降血压肽对人脐静脉内皮细胞eNOS、iNOS 、ET-1 mRNA表达的影响

目的和方法:采用RT-PCR技术分析降血压肽(ACEIP)对人脐静脉内皮细胞内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)、内皮素-1(ET-1)mRNA表达水平的影响,从而探讨降血压肽降血压的部分作用机制。结果:与对照组比较,不同剂量的降血压肽可降低ET-1、iNOSmRNA的表达;升高eNOSmRNA的表达,均以ACEIP中剂量组效果最明显(P<0.01)。结论:降血压肽降低血压的机制可能与其降低人脐静脉内皮细胞ET-1、iNOS的基因表达;诱导eNOS基因表达有关。     

硒对氟诱导人脐静脉血管内皮细胞iNOS和eNOS表达的影响

目的研究一定浓度的硒对氟诱导人脐静脉血管内皮细胞(ECV-304)内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)表达的影响。方法体外培养人脐静脉血管内皮细胞株,加氟组培养液中分别加入400、1 000、2 000μmol/L的氟化钠;加氟加硒组培养液中分别加入400、1 000、2 000μmol/L的氟化钠,同时加入100 nmol/L的亚硒酸钠;对照组细胞在无血清培养液中培养;连续培养24 h,检测培养液中的iNOS、eNOS的活力;采用逆转录聚合酶连反应(RT-PCR)测定iNOS mRNA和eNOS mRNA的表达。结果与对照组比较,400、1 000、2 000μmol/L加氟组iNOS活力和iNOS mRNA的表达增强(P<0.01);而400、1 000、2 000μmol/L加氟组eNOS的活力和1 000、2 000μmol/L加氟组eNOSmRNA的表达降低(P<0.05,P<0.01);与相应剂量的加氟组(400、1 000μmol/L)比较,加硒加氟组iNOS活力和iNOS mRNA的表达降低(P<0.05),而eNOS的活力和eNOS mRNA的表达升高(P<0.05);而加硒加氟组(氟剂量:2 000μmol/L、硒剂量:100 nmol/L)的iNOS与eNOS的表达与相应剂量的加氟组(2 000μmol/L)比较,差异无统计学意义(P>0.05)。结论硒对氟诱导人脐静脉血管内皮细胞iNOS和eNOS表达产生影响,但是对高剂量氟组作用不强。     

降血压肽对人脐静脉内皮细胞功能的影响

目的研究降血压肽ACEIP对脐静脉内皮细胞释放内皮素(ET)、一氧化氮(NO)的影响。方法按150，300，600μg／ml不同剂量的降血压肽作用于脐静脉内皮细胞，进行细胞生长试验及细胞培养液中NO、ET含量的检测。结果与空白对照组比较，不同剂量的降血压肽可升高培养液中NO含量。降低ET含量；去甲肾上腺素的促进脐静脉内皮细胞的生长以及其促进脐静脉内皮细胞分泌ET、降低培养液中NO含量的作用可被降血压肽拮抗。结论降血压肽对血管内皮细胞功能具有保护作用。     

临床营养

抗性淀粉干预糖尿病胰岛素抵抗的临床随机对照研究；高蛋白膳食对限食大鼠肠屏障功能的保护作用；降血压肽对人脐静脉内皮细胞eNOS、iNOS、ET-1mRNA表达的影响；NF-κBP65及Bcl-2蛋白在植酸诱导人胃癌细胞凋亡中的作用     

