Cinaciguat对高糖损伤血管内皮细胞功能的影响

目的 糖尿病状态下，一氧化氮(NO)与可溶性鸟苷酸环化酶(soluble guanylatecyclase,sGC)均被氧化，使NO-sGC-环磷酸鸟苷(cGMP)信号通路传导异常，NO-sGC-cGMP信号通路异常是糖尿病血管内皮功能障碍的重要病理基础。可溶性鸟苷酸环化酶激活剂(cinaciguat，CIN)可激活失活的sGC进而起到保护血管内皮细胞功能的作用。本研究通过体外实验制造高糖内皮细胞损伤模型，观察CIN对人脐静脉内皮细胞功能的影响。 方法 培养人脐静脉内皮细胞，并将细胞分为正常对照组、高糖组、CIN干预组，每组6例。高糖组及CIN干预组均加入33.3 mmol/L的葡萄糖制造高糖细胞损伤模型，而CIN组同时加入0.1 μmol/L CIN干预。各组细胞在培养6、24、48 h时，检测上清液中的NO水平及细胞内一氧化氮合酶(NOS)浓度；培养48 h后RT-PCR检测细胞内诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)mRNA的表达；培养48 h后Western blotting检测细胞间粘附分子-1(ICAM-1)及血管粘附分子-1(VCAM-1)蛋白的表达。 结果 与正常对照组相比，高糖组NO、NOS含量呈现早期升高晚期下降的趋势，而CIN组上述趋势消失；与正常组比较，高糖组内皮细胞的eNOS mRNA表达水平降低(P＜0.05)，iNOS mRNA、ICAM-1与VCAM-1蛋白表达明显升高(P＜0.05)，而CIN组eNOS mRNA表达水平较高糖组明显升高(P＜0.05)，iNOS、ICAM-1与VCAM-1蛋白的表达水平明显降低(P＜0.05)。 结论 CIN可平衡NOS的活性，调节NO的生成及ICAM-1与VCAM-1的表达，改善高糖状态下内皮细胞的功能。      

抵抗素诱导脐静脉内皮细胞功能异常

 【目的】研究抵抗素（resistin）对脐静脉内皮细胞功能的影响，以探讨抵抗素在粥样硬化性疾病中的作用及其机制。【方法】原代培养人脐静脉内皮细胞，不同浓度人抵抗素（0、50、100ng／mL）培养24h。流式细胞检测抵抗素对内皮细胞间黏附分子（ICAM-1）、血管细胞间黏附分子（VCAM-1）与活性氧簇（ROS）表达的影响；RT-PCR检测抵抗素对内皮素（ET-1）、内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）mRNA表达的影响。【结果】人脐静脉内皮细胞经人抵抗素（50ng／mL、100ng／mL）处理24h后ICAM-1和ET-1mRNA表达显著增高，而各组间VCAM-1表达、ROS生成，以及eNOS和iNOS mRNA表达无明显差别。【结论】抵抗素可通过增加ICAM-1表达，上调ET-1表达直接促进内皮细胞激活，提示脂肪细胞与内皮细胞的相互作用可能是动脉粥样硬化性疾病的发病因素之一。     

抵抗素诱导脐静脉内皮细胞功能异常

【目的】研究抵抗素（resistin）对脐静脉内皮细胞功能的影响，以探讨抵抗素在粥样硬化性疾病中的作用及其机制。【方法】原代培养人脐静脉内皮细胞，不同浓度人抵抗素（0、50、100ng／mL）培养24h。流式细胞检测抵抗素对内皮细胞间黏附分子（ICAM-1）、血管细胞间黏附分子（VCAM-1）与活性氧簇（ROS）表达的影响；RT-PCR检测抵抗素对内皮素（ET-1）、内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）mRNA表达的影响。【结果】人脐静脉内皮细胞经人抵抗素（50ng／mL、100ng／mL）处理24h后ICAM-1和ET-1mRNA表达显著增高，而各组间VCAM-1表达、ROS生成，以及eNOS和iNOS mRNA表达无明显差别。【结论】抵抗素可通过增加ICAM-1表达，上调ET-1表达直接促进内皮细胞激活，提示脂肪细胞与内皮细胞的相互作用可能是动脉粥样硬化性疾病的发病因素之一。     

