Nrf2活化在防治小鼠糖尿病肾病过程中的作用

发布时间：2018-03-19 20:43

本文选题：糖尿病肾病　切入点：核因子相关因子2　出处：《吉林大学》2014年博士论文　论文类型：学位论文

【摘要】：氧化应激是导致糖尿病肾病(DN)的一个重要原因。上调抗氧化因子核因子相关因子2(Nrf2)可以延缓DN的病情进展。本研究旨在探讨Nrf2激动剂萝卜硫素(SFN)和小剂量蛋白酶体抑制剂MG132对小鼠DN的防治作用。为探讨SFN对DN的预防作用，我们采用小剂量多次注射链脲佐菌素的方法建立1型糖尿病小鼠模型，并与模型建立成功的同时给予糖尿病小鼠及同龄对照小鼠SFN干预3个月(0.5mg/kg/day，每周5天)，处死第一批小鼠(3个月组)，收集血液、尿液及肾脏标本；第二批小鼠停止SFN干预并继续喂养3个月后处死(6个月组)，收集相应标本。为探讨蛋白酶体抑制剂MG132对DN的治疗作用，我们给予3月龄转基因1型糖尿病(OVE26)小鼠腹腔注射MG132(10μg/kg/day)，此时OVE26小鼠已表现为肾功能异常(尿蛋白增加)。MG132治疗3个月后，处死小鼠，，收集相应标本。我们应用Real-time PCR、Westernblot、免疫组织化学以及相关试剂盒检测肾脏功能学、形态学及生物化学等指标。此外，我们尚在体外培养人肾小管上皮细胞(HK11)，通过给予高糖/高脂和SFN、MG132等处理模拟体内实验，并应用Nrf2特异性siRNA沉默HK11细胞的Nrf2基因，从而探讨Nrf2在SFN和MG132防治DN过程中的核心作用。 SFN预防实验结果显示，与糖尿病(3个月)组小鼠相比，糖尿病+SFN (3个月)组小鼠肾脏氧化损伤、炎症、纤维化以及肾脏结构和功能的改变均显著减轻，并伴随Nrf2表达增加和活性增强；而SFN的这种肾脏保护作用以及Nrf2的上调在停止SFN干预的6个月组小鼠中未能持续。MG132治疗实验结果显示，与同龄未治疗的糖尿病小鼠相比，经MG132治疗的糖尿病小鼠肾脏病理和功能均明显改善。MG132的治疗作用伴随着肾脏Nrf2蛋白表达增加以及Nrf2下游抗氧化基因转录水平上调。此外，我们尚发现糖尿病时肾脏蛋白酶体活性显著增加，MG132治疗3个月可以使其降低。体外实验结果表明，经Nrf2特异性siRNA预处理，SFN和MG132对高糖/高脂导致的Nrf2及其下游抗氧化基因的上调作用均明显减弱，并且高糖/高脂诱导的HK11细胞高结缔组织生长因子表达被完全逆转。以上结果表明，SFN干预3个月可以预防1型糖尿病所致的肾脏损伤，但具有非持续性；小剂量MG132治疗3个月可以减轻1型糖尿病所致的肾脏氧化损伤、炎症以及纤维化；Nrf2激动剂SFN对DN预防作用可能系通过上调Nrf2的蛋白表达，增强Nrf2活性；MG132通过上调Nrf2而发挥其对DN的治疗作用可能依赖于抑制蛋白酶体活性。
[Abstract]:Oxidative stress is an important cause of diabetic nephropathy. Upregulation of antioxidant nuclear factor-associated factor 2nrf2 can delay the progression of DN. The purpose of this study was to investigate the effects of Nrf2 agonist sulforaphane (SFN) and low-dose protease. Preventive and therapeutic effects of body inhibitor MG132 on mouse DN. In order to investigate the preventive effect of SFN on DN, we established the model of type 1 diabetes mellitus by small dose multiple injection of streptozotocin. At the same time, the diabetic mice and the control mice were treated with SFN for 3 months, 0.5 mg / kg / day, 5 days a week, and the first group of mice were killed (3 months group) to collect blood, urine and kidney samples. The second group of mice were killed after stopping SFN intervention and continuing feeding for 3 months (6 months group) and collected corresponding specimens. To investigate the therapeutic effect of proteasome inhibitor MG132 on DN, We gave 3-month-old transgenic type 1 diabetes mellitus (OVE26) mice intraperitoneal injection of MG132(10 渭 g / kg 路kg / day.At this time, the OVE26 mice showed abnormal renal function. We used Real-time PCR Western blot, immunohistochemistry and related kits to detect renal function, morphology and biochemistry. Human renal tubular epithelial cells were cultured in vitro. We treated them with high glucose / high fat and SFN MG132, and silenced the Nrf2 gene of HK11 cells by Nrf2 specific siRNA, so as to explore the central role of Nrf2 in the prevention and treatment of DN by SFN and MG132. The results of SFN prophylaxis test showed that the oxidative damage, inflammation, fibrosis and changes of renal structure and function in diabetic SFN (3-month) mice were significantly reduced compared with diabetic (3-month) mice. The renal protective effect of SFN and the up-regulation of Nrf2 were not sustained in the 6-month group treated with SFN. The results showed that compared with untreated diabetic mice of the same age, MG132 could not be sustained. The renal pathology and function of diabetic mice treated with MG132 were significantly improved. MG132 was accompanied by increased expression of Nrf2 protein in kidney and up-regulation of antioxidant gene transcription level downstream of Nrf2. We also found that renal proteasome activity was significantly increased after 3 months of treatment with diabetes. The up-regulation of Nrf2 and its downstream antioxidant genes induced by high glucose / high fat was significantly decreased by pretreatment with Nrf2 specific siRNA, and the expression of high connective tissue growth factor in HK11 cells induced by high glucose / high fat was reversed completely. These results suggest that MG132 can prevent renal injury induced by type 1 diabetes for 3 months, but it is not sustainable, and small dose of MG132 can reduce renal oxidative damage caused by type 1 diabetes for 3 months. The prophylaxis of inflammation and fibrotic nrf2 agonist SFN on DN may depend on the inhibition of proteasome activity by up-regulating the expression of Nrf2 protein and enhancing the activity of Nrf2 MG132 by upregulating Nrf2.
【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

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