白藜芦醇对糖尿病肾病炎症及系膜细胞增殖的影响及其机制研究

发布时间：2018-05-05 06:27

本文选题：糖尿病肾病 + 白藜芦醇　；参考：《吉林大学》2014年博士论文

【摘要】：糖尿病肾病(diabetic nephropathy, DN）是糖尿病最重要的微血管并发症之一。据发达国家统计资料表明，DN已跃升为终末期肾病的首位病因。近年来我国糖尿病患者人数日益增多，DN已成为我国导致慢性肾功能衰竭的重要原因之一。目前认为DN与糖代谢紊乱、血流动力学异常、氧化应激、糖基化终末产物堆积、细胞因子异常表达等多种因素有关。其中炎症因子与氧化应激、免疫反应相互作用是导致糖尿病肾损害，造成系膜外基质积聚的重要原因。白藜芦醇具有抗炎、抗氧化、抗癌、心脏保护等作用，作用机理涉及包括环氧酶、脂氧酶、蛋白激酶B(protein kinase B，PKB/Akt)、丝裂原活化蛋白激酶家族、去乙酰化酶等多个信号传导通路。最新的研究发现白藜芦醇还具有降低血糖，改善胰岛素抵抗，调节异常脂代谢，减少炎症因子分泌和表达等作用。Akt能够通过对葡萄糖转运、糖原合成、糖酵解和糖异生的调节及相关基因的突变、沉默、敲除影响葡萄糖的代谢及糖尿病的发生和发展,在糖尿病血管并发症产生、发展的病理生理过程中也起到关键作用。核因子κB (nuclear factor-κB， NF-κB)是参与氧化应激、炎性反应的关键因子之一,与血管内皮细胞损伤、组织细胞凋亡等糖尿病血管并发症的多个病理生理过程相关。多项研究提示白藜芦醇可能成为治疗糖尿病及其并发症的新型药物，而这些作用的发挥可能与该药可以对细胞内多个信号转导通路产生影响有关，其中Akt、NF-κB可能发挥了重要作用。本研究旨在探讨白藜芦醇对于糖尿病所致的肾脏炎症与系膜细胞增殖的保护作用及相关机制。在体外实验当中，我们应用高糖模拟糖尿病患者的体内环境，应用Western blot、ELISA以及BRDU、CCK8试剂盒检测炎症因子、细胞通路蛋白及细胞增殖情况。通过观察不同浓度、不同处理时间点白藜芦醇对高糖环境下原代大鼠系膜细胞对人纤溶酶原激活物抑制剂-1（plasminogenactivator inhibitor-1，PAI-1）表达和Akt活性的影响确定白藜芦醇的有效作用浓度和有效作用时间。应用白藜芦醇及两种Akt活性抑制剂，作用于Akt上游磷脂酰肌醇-3激酶（PI3K）的LY294002和直接作用于Akt的MK2206，处理高糖环境下原代大鼠系膜细胞。结果显示，与正常对照组相比，高糖处理组中NF-κB、PAI-1表达明显增加(P0.05)；与高糖处理组相比，白藜芦醇和Akt活性抑制剂处理组NF-κB、PAI-1表达明显下降(P0.05)。提示处于NF-κB下游的PAI-1表达变化可能与Akt/NF-κB通路有关。检测应用白藜芦醇及Akt活性抑制剂处理的原代大鼠系膜细胞，在高糖刺激24小时后细胞增殖及同期细胞上清液当中层粘连蛋白（fibronetic,FN）表达情况。结果显示，与正常对照组相比，高糖处理组细胞增殖明显增强(P0.05)，白藜芦醇和Akt活性抑制剂处理组细胞增殖较高糖处理组细胞增殖水平下降(P0.05)。与正常对照组相比，高糖处理组FN表达明显增加(P0.05)，白藜芦醇和Akt活性抑制剂处理组FN表达水平较高糖处理组明显下降(P0.05)。提示白藜芦醇对高糖刺激下原代大鼠系膜细胞增殖及FN分泌表达的影响可能与抑制Akt活性有关。在动物实验当中，我们应用小剂量多次注射链脲佐菌素的方法建立1型糖尿病小鼠动物模型。并在模型建立成功后，给予糖尿病组小鼠白藜芦醇(10mg/kg/d)灌胃，正常对照组给予相同体积生理盐水灌胃，12周后处死小鼠，收集血液、尿液及肾脏标本。我们应用Real-time PCR、Western blot、PAS染色及免疫组织化学法检测肾脏功能学、形态学及生物化学指标。结果显示，在功能学方面，与正常对照组小鼠相比，糖尿病组与白藜芦醇治疗组小鼠空腹血糖、尿素氮、肌酐显著升高(P0.05)，治疗组在应用白藜芦醇干预12周后血糖、尿素氮、肌酐有所下降，但差异没有显著性(P0.05)。与正常对照组小鼠相比，糖尿病组与白藜芦醇治疗组小鼠的尿白蛋白与尿肌酐比值（albumin/creatinine ratio, ACR）显著升高(P0.05)；与糖尿病组相比，白藜芦醇治疗组ACR显著降低(P0.05)。在形态学方面，白藜芦醇治疗组较糖尿病组系膜细胞增生，系膜外基质增多等病理改变显著减轻。在分子生物学方面，反映Akt活性的p-Akt/Akt、NF-κB表达在糖尿病组和白藜芦醇治疗组均显著高于正常对照组(P0.05)。与糖尿病组相比，上述指标在白藜芦醇治疗组明显下降。免疫组织化学显示上述蛋白在肾间质、肾小管及肾小球均有表达，变化趋势与Western blot检测结果一致。炎症因子PAI-1、细胞间粘附分子-1（intercellular adhesionmolecule-1，ICAM-1）蛋白表达在糖尿病组与白藜芦醇治疗组均显著高于正常对照组(P0.05)。与糖尿病组相比，，上述指标在白藜芦醇治疗组明显下降(P0.05)。免疫组织化学显示PAI-1蛋白在肾间质、肾小管及肾小球均有表达，变化趋势与Western blot检测结果一致。在细胞增殖方面，与正常对照组相比，糖尿病组增殖细胞核抗原（PCNA）mRNA水平明显增加(P0.05)，白藜芦醇治疗组PCNA mRNA水平较糖尿病组明显下降(P0.05)。免疫组化结果显示，糖尿病组小鼠肾脏每单位面积当中PCNA阳性细胞明显多于正常对照组(P0.05)，而经过白藜芦醇治疗则明显减少了小鼠肾脏每单位面积当中的PCNA阳性细胞数目(P0.05)。综上所述，本研究证实了：（1）白藜芦醇能够明显减少高糖刺激下细胞因子NF-κB、PAI-1的过表达，降低细胞增殖水平。说明白藜芦醇能够降低高糖环境下原代大鼠系膜细胞的炎症反应和细胞增殖。