【作者】 张风雷；

【导师】 高海青；

【作者基本信息】 山东大学 ， 内科学， 2007， 博士

【摘要】 第一部分葡萄籽原花青素选择性抑制AGEs诱导的内皮细胞黏附分子表达背景随着社会发展及人口老龄化，糖尿病发生率逐年增加，而其血管并发症已成为糖尿病病人致死或致残的主要原因。近年来研究显示尽管强化降糖治疗能改善部分糖尿病微血管并发症的预后，但对糖尿病动脉粥样硬化等大血管并发症影响不大，而且血糖严格控制往往容易导致严重的低血糖反应，病人难以耐受。进一步的研究揭示，此“高血糖记忆现象”与糖尿病病人体内积聚的糖基化终产物(Advanced glycation end products，AGEs)有关。一方面AGEs直接作用于血管壁，发生交联反应，造成血管壁增厚、弹性下降及顺应性减退，也能作用于脂质，导致LDL易于氧化及血管壁沉积，HDL糖基化使其胆固醇逆转运功能降低。然而，AGEs最主要的致病机制是通过与其受体RAGE相互作用介导的，抗RAGE抗体或可溶性RAGE(sRAGE)阻断AGEs与RAGE相互作用能逆转血管高通透性，抑制血管病变的早期发展及动脉粥样硬化病变的进展，抑制血管损伤后的内膜过度增生，改善神经传导，减轻肾肿大、肾小球硬化及蛋白尿等。随着研究的深入，除了严格控制血糖外，以AGEs-RAGE系统为靶点的药物治疗将成为糖尿病血管并发症防治的重点之一。目的大量证据证实AGEs与其受体RAGE相互作用在糖尿病血管并发症的发生和发展中起了至关重要的作用，AGEs与RAGE结合能诱导细胞内氧化应激，活性氧簇(ROS)产生增加，增加的ROS反过来激活一系列复杂的信号传导途径及随后的转录因子NF-κB等，后者进一步促进多种基因的表达，其中大多数是与炎症、免疫和动脉粥样硬化相关的，如黏附分子、趋化蛋白、生长因子、组织因子等。葡萄籽原花青素(Grape seed proanthocyanidin extracts，GSPE)是从葡萄籽中提取的多酚类化合物，拥有很强的抗氧化活性。本研究以体外培养的人脐静脉内皮细胞(Human umbilical vein endothelial cells，HUVEC)为研究对象，观察GSPE是否能通过抗氧化机制抑制AGEs诱导的内皮细胞黏附分子表达。方法牛血清白蛋白(BSA)与高浓度的葡萄糖在37℃下孵育12周以制备糖基化终产物AGE-BSA。激光共聚焦显微镜和流式细胞仪评价细胞内ROS产生，流式细胞仪测定细胞表面VCAM-1及ICAM-1蛋白表达，RT-PCR检测细胞内VCAM-1及ICAM-1 mRNA水平。首先用200μg／ml AGE-BSA与内皮细胞孵育不同时间，观察AGEs对内皮细胞ROS产生及VCAM-1、ICAM-1蛋白及基因表达的时间刺激强度。接下来，先用不同浓度(5、15、25μg／ml)GSPE预处理内皮细胞4h，然后加用200μg／ml AGE-BSA刺激内皮细胞不同时间，观察GSPE是否能抑制细胞内ROS产生，进而抑制VCAM-1及ICAM-1蛋白及基因的表达。结果1．200μg／ml AGE-BSA与内皮细胞孵育3h时VCAM-1及ICAM-1蛋白表达开始增强，12h时达到高峰，24h表达降低，但仍未到达基础水平。对照组未处理的内皮细胞VCAM-1及ICAM-1蛋白表达很低，未糖基化修饰的BSA与25μg／mlGSPE均没有影响VCAM-1及ICAM-1蛋白表达。而不同浓度(5、15及25μg／ml)GSPE与内皮细胞预孵育4h显著抑制200μg／ml AGE-BSA刺激的VCAM-1蛋白表达，并呈剂量依赖性，但却没有影响增高的ICAM-1蛋白水平。在不同剂量组，VCAM-1蛋白表达分别降低到79.90％±8.49％(5μg／ml GSPE组)、52.09％±5.19％(15μg／ml GSPE组)、20.53％±5.21％(25μg／ml GSPE组)(与AGE-BSA诱导组相比，P＜0.01)。2．AGE-BSA处理内皮细胞2h时VCAM-1及ICAM-1 mRNA水平均开始增加，6h时达到高峰，12h时有所下降，但未接近基础水平。不同剂量(5、15及25μg／ml)GSPE与内皮细胞预孵育4h，再用200μg／ml AGE-BSA刺激6h，与VCAM-1及ICAM-1表面蛋白表达相一致，GSPE显著降低了VCAM-1 mRNA水平，并呈剂量依赖性，而增强的ICAM-1 mRNA水平却没有改变。不同剂量的GSPE分别将VCAM-1 mRNA水平从0.388±0.035(AGE-BSA诱导组)降低到0.275±0.015(5μg／ml GSPE组)、0.146±0.031(15μg／ml GSPE组)、0.077±0.009(25μg／ml GSPE组)(与AGE-BSA诱导组相比，P＜0.01)。3．激光共聚焦显微镜检测结果显示，AGE-BSA与内皮细胞孵育0min时细胞内荧光强度较弱，ROS产生较低，15min时ROS产生增加，30min时达到高峰，以后逐渐下降，60min时接近基础水平。5、15、25μg／ml的GSPE与HUVEC预孵育4h明显抑制了200μg／ml AGE-BSA诱导的ROS产生，抑制作用随GSPE剂量的增加而增强(AGE-BSA诱导组：3.12±0.31、5μg／ml GSPE组：2.36±0.34、15μg／ml GSPE组：1.95±0.41、25μg／ml GSPE组：1.14±0.23，与AGE-BSA诱导组相比，P＜0.01)。流式细胞仪测定细胞内ROS也得出相似的结果。结论1．体外制备的AGE-BSA与内皮细胞孵育能诱导细胞内氧化应激，ROS产生增加，进而促进内皮细胞VCAM-1及ICAM-1蛋白与mRNA表达。2．不同剂量的GSPE预处理内皮细胞选择性抑制了AGE-BSA诱导的VCAM-1蛋白及mRNA表达，并呈剂量依赖性，但没有影响ICAM-1水平。3．通过其抗氧化特性，GSPE以剂量依赖的方式抑制了细胞内ROS产生，这可能涉及GSPE对AGEs诱导的黏附分子表达的选择性抑制作用。4．GSPE能选择性抑制AGEs诱导的内皮细胞VCAM-1的表达，因此，GSPE有助于防止糖尿病早期血管病变的发生，这将为GSPE在糖尿病病人中的应用提供理论依据。第二部分葡萄籽原花青素对AGEs诱导的内皮细胞RAGE基因表达的影响背景研究证实RAGE是一信号传导受体，属于免疫球蛋白超家族成员，是介导AGEs致糖尿病血管病变的主要结合蛋白。RAGE与AGEs结合能诱导细胞内氧化应激，激活下游信号传导，促进炎症反应等。多种细胞如内皮细胞、单核细胞、平滑肌细胞及淋巴细胞等均能表达RAGE，正常情况下表达量很低，但在某些病理条件下表达增加并能持续数年，且AGEs与RAGE表达之间存在正反馈调节机制，在血管系统AGEs增多的病变部位往往伴随着RAGE表达的增加，这种正反馈调节被认为能促进并延长RAGE与配体的致病作用。另外，内皮细胞表面RAGE还可作为黏附受体与白细胞β2整合素作用，直接参与血管壁炎症细胞的募集。因此，限制RAGE表达将有助于抑制AGEs-RAGE系统的致病作用，并能阻断其恶性循环。目的葡萄籽原花青素(GSPE)是从葡萄籽中提取的天然抗氧化物质。