较低剂量甲醛诱导人脐静脉内皮细胞损伤的时相特征及机制

目的观察较低剂量甲醛(formaldehyde,FA,浓度为40μmol/L)诱导人脐静脉内皮细胞损伤时相特征及一氧化氮(NO)含量变化的时相规律,分析甲醛在诱导内皮细胞损伤中的作用机制。方法将人脐静脉内皮细胞(HU-VEC-12株)与甲醛浓度为40μmol/L的DMEM培养基孵育。分别处理一定时间后(0,4,12,24,48,72h)H-E染色-光学显微镜观察细胞形态变化;MTT法测定细胞的活性;丙酮酸二硝基苯腙比色-分光光度法检测细胞上清液乳酸脱氢酶(LDH)漏出量;硫代巴比妥法检测细胞上清液中丙二醛(MDA)浓度;硝酸还原酶法测定培养液中NO浓度;生化分析法测定内皮细胞一氧化氮合酶活性;免疫细胞化学法测定内皮型一氧化氮合酶(eNOS)和诱生型一氧化氮合酶(i NOS)的蛋白表达;RT-PCR法测定eNOS和i NOS的mRNA表达。结果较低浓度甲醛呈时间依赖性地促进细胞LDH漏出,以及内皮细胞上清液MDA和NO的产生;降低eNOS的活性,抑制eNOS在蛋白和基因水平的表达;增加i NOS活性,促进i N-OS在蛋白和基因水平的表达。结论甲醛诱导HUVEC-12细胞损伤,机制可能与其抑制eNOS的活性及表达,诱导i NOS的活性及表达,增加病理性NO产生,从而导致内皮细胞损伤。     

硒对体外培养血管内皮细胞的影响

目的 研究不同浓度的硒对体外培养人脐静脉血管内皮细胞(HUVEC-304)的影响.方法 体外培养人脐静脉血管内皮细胞株,在培养液中加入不同浓度的亚硒酸钠(50、100、500、1 000、2000 nmoL/L),连续培养48 h后,以瑞氏-吉姆萨染色观察细胞形态,以四唑氮蓝(MTT)比色法检测细胞活性,并检测培养液中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)的活力,丙二醛(MDA)、NO的含量.结果 硒浓度为1 000、2000nmoL/L时,细胞出现损伤样改变.硒浓度为100nmol/L时,细胞活性高于正常对照组(P＜0.05);硒浓度为1 000、2000 nmol/L时,细胞活性降低.与对照组比较,硒浓度为50～500 nmol/L时,培养液中的SOD、GSH-Px活力升高,差异有统计学意义(P＜0.05);硒浓度达2000nmoL/L时,SOD、GSH-Px活力明显降低(P＜0.01).与对照组比较,MDA在硒低浓度时无显著变化,当硒浓度升高至1 000、2 000 nmol/L时显著增高(P＜0.05,P＜0.01).低浓度的硒增强eNOS活力,降低iNOS活力,降低NO浓度;高浓度的硒降低eNOS活力,增强iNOS活力,升高NO浓度.结论 低浓度的硒对脐静脉血管内皮细胞有促进增生作用,高浓度的硒有损伤作用,并随剂量的增高而加重.     

乌司他丁对庆大霉素损伤的大鼠肾小管上皮细胞的保护作用

摘要:目的　观察乌司他丁对庆大霉素损伤的大鼠肾小管上皮细胞表达内皮素-1（ET-1）和一氧化氮（NO）的影响。方法　传代培养大鼠肾小管上皮细胞（NRK-52E）,分为对照组、损伤组（2 mg/ml庆大霉素）、治疗组A（2 mg/ml庆大霉素+160 U/ml乌司他丁）、B（2 mg/ml庆大霉素+320 U/ml乌司他丁）和C（2 mg/ml庆大霉素+640 U/ml乌司他丁）,观察各组细胞增殖能力、乳酸脱氢酶（LDH）水平、细胞凋亡率、ET-1和NO含量、内皮型一氧化氮合酶（eNOS）和诱生型一氧化氮合酶（iNOS）活性以及ET-1、eNOS和iNOS mRNA表达情况。结果　与对照组相比,损伤组细胞增殖能力下降,LDH、细胞凋亡率、ET-1、ET-1 mRNA、NO、iNOS和iNOS mRNA表达均增多（P均＜0.05）。与损伤组相比,治疗组细胞增殖能力增高,LDH、细胞凋亡率、ET-1、ET-1 mRNA、NO、iNOS和iNOS mRNA表达均减少;这些改变随剂量增加而明显（P均＜0.05）。eNOS活性和mRNA表达在5组间的差异无统计学意义（P均＞0.05）。结论　乌司他丁通过抑制ET-1、NO和iNOS减轻庆大霉素损伤大鼠肾小管上皮细胞,这些作用在一定范围具有剂量依赖性。     

