切应力通过Pim1/Akt调节人脐静脉内皮细胞内皮型一氧化氮合酶Ser633和Ser1177位点磷酸化

目的 探讨不同模式的血流切应力对人脐静脉内皮细胞(HUVECs)Pim1表达的影响，以及对内皮型一氧化氮合酶(eNOS)Ser1177和Ser633位点磷酸化的调控作用。方法 体外原代培养的HUVECs,采用平行平板流动腔系统给HUVECs分别施加15 dyn/cm²层流切应力(LSS)和(0.5±4)dyn/cm²振荡切应力(OSS),Western blotting法检测Pim1、Akt和Akt Ser473磷酸化、eNOS、eNOS Ser1177和Ser633位点磷酸化蛋白表达。用特异性小干扰RNA(siRNA)技术分别敲低HUVECs中Pim1和Akt进行干预。结果 与OSS刺激相比较，LSS显著上调HUVECs中Pim1蛋白表达水平(P<0.01),同时，Akt Ser473磷酸化、eNOS Ser633和Ser1177位点磷酸化蛋白表达增加(P<0.05或P<0.01); siPim1和Pim1特异性抑制剂SMI-4a干预后，LSS诱导的Pim1蛋白表达显著被抑制(P<0.05),同时Akt Ser473...     

蛋白磷酸酶4在棕榈酸降低人脐静脉内皮细胞eNOS Ser633位点磷酸化中的作用

目的:探讨蛋白磷酸酶4(protein phosphatase 4,PP4)在棕榈酸(palmitic acid,PA)引起内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)Ser633位点磷酸化水平降低中的调控作用。方法:选用人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)为研究对象,分别用终浓度为25μmol/L、50μmol/L、100μmol/L和200μmol/L的PA处理HUVECs 36 h,另用100μmol/L PA处理HUVECs 12 h、24 h、36 h和48 h,用蛋白磷酸酶2A(protein phosphatase 2A,PP2A)家族抑制剂福司曲星(fostriecin,FST)20 nmol/L或冈田酸(okadaic acid,OA)5 nmol/L分别预处理细胞30 min,然后用蛋白磷酸酶4催化亚基(protein phosphatase 4 catalytic subunit,PP4c)小干扰RNA(small interfering RNA,siRNA)和蛋白磷酸酶2A催化亚基(protein phosphatase 2A catalytic subunit,PP2Ac)siRNA分别转染HUVECs。用Western blot法检测eNOS总蛋白、PP4c和PP2Ac蛋白表达水平及eNOS Ser633磷酸化水平,用DAF-FM DA荧光探针检测细胞内一氧化氮(nitric oxide,NO)的含量。结果:(1)与control组比较,PA(终浓度25μmol/L、50μmol/L、100μmol/L和200μmol/L)处理HUVECs 36 h后,eNOS Ser633磷酸化水平均显著降低(P<0.05),100μmol/L PA处理HUVECs 24 h、36 h和48 h后eNOS Ser633磷酸化水平均显著降低(P<0.05);各组间eNOS总蛋白表达无显著差异。(2)与control组相比,FST和OA预处理均可逆转PA处理引起的eNOS Ser633磷酸化水平降低(P<0.05)和细胞内NO产量减少(P<0.05);各组间eNOS总蛋白表达无显著差异。(3)与siControl组相比,si-PP4c转染组PP4c蛋白表达水平显著降低(P<0.05),同时eNOS Ser633磷酸化水平显著增高(P<0.05);si-PP2Ac转染组PP2Ac蛋白表达水平显著降低(P<0.05),但eNOS Ser633磷酸化水平表达无显著差异;各组间eNOS总蛋白表达无显著差异。结论:PA可显著降低HUVECs中eNOS Ser633磷酸化水平及NO产量,其机制可能是由于PA诱导PP2A家族中的PP4而不是PP2A激活所致。     