（2）白藜芦醇和Akt活性抑制剂均能够有效降低原代大鼠系膜细胞在高糖刺激下异常增高的Akt活性，减少细胞因子NF-κB、PAI-1的过表达。说明白藜芦醇与Akt/NF-κB通路关系密切。 (3)在1型糖尿病小鼠模型成功建立后，连续应用白藜芦醇干预12周，白藜芦醇处理组小鼠ACR下降，PAI-1、ICAM-1表达减少，病理改变减轻，肾组织PCNA阳性细胞数目下降。说明白藜芦醇可以显著减轻DN所致的蛋白尿、炎症和系膜细胞增生。 (4)白藜芦醇对于糖尿病肾病的保护作用可能是通过抑制Akt活性，下调NF-κB蛋白表达，进而减轻炎症反应、抑制系膜细胞增殖来实现的。本研究的主要创新点为： (1)首次在动物实验当中证实了白藜芦醇可以通过Akt/NF-κB路径抑制肾小球系膜细胞增殖。 (2)通过体内与体外两方面实验阐明了白藜芦醇以及Akt活性抑制剂与DN中炎症介质PAI-1、ICAM-1的关系,并提出上述影响可能与Akt/NF-κB通路有关。
[Abstract]:Diabetic nephropathy (DN) is one of the most important microvascular complications of diabetes. According to the statistics of developed countries, DN has jumped to the first cause of end-stage renal disease. In recent years, the number of diabetic patients in China is increasing, and DN has become one of the important causes of slow renal failure in China. At present, DN is considered to be a major cause of chronic renal failure. It is related to many factors such as disorder of sugar metabolism, abnormal hemodynamics, oxidative stress, accumulation of end products of glycosylation, abnormal expression of cytokine and other factors. The interaction of inflammatory factors and oxidative stress and immune response is the main cause of diabetic renal damage, which causes the accumulation of extracellular matrix. Resveratrol has anti-inflammatory, antioxidant and anti-cancer. Cardiac protection and so on, the mechanism of action involves multiple signal transduction pathways including epoxidase, lipoxygenase, protein kinase B (protein kinase B, PKB/Akt), mitogen activated protein kinase family, deacetylase and so on. The latest research has found that resveratrol also reduces blood sugar, improves insulin resistance, regulates abnormal lipid metabolism, and reduces inflammation. .Akt can play a key role in the production and development of diabetic vascular complications and development. Nuclear factor kappa B (n) can also play a key role in the development of diabetic vascular complications and development of diabetes by the transfer of glucose, glycogen synthesis, glycolysis and sugar isogenesis and the mutation of related genes. Uclear factor- kappa B, NF- kappa B) is one of the key factors involved in oxidative stress and inflammatory response. It is related to multiple pathophysiological processes of vascular endothelial cell injury, tissue cell apoptosis and other diabetic vascular complications. Many studies suggest that resveratrol may be a new drug for the treatment of diabetes and its complications. Akt, NF- kappa B may play an important role in the effects of the drug on multiple signal transduction pathways in cells. This study aims to explore the protective effect of resveratrol on renal inflammation and mesangial cell proliferation induced by diabetes and its mechanism.
