C肽对人脐静脉内皮细胞凋亡的影响及其与内皮型一氧化氮合酶表达的相关性分析

目的探讨C肽对高糖状态下人脐静脉内皮细胞(HUVEC)凋亡的影响,并对凋亡与内皮型一氧化氮合酶(eNOS)表达进行相关性分析。方法内皮细胞凋亡定性用磷脂酰丝氨酸外翻分析(AnnexinV法),凋亡定量采用细胞DNA片段酶联免疫吸附测定法(ELISA)。检测内皮细胞eNOS表达采用半定量逆转录聚合酶链反应(SQRTPCR)法。结果高浓度葡萄糖(33.3mmol/L)使HUVEC凋亡增加(P<0.01),eNOS表达减少,加入生理浓度(1.0nmol/L)或高浓度(50.0nmol/L)C肽后,凋亡显著减少(P<0.05),eNOS表达增加,HUVEC凋亡与eNOS表达相关(r=-0.845,P<0.01)。结论高糖可导致HUVEC凋亡增加;高浓度或生理浓度的C肽可抑制高糖引起的HUVEC凋亡;HUVEC凋亡与eNOS表达呈负相关。     

α-硫辛酸对高糖诱导的血管内皮细胞凋亡的保护作用

目的 观察α-硫辛酸对高糖诱导的血管内皮细胞凋亡是否有保护作用.方法 以不同浓度α-硫辛酸干预高糖作用下的血管内皮细胞(ECV304),共同孵育72h,以Annexin-FITC/PI双染流式细胞术和TUNEL法检测细胞凋亡.结果 高糖明显增加内皮细胞凋亡(P＜0.01);以不同浓度α-硫辛酸干预,内皮细胞凋亡明显减少(P＜0.01),这种作用具有浓度依赖性.结论 α-硫辛酸对高糖诱导的血管内皮细胞凋亡有保护作用.     

艾塞那肽抑制高糖诱导内皮细胞凋亡的作用机制

在正常糖浓度(5.5 mmol/L)和高糖(33 mmol/L)培养的人脐静脉内皮细胞(HUVECs)分别加入不同浓度的艾塞那肽并干预不同时间后,噻唑蓝(MTF)法检测细胞活力,应用流式细胞仪检测细胞早期凋亡率,Western印迹法检测蛋白激酶B(Akt)磷酸化、Bcl-2和Bax蛋白表达水平.结果显示,HUVECs经高糖培养48和72 h后,细胞活力明显下降(P＜0.01).经1、10和100 nmol/L艾塞那肽干预48 h后,细胞活力明显增加,并呈浓度依赖性(P＜0.01).与正常糖浓度组比较,高糖组细胞凋亡率升高,Akt磷酸化和Bcl-2蛋白表达水平下降,Bax蛋白表达增加,Bcl-2/Bax比值降低(P＜0.01).艾塞那肽干预后,细胞凋亡率下降,Akt磷酸化和Bcl-2蛋白表达增加,Bax蛋白表达下降,Bcl-2/Bax比值升高(P＜0.01),而艾塞那肽的作用可被磷酸肌醇3激酶(PI3K)抑制剂LY294002所对抗(P＜0.01).提示艾塞那肽可通过PI3 k/Akt信号通路调节Bcl-2/Bax蛋白表达来抑制高糖诱导的内皮细胞凋亡,起到保护内皮细胞的作用.     

丹酚酸B作用不同时间对高糖高脂内皮细胞凋亡的抑制作用

目的观察丹酚酸B抑制高糖高脂诱导的内皮细胞凋亡的作用。方法以糖基化终末产物(AGEs)和氧化低密度脂蛋白(ox-LDL)联合作用诱导人脐静脉内皮细胞凋亡,通过Hochest33258染色,观察并比较丹酚酸B及洛伐他汀作用不同时间(12、24、36、48、72h),抑制高糖高脂诱导的内皮细胞凋亡的效果。结果研究表明,洛伐他汀作用12、24h时,抑制内皮细胞凋亡效果明显,而丹酚酸B作用36、48h及72h抑制内皮细胞凋亡效果显著。结论与洛伐他汀相比,丹酚酸B抑制高糖高脂诱导的内皮细胞凋亡的作用发挥得较迟缓,但效果持久。     

