Effect of PGEI,on Production of NO and the Activity of eNOS in Human Umbilical Vein Endothelial Cells.
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Objective To evaluate the effects of PGE_1 on Production of No and the activity of eNOS in human umbilical vein endothelial cells(HUVECs).Methods Nitric oxide(NO) production and the activity of nitric oxide synthase(NOS) in HUVECs were measured by No and NOS assay kits,after HUVECs incubated with PGE_1 alone at different concentrations(0.04-0.4 μg/ml) and different times(2-4h) or HUVECs incubated with PGE_1 as pretreatment before TNF-α 10ng/ml.Results(1) Activity of eNOS and the expression of NO were up-regulated by PGE_1 in concentration-dependent manner.(2) Short time intervention with PGE_1 had little effects on both activity of eNOS and the expression of NO.However,the expression of NO was increased after 12h incubation and the activity of eNOS was enhanced after 24h treatment.(3) PGE_1 could prevent eNOS activity inhibition induced by TNF-α.Conclusions PGE_1 has a protection effect on endothelial cells by enhancing eNOS activity and increasing NO production,besides,it could prevent eNOS activity inhibition induced by TNF-α.Cite
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Objective:To explore the effects of Xiaokening in high glucose on nitric oxide synthase(NOS),nitric oxide(NO) and peroxynitrite(ONOO-) produced by vein endothelial cells.Methods:Human umbilical vein endothelial cells strain ECV-304 were divided into seven groups:normal groups:high-glucose group(30mM) and the Xiaokening groups(20,40,60,80,100g/L).The eNOS,iNOS,NO and nitrotyrosine(NT) were detected at the end of the experiment.Results:Compared with high-glucose group,eNOS activity increased in Xiaokening groups,(P 0.05).The change in 100g/L was greater than that in 20g/L(P 0.05).While iNOS,NO and NT decreased.Conclusion:Xiaokening can take more effect on inducing eNOS and inhibiting iNOS,NO and ONOO-release from vein endothelial cells than that of high glucose.
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Objective To investigate the effect of uric acid(UA) on the expression of endothelial nitric oxide synthase(eNOS) and the production of nitric oxide in cultured human umbilical vein endothelial cells(HUVECs).Methods HUVECs were incubated with different concentrations of UA(0,0.5,1,1.5,2 mg/L) and 50 mg/L ox-LDL(as positive control) for 24,48 and 72 h.Then,eNOS mRNA was detected by RT-PCR.The expression of eNOS protein was observed by Western blot.NO in supernatant medium was analyzed by enzymic method.ResultsUA 5 mg/dl group increased the expression of eNOS mRNA of HUVECs as compared with that from placebo control group(P0.05).While the concentration of UA increasing and time runing,the expression of eNOS and the production of NO and ox-LDL decreased significantly(72 h NO2-/NO3-is 0.52±0.18 versus 1.00±0.1 for 2 mg/L versus control,P0.05).Conclusion UA inhibits the expression of eNOS and the production of NO of HUVECs in a dose-dependent and time-dependent manner.It suggests that UA may damage the function of endothelial cells and may influence the genesis and development of cardiovascular diseases.
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The present study investigated the relationship between uncoupling of endothelial nitric oxide synthase (eNOS) and vascular endothelial cell (VEC) oxidative stress (OS) during sepsis and the role of eNOS glutathionylation in eNOS uncoupling of septic VECs. Human umbilical vein endothelial cells (HUVECs) cultured in vitro (EA.hy269 cell line) were incubated with Dulbeccos modified Eagles medium (DMEM) (normal control group), lipopolysaccharide (LPS) (sepsis group), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (glutathionylation group), and LPS+ dithiothreitol (DTT) (deglutathionylation sepsis group). As result, compared with the DMEM group, malondialdehyde (MDA) level and uncoupling eNOS activity significantly increased in the LPS and BCNU groups. However, in the LPS + DTT group, only the NO level increased. Compared with the LPS group, MDA level, NO concentration, and normal functional eNOS activity significantly decreased, and uncoupling eNOS activity significantly increased in the BCNU group. In the LPS + DTT group, MDA level and uncoupling eNOS activity significantly decreased, and NO concentration and normal functional eNOS activity significantly increased. During sepsis, the main mechanism for VEC OS was eNOS uncoupling mediated by eNOS glutathionylation.
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Dithiothreitol
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Objective:To investigate the effect of fluvastatin on the intracellular Ca2+ level and endothelial nitric oxide synthase(eNOS)activity in cultured human umbilical vein endothelial cells(HUVECs).Method:Cultured HUVECs were randomly divided into 5 groups:control,fluvastatin(10-8-10-5 mol/L).Nitric oxide(NO)was measured by colorimetry.L-[3H]-arginine and L-[3H]-citrulline were measured by scinillation counting.[Ca2+]i was observed by laser-scanning confocal microscope.Result:Incubation HUVECs with 10-8-10-5 mol/L fluvastatin for 12 h increased intracellular Ca2+ level,eNOS activity and NO release in a concentration-dependent manner.In addition,10-5 mol/L fluvastatin improved eNOS activity in a time-dependent way at different time(0,6,12 h),and eNOS activity was most promoted by fluvastatin after 12 h incubation.Conclusion:Fluvastatin dose-dependently increases HUVECs eNOS activity and NO release,which is related to increased intracellular Ca2+ level.