In vitro experiments, we used high glucose to simulate the internal environment of diabetic patients. We used Western blot, ELISA and BRDU, CCK8 kit to detect the inflammatory factors, cell pathway proteins and cell proliferation. By observing different concentrations and different treatment time points, resveratrol was used for the human fibrinolytic enzyme in the primary rat mesangial cells under high glucose environment. The effect of the expression of the original activator inhibitor -1 (plasminogenactivator inhibitor-1, PAI-1) and the activity of Akt to determine the effective concentration and effective time of resveratrol. The use of resveratrol and two kinds of Akt activity inhibitors, the LY294002 of the phosphatidyl inositol -3 kinase (PI3K) in the upstream of Akt and the direct action on Akt MK2206, and the treatment of high sugar Compared with the normal control group, the expression of NF- kappa B and PAI-1 increased significantly (P0.05), and the expression of NF- kappa B and PAI-1 in the treatment group of resveratrol and Akt activity decreased significantly compared with the high glucose treatment group, suggesting that the expression of PAI-1 expression in the lower reaches of NF- kappa B may be associated with those of NF- kappa B downstream. Primary rat mesangial cells treated with resveratrol and Akt activity inhibitor were used to detect the proliferation of cells and the expression of fibronetic (FN) in the cell supernatant after 24 hours of high glucose stimulation. The results showed that the cell proliferation of the high glucose treatment group was significantly enhanced (P0.05), resveratrol, compared with the normal control group (P0.05). Compared with the normal control group, the expression of FN in the high glucose treatment group was significantly increased (P0.05), and the expression level of FN in the resveratrol and Akt active inhibitor treated group was significantly lower than that in the high glucose treatment group (P0.05). The results suggested that resveratrol was stimulated by high glucose in the original generation, compared with the normal control group (P0.05). The influence of mesangial cell proliferation and FN secretion expression may be related to inhibition of Akt activity.