脱氢表雄酮对血管内皮细胞损伤后单核细胞与内皮细胞相互作用的影响

目的脱氢表雄酮(dehydroepiandrosterone,DHEA)是成人体内最为丰富的来源于肾上腺的甾体激素,文中探讨DHEA对氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)所致血管内皮细胞损伤后单核细胞与内皮细胞相互作用的影响。方法以培养的人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)作为靶细胞,在内皮细胞培养基中加入ox-LDL或在试验体系中加入不同浓度的DHEA,测定人单核细胞系U937与HUVEC的黏附,检测内皮细胞条件培养基中一氧化氮(nitric oxide,NO)及单个核细胞趋化蛋白-1(monocyte chemoattractant protein,MCP-1)的含量,应用流式细胞仪检测内皮细胞表面黏附分子细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)、血管细胞黏附分子-1(vascularcell adhesion molecule-1,VCAM-1)及E-选择素的表达。将内皮细胞条件培养基作用于U937细胞,应用RT-PCR检测U937细胞CCR2、LFA-1以及VLA-4 mRNA的表达。结果 DHEA可明显抑制单核U937细胞与内皮细胞之间的相互黏附,明显促进内皮细胞NO合成,抑制MCP-1分泌,降低内皮细胞表面ICAM-1、VCAM-1的表达,且经DHEA作用后的内皮细胞条件培养基可明显抑制单核U937细胞CCR2、淋巴细胞功能相关抗原(lymphocyte function-associated antigen,LFA)-1以及非常晚期抗原(very late antigen,VLA)-4 mRNA的表达。结论 DHEA可通过抑制内皮细胞分泌趋化因子、下调内皮细胞表面黏附分子的表达,抑制内皮细胞对单核细胞的趋化及相互黏附,据此认为DHEA可能对血管内皮细胞具有保护作用。     

ox-LDL对PLGF作用内皮细胞释放NO和内皮细胞黏附分子的影响

目的：探讨氧化型低密度脂蛋白对胎盘生长因子-1作用人脐静脉内皮细胞释放一氧化氮和内皮细胞黏附分子的影响。方法：在ox-LDL（25μg／mL）条件下,应用不同浓度的PLGF-1（20、40、80ng／mL）孵育ECV-304,对照组为内皮细胞未受损时,相同浓度梯度的PLGF-1孵育ECV-304,3h、6h、12h、24h后应用ELISA法检测内皮细胞黏附分子-可溶性细胞间黏附因子-1、可溶性血管内皮细胞黏附因子-1的表达,硝酸盐还原酶法检测NO的表达。结果：正常生理条件下,PLGF诱导NO和内皮细胞黏附分子的表达,且呈时间（当t=6h达到最理想的分泌量）、浓度依赖关系。在氧化损伤条件下,PLGF诱导NO的表达下降,而内皮细胞黏附分子的表达则增高,以VCAM-1表达增多更为明显。结论：氧化损伤是动脉粥样硬化的独立危险因素,而PLGF诱导内皮的活化,上调内皮黏附分子的表达,可进一步促使疾病的恶化。故认为抑制PLGF的生物活性可达到控制炎症反应的作用。     