IL-1β通过抑制AKT下调人脐静脉内皮细胞eNOS Ser1177磷酸化水平

目的:探讨白细胞介素1β(interleukin-1β,IL-1β)能否调控人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)中内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)第1 177位丝氨酸(Ser1177)磷酸化水平及其可能的机制。方法:将HUVECs随机分为正常对照组、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)处理组、IL-1β处理组、IL-6处理组、单纯SC79[蛋白激酶B(protein kinase B,PKB/AKT)特异性激动剂]处理组和SC79+IL-1β处理组。用Western blot方法检测HUVECs中eNOS、p-eNOS-Ser1177、AKT和pAKT-Ser473的蛋白水平。采用化学比色法检测HUVECs培养液中一氧化氮(nitric oxide,NO)的含量。结果:与正常对照组相比,TNF-α和IL-6处理对HUVECs中p-eNOS-Ser1177的蛋白水平均无显著影响(P0.05),而IL-1β处理则显著降低细胞中p-eNOS-Ser1177蛋白水平和培养液中NO含量(P0.05),同时伴有p-AKT-Ser473的下调(P0.05)。IL-1β对细胞中p-eNOS-Ser1177蛋白水平和培养液中NO含量的调节能被SC79所阻断(P0.05)。结论:IL-1β可下调HUVECs中p-eNOS-Ser1177蛋白水平进而影响eNOS活性,这种效应可能涉及到AKT/eNOS通路。     

AngⅡ/AT_1R通路通过激活人脐静脉内皮细胞PP2A导致eNOS Ser1177磷酸化水平下调

目的:初步探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)/血管紧张素Ⅱ1型受体(angiotensinⅡtype1 receptor,AT_1R)通路是否通过激活人脐静脉内皮细胞蛋白磷酸酶2A(protein phosphatase 2A,PP2A)导致内皮型一氧化氮合酶(eNOS)Ser1177磷酸化水平下调。方法:将人脐静脉内皮细胞随机分为正常对照(control)组、AngⅡ处理组、单纯坎地沙坦(candesartan,CAN;AT_1R特异性阻断剂)组和CAN预处理+AngⅡ组。用Western blot方法检测各组eNOS总蛋白表达、eNOS Ser1177磷酸化水平、PP2Ac蛋白表达、PP2Ac-Tyr307磷酸化水平和PP2A内源性抑制蛋白I_2~(PP2A)表达水平。采用化学比色法检测各组细胞培养基中的NO含量。结果:与control组相比,AngⅡ处理后eNOS Ser1177磷酸化水平及细胞培养基中的NO含量降低(P0.05);与同一浓度AngⅡ组相比,CAN预处理可增加eNOS Ser1177磷酸化水平及细胞培养基中的NO含量(P0.05);各组间eNOS蛋白表达差异无统计学显著性。与control组比较,AngⅡ处理后PP2Ac Tyr307磷酸化水平和I_2~(PP2A)表达降低(P0.05);与同一浓度AngⅡ组相比,CAN预处理可增加PP2Ac Tyr307磷酸化水平和I_2~(PP2A)表达(P0.05);各组间PP2Ac蛋白表达差异无统计学显著性。结论:AngⅡ可通过AT_1R通路导致人脐静脉内皮细胞eNOS Ser1177磷酸化水平下调,NO合成减少,这一效应可能与AngⅡ/AT_1R通路降低PP2Ac Tyr307磷酸化水平和I_2~(PP2A)表达水平、导致PP2A活性增强有关。特异性AT_1R阻断剂CAN预处理可通过增加PP2Ac Tyr307磷酸化水平和I_2~(PP2A)表达水平而降低PP2A活性,最终上调eNOS Ser1177磷酸化水平,恢复eNOS活性。     

