuntura , Animales , Encéfalo/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Masculino , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.