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OBJECTIVE@#To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on cardiac function of ventriculus sinister in rats with spontaneously hypertensive (SHR), and to explore the mediation effect of endothelin-1 (ET-1)/endothelial nitric oxide synthase (eNOS).@*METHODS@#Six 12-week-old male Wistar Kyoto (WKY) rats were taken as the normal group. Eighteen 12-week-old SHR were randomly divided into a model group, an EA group and a sham EA group, 6 rats in each group. The rats in the EA group were treated with EA (disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity) at "Neiguan" (PC 6), 30 min each time, once a day for 8 weeks. The rats in the sham EA group were treated with superficial needling at "Neiguan" (PC 6) with no electrical stimulation applied. After treatment, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were tested by echocardiographic analysis. The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), the maximum rate of increase/decrease of left ventricular pressure (±dp/dtmax) were detected. The serum content of ET-1 was detected by ELISA. Western blot was used to evaluate the expression of ETAR, eNOS in myocardial tissue of left ventricular.@*RESULTS@#Compared with the normal group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were decreased (P<0.01, P<0.05), while LVSP, LVEDP, +dp/dtmax and -dp/dtmax were increased (P<0.01) in the model group. Compared with the model group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were increased (P<0.01, P<0.05), and LVSP and LVEDP were decreased (P<0.01) in the EA group. Compared with the normal group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were increased (P<0.01), whereas expression of eNOS was decreased (P<0.01) in the model group. Compared with the model group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were decreased (P<0.05), whereas expression of eNOS was increased (P<0.05) in the EA group.@*CONCLUSION@#EA intervention may alleviate hypertensive cardiac function damage by up-regulating the expression of eNOS protein in myocardial tissue, down-regulating the serum content of ET-1 and the expression of ETAR protein in myocardial tissue.
Subject(s)
Animals , Male , Rats , Electroacupuncture , Endothelin-1/genetics , Heart Diseases , Hypertension/therapy , Nitric Oxide Synthase Type III/genetics , Rats, Inbred SHR , Rats, Inbred WKY , Stroke Volume , Ventricular Function, LeftABSTRACT
Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.
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Objective:To investigate the effect of Danggui Buxuetang（DGBX）on the functional activity of rat endothelial progenitor cells（EPCs）exposed to different luminar shear stress （SS）. Method:EPCs isolated from rat bone marrow were incubated on a parallel plate flow chamber at a steady SS of 0， 0.12， 1.2， 2.4 Pa for 6 h，then the cells exposed to different SS were randomly divided into 8 groups： control group （perfused with serum free medium），simvastatin group（0.1 μmol·L<sup>-1 </sup>simvastatin），3 DGBX groups（low，medium，high-dose DGBX）and 3 inhibitor groups（3 DGBX groups with LY294002）. After 12 h，the samples were collected for the detection of cell proliferation ，migration，tubule formation ，the secretion of nitric oxide （NO） ，and the expressions of endothelial nitric oxide synthase（eNOS） mRNA and protein kinase B（Akt），respectively. Result:Compared with the control group，simvastatin and DGBX（high-dose）could both promote the functional activities and NO secretion，and up-regulate the expressions of eNOS mRNA and Akt protein in EPCs exposed to different SS（<italic>P</italic><0.05），while DGBX（mid-dose）could do these only at 0 Pa. However，LY294002 could inhibit all effects of DGBX on EPCs. Conclusion:SS seems to play an important role in the effect of DGBX on EPCs，and DGBX could promote the functional activity of EPCs exposed to SS by up-regulating the expressions of NO/eNOS/Akt.