miRNA-31在高糖诱导内皮细胞功能障碍中的作用

目的 探讨miRNA-31在高糖诱导的内皮细胞功能障碍中的作用机制.方法 体外培养人冠状动脉内皮细胞系,不同浓度高糖刺激24h后检测细胞内皮型一氧化氮合酶(eNOS)蛋白表达及培养上清液NO含量;Real-time PCR检测高糖刺激后miRNA-31表达变化;microRNA在线分析软件预测并应用双荧光素酶报告基因系统和Western blot验证eNOS是否为miRNA-31的直接靶基因;通过转染miRNA-31 antagomir观察miR-NA-31对内皮细胞功能障碍的影响.结果 不同浓度的高糖刺激内皮细胞24h后,eNOS蛋白表达和培养液NO含量呈剂量依赖性下降.高糖刺激内皮细胞miRNA-31表达明显增加.microRNA在线分析软件及双荧光素酶报告基因实验提示eNOS的翻译水平受miRNA-31直接调控.转染miRNA-31 antagomir明显上调高糖刺激后内皮细胞eNOS蛋白和培养液NO含量.结论 miRNA-31上调进而抑制eNOS表达可能是高糖诱导内皮细胞功能障碍的作用机制之一.     

波动性高糖对人脐静脉内皮细胞中NO 合成的影响及作用机制

目的：探索波动性与持续性高糖对人脐静脉内皮细胞合成血管舒张因子一氧化氮（NO）的影响及其作用机制。方法：以体外培养人脐静脉内皮细胞（HUVECs）为研究对象,分别测定波动性高浓度葡萄糖(5.5 或20 mmol/L) 与持续性高浓度葡萄糖(20 mmol/L)环境下NO浓度,RT-PCR及Western印迹方法检测磷脂酰肌醇-3激酶(PI3K)、蛋白激酶B（PKB/AKT）、内皮型一氧化氮合酶(eNOS）mRNA及蛋白表达水平。结果：波动性高糖组NO均明显低于持续高糖组和正常组（P<0.05）;波动组,持续高血糖组的PI3K,PKB,eNOS mRNA及蛋白表达水平均较正常组下调（P<0.01）,而波动组又低于持续组（P<0.05）。结论：波动性高血糖较持续性高血糖可能对血管内皮细胞具有更强的损伤效应,并可能通过影响信号通路PI3K/PKB/eNOS导致NO合成减少。     

高浓度葡萄糖对培养血管内皮细胞活力和黏附分子表达的影响

目的探讨高浓度葡萄糖作用于脐静脉内皮细胞后,对脐静脉血管内皮细胞系的活力和白细胞黏附分子〔血管细胞黏附分子-1(VCAM-1)、细胞间黏附分子-1(ICAM-1)〕表达的影响,为阐明高血糖在糖尿病血管病变的早期发病中作用与机制提供依据。方法采用胎盘蓝染色法及流式细胞仪检测细胞活力和黏附分子表达。结果高浓度葡萄糖可使内皮细胞的死亡率增加,ICAM-1表达显著上调,但VCAM-1表达无明显变化。结论减少高糖诱导的ICAM-1、VCAM-1表达增加,对减少糖尿病患者慢性并发症的发生概率是一条有益的途径。     

依帕司他对高糖诱导的血管内皮损伤的保护作用

目的初步研究依帕司他保护高糖诱导的血管内皮细胞损伤的作用。方法体外培养脐静脉血管内皮细胞HUVEC,高糖刺激8 h后利用化学发光法检测细胞培养上清中NO的含量,利用Real time PCR和Western blot分别检测细胞中内皮型一氧化氮合酶(eNOS)的mRNA和蛋白表达水平,以同浓度的甘露醇作为对照。给予依帕司他(0.1μmol/L)预处理30 min,随后再进行高糖培养,检测NO的含量以及eNOS的mRNA和蛋白表达,同时检测依帕司他的靶向基因醛糖还原酶(AR)以及NO的上游基因NADPH氧化酶4(NOX4)的蛋白表达水平。结果高糖培养8 h之后,血管内皮细胞分泌NO下降,eNOS的mRNA和蛋白表达水平下降,与甘露醇对照组相比,具有统计学差异(P<0.05);预先给予依帕司他处理后再进行高糖培养,细胞中eNOS的mRNA和蛋白表达水平均升高,NO表达上调,与单独高糖培养组相比,具有统计学差异(P<0.05)。同时,细胞中AR和NOX4的蛋白表达下降。结论高糖可以诱导内皮细胞损伤,表现为NO的分泌水平降低。依帕司他可能是通过抑制AR和NOX4的表达而发挥对血管内皮细胞损伤的保护作用。     

