过氧化氢对内皮祖细胞凋亡相关蛋白表达的影响

目的:比较不同浓度过氧化氢(hydrogen peroxide,H2O2)对人外周血来源的内皮祖细胞(endothelial progenitor cells,EPCs)生存能力的改变及其对凋亡相关蛋白表达的影响。方法:采用密度梯度离心法和差速贴壁法从人外周静脉血中分离培养内皮祖细胞。选取传代后第3代EPCs作为研究对象,以终浓度分别为50μmol/L、100μmol/L、150μmol/L、200μmol/L、300μmol/L和400μmol/L的过氧化氢处理内皮祖细胞12 h,同时设立正常处理对照组。CCK-8法检测各组内皮祖细胞生存能力的差异;Western blot分析各组内皮祖细胞中凋亡相关蛋白Bax、Bcl-2和p53的蛋白表达情况。结果:与对照组相比,在浓度为50μmol/L、100μmol/L和150μmol/L过氧化氢处理组中细胞存活能力逐渐增强,促凋亡蛋白Bax、p53随之下调,抗凋亡蛋白Bcl-2则显著上调;在浓度为200μmol/L、300μmol/L和400μmol/L过氧化氢处理组中细胞生存能力相对于正常对照组逐渐减弱,促凋亡蛋白Bax、p53表达水平逐渐增加,抗凋亡蛋白Bcl-2表达水平下降。结论:过氧化氢对内皮祖细胞存活能力和凋亡相关蛋白Bax、Bcl-2、p53表达的影响均呈双相性变化。     

Selective Degeneration of Oligodendrocytes Mediated by Reactive Oxygen Species

The mechanism underlying demyelination in inflammatory canine distemper encephalitis is uncertain. Macrophages and their secretory products are thought to play an important effector role in this lesion. Recently, we have shown that anti-canine distemper virus antibodies, known to occur in chronic inflammatory lesions, stimulate macrophages leading to the secretion of reactive oxygen species (ROS). To investigate whether ROS could be involved in demyelination, dog glial cell cultures were exposed to xanthine/xanthine oxidase (X/XO), a system capable of generating O,. This treatment resulted in a specific time-dependent degeneration and loss of oligodendrocytes, the myelin producing cells of the central nervous system. Initial degeneration was not associated with a decrease in viability of oligodendrocytes as judged by trypan blue and propidium iodide exclusion. Astrocytes and brain macrophages were not affected morphologically by this treatment. Further, an evaluation of the effect of several ROS scavengers, transition metal chelators and inhibitors of poly (ADP-ribose) polymerase suggests that a metal dependent formation of OH or a similar highly oxidizing species could be responsible for the observed selective damage to oligodendrocytes.     

Effect of Reactive Oxygen Species on Myelin Membrane Proteins

Fresh myelin, isolated from brainstems of adult rats, was incubated in the presence of Cu2+ and H2O2. Electrophoretic analysis of the reisolated myelin membrane revealed a gradual loss of the protein moiety from the characteristic pattern and an increase in aggregated material appearing at the origin of the gel. The aggregation of proteins was time-dependent and was concomitant with the accumulation of lipid peroxidation products reactive with thiobarbituric acid. Furthermore, during the course of incubation, there was a gradual decrease in the amount of recovered light myelin and a quantitatively similar increase in heavier myelin subfractions. The aggregation of proteins seems not to be directly related to the buoyant densities of myelin fragments. The peroxidative damage to the myelin proteins may be an important contributor to pathochemistry of myelin sheath, in particular, and in general it implies the susceptibility of the protein moiety of cell membranes to oxygen-induced deterioration.     

Cultured Astrocytes Release a Factor that Decreases Endothelin-1 Secretion by Brain Microvessel Endothelial Cells

Abstract: Endothelin-1 (ET-1), originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells, has now been described to possess a wide range of biological activities within the cardiovascular system and in other organs. Brain microvessel endothelial cells, which, together with perivascular astrocytes, constitute the blood-brain barrier, have been shown to secrete ET-1, whereas specific ET-1 receptors are expressed on astrocytes. It is reported here that conditioned medium from primary cultures of mouse embryo astrocytes could significantly, and reversibly, attenuate the accumulation of both ET-1 and its precursor big ET-1 in the supernatant of rat brain microvessel endothelial cells by up to 59 and 76%, respectively, as assessed by immunometric assay. This inhibitor of ET-1 production was purified by gel-exclusion and ion-exchange chromatography as a 280-Da iron-containing molecule, able to release nitrites upon degradation. These results suggest that astrocytes, via release of an iron-nitrogen oxide complex, may be involved in a regulatory loop of ET-1 production at the level of the blood-brain barrier.     

