α-硫辛酸对硫酸吲哚酚致脐静脉内皮细胞氧化损伤的影响

发布时间：2018-04-20 06:15

本文选题：α-硫辛酸 + 硫酸吲哚酚　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的:慢性肾脏疾病(chronic kidney disease,CKD)患者尤其是终末期肾衰患者的心血管发病率和死亡率远高于普通人群。加速性动脉粥样硬化是尿毒症患者心血管并发症的主要病理机制之一，起始于血管内皮细胞损伤。CKD患者随着肾小球滤过率降低，肾毒性物质在体内蓄积，可诱导氧化应激反应,促进动脉血管产生粥样硬化病变。硫酸吲哚酚（indoxylsulfate,IS）即是其中一种代表性的蛋白结合毒素，诱发氧化应激使心血管系统受损并加速慢性肾脏病进程。α-硫辛酸(α-lipoic acid,LA)是天然存在的最强抗氧化剂，目前在糖尿病、糖尿病并发症、缺血再灌注损伤等疾病的防治方面应用较广泛，而在CKD患者应用较少。本研究以人脐静脉内皮细胞(human umbilical vein endothelial cell，HUVEC)为对象,探讨α-硫辛酸能否对硫酸吲哚酚致血管内皮细胞产生的氧化损伤起到保护作用及其可能的作用机制，，为α-硫辛酸在慢性肾脏病中应用提供实验依据。方法:培养人脐静脉内皮细胞,将其分成对照组（CONTROL组），α-硫辛酸组（LA组），硫酸吲哚酚组（IS组）和硫酸吲哚酚+α-硫辛酸组（IS+LA组）。用MTS法检测细胞存活率；硫代巴比妥酸法测定丙二醛（MDA）的含量；用流式细胞术检测细胞内活性氧自由基(reactiveoxygen species,ROS)的变化；应用RT-PCR法测定NADPH氧化酶4（NOX4）mRNA的表达。统计学处理采用GraphPad Prism5软件，实验数据中计量资料用均数±标准差表示。多组间均数比较采用单因素方差分析（ANOVA）,均数两两比较采用Bonferroni法检验，P＜0.05具有统计学意义。各组实验至少重复3次。结果: 1IS呈浓度（100、200、500μm/L）依赖性的降低细胞存活率，大于等于200μm/L时与对照组相比具有统计学意义（P0.001）；而LA呈浓度（20、50、100、250、500μm/L）依赖性的提高细胞存活率，大于等于250μm/L时与对照组有显著性差异（P0.01）；250μm/L LA预处理细胞再与500μm/L IS共培养，与IS组（500μm/L）相比，细胞存活率显著提高（P0.01）。选定500μm/L和250μm/L分别作为IS和LA继续实验浓度。 2与对照组（1.29±0.40nmol/ml）相比，IS组（2.87±0.66nmol/ml）细胞MDA含量升高（P0.001）；细胞经LA预处理，IS+LA组（2.04±0.46nmol/ml）较IS组（2.87±0.66nmol/ml） MDA含量降低（P0.05）。 3与对照组(62.07±1.60%)相比，IS组(76.17±2.26%)ROS阳性细胞率增加(P0.001)；细胞经LA预处理，IS+LA组(69.70±3.01%)较IS组(76.17±2.26%)ROS阳性细胞率降低（P0.05）。 4与对照组（1.00±0.24）相比，IS组（1.65±0.18）细胞NOX4mRNA相对表达量升高（P0.05）；细胞经LA预处理, IS+LA组（1.11±0.41）较IS组（1.65±0.18）NOX4mRNA相对表达量降低（P0.05）。结论： 1硫酸吲哚酚诱导氧化应激使内皮细胞受损 2α-硫辛酸可能通过下调NADPH氧化酶，降低ROS水平，改善硫酸吲哚酚对内皮细胞的氧化损伤
[Abstract]:Objective: cardiovascular morbidity and mortality in patients with chronic kidney disease, especially in patients with end-stage renal failure, were significantly higher than those in the general population. Accelerated atherosclerosis is one of the main pathological mechanisms of cardiovascular complications in uremic patients. It begins with the decrease of glomerular filtration rate and accumulation of nephrotoxic substances in patients with vascular endothelial cell injury. Promote atherosclerotic lesions in the arteries. Indole sulfate indoxyl sulfate is one of the typical protein-binding toxins, which induces oxidative stress to damage the cardiovascular system and accelerates the progression of chronic kidney disease. 伪 -lipoic acid (伪 -lipoic acid) is the strongest natural antioxidant in diabetes. Diabetes complications, ischemia reperfusion injury and other diseases are widely used in the prevention and treatment, but less in patients with CKD. In this study, human umbilical vein endothelial cells (HUVECs) were used to investigate whether 伪 -lipoic acid could protect endothelial cells from oxidative damage induced by indole sulfate and its possible mechanism. To provide experimental basis for the application of 伪-lipoic acid in chronic kidney disease. Methods: human umbilical vein endothelial cells were cultured and divided into control group (control group), 伪 -lipoic acid group (LA group), indoleol sulfate group (is group) and indolol sulfate 伪 -lipoxylic acid group (ISLA group). MTS assay was used to detect cell survival rate, thiobarbituric acid method to determine the content of malondialdehyde (MDA), flow cytometry to detect the changes of reactive oxygen species (Ros), and RT-PCR method to detect the expression of NADPH oxidase 4(NOX4)mRNA. GraphPad Prism5 software was used to process statistics, and the measurement data in experimental data were expressed as mean 卤standard deviation. Single factor analysis of variance (ANOVAX) was used to compare the mean of the two groups, and the Bonferroni method was used to test the difference between the two groups (P < 0. 05, P < 0. 05). The experiments in each group were repeated at least 3 times. Results: 1IS decreased the cell survival rate in a concentration-dependent manner (> 200 渭 m / L) and increased the cell survival rate in a dose-dependent manner compared with the control group (P 0.001 / L), while LA increased the cell survival rate in a concentration-dependent manner. Compared with the control group, there was a significant difference when the concentration of LA was greater than or equal to 250 渭 m / L, and the cell survival rate was significantly higher than that in the is group (P < 0.01), and then co-cultured with 500 渭 m 路L ~ (-1) L 路L 路L ~ (-1) LA, compared with 500 渭 m 路L ~ (L) in the is group (P _ (0.01) 路L ~ (-1)). 500 渭 m / L and 250 渭 m / L were selected as the continued concentration of is and LA, respectively. 2Compared with the control group (1.29 卤0.40 nmol / ml), the MDA content of the cells in the is group increased (2.87 卤0.66 nmol / ml), and the MDA content in the ISLA group decreased by 2.04 卤0.46 nmol / ml (2.87 卤0.66 nmol / ml) after LA preconditioning, compared with the control group (2.87 卤0.66 nmol / ml). 3Compared with the control group (62.07 卤1.60), the percentage of positive cells of 76.17 卤2.26%)ROS was increased in the is group, and the percentage of positive cells in the LA group (69.70 卤3.01) was significantly lower than that in the is group (76.17 卤3.01). 4Compared with the control group (1.00 卤0.24), the relative expression of NOX4mRNA in the is group (1.65 卤0.18) was higher than that in the control group (P 0.05), and the relative expression of NOX4mRNA in the is LA group was 1.11 卤0.41) lower than that in the is group (1.65 卤0.18). Conclusion: 1. Oxidative stress induced by indole sulfate damaged endothelial cells 2 伪 -lipoic acid may decrease the level of ROS by down-regulating NADPH oxidase, and improve the oxidative damage of endothelial cells induced by indole sulfate.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

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