苯并[a]芘对血管内皮细胞一氧化氮合酶表达的影响

目的　探讨苯并 [a]芘 (BaP)对内皮细胞一氧化氮合酶 (NOS)表达的影响。方法　培养猪主动脉血管内皮细胞 ,分别以不同浓度的BaP (0 ,0 1× 10 -6,0 5× 10 -6,1× 10 -6,5× 10 -6,10× 10 -6mol/L)染毒2 4h ,用Westernblot法检测诱导型一氧化氮合酶 (iNOS)和内皮型一氧化氮合酶 (eNOS)的改变。结果　基础状态下 ,主动脉血管内皮细胞iNOS有微量表达 [蛋白质条带扫描定量的光密度 (A)为 1 0 87],随BaP浓度 (0 1× 10 -6,0 5× 10 -6,1 0× 10 -6,5 0× 10 -6mol/L)的升高 ,iNOS表达量逐渐升高 (A值分别为2 0 784 ,19 4 15 ,33 714 ,5 5 2 19) ,iNOS表达水平与BaP浓度呈一定的线性剂量 -反应关系。而eNOS (A值为 7 6 2 5 )在基础状态下的表达水平高于iNOS ,随BaP浓度 (0 1× 10 -6,0 5× 10 -6mol/L)的升高eNOS表达量也逐渐升高 (A值分别为 8 5 36 ,17 2 5 3) ,在BaP浓度为 1 0× 10 -6mol/L时eNOS的表达呈高峰 (A值为4 9 2 4 2 ) ,然后随BaP浓度 (5 0× 10 -6,10 0× 10 -6mol/L)增高而快速降低 (A值分别为 33 16 3,14 4 2 4 ) ,其表达水平与BaP浓度呈“偏峰”型。结论　BaP可激活NOS ,iNOS表达水平与BaP浓度呈一定的剂量 -反应关系 ,高浓度BaP抑制eNOS的表达。     

一氧化氮合酶在心肌缺血再灌注过程中的动态变化

目的　测定心肌缺血再灌注过程中不同时点一氧化氮合酶 (nitrieoxidesynthase ,NOS)活性、基因表达水平及一氧化氮 (nitricoxide,NO)产量。方法　结扎新西兰白兔冠状动脉左前降支建立在体心肌缺血再灌注模型。测定正常、缺血 3 0min、 60min、缺血 60min再灌注 3 0min、 60min、 12 0min心肌NOS活性、内皮性一氧化氮合酶 (endothelialnitricoxidesynthase,eNOS)、诱生性一氧化氮合酶 (induciblenitricoxidesynthase ,iNOS)基因表达水平及NO产量。结果　正常心肌和缺血 60min再灌注 12 0min心肌NOS活性高于缺血心肌 ,(P <0 0 1)。正常心肌和缺血 60min再灌注 12 0min心肌NO含量、eNOS基因表达水平高于其余各时点心肌 (P<0 0 5 )。各时点心肌均未见到iNOS基因的表达。结论　 ( 1)在缺血期及再灌注的初期 (≤ 1小时 ) ,心肌eNOS活性 ,基因表达水平及NO含量均明显降低。 ( 2 )至恢复灌注 2小时 ,心肌eNOS活性、基因表达水平及NO含量均已接近基础水平。 ( 3 )缺血 1小时 ,恢复灌注 2小时并无iNOS活性或基因表达。     