KCa3.1在藏红花素促进人脐静脉内皮细胞NO生成过程中的调节作用

目的观察藏红花素(crocin)对人脐静脉内皮细胞(HUVECs)体外eNOS表达及NO生成的影响以及。方法用crocin及KCa3.1选择性阻断剂tram34分别处理HUVECs,NO荧光探针DAF-FM DA检测HUVECs细胞中NO的表达, Western blot法检测磷酸化一氧化氮合酶(peNOS)和总一氧化氮合酶(t-eNOS)的表达。结果 crocin明显促进细胞内NO的生成,提高p-eNOS和t-eNOS蛋白的表达水平;在KCa3.1选择性阻断剂tram34的干预下,crocin对于HUVECs中NO及eNOS的上调作用被明显抑制。结论藏红花素促进内皮细胞eNOS介导的NO生成与KCa3.1信号通路相关。     

氧化低密度脂蛋白下调血管内皮细胞表达Rictor

目的探讨氧化低密度脂蛋白对培养血管内皮细胞表达基因Rictor的影响。方法分离并培养人脐静脉内皮细胞,用不同浓度(10、20、40和80 mg/L)氧化低密度脂蛋白作用24 h后,用RT-PCR及Western blot检测Rictor的表达情况,并检测转染Rictor后,蛋白激酶Akt及eNOS磷酸化的变化,用硝酸还原酶还原法测定NO释放量。结果氧化低密度脂蛋白显著降低Rictor的mRNA及蛋白的表达量(P<0.01),使Rictor-mTOR复合物形成减少。转染Rictor后,不仅Rictor表达增加而且使蛋白激酶Akt及eNOS磷酸化增加;内皮细胞NO释放量增多(P<0.05)。结论影响内皮细胞表达Rictor,是氧化低密度脂蛋白诱发血管内皮细胞功能不良的重要机制之一。     

棕榈酸激活人脐静脉内皮细胞PP2C降低eNOS Ser1177的磷酸化

目的探讨棕榈酸(PA)激活人脐静脉内皮细胞(HUVEC)蛋白磷酸酶2C (PP2C)对内皮一氧化氮合酶第1177位丝氨酸(eNOS Ser1177)磷酸化的调控作用。方法 HUVEC随机分为对照组、 PA组、特异性PP2C抑制剂血根碱(San)联合PA组以及转染PP2Cα特异性小干扰RNA(siRNA)组。采用Western blot法检测eNOS总蛋白、 eNOS Ser1177磷酸化水平和PP2Cα蛋白水平,二氨基荧光素-FM二乙酸酯(DAF-FM DA)负载法检测细胞内一氧化氮(NO)含量,免疫共沉淀法观察eNOS与PP2C之间的共定位关系。结果与对照组相比, PA处理组eNOS Ser1177磷酸化水平及NO含量均降低, San预处理组可逆转以上变化。敲低PP2Cα蛋白表达水平后, eNOS Ser1177磷酸化水平增加。eNOS与PP2C在细胞内共定位。结论 PA通过激活HUVEC的PP2C引起eNOS Ser1177磷酸化水平降低。     

罗格列酮对大鼠急性心肌梗死再灌注后无复流的影响

目的 探讨罗格列酮对急性心肌梗死(AMI)再灌注后无复流及内皮型一氧化氮合酶（eNOS）表达的影响。方法 结扎大鼠左冠状动脉前降支3 h后松解2 h建立AMI再灌注后无复流动物模型,将60只大鼠随机分为对照组、假手术组、罗格列酮组、罗格列酮+L-NNA（NOS抑制剂）组、L-NNA组;以伊文思蓝-硫磺素染色法检测大鼠心肌组织无复流范围;Western blot检测丝氨酸1177磷酸化内皮型一氧化氮合酶(p-eNOS Ser1177)蛋白表达,RT-PCR检测eNOS mRNA的表达。结果 （1）与对照组相比,罗格列酮组大鼠心肌组织无复流范围明显减小（P<0.05）,而L-NNA+罗格列酮组无复流范围无显著差异;（2）罗格列酮组eNOS mRNA表达较对照组无显著差别,而p-eNOS Ser1177蛋白表达显著增强(P<0.05);结论 罗格列酮显著减小AMI再灌注后无复流范围,其机制与其促进再灌注后eNOS磷酸化,增加NO释放有关。     

