盐酸川芎嗪对血管紧张素Ⅱ损伤内皮细胞的保护作用

以体外培养人脐静脉内皮细胞系EA.hy926为研究模型,从细胞水平上分析了血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)损伤过程及中药川芎的一种有效成分——盐酸川芎嗪(tetramethylpyrazine hydrochloride,TMP·HCl)保护过程中内皮细胞的相关酶活性变化及信号转导规律。结果表明,体外培养内皮细胞在AngⅡ刺激下,细胞活力降低;且ERK、JNK的磷酸化程度,内皮型一氧化氮合成酶(eNOS)和诱生型一氧化氮合成酶(iNOS)表达量,胞内抗氧化酶的活力分别发生变化;而TMP·HCl预保护后,发生相反变化,同时提高了细胞活力,证实ERK、JNK、eNOS、iNOS等信号分子以及活性氧参与细胞损伤及存活过程。     

呼吸道合胞病毒感染人肺上皮细胞一氧化氮的产生及其作用

本文探讨呼吸道合胞病毒(RSV)感染人肺上皮细胞(A549细胞)后, 一氧化氮(NO)的水平变化及其在RSV感染中的氧化损伤和抗病毒作用。RSV以不同时间感染A549细胞, 并给予NO合成的抑制剂氨基胍(AG)处理。收集细胞培养上清, 分别用硝酸还原酶法和硫代巴比妥酸法检测NO和丙二醛(MDA)含量, 化学法检测羟自由基(OH·)与超氧阴离子(O2.—)水平, 空斑形成试验测定病毒复制滴度(PFU)。结果显示在RSV感染4 h后即上调NO、OH·、O2.—和MDA的表达水平。当RSV感染中给予AG处理以抑制iNOS合成NO时, 则降低OH·、O2.—和MDA含量, 但病毒PFU升高。各指标的变化与相应时间点的感染组相比, 差异均有显著性。提示RSV感染肺上皮细胞诱导生成的NO与细胞内自由基水平升高和加重细胞的自由基损伤程度有关; 但在一定程度上可抑制病毒的增殖水平。     

β淀粉样蛋白1-42对BV-2细胞产生一氧化氮功能的刺激作用

目的应用β淀粉样蛋白1-42(β-Amyloid,Aβ1-42)作用于小胶质细胞(microglia,MG),对MG产生一氧化氮(nitric oxide,NO)的作用进行研究.方法应用高度纯化的BV-2小胶质细胞作为体外小胶质细胞模型,测定加入Aβ1-42后细胞上清NO含量及细胞iNOS酶活力;Western blot法测定Aβ对BV-2细胞iNOS蛋白表达的影响,免疫细胞化学方法对iNOS蛋白的表达情况进行观察.结果 Aβ1-42可以刺激BV-2细胞产生NO、提高细胞iNOS酶活性、增加iNOS蛋白质表达,以上作用均具有时间及浓度依赖性.结论 Aβ1-42在体外可通过提高细胞iNOS酶活性、增加iNOS蛋白质表达而增加NO的分泌,为NO发挥神经元毒性作用创造了条件.     

不同尿素氮水平尿毒血清对人脐静脉内皮细胞的影响

目的:观察不同BUN水平尿毒血清对体外培养人脐静脉内皮细胞(HUVEC)的影响,并探讨尿毒症患者动脉粥样硬化发病的可能机制.方法:比色法测定HUVEC上清液NO、MDA的水平;免疫细胞化学法测定胞浆iNOS表达;酶联免疫吸附法(ELISA)测定上清液iNOS蛋白水平.结果:尿毒血清各组均可上调HUVEC的MDA生成,且以BUN≥50 mmol/L的尿毒血清组对MDA上调最明显(P<0.01);尿毒血清各组可下调HUVEC的NO生成,呈BUN浓度依赖性关系(P<0.01);尿毒血清各组可激活HUVEC的iNOS,诱导细胞生成大量NO,但随着BUN水平的升高,NO、iNOS呈下降趋势,BUN≥50 mmol/L尿毒血清组下降显著(P<0.01).结论:尿毒血清对HUVEC有毒性作用,呈BUN浓度依赖性关系,尤其是BUN≥50 mmol/L时,细胞损伤更为加重.     