本研究以体外培养的人脐静脉内皮细胞为研究对象，观察GSPE是否能抑制AGEs诱导的内皮细胞RAGE基因表达。方法激光共聚焦显微镜及流式细胞仪检测细胞内ROS的改变，RT-PCR测定RAGE mRNA水平。首先用200μg／ml AGE-BSA与内皮细胞孵育不同时间，观察AGEs对内皮细胞RAGE基因表达的刺激作用，然后用不同浓度(5、15及25μg／ml)GSPE与内皮细胞预孵育4h，再用200μg／ml AGE-BSA刺激内皮细胞，观察GSPE是否抑制AGEs诱导的内皮细胞RAGE基因表达。结果1．200μg／ml AGE-BSA与内皮细胞孵育2h时RAGE mRNA的表达有增加趋势，4h时表达明显增强，6h时达到峰值，以后RAGE mRNA表达逐渐降低，但12h时未接近正常水平。BSA及GSPE单独没有影响细胞内RAGE mRNA的水平。不同剂量GSPE与内皮细胞预孵育4h能以剂量依赖的方式抑制AGE-BSA诱导的内皮细胞RAGE mRNA表达，5、15、25μg／ml GSPE降低细胞内RAGEmRNA水平分别从1.12±0.15(AGE-BSA诱导组)到0.75±0.11、0.48±0.08、0.27±0.10(与AGE-BSA诱导组相比，P＜0.01)。2．激光共聚焦显微镜测定结果显示，与正常对照组相比较，BSA及GSPE单独没有增强细胞内ROS产生，同样的，GSPE与内皮细胞预孵育4h以剂量依赖的方式抑制了细胞内ROS的产生。细胞内ROS水平分别从3.02±0.29(AGE-BSA诱导组)降低到2.49±0.42(5μg／ml GSPE组)、1.95±0.24(15μg／mlGSPE组)、1.26±0.41(25μg／ml GSPE组)(与AGE-BSA诱导组相比，P＜0.01)。另外，流式细胞仪测定细胞内ROS也得出相一致的结果。结论1．体外制备的AGE-BSA能诱导细胞内氧化应激，ROS产生增加，并能导致RAGE mRNA表达增强，未修饰的BSA和GSPE单独没有影响细胞内ROS及RAGE mRNA水平。2．不同剂量GSPE与内皮细胞预孵育明显抑制AGE-BSA刺激的内皮细胞RAGE mRNA表达，并呈剂量依赖性，这有助于阻止AGEs-RAGE相互作用，阻断其致病过程的恶性循环，进一步为GSPE防治糖尿病血管并发症提供理论依据。3．GSPE同样以剂量依赖的方式抑制AGEs诱导的细胞内ROS产生，因此，GSPE对ROS的抑制可能也涉及其对RAGE mRNA的影响作用。4．GSPE对AGEs诱导的内皮细胞黏附分子的选择性抑制作用可能也与其降低RAGE表达有关。更多还原

【Abstract】 Part oneSelective inhibition by Grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by Advanced glycation end products in endothelialcellsBackgroundAlong with social development and population aging, the incidence of diabetes is steadily increasing every year and diabetic vascular complications are the major cause of morbidity and mortality for patients with diabetes. Recent work has demonstrated that strict glucose control can ameliorate certain microvascular diseases, but has few influences on atherosclerosis. Moreover, close control of blood sugar often results in serious hypoglycemic episodes, which is unacceptable for patients. Further studies have indicated this phenomenon of "hyperglycemic memory" is close associated with advanced glycation end products (AGEs) accumulated in patients with diabetes. AGEs directly cause vessel wall thickening, decreased elasticity and compliance, and have an influence on lipid as showed by increased susceptibility of LDL to oxidative modification and diminished role of HDL in reverse cholesterol transport. However, a major means by which AGEs exert their detrimental effects on the vasculature is through interacting with their signal-transduction receptor RAGE. The blockage of AGEs-RAGE interaction by administration of anti-RAGE IgG or sRAGE reversed the enhanced vascular hyperpermeability, suppressed the accelerated early lesions expansion or the progression of established atherosclerotic lesions, inhibited neointimal expansion after vessel injury, improved nerve conduction and mitigated renomegaly, glomerular sclerosis and proteinuria. Hence, in addition to close glucose control, drugs targeting AGEs-RAGE system become an emphasis on the prevention and treatment of patients with diabetic vascular complications accompanying research deepening.ObjectiveA growing body of evidences have demonstrated that interaction of AGEs with RAGE in endothelium