氟对体外培养血管内皮细胞影响及硒干预作用研究

目的研究不同浓度氟对体外培养血管内皮细胞的影响及硒的干预作用。方法人脐静脉血管内皮细胞(HUVEC)置于37℃,5%CO2,饱和湿度条件下培养。在培养液中加入氟化钠,氟浓度分别为0、100、400、700、1000、2000μmol/L,同时加入硒,浓度为100 nmol/L。连续培养48 h后,收集细胞培养液与细胞。应用瑞氏-吉姆萨染色观察细胞形态,吖啶橙荧光染色测定细胞凋亡,四唑氮蓝(MTT)比色法检测细胞活性;检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量;检测细胞培养液诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)活性、细胞iNOSmRNA、eNOSmRNA表达;检测细胞间粘附分子-1(ICAM-1)、血管细胞粘附分子-1(VCAM-1)含量。结果在氟浓度100~700μmol/L加硒100 nmol/L,可明显减轻氟对细胞生长的抑制,恢复细胞形态的改变;使SOD、GSH-Px活性升高(P<0.05)、MDA含量下降(P<0.05);降低iNOS活性和iNOSmRNA表达(P<0.05),升高eNOS的活性和eNOSmRNA表达(P<0.05);减少CAM-1和VCAM-1含量(P<0.05,P<0.01)。结论氟可致血管内皮细胞形态改变、功能异常。适宜剂量硒补充可通过提高抗氧化功能,调整NO代谢,抑制粘附分子异常表达等途径拮抗高氟所致的血管内皮损伤。     

维生素E抑制子痫前期脐血清诱导脐静脉内皮细胞中NOSTRIN表达上调的体外研究

目的:探讨子痫前期患者脐血清诱导脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)中人内皮型一氧化氮合酶运输介导物(endothelial nitric oxide synthase traffic inducer,NOSTRIN)的表达及维生素E对其表达的调节作用。方法:采用消化培养法培养正常晚孕妇女的HUVEC,传代后待细胞长满至70%~80%后,加或不加维生素E作用30min后分别加入正常妊娠(正常组)及子痫前期(子痫前期组)脐静脉血清,培养48 h后,MTT测定细胞活力,RT-PCR测定NOSTRINmRNA的表达,分光光度法测定细胞上清中eNOS的活性。结果:子痫前期患者脐静脉血清培养的HUVEC,其细胞的活力明显低于正常组(P<0.05),细胞中NOSTRINmRNA的表达量明显高于正常妊娠组(P<0.01),细胞上清中eNOS的活性明显低于正常组(P<0.01);加维生素E预处理后,子痈前期组细胞的活力明显升高(P<0.05),其细胞中NOSTRIN的表达水平明显下降(P<0.01),eNOS活性明显升高(P<0.01)。结论:子痫前期患者脐静脉血清可使HUVEC细胞活力降低、NOSTRIN表达增加以及eNOS活性降低,维生素E可抑制子痫前期患者脐静脉血清诱导的NOSTRIN表达的增加以及eNOS活性降低。     

裙带菜膳食纤维对高血脂大鼠血管内皮功能的影响

目的研究裙带菜可溶性膳食纤维对高脂血症大鼠内皮功能的影响。方法40只大鼠分为4组(n=10):正常对照组,高脂模型组,膳食纤维低剂量组(5%),膳食纤维高剂量组(10%);经裙带菜可溶性膳食纤维处理8w后,检测离体血管的内皮依赖性舒张效应,血浆丙二醛(MDA),一氧化氮(NO),内皮素-1(ET-1)水平,并用Western blotting检测内皮型一氧化氮合酶(eNOS)蛋白表达水平。结果裙带菜可溶性膳食纤维显著降低MDA,ET-1水平,显著改善内皮功能及血浆NO的水平并伴随着eNOS蛋白表达水平的上调。结论裙带菜可溶性膳食纤维能改善高脂血症大鼠的血管内皮功能,这可能与其降低ET-1水平,增加NO的产生有关。     