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<b>Objective::To study whether Sanhuang Xiexintang (SHXXT) can restore endothelial function by inhibiting the activation of NOD-like receptor protein 3 (NIRP3) induced by 7-ketocholesterol (7-keto) in vascular endothelial cells. <b>Method::The aortic rings of mice were cultured in normal group, model (7-keto) group, SHXXT groups (1%, 2% and 5% drug-containing serum). Vasodilation function of mice was observed. Microvascular endothelial cells were cultured according to the above experimental groups, and NIRP3 inhibitor isoglycyrrhizin (ISO) group, was also set. Western blot was used to detect the expressions of endothelial nitric oxide synthase (eNOS), NIRP3, cysteinyl aspartate specific proteinase-1 (Caspase-1), interleukin-1<italic>β</italic> (IL-1<italic>β</italic>) protein. In addition, nitric oxide (NO) quantitative kit was used to detect the concentration of NO. <b>Result::Compared with the normal group, the endothelium-dependent vasodilation function of vascular rings was significantly reduced in model group (<italic>P</italic><0.01), and the drug group significantly restored the endothelium-dependent vasodilation function in a concentration-dependent manner (<italic>P</italic><0.05, <italic>P</italic><0.01). Meanwhile, microvascular endothelial cells were also studied. Compared with the normal group, the content of eNOS protein in the model group decreased (<italic>P</italic><0.05), while the concentration of NO decreased significantly (<italic>P</italic><0.01). After treatment with SHXXT serum, eNOS and NO could be restored, with significant differences in the concentration of NO with 5% (<italic>P</italic><0.05) and 10% (<italic>P</italic><0.01) SHXXT serum. At the same time, the expressions of NIRP3 (<italic>P</italic><0.05), cle-Caspase-1 activation (<italic>P</italic><0.01) and IL-1<italic>β</italic> production (<italic>P</italic><0.01) in endothelium were significantly increased under 7-keto stimulation, and the SHXXT serum could significantly inhibit the expression and activation of relevant proteins. Subsequently, endothelial cells were treated with NIRP3 inhibitor ISO. Compared with the model group, eNOS expression increased, and NO concentration increased significantly (<italic>P</italic><0.01) after treatment with ISO, but ISO had no synergistic effect on SHXXT serum. <b>Conclusion::SHXXT can improve endothelium-dependent vascular dysfunction induced by 7-keto, which is achieved by NO signaling pathway mediated by inhibiting the activation of endothelial NIRP3-related proteins.
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PURPOSE: To investigate the effects of valproic acid on the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0.25, 0.5, and 1.0 mM valproic acid for 6, 12, and 24 hours. Expression of eNOS mRNA was assessed with Reverse transcription-polymerase chain reaction, and production of NO was assessed with Griess assay. Cellular survival was assessed with the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Valproic acid at concentrations of 0.25, 0.5, 1.0 mM did not affect the cellular survival of HTMC significantly after exposure for 24 hours. Valproic acid increased NO production in a dose- and time-dependent manner. Also, valproic acid increased the degree of eNOS mRNA expression in a dose-dependent manner in HTMC. CONCLUSIONS: Valproic acid increases production of NO and expression of eNOS mRNA in HTMC. Thus, valproic acid might increase aqueous outflow through the trabecular meshwork.
Subject(s)
Humans , Nitric Oxide Synthase Type III , Nitric Oxide Synthase , Nitric Oxide , RNA, Messenger , Trabecular Meshwork , Valproic AcidABSTRACT
Objective To further investigate the molecular mechanism of microRNA(miRNA)-24 on endothelial nitric oxide synthase(eNOS)gene expression,activity and the impact on its metabolites. Methods The plasmid of miRNA-24 was constructed and transfected to human umhilical vein endothelial cells(HUVECs). The proliferation of cells was detected by MTT test. Migration ability was measured by scratch wound model. The mRNA transcription and protein expressions of eNOS were determined by RT-PCR and Western blot respectively. The eNOS activity was detected by ELISA. The metabolite NO production in cell culture supernatant was detected by nitrate reduction. Results Compared with control group,the proliferation of endothelial cells in the miRNA-24 group was decreased by 54.32%;the migration of HUVECs was reduced by 48.62%;the expressions of eNOS mRNA and protein were de?creased by 43.92%and 42.71%,respectively. miRNA-24 suppressed eNOS activity by 73.20%. Meanwhile,the NO production was decreased by 55.29%. Conclusion miRNA-24 inhibits the synthesis and release of metabolite NO,which may be a molecular target of prevention and treatment in cardiovascular diseases.
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Objective To further investigate the molecular mechanism of microRNA(miRNA)-24 on endothelial nitric oxide synthase(eNOS)gene expression, activity and the impact on its metabolites. Methods The plasmid of miRNA-24 was constructed and transfected to human umhilical vein endothelial cells(HUVECs). The proliferation of cells was detected by MTT test. Migration ability was measured by scratch wound model. The mRNA transcription and protein expressions of eNOS were determined by RT-PCR and Western blot respectively. The eNOS activity was detected by ELISA. The metabolite NO production in cell culture supernatant was detected by nitrate reduction. Results Compared with control group, the proliferation of endothelial cells in the miRNA-24 group was decreased by 54.32%;the migration of HUVECs was reduced by 48.62%;the expressions of eNOS mRNA and protein were de? creased by 43.92% and 42.71%, respectively. miRNA-24 suppressed eNOS activity by 73.20%. Meanwhile, the NO production was decreased by 55.29%. Conclusion miRNA-24 inhibits the synthesis and release of metabolite NO, which may be a molecular target of prevention and treatment in cardiovascular diseases.