营养代谢疾病

20052721 血浆抗素水平与胰岛素抵抗的关系研究;20052722 糖耐量低减患者红细胞胰岛素酶活性与胰岛素白抵抗；20052723 糖尿病并发高血压男性患者的血清性腺激素水平分析；20052724 血清脂联素、TNF-α与胰岛素抵抗的相关性研究；20052725 C肽和胰岛素对高糖诱导的内皮细胞凋亡的影响。     

自由基清除剂MCI-186对高糖环境中内皮细胞凋亡的干预作用

观察内皮细胞在高糖环境及经肿瘤坏死因子α(TNF-α)诱导后的细胞凋亡情况，并加入自由基清除剂MCI-186予以干扰。检测细胞DNA裂解片段及凋亡信号蛋白(caspase)的表达量，以期探讨MCI-186对高糖环境中血管内皮细胞凋亡的干预作用及凋亡机制。发现高糖环境可增加内皮细胞凋亡率，并上调TNF-α诱导的调亡。MCI-186可抑制高糖诱导的细胞凋亡。但无法干预高糖环境中TNF—a诱导的凋亡。     

酰基化ghrelin抑制高糖诱导的人血管内皮细胞凋亡

目的近年来研究证明高糖引起的血管内皮细胞凋亡可能加重糖尿病动脉粥样硬化,本研究旨在探讨酰基化ghrelin能否抑制高糖诱导的人血管内皮细胞凋亡。方法应用吖啶橙形态学染色及TUNEL、流式细胞分析仪、分光光度计研究高糖及ghrelin预处理后内皮细胞凋亡及caspase-3活性情况。结果内皮细胞暴露于高浓度葡萄糖(33.3mmol/L)72h与正常水平葡萄糖(5.5mmol/L)相比凋亡细胞数量显著增加,酰基化ghrelin(10-7mol/L)预处理24h后显著降低高糖诱导的凋亡。同时高糖环境下凋亡蛋白caspase-3活性增高,酰基化ghrelin预处理后明显降低caspase-3活性。结论酰基化ghrelin可以抑制高糖诱导的血管内皮细胞凋亡及凋亡蛋白caspase-3的表达,可能在防治糖尿病动脉粥样硬化的过程中起到一定作用。     

神经酰胺在高糖诱导的人脐静脉内皮细胞凋亡中的作用

目的 观察神经酰胺（CER）在高糖诱导的人脐静脉内皮细胞凋亡中的作用。方法分别以流式细胞术和TUNEL法、高压液相色谱法等检测细胞凋亡、CER和酸性鞘磷酯酶（ASM）。结果（1）高糖呈浓度依赖性引起细胞内CER和酸性ASM活性增加,左旋葡萄糖无类似效应;（2）地昔帕明可抑制高糖诱导的内皮细胞凋亡,外源性（CER）可逆转其抑制作用;（3）外源性CER及酸性ASM可诱导内皮细胞凋亡;（4）高糖诱导的细胞内CER增加与高糖浓度及细胞凋亡均正相关。结论由酸性ASM水解引起的细胞内神经酰胺增加参与了高糖诱导的内皮细胞凋亡。     

抑制p22phox表达对高糖所致的内皮细胞活性氧生成和细胞凋亡的影响

目的 探讨p22phox基因沉默在高浓度葡萄糖诱导的内皮细胞活性氧生成和细胞凋亡中的作用.方法 将原代培养的人脐静脉内皮细胞分为对照组、高糖组、高糖+siRNA转染组和siRNA转染组,采用Western-blot检测p22phox蛋白的表达,流式细胞仪检测细胞内活性氧水平及细胞凋亡率.结果 siRNA能有效抑制p22phox表达,高糖能诱导内皮细胞p22phox表达增加.高糖+siRNA转染组的细胞内活性氧水平和细胞凋亡率明显低于高糖组(P<0.05).结论 抑制p22phox表达能降低高糖所致的内皮细胞活性氧生成和细胞凋亡.     