辛伐他汀促进模型大鼠心肌梗死后局部血管新生

目的 研究辛伐他汀对急性心肌梗死（AMI）后血管生成素-1（Ang-1）及内皮型一氧化氮合酶（eNOS）的表达调控与其促血管新生作用的关系。方法 健康成年SD大鼠60只,随机分为假手术组、对照组、辛伐他汀组、辛伐他汀+ L-NAME(NOS抑制剂)组、辛伐他汀+ AMG386 (Ang-1抑制剂)组;结扎大鼠冠状动脉左前降支建立急性心肌梗死动物模型。术后2 d分别给予辛伐他汀(1 mg/(kg·d) ),辛伐他汀+L-NAME(40 mg/(kg·d) ),辛伐他汀+AMG386（10 mg/(kg·wk）),均为2 周,以CD31染色新生血管并检测新生血管密度;以Western blot及RT-PCR检测缺血区心肌Ang-1、eNOS、丝氨酸1177磷酸化内皮型一氧化氮合酶(p-eNOS)的表达。结果（1）辛伐他汀使AMI后缺血区心肌新生血管密度显著增加（P<0.05 ）,而L-NAME 、AMG386则显著抑制了辛伐他汀的促心肌血管新生作用（P<0.05）。（2）辛伐他汀使AMI后缺血区心肌Ang-1、eNOS、p-eNOS表达均显著增强（P<0.05）,而AMG386使辛伐他汀上调p-eNOS表达的作用被显著抑制（P<0.05）。结论 辛伐他汀促心肌血管新生作用可能与其上调Ang-1、eNOS的表达及促进eNOS磷酸化有关,其中eNOS磷酸化可能是介导Ang-1促血管新生作用的下游机制。     

下调牛磺酸调节基因1减轻氧化低密度脂蛋白诱导的血管内皮细胞氧化损伤

目的研究牛磺酸调节基因1(TUG1)对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)氧化损伤的影响。方法将HUVECs分为:对照组、ox-LDL组(含有100 g/mL的ox-LDL细胞培养液)、si-NC+干预组(转染siRNA阴性对照)和si-TUG1+干预组(转染TUG1 siRNA)。实时定量PCR检测HUVECs中TUG1表达;DCFH-DA法检测细胞活性氧(ROS)水平;WST法检测细胞超氧化物歧化酶(SOD)活性;TBA比色法检测培养液中丙二醛(MDA)含量;LD-P法检测培养液中乳酸脱氢酶(LDH)活性;硝酸还原酶法检测培养液中一氧化氮(NO)含量;流式细胞计量术检测细胞凋亡,Western blot检测细胞中c-caspase-3蛋白水平。结果 ox-LDL组HUVECs中TUG1表达水平较对照组升高(P0.05)。TUG1 siRNA转染可下调TUG1的表达(P0.05)。ox-LDL组HUVECs中的ROS水平升高(P0.05),SOD活性降低(P0.05),培养液中MDA含量升高(P0.05),LDH活性升高(P0.05),NO含量降低(P0.05),细胞凋亡率升高(P0.05),细胞中c-caspase-3蛋白水平升高(P0.05)。si-TUG1+干预组显著减轻ox-LDL组的上述变化(P0.05)。结论下调TUG1减轻ox-LDL诱导的HUVECs氧化损伤,减少HUVECs凋亡。     

Effects of pulsatile shear stress on signaling mechanisms controlling nitric oxide production, endothelial nitric oxide synthase phosphorylation, and expression in ovine fetoplacental artery endothelial cells.