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[Objective] To investigate the effects and mechanisms of Nrf2-ARE (nuclear factor erythroid-2 related factor-anti-oxidant response element) pathway on uremic serum-mediated endothelial dysfunction in human aortic endothelial ceils.[Methods] Human aortic endothelial cells were incubated in endothelial cell medium containing 10％ normal serum,10％ non-diabetic nuremic serum or 10％ diabetic uremic serum respectively,and 20 μmol/L tertiary butyl hydroquinone (tBHQ) were pretreated with cells to active Nrf2-ARE pathway.The cells apoptosis rate were measured by flow cytometry,and the synthesis of NO was detected by flow cytometry and immune fluorescent confocal,while the expression of P-eNOSer1177/eNOS,and quinone oxidoreductase-1 (NQO1) were measured by western blotting.The levels of malondialdehyde,superoxide dismutase,eatalase,and glutathione in these cells were also measured with kits.[Results] Aortic endothelial cells incubated with uremic serum had a higher level of apoptosis rate and MDA (P ＜ 0.05),and a lower level of NO systhesis,P-eNOSSer1177/eNOS expression,CAT,SOD,GSH (P ＜ 0.05).Pretreated with tBHQ can reduce the apoptosis rate and MDA level (P ＜ 0.05),improve the amount of NO systhesis,the expression of P-eNOSSer1177/eNOS,the levels of CAT,SOD,and GSH in these cells (P ＜ 0.05).[Conclusion] Activation of Nrf2-ARE pathway can improve endothelial dysfunction in aortic endothelial cells induced by uremic serum,and its mechanism might be related with enhancement of the antioxidant stress.
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PURPOSE: To investigate the effects of Rho kinase (ROCK) inhibitor on the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0 µM, 10 µM or 100 µM Y-27632 for 3 days and NO production was assessed using Griess assay. After 24 hours, the effect of Y-27632 on the contraction of collagen matrix and the permeability of the HTMC monolayer was determined. The expression of eNOS mRNA was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and cellular survival with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: In HTMC, 10 µM and 100 µM Y-27632 significantly increased NO production after 1 day and 3 days (p = 0.020 and 0.001, respectively). At 1 day after exposure, Y-276320 significantly relaxed the collagen matrix and increased the permeability of the HTMC monolayer (all p = 0.001) and the eNOS mRNA expression (p = 0.039). CONCLUSIONS: Increased NO production may play a role in the mechanism of increased trabecular outflow associated with ROCK inhibitor.
Subject(s)
Humans , Collagen , Nitric Oxide Synthase Type III , Nitric Oxide , Permeability , rho-Associated Kinases , RNA, Messenger , Trabecular MeshworkABSTRACT
Background & objectives Endothelial nitric oxide is a potent vasodilator and impairment of its generation brought about by gene polymorphism is considered a major predictor for several diseases. A single nucleotide polymorphism G894T within exon 7 of endothelial nitric oxide synthase (eNOS-7) gene, resulting in a replacement of glutamic acid by aspartic acid, has been studied as a putative candidate gene for cardiovascular diseases. The pattern of eNOS-7 Glu298Asp variant in the Indian population is poorly known. The present study was planned to determine the prevalence of the variant of this gene among tea garden community in Assam, North-East India with high prevalence of hypertension. Methods Study participants of both sex aged ≥18 yr were recruited randomly from temporary field clinics established in tea gardens of Dibrugarh, Assam. Genomic DNA was extracted from 409 subjects by the conventional phenol-chloroform method. The prevalence of the eNOS exon 7 Glu298Asp variant was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. Results The study population was in Hardy-Weinberg Equilibrium. The frequency of the eNOS GG, GT and TT genotypes was found to be 75, 22 and 3 per cent respectively and did not show any significant difference in gender wise analysis. Interpretation & conclusions Our results showed that the prevalence of the homozygous GG genotype was high (75%) and the rare mutant genotype (homozygous, TT) was 3 per cent in a population at risk with cardiovascular disease. Such population-based data on various polymorphisms can ultimately be exploited in pharmacogenomics.