cAMP反应元件结合蛋白在高糖所致内皮细胞损伤中的作用

目的探讨cAMP反应元件结合蛋白(CREB)在高糖致内皮细胞损伤中的作用。方法建立高糖诱导的血管内皮细胞损伤模型,将内皮细胞ECV304分为对照组、高糖组、转染PCI组、转染PCI+高糖组、转染PCI-CREB组和转染PCI-CREB+高糖组,采用MTT法检测细胞存活率,流式细胞仪检测细胞凋亡率,硫代巴比妥酸法检测丙二醛(MDA)含量,黄嘌呤氧化酶法检测细胞超氧化物歧化酶(SOD)活性。结果转染PCI-CREB组和转染PCI组内皮细胞存活率、SOD活性、MDA含量和细胞凋亡率与对照组相比差异无统计学意义(P>0.05);与对照组相比,高糖组内皮细胞存活率和SOD活性明显下降,而MDA含量和细胞凋亡率明显增加(P<0.05);转染PCI+高糖组内皮细胞存活率、SOD活性、MDA含量和细胞凋亡率与高糖组相比差异无统计学意义(P>0.05);与高糖组相比,转染PCI-CREB+高糖组细胞存活率和SOD活性明显高于高糖组(P<0.05),而MDA含量和细胞凋亡率明显低于高糖组(P<0.05)。结论过表达CREB对高糖诱导的内皮细胞损伤具有保护作用。     

Effect of erythropoietin on endothelial cell apoptosis induced by high glucose

Erythropoietin (Epo) has been reported to inhibit apoptosis of neuron and erythroid cells. In this study, we examined an effect of high glucose on apoptosis of endothelial cells and investigated an anti-apoptotic effect of Epo. Human aortic endothelial cells were incubated with normal or high glucose for 72 h, and apoptotic cells were detected by TUNEL assay. Simultaneously, Epo (100 U/ml) was added to the high glucose medium to examine an inhibitory effect on the apoptosis induced by high glucose. Activity of caspase-3 was also measured using a specific substrate. To investigate a possible mechanism of Epo's action on apoptosis, phosphorylation of Akt was examined by applying Epo. Incubation with high glucose increased apoptosis of endothelial cells, whereas this effect was prevented by co-incubation with Epo. Caspase-3 activity was also increased (1.4-fold) by incubation with high glucose, and the activation of caspase-3 was normalized to the control level by co-incubation with Epo. Furthermore, Epo-induced phosphorylation of Akt in dose-dependent manner. In conclusion, we demonstrated that incubation with high glucose activated caspase-3 and induced apoptosis of endothelial cells. Epo was shown to phosphorylate Akt, leading to the inhibition of caspase-3 activation and apoptosis induced by high glucose. These results suggest that reduced production of Epo in patients with end-stage of nephropathy may accelerate diabetic angiopathy and that replacing therapy with Epo might inhibit endothelial cell apoptosis and diabetic angiopathy.     

不同浓度葡萄糖对人血管内皮细胞的影响

目的研究高浓度葡萄糖对人血管内皮细胞的影响。方法在人脐静脉内皮细胞株ECV304培养基中分别加入5.5、20、40 mmol/L葡萄糖,作用24~72 h。采用四甲基偶氮唑盐微量酶反应比色（MTT）法观察细胞增殖情况,倒置相差显微镜观察细胞形态,透射电镜观察内皮细胞V304凋亡情况;流式细胞术检测细胞凋亡率。结果高浓度葡萄糖能明显抑制血管内皮细胞的增殖,诱导培养的内皮细胞发生凋亡,细胞凋亡率增加,并随浓度的增高、时间的延长作用明显。结论高浓度葡萄糖能抑制培养的人血管内皮细胞增殖,促进细胞凋亡。     