Enhancement of Enzyme-Catalyzed Production of Reactive Oxygen Species by Suspensions of “Crocidolite” Asbestos Fibres

Ethylene release from methylthio-ketobutyric acid is an indicator for activated oxygen species of the OH '-radical type. Xanthine oxidase plus xanthine or diaphorase in the presence of NADH and j uglone produce OH'-type oxy-radicals. The production of reactive oxygen species in these enzymatic systems is enhanced by “crocidolite” asbestos fibres.     

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91^phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91^phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91^phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.     

Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells

Although it is understood that hydrogen peroxide (H₂O₂) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H₂O₂ in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H₂O₂. The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0  ±  4.7 h versus 28.6  ±  0.8 h, p < 0.05), consistent with a diminished growth rate and H₂O₂ release. Incubation with aminotriazole, a catalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G₀/G₁ phase in hCatTg MAECs compared to the wild-type cells (25.0  ±  0.9 h versus 15.9  ±  1.4 h, p  <  0.05). The hCatTg MAECs also exhibited decreased activities of the cyclin-dependent kinase (Cdk) complexes responsible for G₀/G₁- to S-phase transition in the cell cycle, including the cyclin D–Cdk4 and cyclin E–Cdk2 complexes. Moreover, the reduction in cyclin–Cdk activities in hCatTg MAECs was accompanied by increased protein levels of two Cdk inhibitors, p21 and p27, which inhibit the Cdk activity required for the G₀/G₁- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H₂O₂ scavenger, suggest that endogenously produced H₂O₂ mediates MAEC proliferation by fostering the transition from G₀/G₁ to S phase.     

TNF-alpha induces interleukin-8 and endothelin-1 expression in human endothelial cells with different redox pathways

We investigated the effect of TNF-alpha on interleukin-8 (IL-8) and endothelin-1 (ET-1) expression, and their different signal transduction pathways. Human dermal microvascular endothelial cells (HMECs) were treated with TNF-alpha. By Northern blot analysis, TNF-alpha at 50, 100, 200, and 400 U/ml significantly induced IL-8 mRNA expression by 206%, 252%, 211%, and 158%, respectively, as compared to controls (p < 0.05). Overexpression of human superoxide dismutase (SOD) by adenovirus-mediated gene transfer or addition of exogenous hydrogen peroxide (H(2)O(2)) significantly enhanced TNF-alpha-induced IL-8 mRNA expression. Furthermore, HMECs treated with TNF-alpha at 50, 100, and 200 U/ml significantly increased ET-1 mRNA expression by 71%, 82%, and 66%, respectively (p < 0.05). By contrast, SOD gene transfer and exogenous H(2)O(2) significantly inhibited TNF-alpha-induced ET-1 mRNA expression. Thus, TNF-alpha significantly induces both IL-8 and ET-1 gene expression in HMECs possibly through different redox signaling pathways. H(2)O(2) enhances TNF-alpha-induced IL-8 expression, but inhibits TNF-alpha-induced ET-1 expression.     

Inhibition of Glutamate Transport in Synaptosomes by Dopamine Oxidation and Reactive Oxygen Species

Abstract: Dopamine can form reactive oxygen species and other reactive metabolites that can modify proteins and other cellular constituents. In this study, we tested the effect of dopamine oxidation products, other generators of reactive oxygen species, and a sulfhydryl modifier on the function of glutamate transporter proteins. We also compared any effects with those on the dopamine transporter, a protein whose function we had previously shown to be inhibited by dopamine oxidation. Preincubation with the generators of reactive oxygen species, ascorbate (0.85 m M ) or xanthine (500 µ M ) plus xanthine oxidase (25 mU/ml), inhibited the uptake of [³H]glutamate (10 µ M ) into rat striatal synaptosomes (−54 and −74%, respectively). The sulfhydryl-modifying agent N -ethylmaleimide (50–500 µ M ) also led to a dose-dependent inhibition of [³H]glutamate uptake. Preincubation with dopamine (100 µ M ) under oxidizing conditions inhibited [³H]glutamate uptake by 25%. Exposure of synaptosomes to increasing amounts of dopamine quinone by enzymatically oxidizing dopamine with tyrosinase (2–50 U/ml) further inhibited [³H]glutamate uptake, an effect prevented by the addition of glutathione. The effects of free radical generators and dopamine oxidation on [³H]glutamate uptake were similar to the effects on [³H]dopamine uptake (250 n M ). Our findings suggest that reactive oxygen species and dopamine oxidation products can modify glutamate transport function, which may have implications for neurodegenerative processes such as ischemia, methamphetamine-induced toxicity, and Parkinson's disease.     