肝细胞生长因子抑制糖基化终产物诱导人脐静脉内皮细胞的凋亡作用

目的探讨肝细胞生长因子(HGF)抑制糖基化终产物(AGEs)诱导人脐静脉内皮细胞凋亡的作用及其相关分子机制。方法体外培养ECV-304人脐静脉内皮细胞,采用噻唑蓝(MTT)法测定HGF对AGEs作用后ECV-304细胞生长抑制率的影响;通过Hoechst33258荧光染色观察细胞形态学改变、流式细胞术测定AnnexinV-FITC/PI双染标记的细胞凋亡率,检测HGF对AGEs诱导ECV-304细胞凋亡的影响;Western印迹法检测Bax、Bcl-2蛋白的表达。结果HGF能明显降低AGEs对ECV-304细胞生长的抑制作用;AGE诱导培养的ECV-304细胞出现明显的凋亡形态学改变,在一定浓度范围内,ECV-304细胞凋亡率与AGEs的浓度和作用时间呈依赖关系,加入HGF处理后可显著降低不同时间的内皮细胞凋亡率;HGF作用ECV-304细胞后Bcl-2蛋白表达明显升高,而Bax蛋白表达无明显变化。结论AGEs能诱导内皮细胞凋亡,而HGF能部分抑制AGEs诱导的内皮细胞凋亡,其作用机制可能与上调Bcl-2蛋白的表达水平有关。     

孕酮通过抑制iNOS的表达和NO的生成保护新生鼠缺氧缺血性脑损伤

目的:观察孕酮对缺氧缺血性脑病新生鼠皮层和海马组织中一氧化氮(NO)的含量、诱导型一氧化氮合成酶(iNOS)活性和iNOS表达的影响。方法:建立新生鼠缺氧缺血脑病动物模型,孕酮处理组于建立模型前腹腔注射孕酮溶液,24 h后动物被全部处死,采用硝酸还原酶法检测NO水平和iNOS活性的变化,免疫组化检测各组iNOS的阳性细胞数,RT-PCR检测iNOS mRNA的表达。结果:缺氧缺血组新生鼠皮层和海马组织中NO水平、iNOS活性、iNOS的阳性细胞数、iNOS mRNA的表达明显高于假手术组,孕酮处理组明显低于缺氧缺血组(P<0.05)。结论:孕酮可以保护新生鼠缺氧缺血后引起的皮层和海马损伤,其作用机制与抑制iNOS的活性,抑制iNOS的表达,降低NO水平有关。     

尾加压素Ⅱ对人脐静脉内皮细胞iNOS表达的影响

目的：探讨尾加压素Ⅱ（UⅡ）对人脐静脉内皮细胞（HUVEC）诱导型一氧化氮舍酶（iNOS）mRNA表达及一氧化氮（NO）合成的影响。方法：用不同浓度的UⅡ（10^-9～10^-7mol／L）干预体外培养的HUVEC,用硝酸酶还原法及比色法检测细胞培养上清液中NO的水平及iNOS的活性,半定量逆转录一聚合酶联反应（RT—PCR）法检测内皮细胞iNOSmRNA的表达。结果：UⅡ干预24h后,与空白对照组相比,UⅡ呈浓度依赖性显著刺激NO的合成（P〈0．05）,增加iNOS的活性（P〈0．05）,上调iNOSmRNA的表达（P〈0．05）。结论：UⅡ能刺激HUVEC的iN—OSmRNA的表达和NO的合成,提示UⅡ可能通过激活iNOS／NO途径而发挥舒张血管的作用。     

秦皮甲素对AGEs致损的ECV304细胞增殖及氧化应激的影响

为研究秦皮甲素对血管内皮细胞的保护作用,采用CCK-8法观察秦皮甲素对体外AGEs培养的人脐静脉内皮细胞增殖的影响。检测不同浓度AGEs以及秦皮甲素作用后对内皮细胞一氧化氮(NO)、不对称二甲基精氨酸(ADMA)水平的影响以及内皮细胞氧化应激有关指标:活性氧簇(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD);脂肪代谢相关指标:乳酸脱氢酶(lactic dehydrogenase,LDH)、总胆固醇(total cholesterol,CHO)、甘油三酯(triglyceride,TG)和低密度脂蛋白(low density lipoprotein,LDL),同时分别检测粘附相关因子:血管细胞粘附分子-1(VCAM-1)和细胞间粘附分子-1(ICAM-1)的表达水平。结果显示200 mg/L AGEs对人内皮细胞ECV304增殖有显著抑制作用,秦皮甲素可对抗AGEs导致的内皮细胞增殖抑制,并呈浓度依赖性。在25 mg/L时,保护效应达到最高。秦皮甲素可抵抗ROS生成。同时可改善细胞的脂类代谢:胆固醇、LDL以及TG含量在秦皮甲素作用后改善明显。秦皮甲素可显著抑制内皮粘附因子VCAM-1的表达。秦皮甲素还可上调NO水平,下调ADMA水平。总之,秦皮甲素可有效促进人血管内皮细胞增殖并在改善氧化应激,脂代谢,粘附因子和NO释放等方面发挥作用。     