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Objective To study the effect of recombinant human C-reactive protein (rhCRP) on platelet endo- thelial nitric oxide synthase (eNOS) activity.Methods Peripheral venous blood was collected and platelets were i- solated with gel-filtration chromatography and incubated with histamine.L-NAME and different concentrations of rhCRP(5,10,25,50,100 mg/L) were co-cultured with platelet for 30 minutes,eNOS activity was measured by production of ~3H L-citrulline from ~3 H-L-arginine.Results Platelet eNOS activity was significantly inhibited after incubated platelets with L-NAME and increased after incubated with histamine (P0.05).rhCRP significantly in- hibited eNOS activity increase by histamine in a concentration-dependent manner(P0.05).Conclusion rhCRP significantly inhibits eNOS activity induced by histamine in platelets in a concentration-dependent manner.
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Objective To study the effects of mitiglinide on nitric oxide(NO)production and nitric oxide synthase(NOS)activity in human aortic endothelial cells(HAECs).Methods HAECs were cultured and incubated with glucose 30mmol/L(group A),glucose 30mmol/L+mitiglinide10μmol/L(group B)and glucose 5.5mmol/L(group C),respectively.Cell lysate was collected and the contents of NO,endothelial nitric oxide synthase(eNOS)and inducible nitric oxide synthase(iNOS)were measured by ELISA.The expressions of eNOS mRNA and protein were analyzed by RT-PCR and Western blot,respectively.Results Compared with group C,the content of NO was increased at 24hours and decreased at 48hours and 72hours after incubation in group A(P0.05).Compared with group A,the content of NO was decreased at 24hours and increased at 48hours and72hours after incubation in group B(P0.05).Compared with group A,the content of iNOS was decreased and the expressions of eNOS mRNA and protein were increased in group B(P0.05).Conclusion Mitiglinide is able to decrease iNOS activity,increase eNOS activity and its expression,and improve NO production in cultured HAECs with high glucose.
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This study examined the effect of acetate on endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) by immunoblotting assay and the ability of acetic acid to upregulate flow-mediated vasodilatation in humans. In HUVECs, acetate induced a biphasic increase in the phosphorylated form of eNOS. The amount of phosphorylated eNOS was significantly increased by exposure to 200 μmol/l acetate for 20 min (early phase) and for 4 h (late phase). The inhibitors of cAMP-dependent protein kinase (PKA) and AMP-activated protein kinase (AMPK) blocked acetate-induced eNOS phosphorylation in the early and the late phase respectively. Furthermore, in postmenopausal women, maximum forearm blood flow (FBF) in response to shear stress increased in the vinegar (acetic acid) administered group compared to the placebo group. These results suggest that acetic acid-induced eNOS phosphorylation contributes to upregulation of flow-mediated vasodilatation in humans.
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Objective To investigate the effect of TNF-α on production of nitric oxide (NO) and activity of nitric oxide synthase(eNOS) in human umbilical vein endothelial cells(HUVECs). Methods HUVECs were incubated with TNF-α at different concentrations (2-20ng/ml) and different times(2-48h) or pretreatment with L-NMMA or DXM. Nitric oxide production and activity of nitric oxide synthase in HUVECs were measured by NO and NOS assay kits. Results (1) Activity of eNOS was down-regulated by TNF-α in a concentration-dependent manner, while production of NO was upregulated. (2) The activity of eNOS attenuated after long-time treatment with TNF-α, but marked increased production of NO was detected after 24h. (3) The induction of NO by TNF-α can be inhibited by L-NMMA and DXM. Conclusion TNF-α can reduce activity of eNOS and induce iNOS, so that production of NO is increased.
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【Objective】To investigate the effects of fenofibrate on nitric oxide(NO) production and endothelial nitric oxide synthase(eNOS) mRNA and protein expression induced by lysophosphatidylcholine in cultured human umbilical vein endothelial cells(HUVECs).【Methods】HUVECs were cultured in vitro.The study was designated to 5 groups: ①normal control,②LPC group,③low-concentration fenofibrate(10μmol/L)control,④ middle-concentration fenofibrate(50 μmol/L)control,⑤high-concentration fenofibrate(100μmol/L)control.Expression of eNOS mRNA and protein were observed by real-time polymerase chain reaction(real-time PCR) and flow cytometry(FCM) respectively.NO was determined by nitrate reductase method.【Results】Compared with control group,LPC could down-regulate eNOS mRNA and its protein expression and decrease NO production in HUVECs.Fenofibrate could increase eNOS mRNA and its protein expression and enhance NO production in a dose-and time-dependent manner in HUVECs.【Conclusion】Fenofibrate could improve eNOS mRNA and its protein expression induced by lysophosphatidylcholine in HUVECs,that may play a very important role in the prevention and treatment of atherosclerosis.
Fenofibrate
Lysophosphatidylcholine
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