Enhancement of Enzyme-Catalyzed Production of Reactive Oxygen Species by Suspensions of “Crocidolite” Asbestos Fibres

Ethylene release from methylthio-ketobutyric acid is an indicator for activated oxygen species of the OH '-radical type. Xanthine oxidase plus xanthine or diaphorase in the presence of NADH and j uglone produce OH'-type oxy-radicals. The production of reactive oxygen species in these enzymatic systems is enhanced by “crocidolite” asbestos fibres.     

Effects of reactive oxygen metabolites on norepinephrine-induced vasoconstriction

Aortic rings, 4 mm in length, were obtained from rats and placed on isometric force transducers in oxygenated Krebs buffer. Following a period of stabilization, the cumulative dose response relationship to norepinephrine was assessed. The vessels were washed and allowed to return to baseline in Krebs buffer containing xanthine (0.5 mM). Xanthine oxidase (0.1 U/ml) was then added to the bath and vessels incubated for 30 min. The vessels were resuspended in Krebs buffer and cumulative dose-response curves to norepinephrine reevaluated. The results indicate that generation of reactive oxygen metabolites by xanthine/xanthine oxidase decreases the pD₂ from 7.80 ± 0.04 to 7.40 ± 0.09 with the endothelium intact. Removal of the endothelium did not attenuate the contractile dysfunction, indicating that endothelial-derived metabolites were not mediating the loss of vasoconstrictor effectiveness. Maximal tension development did not differ between normal and oxidized vessel rings. Introduction of oxypurinol (0.2 mg/ml) to the bath prevented the loss of constrictor responsiveness, thereby confirming that all of the oxidants were derived from the xanthine/xanthine oxidase reaction. Superoxide dismutase (200 U/ml) partially prevented the loss of norepinephrine responsiveness produced by xanthine oxidase-derived radicals. The pD₂ in the SOD + xanthine/xanthine oxidase-treated vessels rings (7.19 ± 0.11) was significantly lower tan control vessel rings (7.49 ± 0.04) and significantly higher than xanthine/xanthine oxidase-treated vessels (6.89 ± 0.06). Catalase (1000 U/ml) also partially attenuated the loss of vascular norepinephrine responsiveness. The pD₂ for the catalase + xanthine/xanthine oxidase-treated vessels (7.15 ± 0.02) was significantly lower than control vessels (7.39 ± 0.07)and significantly higher than the xanthine/xanthine oxidase-treated vessels (6.82 ± 0.11). The pD₂ of vessels treated with a combination of SOD and catalase (7.40 ± 0.10) did not differ from control vessels (7.49 ± 0.12). The results of this study indicate that reactive species produced by the interaction of xanthine with xanthine oxidase depress norepinephrine-induced vasoconstriction. The loss of vasoconstrictor responsiveness appears to involve both superoxide and hydrogen peroxide.     

Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H₂O₂), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1^+/+) or TRPA1-deficient (Trpa1^−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H₂O₂ generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H₂O₂ in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H₂O₂-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H₂O₂ mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H₂O₂ production as potential targets for treatment of acute gout attacks.     

辛伐他汀通过上调sirt1抵抗氧化低密度脂蛋白诱导的内皮细胞衰老

目的:探讨辛伐他汀对氧化低密度脂蛋白诱导的内皮细胞衰老的作用及其可能机制.方法:体外培养人脐静脉内皮细胞,给予不同浓度(0、25、50、100 μg/ml)氧化低密度脂蛋白(ox-LDL)培养24小时,观察细胞β-半乳糖苷酶染色及SIRT1蛋白表达的变化;给予不同浓度辛伐他汀(1、5、10 μmol/L)预处理内皮细胞l小时后加入100μg/ml ox-LDL培养人脐静脉内皮细胞23小时,检测细胞β-半乳糖苷酶染色及SIRT1蛋白表达的变化.结果:随着ox-LDL作用浓度的增加,细胞内β-半乳糖苷酶染色的阳性细胞百分率逐渐升高,与空白对照组相比差异均有统计学意义(P＜0.05),在ox-LDL(100 μg/mll)组达到最高,显著高于ox-LDL(25 μg/ml)组(P＜0.001).而不同浓度ox-LDL处理的细胞内SIRT1的表达较空白对照组相比逐渐下降,ox-LDL(50、100 μg/ml)组SIRT1的表达显著低于ox-LDL(25 μg/ml)组(P＜0.05).10 μmol/L辛伐他汀预处理能明显降低100μg/ml ox-LDL处理的内皮细胞内β-半乳糖苷酶染色的阳性细胞百分率(P＜0.001),并显著抑制细胞内SIRT1的蛋白表达(P＜0.001).结论:辛伐他汀可以抵抗ox-LDL诱导的内皮细胞衰老,可能与增加内皮细胞内SIRT1的表达有关.     

