白藜芦醇对高糖“代谢记忆”介导的人脐静脉内皮细胞增殖及氧化应激的影响

发布时间：2018-03-27 07:45

本文选题：白藜芦醇　切入点：人脐静脉内皮细胞　出处：《河北医科大学》2015年硕士论文

【摘要】：目的:糖尿病是一种代谢紊乱性疾病,可以导致糖尿病特有的微血管并发症(视网膜病变、肾病、神经病变)和大血管并发症(心血管疾病和中风),最终导致患者寿命的缩短。我国2010年糖尿病流行病学调查显示,成人糖尿病的患病率为9.7%,如果将Hb A1c≥6.5%也作为糖尿病的诊断标准,则糖尿病的患病率达11.6%。糖尿病是心脑血管疾病的独立危险因素,据报道糖尿病患者发生心血管疾病的风险是非糖尿病患者的2-4倍。大多数的糖尿病患者死于心脑血管疾病及糖尿病肾病,可见糖尿病慢性并发症的治疗对于糖尿病患者的重要性。高血糖的糖毒性参与了糖尿病慢性并发症的发生,但是从临床治疗和一些临床实验来看,通过药物使血糖控制在理想范围内的糖尿病患者仍可能发生糖尿病并发症,且这些并发症持续进展。为此人们进行了一些大型临床试验研究,提出高血糖代谢记忆效应,很好的解释了这一现象。糖尿病患者早期高血糖状态可对机体各个器官造成损害,即使后期血糖达到理想水平,这种损伤仍然持续存在,这种现象称为“代谢记忆”现象。多项研究表明,氧化应激在糖尿病患者心血管病变的发病机制中起关键作用。机体在高血糖刺激下出现氧化应激反应,从而引起糖尿病的发生、发展。当前,已被证实有4种主要机制参与了高血糖引起的组织损伤:多元醇通路的增加、细胞内糖基化终产物(AGEs)的增加、蛋白激酶C(PKC)的激活、己糖胺通路的过度激活。高血糖可以通过激活多元醇通路和己糖胺通路使机体产生过量的ROS,诱导糖基化终产物(AGEs)形成,并使其与特异性受体(RAGE)结合。反过来,AGEs与其特异性受体(RAGE)的结合,也促进了活性氧簇(ROS)的产生,进而激活核转录因子NF-κB通路,使细胞长期处于炎症状态,加速血管动脉粥样硬化的形成。并且,细胞外基质中的AGEs,可以使血管弹性下降、一氧化氮的活性降低、血管内皮依赖性舒张功能减低。此外,慢性高血糖增加了循环血中细胞因子、生长因子、内皮素-I、血管紧张素II含量,这些物质与相应的细胞表面受体结合,激活蛋白激酶C(PKC)通路,而PKC通路的激活可诱导血管损伤,抑制内皮细胞中一氧化氮合酶(e NOS)的表达,抑制平滑肌细胞中一氧化氮的产生,提高纤溶酶原激活物(PAI-I)的表达,增加氧化还原敏感的核转录因子NF-κB表达,提高促氧化剂NADPH氧化酶的活性,进而对血管内皮细胞造成损伤。白藜芦醇(Resveratrol,Res),化学名为3,5,4-三羟基-1,2-二苯乙烯,是一种天然的多酚类化合物。白藜芦醇在葡萄皮及红酒中含量丰富,可以作为活性氧(ROS)的清除剂、金属螯合金、酶调节剂。一些研究表明红酒或白藜芦醇可以使糖尿病大鼠的不同大脑区域的脂质过氧化降低,同时增加抗氧化酶水平。并且最近的研究也表明,白藜芦醇对不同的组织以及心血管疾病、炎性反应、癌症、糖尿病等病理疾病有抗氧化应激保护作用。本研究旨在通过体外培养人脐静脉内皮细胞,探讨高血糖代谢记忆对于体外培养的血管内皮细胞的氧化应激作用,并且探讨白藜芦醇在高糖代谢记忆中对血管内皮细胞的保护作用。方法:人脐静脉内皮细胞株(HUVECs)体外培养贴壁后随机分组:正常对照组:NG,5.5mmol/L D-葡萄糖×3天;甘露醇对照组:MA,5.5mmol/L D-葡萄糖、24.5mmol/L D-甘露醇×3天;高糖持续组:HG,30mmol/L D-葡萄糖×3天;高糖记忆组:TG,30mmol/L D-葡萄糖×1天更换为5.5mmol/L D-葡萄糖×2天;高糖记忆+白藜芦醇组:TG+100μmol/L、10μmol/L、1μmol/L、0.1μmol/L Res×1天更换为5.5mmol/L D-葡萄糖×2天。NG组、MA组、HG组、TG组、TG+Res组分别于培养24h、48h和72h用MTT法检测细胞增殖情况,用wst-1法检测细胞内超氧化物歧化酶(SOD)的活力,用TBA法检测细胞内丙二醛(MDA)的含量。结果:1与正常对照组比较,高糖组明显抑制脐静脉内皮细胞增殖活性(P0.01)。更换为正常糖浓度后,内皮细胞增殖活性部分恢复(P0.05),但仍低于正常对照组(P0.01)。白藜芦醇以剂量依赖形式增加代谢记忆组细胞增殖活性(P0.05)。甘露醇对照组对内皮细胞的增殖也有一定的抑制作用(P0.05)。2甘露醇组细胞内MDA含量高于正常对照组(P0.05)。与正常对照组比较,高糖组细胞内MDA含量明显升高(P0.01)。高糖代谢记忆组细胞内MDA含量低于高糖组(P0.05),但高于正常对照组(P0.01)。白藜芦醇以浓度依赖形式降低高糖记忆组内皮细胞内MDA含量(P0.05)。3与正常对照组相比,甘露醇组细胞内SOD水平下降(P0.05),高糖组、高糖记忆组和白藜芦醇干预组细胞内SOD水平明显下降(P0.01)。高糖持续组细胞内SOD水平明显降低高糖代谢记忆组(P0.01)。白藜芦醇呈浓度依赖性恢复代谢记忆组内皮细胞内SOD水平(P0.05)。结论:1高糖抑制体外HUVECs增殖、诱发HUVECs氧化应激,去除高糖刺激后,细胞增殖抑制作用和氧化应激持续存在。提示代谢记忆效应参与糖尿病的血管内皮损伤机制。2白藜芦醇剂量依赖地恢复高糖代谢记忆效应介导的细胞增殖抑制及氧化应激效应。提示白藜芦醇可能对代谢记忆效应介导的内皮细胞损伤具有潜在的保护作用。
[Abstract]:Objective: diabetes mellitus is a metabolic disorder, microvascular complications can lead to diabetes specific (retinopathy, nephropathy, neuropathy) and macrovascular complications (cardiovascular disease and stroke), eventually lead to shorten the patient life. 2010 survey of diabetes epidemiology in China, adult prevalence rate of diabetes was 9.7%, if the Hb A1c = 6.5% as the diagnostic standard of diabetes, the diabetes prevalence rate of 11.6%. diabetes is the independent risk factor for cardiovascular disease risk, according to reports of cardiovascular disease in patients with diabetes is 2-4 times higher than the non diabetes patients. Most of the diabetic patients died of cardiovascular and cerebrovascular diseases and diabetic nephropathy, treatment of chronic complications of diabetes mellitus for visible the importance of diabetes. High blood sugar glucose toxicity in chronic diabetic complications, but from