氰酸盐诱导人脐静脉内皮细胞氧化应激损伤

该研究探讨尿素水解产物氰酸盐(cyanate)对体外培养的血管内皮细胞氧化应激和功能障碍的作用。培养人脐静脉内皮细胞系(human umbilical vein endothelial cells,HUVECs),通过CCK8法检测氰酸盐对内皮细胞活力的影响;采用DCFH-DA法检测ROS水平;用比色法测定NO水平;分别用细胞免疫荧光和Western blot检测ICAM-1(intercelluar adhesion molecule-1)、e NOS(endothelial nitric oxide synthase)表达。氰酸盐呈浓度依赖性影响内皮细胞活力,与对照组相比,当氰酸盐浓度为1.00 mmol/L时,细胞活力受到明显抑制(P0.05);与正常组和阴性对照组(甘露醇组)相比,氰酸盐诱导内皮细胞内ROS水平明显升高;NO水平明显减少(P0.05)。免疫荧光结果显示,氰酸盐作用内皮细胞24 h后,ICAM-1荧光明显增强,e NOS明显减弱(P0.05)。Western blot结果显示,内皮细胞内ICAM-1水平随氰酸盐浓度升高和负荷时间延长上调,而e NOS水平下调(P0.05)。氰酸盐诱导血管内皮细胞氧化应激产生和功能障碍。     

羟基红花黄色素A对脂多糖作用后血管内皮细胞诱导型一氧化氮合酶表达的影响

旨在探讨羟基红花黄色素A(hydroxysafflor yellow A,HSYA)对脂多糖(lipopolysaccharide,LPS)作用后人脐静脉内皮细胞株HUVECs细胞株诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达的影响.培养HUVECs细胞株,用 1 mg/L LPS及不同浓度的HYSA处理细胞24 h,MTT法检测细胞增殖情况,硝基还原酶法检测培养液中一氧化氮(NO)含量,RT-PCR及Western blotting检测iNOS表达.结果表明0.01、0.1 mmol/L HYSA对LPS引起的iNOS升高无明显作用,但1 mmol/L HYSA能明显抑制LPS作用后高度表达的iNOS量.因此,HYSA能下调LPS所致iNOS的异常表达,这可能有助于临床治疗血管炎症疾病.     

Inhibition of nitric oxide synthase activity by early and advanced glycation end products in cultured rabbit proximal tubular epithelial cells

Nitric oxide (NO) is important in the regulation of renal tubular function. We have investigated whether glycated proteins could impair the NO production by examining the effects of Amadori products (AP-BSA) and advanced glycation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by measurement of the conversion of L-arginine to L-citrulline and by production of NO, after short-term (30 min) or long-term (1 or 3 days) incubation. Short incubations of PTE cells with either 200 microg/ml AP-BSA or 40 microg/ml AGE-BSA significantly decreased NO production. AP-BSA (3000 microg/ml) inhibited the Ca(2+)-dependent NOS activity even though above 50 microg/ml it increased Ca(2+)-independent NOS activity. In contrast, 40 microg/ml AGE-BSA inhibited both isoforms of NOS. Longer incubations with 200 microg/ml AP-BSA or 250 microg/ml AGE-BSA decreased NO release and inhibited Ca(2+)-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso-N-acetyl-penicillamine (SNAP), while 250 microg/ml AGEs decreased it. After 3 days incubation, glycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, suggesting that the fall in NO production was not due to there being fewer cells. These data indicate that APs and AGEs directly inhibit NOS activity, and additionally that AGEs quench released NO. Thus, both types of glycated proteins alter the production of NO by PTE cells and could participate in the renal tubule dysfunction associated with aging and diabetes.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Extracellular histones disarrange vasoactive mediators release through a COX‐NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.     