Synthesis of Apolipoprotein A-1 in Pig Brain Microvascular Endothelial Cells

In an approach toward the identification of hitherto unknown proteins involved in the function of the blood-brain barrier, we constructed a pig brain microvessel-derived cDNA library that is enriched in blood-brain barrier specific sequences by means of subtractive cloning. Sequence analysis of selected clones revealed that one of the cDNAs encoded porcine apolipoprotein (apo) A-1. The identity of apo A-1 mRNA was further confirmed by in vitro translation of RNA from brain microvascular endothelial cells and subsequent immunoprecipitation with an antibody against human apo A-1. We further investigated the expression of apo A-1 mRNA in several tissues and in endothelial cells of the pig. It is shown that cultured brain microvascular endothelial cells provide an in vitro model to study the expression and function of apo A-1 in the microvasculature of the brain.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

NMDA Receptor Activation Produces Concurrent Generation of Nitric Oxide and Reactive Oxygen Species: Implications for Cell Death

Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescent dye 2,7-dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µM) produced a rapid generation of oxidants that was blocked ～70% by MK-801 (a noncompetitive NMDA-receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. NO production was inhibited with N^G-nitro-l -arginine methyl ester (l -NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismutase (SOD) and catalase, which selectively metabolize O₂^?· andH₂O₂. Concurrent inhibition of O₂^?· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A₂ inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca²⁺-free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate-induced death and oxidant generation. Glutamate-induced cytotoxicity was blocked by MK-801 and attenuated by treatment with l -NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non-NMDA receptors through a Ca²⁺-mediated process. Activation of NO synthase and phospholipaseA₂ contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.     

PTEN基因通过调控ROS抑制结肠癌细胞增殖

为了探讨PTEN基因在结肠癌中的作用以及其机制研究,MTT法检测结肠癌细胞细胞增殖;蛋白免疫印迹检测结肠癌细胞中Ki67蛋白的表达;DCFDA染色流式细胞仪检测结肠癌细胞中ROS水平。结果表明PTEN基因能明显抑制结肠癌细胞细胞增殖;PTEN基因能显著降低结肠癌细胞中Ki67蛋白的表达;细胞内ROS水平在PTEN基因处理组中明显高于空质粒结肠癌细胞组;NAC预处理可明显抑制PTEN基因抑制的细胞增殖;NAC预处理可显著抑制PTEN基因对结肠癌细胞Ki67蛋白的降低作用。PTEN基因能够抑制结肠癌细胞增殖并上调结肠癌细胞内ROS水平。     

二苯乙烯苷对H202诱导血管内皮细胞ICAM-1及VCAM-1表达的影响

目的：通过研究二苯乙烯苷（TSG）对H20：诱导的人脐静脉内皮细胞ICAM-1、VCAM-1表达的影响,探明二苯乙烯苷抗氧化保护内皮细胞的作用机制。方法：体外培养人脐静脉内皮细胞,实验分为空白对照组、H20：组、辛伐他汀组、TSG组,运用逆转录聚合酶链式反应和酶联免疫吸附试验分别检测ICAM-1及VCAM-1mRNA与其蛋白的表达。结果：200μmol·L。的H202作用内皮细胞24h后。ICAM．1和VCAM-1的mRNA和蛋白表达水平均明显上调,与空白对照组比较,差异有显著性（P〈0．01）。而在200μmol·L。的H202作用前用1μmol·L^-1二苯乙烯苷预处理体外培养人脐静脉内皮细胞4h,结果显示二苯乙烯苷能抑制H2O2诱导的内皮细胞ICAM-1、VCAM-1的mRNA和VCAM-1的蛋白水平表达,与H2O2组比较差异有显著性（P〈0．01）;而ICAM-1的蛋白表达水平与H202组比较差异有统计学意义（P〈0．05）;辛伐他汀组ICAM-1和VCAM-1的mRNA及其蛋白水平表达降低,与H20：组比较差异均有显著性（P〈0．01）。实验结果表明二苯乙烯苷可抑制H2O2诱导的内皮细胞粘附分子ICAM-1、VCAM-1表达。结论：二苯乙烯苷可通过降低细胞粘附分子ICAM-1和VCAM-1的表达保护氧化应激引起的人脐静脉内皮细胞损伤。     

Plasminogen Activator Inhibitor-1 Expression by Brain Microvessel Endothelial Cells Is Inhibited by Elevated Glucose

Abstract: Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 m M glucose compared with BBEC cultured in media with 5.5 m M glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.