高浓度葡萄糖促脂质氧化效应及其与低密度脂蛋白诱导血管内皮细胞凋亡的协同作用

以培养的人脐静脉内皮细胞株内皮细胞V30 4为对象 ,研究高浓度葡萄糖有无促进细胞介导的低密度脂蛋白脂质过氧化效应及高浓度葡萄糖和低密度脂蛋白协同诱导凋亡及其可能机制。采用TBARS法检测培养基丙二醛含量 ;Giemsa染色后采用光镜及电镜对凋亡细胞定性观察 ,流式细胞仪检测凋亡率。结果显示 ,高浓度葡萄糖促进细胞介导的低密度脂蛋白脂质过氧化效应 ,并呈量—效关系 ;高浓度葡萄糖和低密度脂蛋白协同诱导血管内皮细胞凋亡 ,很可能与前述效应以及通过产生氧化应激而延长细胞周期转换有关。结果提示 ,高浓度葡萄糖和低密度脂蛋白协同诱导血管内皮细胞凋亡作用促进了糖尿病状态下动脉粥样硬化的发生发展 ,可能是糖尿病易发生动脉粥样硬化的部分机制     

六味地黄丸对糖尿病大鼠胰岛β细胞的保护作用

[目的]观察六味地黄丸对糖尿病大鼠胰岛细胞的保护作用。[方法]将自发性2型糖尿病模型鼠——OLETF鼠分为阳性对照组、六味地黄丸组;非糖尿病对照鼠——LETO鼠为阴性对照组。给药32周后,对各组大鼠观察胰岛,检测胰岛β细胞、胰岛中核因子-κB（NF-κB）的表达。[结果]与阴性对照组比较,阳性对照组、六味地黄丸组大鼠均血糖升高（P〈0．05）、胰岛口细胞减少（P〈0．05）、NF-κB表达增加（P〈0．05）;与阳性对照组比较,六味地黄丸组大鼠胰岛β细胞增加（P〈0．05）,NF-κB表达减少（P〈0．05）。[结论]六味地黄丸可能是通过抑制胰岛中NF-κB的表达保护口细胞。     

Statins protect diabetic myocardial microvascular endothelial cells from injury

To study the protective effect and mechanism of statins on myocardial microvascular endothelial cells injury in diabetic rats. The rats were administrated with atorvastatin (10 mg/kg/day), rosuvastatin (5 mg/kg/day), or placebo for 1 week. The expression of small GTP-binding protein dissociation stimulator (SmgGDS) was analyzed using western blot. The expression of SmgGDS was also analyzed in cultured rat cardiac microvascular endothelial cells using western blot. The cells were divided into normal glucose group, hyper glucose group, and atorvastatin-treated group. The superoxide dismutase (DHE) staining was used to detect the oxidative stress in the rat cardiac microvascular endothelial cells. The expression of SmgGDS was detected using TUNEL staining after knockout of Akt1 and β1-integrin in cardiac microvascular endothelial cells. The followings were the significant findings of this study: (1) SmgGDS was expressed in aorta endothelium of diabetic rats and cardiac microvascular endothelial cells cultured with high glucose, (2) high glucose increased the production of ROS, the activity of NADPH, and the rate of apoptosis in cardiac microvascular endothelial cells, but after atorvastatin pretreatment, the production of ROS, the activity of NADPH, and the rate of apoptosis in cardiac microvascular endothelial cells decreased, (3) the expression of SmgGDS decreased and the oxidative stress increased after knockdown of Akt1 or β1-integrin. Statins can protect diabetic microvascular endothelial cells from oxidative stress and apoptosis by upregulating SmgGDS partly through β1-integrin/Akt1 pathway.     