Subject(s)
Adult , Aged , Aged, 80 and over , Cardiovascular Diseases/enzymology , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/genetics , Chromosomes, Human, Pair 7/genetics , Female , Genetic Predisposition to Disease/epidemiology , Genetic Predisposition to Disease/genetics , Genotype , Haplotypes/genetics , Humans , India/epidemiology , Male , Middle Aged , Mutation, Missense/genetics , Nitric Oxide Synthase Type III/genetics , Pharmacogenetics/methods , Polymorphism, Restriction Fragment Length/genetics , Polymorphism, Single Nucleotide/genetics , PrevalenceABSTRACT
Objective To investigate the effeets of insulin on the apoptosis of vascular endothelial cells cuinduced by burn$eruln in order to explore its possible mechamsm.Method Cultured human ECV304 cells were randomly divided into 33x)ups:control group,the ECV304 cells hured by 15%(V/V)rat normal,qertlm(t=6);bum semm group,the ECV3()4 cells simulated by 15%(V/V)self-made burn semm collected from rats with 30%TBSA full-thickness burns on the,back(n=6);and burn Serum+insulin group.the ECV304 cells cultared by insulin(10-7mol/L)and 15%(V/V) seIf-made rat bum serum(n=6).The transferase mediated nick end labeling(TUNEL) method was employed to measure the apoptosis of endothelial cells at 6 hours after stimulation.Meanwhile.immanohistochemical technique and Western blotting were used to determine the protein expressions of bcl-2 and eNOS.Data are expressed ills mean ±SEM.Statistical comparison was made using oneway analysis of vtriance.Significance was accepted at P<0.05.Results Compswith the control group,bum$erunl induced the apoptusis(18.5±3.1%)and down-regalated bcl·2(O.36±0.12)and p-eNOS(O.55±0.28)protein expressions of HUVECs(P<0.01).Burn 9AJ'unl+insulin significantly decreased the apoptosis(9.6 4-2.8%)and up-regulated bcl-2(0.944-0.25)and p-eNOS(0.89±0.16)protein expressions ofHU-VECs in comparison with the bum serllm group(P<0.01).eNOS showed no significant differences in three groups.Conclusions Insulin could markedly inhibit the apoptosis and up-regalate bcl-2 protein expression of HUVECs induced by bum serum,and its mec,harfism might involve the protein expression ofphosphorylated eNOS.
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@#ObjectiveTo investigate the relationship of the polymorphism of endothelial nitric oxide synthase (eNOS), the 27-bp variable number of tandem repeats (VNTR) in intron 4 with essential hypertension (EH) of the northern Han nationality in China.MethodsGenotypes, the level of plasma nitric oxide metabolites (NOx) and the activity of nitric oxide synthase (NOS) of 207 EH subjects and 231 healthy subjects were measured by polymerase chain-reaction (PCR).ResultsThe frequencies of ecNOS4a/a,ecNOS4b/a, and ecNOS4 b/b in the healthy group were 0.43%, 13.42% and 86.15% respectively. The frequency of the b allele was 92.86%, and the frequency of the a allele was 7.14%. While the frequencies of ecNOS4 a/a, ecNOS4 a/b,and ecNOS4 b/b in the EH group were 0.49%, 19.32% and 80.19% respectively. The frequency of the a allele in EH group (n=42, 10.15%) was significantly higher than that in the healthy group (n=33, 7.14%)(P<0.05). The plasma NOx level of the EH group was 70.04±14.68 mol/L, and significantly lower than that 84.09±27.27 mol/L in the healthy group (P<0.05). Similarly, both the plasma TNOS and iNOS activities of the EH group were 35.49±12.8 U/ml and 14.92±7.93 U/ml, and markedly lower than that 41.47±13.2 U/ml and 10.11±6.21U/ml in the healthy group (P<0.05). But the activities of eNOS in the EH group and healthy group were not significantly different (P>0.05).ConclusionThe variations of ecNOS4 gene locus may be responsible for the decrement of plasma NOx, both plasma NOx level and activity of NOS decreases in EH patients, so it may be a genetic susceptibility marker for EH of the Han nationality in China.
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AIM To investigate the protective action of onychin aganist the growth inhibition of endothelial cell injured by menadione and its mechanism. METHODS The injured model was established by endothelial cell treated with menadione.Protective effect of onychin aganist growth inhibition of injured endothelial cell was determined by MTT assay and cell counting method; NO concentration in the medium was determined by nitrate reductase assay; eNOS and phosph ERK1/2 protein levels were determined by Western blot. RESULT Onychin significantly decreased the growth inhibitory rate of injured endothelial cells,increased NO concentration in the medium and eNOS activity and up regulated phosph ERK1/2 expressing. CONCLUSION Onychin has protective action and against the growth inhibition of endothelial cell injured by menadione may be via NO and ERK1/2 signal pathway.