triggers intracellular oxidative stress and ROS generation, and the increased ROS, in return, activates a complex cascade of signaling pathways and subsequent transcriptional factor NF-κB, which further promotes many genes expressions, the majority of which are highly relevant for inflammation, immunity and atherosclerosis such as cell adhesion molecules, tissue factor, chemokines and cytokines. Grape seed proanthocyanidin extracts (GSPE), a naturally occurring polyphenolic compounds obtained from grape seeds, have been reported to possess potent radical-scavenging and antioxidant properties. The aim of present study is to investigate whether GSPE can inhibit AGEs-induced cell adhesion molecules expression in cultured human umbilical vein endothelial cells through its antioxidant mechanisms.MethodsAGEs was prepared by incubating BSA with high concentration of glucose for 12 weeks at 37 degree C. Intracellular ROS production was detected by laser scanning confocal microscopy (LSCM) and flow cytometer. Surface expression of VCAM-1 and ICAM-1 was determined by flow cytometer. The levels of VCAM-1 and ICAM-1 mRNA were assayed by reverse-transcription polymerase chain reaction (RT-PCR). Firstly, HUVEC were stimulated with 200ug/ml AGE-BSA for different time. The time-ordered stimulatory effect of AGEs on intracellular ROS generation and expressions of VCAM-1 and ICAM-1 protein and mRNA in endothelial cells was observed. Next, after pre-incubation of GSPE of the concentrations of 5, 15, 25μg/ml for 4 hours, the cultured HUVEC was treated with 200μg/ml AGE-BAS for defined periods. We explored whether GSPE can prevent intracellular ROS production, thereby inhibiting the expression of VCAM-1 and ICAM-1 protein and mRNA.Results1. The protein expression of VCAM-1 and ICAM-1 started to enhance when HUVEC were stimulated with 200μg/ml AGE-BSA for 3 hours, reached the peak for 12 hours, was decreased for 24 hours without approach to basal level. The lower expression of VCAM-1 and ICAM-1 proteins was found in untreated endothelial cells. Both unmodified BSA and 25μg/ml GSPE were without effect on VCAM-1 and ICAM-1 expression. However, pre-incubation of 5, 15, 25μg/ml GSPE for 4 hours, in a dose-dependent manner, markedly prevented the up-regulation of VCAM-1 protein induced by 200μg/ml AGE-BSA, but, the enhanced ICAM-1 proteins were not altered. The levels of VCAM-1 expression were reduced to 79.90%±8.49% for 5μg/ml, to 52.09%±5.19% for 15μg/ml, to 20.53%±5.21% for 25μg/ml respectively (P< 0.01, vs AGE-BSA alone).2. The levels of VCAM-1 and ICAM-1 mRNA started to increase at 2 hours of AGE-BSA stimulation of endothelial cells, reached the peak at 12 hours, were moderately decreased at 24 hours without approach to basal level. After endothelial cells were pre-treated with 5, 15, 25μg/ml GSPE for 4 hours, AGE-BSA (200μg/ml) was added for stimulation for 6 hours. Consistent with protein expression, GSPE, in a concentration-dependent fashion, significantly reduced the VCAM-1 mRNA levels, but did not