Thiamine and benfotiamine prevent increased apoptosis in endothelial cells and pericytes cultured in high glucose

BACKGROUND: High glucose induces pathological alterations in small and large vessels, possibly through increased formation of AGE, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. We showed previously that thiamine and benfotiamine correct delayed replication and increase lactate production in endothelial cells subjected to high glucose. We now aim at verifying the effects of thiamine and benfotiamine on cell cycle, apoptosis, and expression of adhesion molecules in endothelial cells and pericytes, under high ambient glucose. METHODS: Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/L) or high (28 mmol/L) glucose, with or without thiamine or benfotiamine, 50 or 100 micro mol/L. Apoptosis was determined by two separate ELISA methods, measuring DNA fragmentation and caspase-3 activity, respectively. Cell cycle and integrin subunits alpha3, alpha5, and beta1 concentration were measured by flow cytometry. RESULTS: Apoptosis was increased in high glucose after 3 days of culture, both in endothelium and pericytes. Thiamine and benfotiamine reversed such effects. Neither cell cycle traversal nor integrin concentrations were modified in these experimental conditions. CONCLUSIONS: Thiamine and benfotiamine correct increased apoptosis due to high glucose in cultured vascular cells. Further elucidations of the mechanisms through which they work could help set the basis for clinical use of this vitamin in the prevention and/or treatment of diabetic microangiopathy.     

Evaluation of gliclazide ability to attenuate the hyperglycaemic 'memory' induced by high glucose in isolated human endothelial cells

BACKGROUND: Patients with long-term exposure to high levels of hyperglycaemia remain more susceptible to diabetes-related complications, even with subsequent lower levels of hyperglycaemia. We sought to confirm the hypothesis that exposure to continuous increased glucose results in a memory of cellular stress in isolated endothelial cells, even when switched back to normal glucose, and to investigate the ability of gliclazide to attenuate this phenomenon. METHODS: Human umbilical vein endothelial cells were incubated for 21 days in normal glucose (5 mmol/L), high glucose (30 mmol/L), or high glucose for 14 days followed by normal glucose for 7 days (memory condition). The effects of gliclazide (10 micromol/L) and glibenclamide (1 micromol/L) were evaluated in the memory condition and added to the culture media early (first 14 days), late (last 7 days), or throughout the study. Oxidative stress and cell apoptosis parameters were investigated. RESULTS: Continuous high glucose increased reactive oxygen species, 8-OHdG, nitrotyrosine, caspase-3, and reduced Bcl-2 expression. These deleterious effects were also observed in the memory condition. Gliclazide applied early or throughout the study improved all parameters. In contrast, glibenclamide showed no relevant effect on study parameters. CONCLUSIONS: Our results suggest that gliclazide prevents endothelial cell apoptosis by reducing oxidative stress. The results appear to confirm the hypothesis that exposure of cells to continuous increased glucose results in a hyperglycaemic cellular memory that remains, even when cells are switched back to normal glucose. Gliclazide attenuated this cellular memory, decreasing oxidative stress and protecting vascular endothelial cells from apoptosis.     

Exogenous BH4/Bcl-2 peptide reverts coronary endothelial cell apoptosis induced by oxidative stress

BACKGROUND: Vascular endothelium undergoes apoptosis when exposed to reactive oxygen species (ROS), including hydrogen peroxide and superoxide radicals. ROS are believed to be the cause of damage to small vessels during ischemia-reperfusion injury and of arterial damage during atherosclerosis. Hydrogen peroxide-induced apoptosis is mediated through the inhibition of Bcl-xl activity and caspase-3 and caspase-9 activation. The BH4 domain of the Bcl-2 family members is responsible for their antiapoptotic activity. The BH4 domains of Bcl-2 and Bcl-xl inhibit cytochrome c release and the loss of mitochondrial membrane potential. METHODS AND RESULTS: The purpose of this project was to study the antiapoptotic effect of cell-permeant derivative of Bcl-2 (BH4 peptide) on endothelial cells exposed to stress conditions. BH4 peptide was conjugated to the cell-permeable peptide TAT and was applied to endothelial cells under conditions of serum starvation and hydrogen peroxide treatment. TAT-BH4 reduced caspase-3 activity and prevented apoptotic cell death. CONCLUSION: Our results indicate that TAT-BH4 peptide can protect endothelial cells from ROS-induced apoptosis.