Effects of dietary polyphenols on gene expression in human vascular endothelial cells

Previous studies have shown that consumption of fruit and vegetables plays a role in preventing the onset of CVD. These beneficial effects have been linked to the presence of polyphenolic compounds in plant-derived foods and their antioxidant capacity. It has been hypothesised that polyphenols may also have a direct effect on vascular endothelial cell growth and the expression of genes involved in angiogenesis and other roles of the endothelium. Previous studies in this area have tended to use concentrations of polyphenols that are supraphysiological (1-100 microm). The effects of more physiological concentrations (0.1 microm) of various individual polyphenols on gene expression were therefore investigated in cultured human umbilical vein endothelial cells (HUVEC) using both microarray and quantitative RT-PCR methodologies. Treatment of HUVEC with ferulic acid, quercetin or resveratrol (0.1 microm) resulted in changes to gene expression that for the three treatments amounted to significant (>2-fold) down-regulation of the expression of 363 genes and significant (>2-fold) up-regulation of 233 genes of the 10 000 genes present on the microarray. The majority of these genes were affected by resveratrol. Quantitative RT-PCR studies indicated that resveratrol (0.1 microm) significantly increased the expression of the gene encoding endothelial NO synthase (eNOS), which synthesises the vasodilator molecule NO, and both resveratrol and quercetin decreased expression of the potent vasoconstrictor, endothelin-1 (ET-1), while ferulic acid had no effect. The effects of resveratrol (0.1 microm) were also investigated when HUVEC were under oxidative stress following treatment with H2O2 (0-50 microm), which dose-dependently increased expression of eNOS and ET-1. Resveratrol stimulated eNOS mRNA in the absence of H2O2 and still allowed the increase with H2O2, but the effects were not additive. In contrast, resveratrol blocked the stimulatory effect of H2O2 on ET-1 expression. Hence, resveratrol has potent effects at a physiological concentration (0.1 microm) that would be expected to result in vasodilation and therefore help reduce blood pressure and the risk of CVD.     

Repeated and long-term treatment with physiological concentrations of resveratrol promotes NO production in vascular endothelial cells

In the present study, we examined the effect of repeated and long-term treatment with resveratrol on NO production in endothelial cells as a model of routine wine consumption. Repeated treatment with resveratrol for 5 d resulted in an increase in endothelial NO synthase (eNOS) protein content and NO production in human umbilical vein endothelial cell (HUVEC) in a concentration-dependent manner. A significant increase in functional eNOS protein content was observed with resveratrol, even at 50 nm. In contrast, eNOS phosphorylation was not stimulated and inducible NO synthase (iNOS) was not detected after resveratrol treatment. Both eNOS protein and mRNA expression were promoted by 50 nm-resveratrol in a time-dependent manner. Increased eNOS mRNA expression in response to resveratrol was not decreased by an oestrogen receptor (ER) antagonist ICI182780, a PPARα inhibitor MK886 or a sirtuin inhibitor Salermide. However, a combination of ICI182780 and MK886 significantly inhibited resveratrol-induced eNOS mRNA expression. Salermide had no effect even in the presence of ICI182780 or MK886. These results demonstrate that resveratrol within the physiological range increases eNOS mRNA and protein expression through ER and PPARα activation, thereby promoting NO production in endothelial cells. eNOS induction might result from the accumulative effect of nanomolar concentrations of resveratrol. The present study results can account in part for the observation that cardiovascular benefits of red wine are experienced with routine consumption, but not with acute consumption.     