influence the increase in ICAM-1 mRNA levels. Reduction differed ranging from 0.388±0.035 to 0.275±0.015 for 5μg/ml, to 0.146±0.031 for 15μg/ml, to 0.077±0.009 for 25μg/ml respectively (P<0.01, vs AGE-BSA alone).3. The results from LSCM demonstrated that intracellular fluorescence intensity was very weak and ROS was generated at a lower level at 0 min of incubation of endothelial cells with AGE-BSA. But, intracellular ROS production started to augment at 15 min, reached the peak at 30 min, subsequently, was gradually decreased and approached to basal level at 60 min. The pretreatment of 5, 15, 25μg/ml GSPE ,in a dose dependent manner , apparently prevented intracellular ROS generation stimulated by 200μg/ml AGE-BSA, which was reduced from 3.12±0.31 to 2.36±0.34 for 5 ug/ml, to 1.95±0.41 for 15 ug/ml, to 1.14±0.23 for 25μg/ml respectively (P<0.01,vs AGE-BSA alone). Meanwhile, the similar results were gained using flow cytometer. Conclusions1. Treatment of endothelial cells with AGE-BSA prepared in vitro could induce intracellular oxidative stress and ROS generation, thereby leading to the expression of VCAM-1 and ICAM-1 protein and mRNA.2. Pre-incubation of GSPE of different concentrations selectively inhibited VCAM-1 protein and mRNA expression induced by AGE-BSA in endothelial cells in a dose-dependent manner, but did not influence ICAM-1 protein and mRNA levels.3. Through its antioxidant properties, GSPE dose-dependently prevented intracellular ROS generation, which might involve in selective inhibitory effect of GSPE on AGEs-induced cell adhesion molecules expression.4. GSPE selectively down-regulated AGEs-induced VCAM-1 expression. Hence, GSPE contribute to the treatment of early diabetic vascular lesion. Our results will provide theoretic evidences for application of GSPE for patient with diabetesPart twoEffects of Grape seed proanthocyanidin extracts on RAGE gene expressioninduced by Advanced glycation end products in endothelial cells BackgroundMany studies have confirmed that RAGE, a member of immunoglobulin superfamily, has already been identified to function predominantly as a signal transduction receptor and mediates diabetic vascular lesions caused by AGEs. The ligation of RAGE by AGEs induces intracellular oxidative stress, activates downstream signal transduction and evokes inflammatory responses. A variety of cell types, including endothelial cells, monocytes, smooth muscle cells and lymphocytes can express RAGE on their cell surface. RAGE is normally expressed at a lower level, but induced and lasted for several years under certain pathological conditions and there exists a positive feedback regulation mechanism between AGEs and RAGE, At the sites of accumulated AGEs in the vascular lesions there is increased RAGE expression. This positive feedback activation is thought to augment and prolong RAGE-ligands mediated detrimental effects. Besides, RAGE on endothelial cells may function as an adhesive receptor that interacts with leukocyte p2-integrins, thereby directly