胰岛素促进血管内皮细胞产生一氧化氮的实验研究

目的：探讨胰岛素对血管内皮细胞增殖、NO产生和NOS基因表达的影响。方法：培养牛主动脉内皮细胞,测定培养上清液中NO氧化产物NO2^－的水平并应用定量RT－PCR技术检测内皮细胞NOS mRNA的表达水平。结果：①胰岛素对大血管内皮细胞无细胞毒作用,也不影响细胞增殖;②在1－15μg/ml浓度范围内,胰岛素加强内皮细胞释放NO,且呈剂量依赖的方式,NOS特异性抑制剂L－NAME可阻抑之;③胰岛素轻度增加NOS mRNA表达水平,但无统计学意义。结论：胰岛素既不影响大血管内皮细胞增殖,也不影响内皮细胞NOS mRNA表达水平,但以剂量依赖的方式加强内皮细胞产生NO,推测其诱导NO产生的机制可能是通过酶活性的诱导,加速NO的合成。     

Possible usefulness of apocynin, an NADPH oxidase inhibitor, for nitrate tolerance: prevention of NO donor-induced endothelial cell abnormalities

The long-term benefits of nitroglycerin therapy are limited by tolerance development. Understanding the precise nature of mechanisms underlying nitroglycerin-induced endothelial cell dysfunction may provide new strategies to prevent tolerance development. In this line, we tested interventions to prevent endothelial dysfunction in the setting of nitrate tolerance. When bovine aortic endothelial cells (BAECs) were continuously treated with nitric oxide (NO) donors, including nitroglycerin, over 2-3 days, basal production of nitrite and nitrate (NO(x)) was diminished. The diminished basal NO(x) levels were mitigated by intermittent treatment allowing an 8-h daily nitrate-free interval during the 2- to 3-day treatment period. Addition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin restored the basal levels of NO(x) that were decreased by continuous nitroglycerin treatment of BAECs. Apocynin caused significant improvement of increased mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in BAECs given nitroglycerin continuously over the treatment period. Apocynin also reduced endothelial production of reactive oxygen species (ROS) after continuous nitroglycerin treatment. These results showed an essential similarity to the effects of a nitrate-free interval. Application of the NOS inhibitor N(omega)-nitro- l-arginine methyl ester caused a recovery effect on basal NO(x) and eNOS expression but was without effect on ROS levels in continuously NO donor-treated BAECs. In conclusion, the present study characterized abnormal features and functions of endothelial cells following continuous NO donor application. We suggest that inhibition of NADPH oxidase, by preventing NO donor-induced endothelial dysfunction, may represent a potential therapeutic strategy that confers protection from nitrate tolerance development.     

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-α, interleukin-1 β and interferon-γ, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25–50 μM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.     

The role of nitric oxide in the development of diabetic angiopathy.

Diabetic angiopathy is the main cause of morbidity and mortality in patients with diabetes mellitus. Clinical manifestations and pathophysiological mechanisms of diabetic angiopathy can be traced back to the development of endothelial cell dysfunction with alterations in the eNOS/NO system production or availability as the primum movens in its natural history. Hyperglycemia per se or through the accumulation of AGEs, increased oxidative stress, leading to NOS uncoupling and NO-quenching by excess superoxide and peroxynitrite, and individual genetic background are thought to be responsible for this NO metabolism imbalance. The complex interplay of these mechanisms results in a perturbation of the physiological properties of NO in the maintenance of endothelial homeostasis, such as vasodilation, anticoagulation, leukocyte adhesion, smooth muscle cell proliferation, and antioxidant capacity. Hence, abnormality in NO availability results in generalized accelerated atherosclerosis, hyperfiltration, glomerulosclerosis, tubulointerstitial fibrosis and progressive decline in glomerular filtration rate, and apoptosis and neovascularization in the retina. Indeed, the parallel development of nephropathy, retinopathy, and macroangiopathy may be considered as manifestations of endothelial dysfunction at distinct vascular sites. Given this scenario, intervention targeting any of the pathways involved in the NOS/NO system cascade may prove potential therapeutic targets in the prevention of long-term diabetic complications.     

Comparison of effects of nitric oxide synthase (NOS) inhibitors on plasma nitrite/nitrate levels and tissue NOS activity in septic organs

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar-King rats. The rats treated with the three iNOS inhibitors (N-(3-(aminomethyl)benzyl)acetamidine (1400W), (1 S, 5 S, 6 R, 7 R )-2-aza-7-chloro-3-imino-5-methylbicyclo [4.1.0] heptane hydrochloride (ONO-1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose-dependent decreases in plasma NOx levels and NOS activity in the lungs. The non-selective NOS inhibitor (N(G)-methyl-L-arginine (L-NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.     

白细胞介素-6通过AMPK/SIRT1通路下调eNOS Thr495/497

该文探讨了白细胞介素-6(interleukin-6,IL-6)对牛主动脉内皮细胞(bovine aortic endo-thelial cells,BAECs)的内皮型一氧化氮合成酶(endothelial nitric oxide synthase,eNOS)的影响及其可能的发生机制.在原代BAECs细胞培养基础上...