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm2, then were exposed to 10, 15, or 25 dynes/cm2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p < .05). Production of NO by OFPAE cells exposed to pulsatile shear stress was de novo; i.e., inhibited by L-NMMA (N(G)-monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm2 (8-fold) exceeded (p < .05) that seen for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). Acute rises in NO production with shear stress occurred by eNOS activation, whereas prolonged NO rises were via elevations in both eNOS expression and enzyme activation. The authors therefore used Western analysis to investigate the signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by "flow-adapted" OFPAE cells. Increasing shear stress from 3 to 15 dynes/cm2 very rapidly increased eNOS Ser1177, ERK1/2 (extracellular signal-regulated kinase 1 and 2) and Akt, but not p38 MAPK (p38 mitogen-activated protein kinase) phosphorylation by Western analysis. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by PI-3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and LY294002, but not the MEK (MAPK kinase) inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels induced by 15 dynes/cm2 shear stress. Blocking of either signaling pathways or p38 MAPK did not change the shear stress-induced increase in eNOS protein levels. Therefore, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibiting MEK. Prolonged shear stress-stimulated increases in eNOS protein levels were not affected by inhibition of MEK- or PI-3K-mediated pathways. In conclusion, pulsatile shear stress greatly induces NO production by OFPAE cells through the mechanisms of both PI-3K-mediated eNOS activation and elevations in eNOS protein levels; bFGF does not further stimulate eNOS expression under flow condition.     

PPARs激动剂上调内皮细胞表达eNOS并增加NO生成

目的： 观察PPARα激动剂非诺贝特及PPARγ激动剂赛格列酮对人脐静脉内皮细胞（HUVECs）血管紧张素Ⅱ（AngⅡ）抑制NO生成的作用。 方法： 体外培养HUVECs，用1×10-7-1×10-4mol/L赛格列酮和10-5、10-4mol/L非诺贝特预处理HUVECs 24 h，再与10-6mol/L AngⅡ 共同孵育12 h，通过RT-PCR和Western blotting分别检测eNOS mRNA和蛋白表达水平；通过Griees反应测定NO2－/NO3－浓度。 结果： 与对照组相比，10-7mol/L AngⅡ刺激HUVEC 12 h下调eNOS mRNA（0.38±0.19 vs 0.13±0.18，P<0.01）和蛋白（35.90±3.18 vs 6.95±2.19，P<0.01）表达，减少NO生成（50.21 μmol/L vs 21.33 μmol/L，P<0.01）。用10-7、10-6、10-5、10-4 mol/L赛格列酮预处理24 h，上调eNOS mRNA表达（分别为0.36±0.03、0.36±0.14、0.37±0.16、0.43±0.06，与AngⅡ组比较，均P<0.01）和蛋白表达（分别为11.60±3.31、11.78±5.45、13.93±2.46、22.93±3.17，与AngⅡ组相比，均P<0.01），增加细胞培养液NO2-/NO3-浓度。非诺贝特也上调eNOS mRNA和蛋白表达，增加细胞培养液NO2-/NO3-浓度（P<0.01）。 结论： AngⅡ减少eNOS表达，从而减少NO生成。赛格列酮和非诺贝特预处理24 h，可拮抗AngⅡ对HUVECs eNOS mRNA和蛋白表达的抑制作用，增加NO的释放。     

NO在脂联素抑制高脂血症大鼠血小板聚集中的作用

目的: 探讨一氧化氮(NO)信号转导通路在脂联素抑制高脂血症血小板聚集机制中的作用。方法: 采用成年大鼠饲以高脂饲料14周,分离其血小板并以重组脂联素(rAPN)孵育。采用免疫荧光、Western blotting等方法观察检测血小板聚集、NO含量、超氧化物含量、内皮型一氧化氮合酶(eNOS)/诱导型一氧化氮合酶(iNOS)的表达和抗氧化物活性。结果: 采用rAPN处理能抑制高脂血症诱导的血小板聚集(P<0.05),并导致血小板NO的生成显著减少。同时,在高脂血症血小板中,采用rAPN处理还能显著减少超氧化物的生成(降低62%, P<0.05) 并增强其抗氧化能力(增加38%, P<0.05)。此外,高脂血症诱导的eNOS磷酸化的降低和iNOS表达的增加在rAPN处理后被显著逆转(P<0.05, P<0.01)。结论: 脂联素是一种抑制高脂血症血小板聚集的脂肪细胞因子,其机制与减少超氧化物水平、增加抗氧化物活性和阻断iNOS的表达有关。     

Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium

This study examines the molecular mechanisms by which hypoxia regulates phosphorylated endothelial nitric oxide synthase (eNOS) activity and NO production in porcine coronary artery endothelial cells (PCAEC). Exposure to hypoxia (pO(2)=10 mmHg) for periods up to 3 h resulted in a time-dependent increase in eNOS protein expression and an early (15 min) and sustained increase in eNOS phosphorylation at Ser-1177. Exposure to hypoxia for 30 min led to a doubling in eNOS activity (control=6.2+/-4.4 vs hypoxia=14.1+/-5.0 fmol cGMP/microg protein, P<0.05) and NO release (control=5.9+/-0.8 vs hypoxia=11.8+/-1.2 nM/microg protein, P<0.05). Hypoxia also led to a significant increase in Akt phosphorylation and upregulation of Hsp90 binding to eNOS. Pretreatment of cells with either 1 microg/ml geldanamycin (a specific inhibitor of Hsp90) or 500 nM wortmannin (a specific PI3 kinase inhibitor) suppressed hypoxia-stimulated Akt and eNOS phosphorylation and significantly attenuated hypoxia-stimulated Hsp90 binding to eNOS. Both eNOS activity and NO production were inhibited by geldanamycin and wortmannin. Although hypoxia led to early activation of p42/44 mitogen-activated protein kinases (MAPK), inhibition of their pathway by PD98059 did not suppress hypoxia-stimulated eNOS phosphorylation and eNOS activity. These data demonstrate that hypoxia leads to increased eNOS phosphorylation via stimulated Hsp90 binding to eNOS and activation of the PI3-Akt pathway. We conclude that a coordinated interaction between Hsp90 and PI3-Akt may be an important mechanism by which eNOS activity and NO production is upregulated in hypoxic heart.     

尿酸抑制人脐静脉内皮细胞合成一氧化氮

目的 研究尿酸(UA)对人脐静脉内皮细胞(HUVEC)表达内皮型一氧化氮合酶(eNOS)及分泌一氧化氮(NO)的影响.方法 不同浓度UA(0、0.5、1、1.5及2 mg/L)及50 mg/L ox-LDL(阳性对照)分别作用HUVEC 24、48及72 h,用real-time PCR法测定HUVEC eNOS mRNA;Western blot法检测细胞eNOS蛋白;酶法检测上清液NO的含量.结果 UA 0.5 mg/L组eNOS mRNA表达水平明显高于对照组(P＜0.05);随着UA浓度升高(1、1.5及2 mg/L组),及其作用时间延长,HUVEC eNOS mRNA及蛋白表达水平及上清液NO分泌最相比对照均明显下降(72 h N02-/N03-2 mg/L组与对照组分别为0.52±0.18与1.00±0.10,P＜0.05),且趋势与ox-LDL组相同.结论 0.5 mg/L以上浓度的UA呈浓度及时间依赖性抑制HUVEC eNOS表达及NO合成,提示高浓度的UA可能损伤血管内皮功能.     

Elevated glucose attenuates agonist- and flow-stimulated endothelial nitric oxide synthase activity in microvascular retinal endothelial cells.

Impaired vasoactive release of opposing vasodilator and vasoconstrictor mediators due to endothelial dysfunction is integral to the pathogenesis of diabetic retinopathy. The aim of this study was to determine the effect of hyperglycemia on the expression of endothelial nitric oxide synthase (eNOS) and the release of nitric oxide (NO) in bovine microvascular retinal endothelial cells (BRECs) under both static (basal and acetylcholine stimulated) and flow (laminar shear stress [10 dynes/cm2 and pulsatile flow 0.3 to 23 dynes/cm2) conditions using a laminar shear apparatus and an in vitro perfused transcapillary culture system. The activity and expression of eNOS, measured by nitrate levels and immunoblot, respectively, were determined following exposure of BRECs to varying concentrations of glucose and mannitol (0 to 25 mM). Under static conditions the expression of eNOS decreased significantly following exposure to increasing concentrations of glucose when compared to osmotic mannitol controls and was accompanied by a significant dose-dependent decrease in nitrate levels in conditioned medium. The acetylcholine stimulated increase in NO release (2.0 +/- 0.3-fold) was significantly reduced by 55% +/- 5% and 65% +/- 4.5% following exposure to 16 and 25 mM glucose, respectively, when compared to osmotic controls. In parallel studies, glucose significantly inhibited both laminar shear stress and pulsatile flow-induced activity when compared to mannitol. We conclude that hyperglycemia impairs agonist- and flow-dependent release of NO in retinal microvascular endothelial cells and may thus contribute to the vascular endothelial dysfunction and impaired autoregulation of diabetic retinopathy.     