clinical treatment Treatment and clinical trials, patients with diabetes control in the ideal range by drugs to blood glucose may still occur in diabetic complications, and the complication of continuous progress. Therefore there were some large clinical trials, the high glucose metabolic memory effect, good explanation of this phenomenon. The early hyperglycemia patients with diabetes damage to various organs of the body, even if the latter glucose reaches the ideal level, the damage still persists, this phenomenon is called "metabolic memory phenomenon. A number of studies have shown that oxidative stress plays a key role in the pathogenesis of diabetic patients with cardiovascular disease. Immune response to oxidative stress stimulated by high glucose, thereby causing diabetes occurrence, development has been confirmed. At present, there are mainly 4 kinds of mechanisms involved in tissue injury caused by hyperglycemia: polyol pathway. Plus, in cells of advanced glycation end products (AGEs) increased, C protein kinase (PKC) activation, excessive activation of the hexosamine pathway. High blood glucose can be through the activation of polyol pathway and hexosamine pathway to produce excessive ROS, induced by advanced glycation end products (AGEs) formation, and the with the specific receptor (RAGE) binding. In turn, the AGEs and its specific receptor (RAGE) binding, but also promote the reactive oxygen species (ROS) generation and activation of nuclear factor kappa B pathway NF-, the cells in the inflammatory state for a long time, the acceleration of vascular atherosclerosis formation. Furthermore, extracellular in the matrix AGEs, can make the blood vessel elasticity decreased, reducing the activity of nitric oxide, endothelium-dependent diastolic function decreased. In addition, chronic hyperglycemia increased cytokine, circulating growth factor, endothelin -I, angiotensin II content, these substances and the corresponding cell surface Binding, activation of protein kinase C (PKC) pathway, and the activation of PKC pathway can induce vascular injury, inhibition of nitric oxide synthase in endothelial cells (E NOS) expression, inhibition of nitric oxide in smooth muscle cells, improve the plasminogen activator (PAI-I) expression of B NF- increased the oxidation reduction of nuclear transcription factor kappa the expression of sensitivity, improve the pro oxidant activity of NADPH oxidase, which caused the injury of vascular endothelial cells. Resveratrol (Resveratrol, Res), the chemical name is 3,5,4- three -1,2- two hydroxy styrene, is a natural polyphenolic compound. Resveratrol rich content in grape skin and red wine, can be used as active oxygen scavenger (ROS), metal chelating alloy, enzyme regulator. Some studies show that red wine or resveratrol can reduce lipid peroxidation in different brain regions in diabetic rats. At the same time, increase the levels of antioxidant enzymes and recently. The study also showed that resveratrol in different tissues as well as cardiovascular disease, inflammation, cancer, diabetes and other pathological disease has a protective effect against oxidative stress. The purpose of this study was to cultured human umbilical vein endothelial cells in vitro, to investigate