being involved in inflammatory cell recruitment. Hence, limiting RAGE expression will contribute to inhibiting AGEs-RAGE caused pathogenic effects, blocking its vicious cycle.ObjectiveGSPE is a naturally occurring antioxidant compound from grape seeds. The present study, taking the cultured HUVEC as study object, examines whether GSPE can inhibit AGEs-induced RAGE expression in endothelial cells.MethodsIntracellular oxidative stress was detected by LSCM or flow cytometry. The RAGE mRNA expression was assayed by RT-PCR. Firstly, endothelial cells were treated with 200μg/ml AGE-BSA for varying time period, and observed AGEs-induced irritant action on RAGE gene expression in endothelial cells. Next, cells were pre-incubated with GSPE of the concentrations of 0, 5, 15, 25μg/ml for 4 hours, and thereafter, stimulated in the presence of 200μg/ml AGE-BSA, and investigated whether GSPE can prevent AGEs-mediated RAGE gene expression in endothelial cells.Results1. RAGE mRNA levels had a tendency to increase at 2 hours of treatment of endothelial cells with 200μg/ml AGE-BSA, were significantly enhanced at 4 hours, reached the peak at 6 hours, were gradually decreased at the following time, but did not approach the normal level at 12hours. Both BSA and GSPE alone could not impact on RAGE mRNA levels. Pretreatment of GSPE of different concentrations for 4 hours, in a dose-dependent manner, inhibited RAGE mRNA expression induced by AGE-BSA in endothelial cells, which differed ranging from 1.12±0.15 to 0.75±0.11 for 5μg/ml, to 0.48±0.08 for 15μg/ml, to 0.27±0.10 for 25μg/ml respectively (P < 0.01, vs AGE-BSA alone).2. The results from LSCM showed that both BSA and GSPE alone did not enhance intracellular ROS generation. Likewise, pre-incubation of GSPE for 4 hours dose-dependently suppressed intracellular ROS production, which was reduced from 3.02±0.29 to 2.49±0.42 for 5μg/ml, to 1.95±0.24 for 15μg/ml, to 1.26±0.41 for 25μg/ml respectively. (P< 0.01, vs AGE-BSA alone) Moreover, similar results from flow cytometer were obtained.Conclusions1. AGE-BSA prepared in vitro could lead to intracellular oxidative stress and ROS generation, and subsequently cause the increase in RAGE mRNA levels. But, both unmodified BSA and GSPE alone did not influence ROS and RAGE mRNA levels.2. The pretreatment of GSPE of different concentrations dose-dependently prevented RAGE mRNA expression stimulated by AGE-BSA in endothelial cells, which contributed to inhibiting the interaction between AGEs and RAGE, interrupted the vicious cycle, further provided theory evidences for application of GSPE for patient with diabetic vascular complications.3. GSPE dose-dependently inhibited AGEs-induced ROS production in endothelial cells as much. Thus, effects of GSPE on RAGE mRNA might have close relation to its inhibitory role in ROS generation.4. We further speculate that selective inhibition by GSPE of cell adhesion molecule expression induced by AGEs in endothelial cells might also involve in down-regulated RAGE expression.更多还原