内皮型一氧化氮合酶活性和基因表达异常与动脉粥样硬化

内皮型一氧化氮合酶(eNOS)产生的一氧化氮(NO)减少将导致内皮细胞功能异常,最终可能导致动脉粥样硬化的产生。eNOS表达降低和酶活性的降低均可以导致NO生成减少。而eNOS活性受eNOS与某些蛋白质相互作用和本身磷酸化的影响。本文主要对eNOS基因表达异常和eNOS活性异常与动脉粥样硬化的关系予以综述。     

Effect of aspirin on high glucose-induced senescence of endothelial cells

Background Endothelial cell senescence is accelerated under high glucose condition, which may contribute to the vascular complications in the diabetics, tt has been proved that aspirin has multiple cytoprotective effects. This study aimed to investigate the effect of aspirin on high glucose-induced endothelial cell senescence and its possible mechanism. Methods Human umbilical venous endothelial cells were cultured in Dulbecco＇s modified Eagle＇s medium （DMEM） with different treatments including the normal glucose （5.5 mmol/L）, high glucose （33 mmol/L） and aspirin （0.01-1.00 mmol/L） with high glucose. And 300 umol/L L-NAME was added to the culture medium when needed. After 48 hours, SA-13-gal staining was used to evaluate the senescence. Total nitric oxide （NO） production and NO synthase （NOS） activity were measured using Griess reaction and molecular probes of 3-amino-4-aminomethyl-2＇, 7＇- difluorescein, diacetate. The level of intracellular reactive oxygen species was monitored by flow cytometry using 2＇, 7＇-dichlorofluorescein diacetate. Endothelial NOS （eNOS）, caveolin-1 protein expressions and caveolin-1/eNOS interaction were analyzed by immunoblotting and immunoprecipitation respectively. Asymmetric dimethylarginine （ADMA） concentration was determined by high-performance liquid chromatography. Results Exposure to 33 mmol/L glucose for 48 hours significantly increased the number of SA-13-gal positive cells. Co-incubation with aspirin markedly inhibited SA-13-gal activity dose-dependently. Aspirin increased NOS activity with eNOS protein expression unchanged and increased NO levels and alleviated oxidative stress. Consistent with these findings, caveolin-1 expression, caveolin-1/eNOS interaction and ADMA accumulation were also decreased. All the inhibitory effects of aspirin on senescence were completely obliterated by L-NAME, the NOS inhibitor. Conclusion The anti-senescent effects of aspirin are fulfilled by increasing NO production via the up-regulation of NOS activity and preventing caveolin-1 expression, caveolin-1/eNOS interaction and ADMA accumulation.     

Selenium inhibits high glucose- and high insulin-induced adhesion molecule expression in vascular endothelial cells