硫化氢通过调节Sirt1/eNOS信号通路延缓人脐静脉内皮细胞衰老

目的:我们既往研究已证实外源性硫化氢能够延缓内皮细胞衰老,本研究拟进一步探讨沉默信息调节因子1(Sirt1)与内皮型一氧化氮合酶(e NOS)系统对硫化氢抗内皮细胞衰老作用的影响。方法:建立葡萄糖(33 mmol/L)诱导的人脐静脉内皮细胞(HUVECs)衰老模型,根据细胞活力、衰老相关β-半乳糖苷酶(SA-β-Gal)染色阳性率和纤溶酶原激活物抑制剂1(PAI-1)的表达水平评估HUVECs衰老;同时采用RNA干扰技术抑制Sirt1蛋白表达,检测e NOS表达及衰老相关指标。结果:HUVECs经高糖处理后,细胞活力减低,SA-β-Gal阳性细胞比例增加,PAI-1表达增高,Sirt1及e NOS表达均明显减少(P0.05);与高糖处理组比较,100μmol/L硫氢化钠(硫化氢供体)处理组的细胞活性增加,SA-β-Gal染色阳性细胞比例及PAI-1蛋白表达显著下降,Sirt1及e NOS蛋白表达增加,NO含量增加(P0.05)。与硫化氢处理组比较,Sirt1 siRNA处理后e NOS表达减少,PAI-1表达增加,SA-β-Gal染色阳性细胞数目增多,NO含量减少(P0.05或P0.01)。结论:硫化氢通过上调Sirt1、增加e NOS表达而促进NO的合成,从而抵抗高糖诱导的HUVECs衰老。     

Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells

OBJECTIVE:

Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs).

METHODS:

HUVECs were divided into four groups: control, treatment with 180 μM hydrogen peroxide (H₂O₂), treatment with 150 μg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H₂O₂ for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction.

RESULTS:

Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs.

CONCLUSION:

Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.     

四逆汤对同型半胱氨酸损伤的EAhy926细胞caveolin-1和eNOS表达的影响

 目的：探讨四逆汤（Sini decoction,SND）防治内皮细胞损伤的机制及陷窝蛋白1（caveolin-1）和一氧化氮（NO）系统在其中的作用。方法：建立同型半胱氨酸(Hcy)损伤的人脐静脉内皮融合细胞（EAhy926细胞）模型,观察四逆汤的保护作用及其对NO系统和caveolin-1的影响。结果：Hcy加入后细胞生长缓慢,贴壁细胞数明显减少,NO浓度明显减低（P<0.05）,caveolin-1 mRNA和蛋白表达明显增强（P<0.05）,内皮型NO合酶(eNOS) mRNA和蛋白表达明显减弱（P<0.05）;四逆汤处理组贴壁细胞及细胞形态明显改善,NO浓度较Hcy模型组明显升高（P<0.05）,caveolin-1 mRNA和蛋白表达较Hcy模型组明显减弱（P<0.05）,eNOS mRNA和蛋白表达较Hcy模型组明显增强（P<0.05）,其中以四逆汤1.0 kg/L +Hcy 4.0 μmol/L组最明显。结论：Hcy可能通过增加caveolin-1表达和抑制eNOS表达而损伤人脐静脉内皮细胞,而四逆汤通过抑制caveolin-1表达和增加eNOS的表达拮抗Hcy对细胞的损伤。