Hcy对脐静脉内皮细胞eNOS和caveolin-1表达影响

目的研究不同浓度同型半胱氨酸(Hcy)对静息和钙离子导体(A23187)激活状态下人脐静脉内皮细胞中内皮型一氧化氮合酶(eNOS)和小凹蛋白-1(caveolin-1)mRNA和蛋白表达的影响。方法人脐静脉内皮细胞(HU-VECs)随机分4组:对照组、Hcy组(20、50、100、300μmol/L)、A23187(1μmol/L)组及上述浓度Hcy分别与A23187(1μmol/L)共同孵育组,测定各组eNOS和caveolin-1蛋白及mRNA表达情况,同时检测各组一氧化氮(NO)、丙二醛(MDA)含量和eNOS、超氧化物歧化酶(SOD)活力。结果对照组caveolin-1蛋白表达为1.13,Hcy各组该蛋白为0.21～2.05,呈浓度依赖性增高,差异有统计学意义(F=30.163,P<0.05);各组eNOS mRNA表达为1.80～2.08,差异无统计学意义;对照组eNOS活力和NO含量分别为1.20 U/mL和122.41μmol/L,Hcy组eNOS活力和NO含量分别为0.65～0.74 U/mL和65.33～98.91μmol/L,均呈浓度依赖性降低,差异均有统计学意义(F=5.12、18.91,P<0.05)。结论 Hcy可能通过增强caveolin-1蛋白表达,使其与eNOS藕联增加,进而抑制了eNOS活力。     

缺氧对脐静脉内皮细胞损伤及可溶性血管内皮生长因子受体-1 mRNA表达的影响

目的:探讨二氯化钴(CoCl2)诱发化学缺氧对培养的人脐静脉内皮细胞(human umbilical vein endothelial cells)损伤和血管内皮生长因子受体-1(soluble vascular endothelial growth factor receptor-1)mRNA表达的影响。方法:采用MTT检测缺氧时人脐静脉内皮细胞的增殖情况,检测上清中LDH量反映脐静脉内皮细胞损伤程度,RT-PCR检测sFlt-1 mRNA表达水平,观察缺氧时间与三者之间的相关关系。结果:CoCl2可诱导脐静脉细胞中sFlt-1 mRNA的表达,导致细胞损伤,降低细胞活性,三者与缺氧时间呈依赖性。结论:缺氧损伤的脐静脉内皮细胞,细胞增殖能力下降,sFlt-1 mRNA表达增加,可能促进妊娠期高血压疾病的发病。     

Red grape berry-cultured cells reduce blood pressure in rats with metabolic-like syndrome

Purpose

Cumulative evidence suggests that moderate red wine consumption protects the cardiovascular system. The effect of cultured cells derived from red grape berry (RGC) on blood pressure (BP) has not been investigated. We therefore studied the antihypertensive effects of oral consumption of RGC in experimental rat model of metabolic-like syndrome and assessed its effect on human umbilical vein endothelial cells (HUVECs).

Methods

Forty male Sprague–Dawley rats were fed for 5 weeks with either a high fructose diet (HFD) (n = 10) or HFD supplemented, during the last 2 weeks, with different doses (200, 400 and 800 mg/kg/day) of RGC suspended in their food (n = 30). BP, plasma triglycerides, insulin and adiponectin levels were measured at the beginning and after 3 and 5 weeks of diet. RGC effect on vasodilatation was evaluated by its ability to affect endothelin-1 (ET-1) production and endothelial nitric oxide synthase (eNOS) expression in HUVECs.

Results

BP, plasma triglycerides, insulin and adiponectin increased significantly in rats fed with a HFD. The increase in BP, plasma triglycerides and insulin was attenuated by RGC supplementation. Incubation of HUVECs with RGC demonstrated a concentration-dependent inhibition of ET-1 secretion and increase in the level of eNOS, signaling a positive effect of RGC on vasodilatation.

Conclusion

In rats with metabolic-like syndrome, RGC decreased BP and improved metabolic parameters. These beneficial effects may be mediated by the cell constituents, highly rich with polyphenols and resveratrol, reside in their natural state.