the role of oxidative stress in high glucose metabolic memory for vascular endothelial cells cultured in vitro, and to explore the protection effect of resveratrol on vascular endothelial cells in high glucose metabolic memory. Methods: human umbilical vein endothelial cells (HUVECs) cultured in vitro adherent were randomly divided into 2 groups: normal control group: NG, 5.5mmol/L * D- glucose for 3 days; mannitol control group: MA, 5.5mmol/L D- 24.5mmol/L D- glucose, mannitol * 3 days; high glucose group: HG, 30mmol/L for D- glucose * 3 days; hyperglycemia group: TG, D- glucose 30mmol/L * 1 days replacement for 5.5mmol/L * D- glucose for 2 days; hyperglycemia + resveratrol group: TG+100 mol/L, 10 m Ol/L, 1 mol/L, 0.1 mol/L Res * 1 days replacement for 5.5mmol/L D- glucose * 2 days in.NG group, MA group, HG group, TG group and TG+Res group were cultured 24h, 48h and 72h cell proliferation was assessed by MTT method, determination of intracellular superoxide dismutase by WST-1 method (SOD) activity, TBA method was used to detect intracellular malondialdehyde (MDA) content. Results: 1 compared with normal control group, high glucose group significantly inhibited the proliferation of human umbilical vein endothelial cell activity (P0.01). The replacement for the normal glucose concentration after restoration of endothelial cell proliferation activity part (P0.05), but still lower than the normal control group. (P0.01). Resveratrol increased proliferation of metabolic memory cell activity in a dose dependent group form (P0.05). The control group of mannitol on endothelial cell proliferation is inhibited (P0.05).2 mannitol group of intracellular MDA content is higher than the normal control group (P0.05). And the normal control group, high glucose content of MDA group of cells Increased significantly (P0.01). The content of MDA memory cells in high glucose metabolism group was lower than that in high glucose group (P0.05), but higher than the normal control group (P0.01). Resveratrol decreased the content of MDA in high glucose group memory in endothelial cells in a concentration dependent form (P0.05.3) compared with the normal control group, mannitol group decreased the intracellular level of SOD (P0.05), high glucose group, high glucose group and resveratrol intervention memory SOD group decreased significantly (P0.01). The level of SOD in high glucose group continued intracellular metabolic memory group decreased significantly (P0.01). Resveratrol showed a concentration dependent recovery of SOD metabolism memory group in endothelial cells (P0.05). Conclusion: 1 high glucose inhibited the proliferation of HUVECs in vitro, HUVECs induced oxidative stress, the removal of high glucose stimulation, cell proliferation inhibition and oxidative stress persists. Suggest that metabolic memory effects in diabetic vascular endothelial injury mechanism of.2 resveratrol dose dependently It can restore cell proliferation inhibition and oxidative stress effect mediated by high glucose metabolism, suggesting that resveratrol may play a potential protective role in endothelial cell injury mediated by metabolic memory effect.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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