In animal experiments, we used a small dose of streptozotocin to establish a model of type 1 diabetic mice. After the establishment of the model, the diabetic mice were fed with resveratrol (10mg/kg/d), and the normal control group was given the same volume of saline. After 12 weeks, the mice were killed and the blood, urine and kidney were collected. We used Real-time PCR, Western blot, PAS staining and immunohistochemical staining to detect renal function, morphology and biochemical indexes. The results showed that the diabetic group and the resveratrol group were significantly higher than the normal control group (P0.05) in the diabetic group and the resveratrol treatment group (P0.05), and the treatment group was significantly higher than the normal control group (P0.05). After 12 weeks of resveratrol intervention, blood sugar, urea nitrogen and creatinine decreased, but the difference was not significant (P0.05). Compared with the normal control group, the ratio of urinary albumin to creatinine (albumin/creatinine ratio, ACR) in the diabetic group and the resveratrol group was significantly higher (P0.05); resveratrol treatment was compared with the diabetic group. ACR in the treatment group was significantly lower (P0.05). In the morphological aspect, the resveratrol treatment group had a significant reduction in the proliferation of mesangial cells and the increase of the extra membrane matrix in the diabetic group. In the molecular biology, the p-Akt/Akt of the Akt activity and the expression of NF- kappa B were significantly higher in the diabetic group and the resveratrol group than in the normal control group (P0.05). Compared with the diabetes group, the above indexes were significantly decreased in the resveratrol treatment group. Immunohistochemistry showed that the above proteins were expressed in the renal interstitium, renal tubules and glomeruli, and the change trend was consistent with the results of Western blot detection. The inflammatory factor PAI-1, the expression of -1 (intercellular AdhesionMolecule-1, ICAM-1) protein of intercellular adhesion molecule (intercellular AdhesionMolecule-1, ICAM-1) was expressed in sugar The urine sickness group and the resveratrol group were significantly higher than the normal control group (P0.05). Compared with the diabetic group, the above indexes were significantly decreased in the resveratrol group (P0.05). The immunohistochemical staining showed that the PAI-1 protein was expressed in the renal interstitium, renal tubules and glomeruli, and the change trend was consistent with the results of Western blot detection. Compared with the normal control group, the level of proliferating cell nuclear antigen (PCNA) mRNA increased significantly in the diabetic group (P0.05), and the level of PCNA mRNA in the resveratrol group was significantly lower than that in the diabetic group (P0.05). The immunohistochemical results showed that the PCNA positive cells in the kidney of the diabetic mice were significantly more than those of the normal control group (P0.05) per unit area of the diabetic group (P0.05). Resveratrol treatment significantly reduced the number of PCNA positive cells per unit area in the kidney of mice (P0.05).
To sum up, this study confirms that:
(1) resveratrol can significantly reduce the overexpression of cytokine NF- kappa B, PAI-1 and the level of cell proliferation under high glucose stimulation. It is said that veratrol can reduce the inflammatory reaction and cell proliferation of primary rat mesangial cells under high glucose environment.
(2) both resveratrol and Akt activity inhibitors can effectively reduce the abnormal increased Akt activity of rat mesangial cells under high glucose stimulation and reduce the overexpression of cytokines NF- kappa B and PAI-1. It is clear that veratrol is closely related to Akt/NF- kappa B pathway.
(3) after the successful establishment of the model of type 1 diabetic mice, the continuous use of resveratrol for 12 weeks, the ACR in the resveratrol treatment group decreased, the expression of PAI-1, ICAM-1 decreased, the pathological changes were reduced, and the number of PCNA positive cells in the renal tissue decreased. It was said that veratrol could significantly reduce the proteinuria, inflammation and mesangial cell proliferation caused by DN.
(4) the protective effect of resveratrol on diabetic nephropathy may be achieved by inhibiting the activity of Akt, reducing the expression of NF- kappa B protein, reducing the inflammatory response and inhibiting the proliferation of mesangial cells.
The main innovations of this study are as follows:
(1) for the first time in animal experiments, resveratrol can inhibit the proliferation of mesangial cells through the Akt/NF- kappa B pathway.
(2) the relationship between resveratrol and Akt activity inhibitors and the inflammatory mediators of DN, PAI-1, ICAM-1, was elucidated through two experiments in vivo and in vitro, and suggested that the above effects may be related to the Akt/NF- kappa B pathway.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R692.9

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