BACKGROUND: Initiation of an atherosclerotic lesion requires endothelial expression of adhesion molecules. Selenium (Se), a biologically essential trace element, can inhibit cytokine (e.g., TNF-alpha)-induced expression of adhesion molecules. Atherosclerosis is accelerated in diabetic patients. This is at least partially caused by hyperglycemia and hyperinsulinemia increasing adhesion molecule expression. These experiments tested whether Se can also alter high glucose- and high insulin-induced expression of adhesion molecules. METHODS: Human umbilical vein endothelial cells (HUVECs) were pretreated with Se and stimulated by high glucose or high insulin. Expression of adhesion molecules was measured by Western blot. RESULTS: Se (100 nmol/L) significantly inhibited glucose (25 mmol/L)-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin. Moreover, Se significantly inhibited insulin (100 nmol/L)-induced VCAM-1 and ICAM-1 expression, whereas high insulin had no inducing effect on E-selectin. Se also inhibited high glucose- and high insulin-induced activation of p38 mitogen-activated protein kinase (p38), which indicated that the preventive effects of Se on adhesion molecules may be associated with p38. The important role of p38 in Se effects was further confirmed using p38 inhibitor SB203580. CONCLUSIONS: These results suggest that Se can inhibit high glucose- and high insulin-induced expression of adhesion molecules. Such antagonism is at least partially mediated through the modulation of p38 pathway. Therefore, Se may be considered as a potential preventive intervention for diabetes-accelerated atherosclerosis.     

异丙酚对大鼠肝一氧化氮合酶的影响

目的　了解异丙酚对大鼠肝脏一氧化氮合酶的影响。方法　 40只SD大鼠 ,随机分为异丙酚组、对照组 ,分别腹腔注射等容积异丙酚 (10ml/kg ,即 10 0mg/kg)和生理盐水 (10ml/kg)。异丙酚组待鼠翻正反射消失后 ,微泵输注异丙酚 ,2 0min后处死 ;对照组鼠腹腔注射 2 0min后处死。检测肝脏组织匀浆中NO水平、NOS酶活性及内皮型NOS(eNOS)在肝脏内的表达与分布 (免疫组化法 )。结果　异丙酚组肝脏组织匀浆中NO水平、NOS酶活性均明显高于对照组 (P <0 0 1)。免疫组化结果显示 :异丙酚组肝脏血管内皮细胞eNOS染色表达强阳性 ,半定量比较有显著差异 (P <0 0 1)。结论　异丙酚可以刺激肝脏中NOS活性 ,升高肝脏内源性NO水平     

胰高血糖素样肽-1对高糖诱导人脐静脉内皮细胞凋亡的影响及相关机制

目的：探讨胰高血糖素样肽-1（glucagon—like peptide-1,GLP—1）对高糖诱导人脐静脉内皮细胞huma numbilica lvein endothelial cells,HUVECs）凋亡的影响及相关机制。方法：HUVECs加入不同处理因素后分组,分别培养48h后,MTT检测细胞活力;流式细胞仪检测细胞早期凋亡率;Westernl印迹测定细胞P-Akt,p-eNOS水平;NO试剂盒检测NO浓度。结果：高糖（33mmol／L）培养48h后HUVECs细胞活力下降p〈0．05）,细胞凋亡率增加（p〈0．01）,细胞p-Akt,p-eNOS,NO水平均下降（P〈0．05）;高糖条件下加人GLP．1（3nmol／L）培养48h,与高糖组相比较,HUVECs细胞活力增加（P〈0．01）,细胞凋亡率减少（p〈O．05）,细胞p-Akt,p-eNOS,NO水平均增／JH（V〈0．05）;P13K抑制剂wortmannine（100nmol／L）可以阻断GLP-1的抗凋亡作用及其对P—Akt蛋白、p-eNOS蛋白及NO水平的影响;eNOS抑制剂L—NAME（100umol／L）仅能阻断GLP-1的抗凋亡作用及对NO水平的影响,不影响p-Akt蛋白的表达。结论：GLP．1可改善高糖诱导的HUVECs凋亡,该抗凋亡作用可能与P13K／Akt／eNOS通路的上调相关。     

Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

Background The vascular endothelial growth factor （VEGF） is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 （ICAM-1） is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells （ECs） is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs. Methods Bovine retinal ECs （BRECs） were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF （100 ng/ml） and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase （eNOS） were detected using Western blotting. Griess reaction was used to detect nitric oxide （NO）. Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C （PKC） altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase （PI3K） or reactive oxygen species （ROS） significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased. Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.