新型姜黄素同源物C66对小鼠糖尿病肾病保护作用的机制研究

发布时间：2018-02-27 12:45

  本文关键词： 糖尿病肾病 氧化应激 核因子相关因子2 微小RNA 姜黄素　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：随着糖尿病发病率的增加，糖尿病肾病(DN)的发病率也呈逐年上升趋势。DN是引起终末期肾脏病的重要原因，导致极高的致死率和致残率，然而目前尚缺乏有效防治手段。因此，寻找有效防治DN的新方法至关重要。氧化应激是DN的重要发病机制。高血糖状态下，肾脏活性氧簇(ROS)产生增多，超过了机体抗氧化防御系统的清除能力，通过多种途径诱导DN的发生、发展。核因子相关因子2(Nrf2)在机体抗氧化系统中起到核心作用。作为转录因子，Nrf2可进入细胞核，结合下游抗氧化基因启动子区的抗氧化反应元件(ARE)，启动这些基因的转录，从而减轻糖尿病引起的氧化应激损伤，延缓DN的病情进展。姜黄素是天然植物提取物，对多种疾病模型具有良好的保护作用，然而其生物利用率低，极大地限制了它的临床应用。在DN动物模型中姜黄素的有效剂量为50-200mg/kg，每日一次；而新型姜黄素同源物C66的有效剂量仅为5mg/kg，隔日一次，与天然姜黄素相比生物利用率显著提高，具有良好的临床应用前景。然而，C66对DN的保护作用机制尚不明确。 我们之前的研究显示C66可显著上调主动脉Nrf2表达，减轻糖尿病引起的大血管病变。为了探讨C66是否通过上调肾脏Nrf2减轻DN，我们在C57BL/6J野生型及C57BL/6J Nrf2敲除小鼠中采用多次小剂量腹腔注射链脲佐菌素的方法诱导糖尿病模型，成模的同时给予C66处理，5mg/kg，隔日一次，持续6个月。结果显示，在野生型小鼠中，C66可显著减轻糖尿病小鼠蛋白尿和肾脏纤维化；而Nrf2敲除部分减弱了C66对DN的保护作用。这些结果一方面证实了C66通过上调Nrf2对DN起重要保护作用；另一方面提示除上调Nrf2外，仍有其他作用机制参与C66对DN的保护作用。 本研究的第二个目的是探讨C66上调Nrf2的机制。在胞浆中，肌动蛋白结合蛋白Kelch样环氧氯丙烷相关蛋白1(Keap1)结合Nrf2，阻止Nrf2入核启动下游抗氧化基因的转录，并介导Nrf2的泛素化降解。因此，下调Keap1是上调Nrf2的有效策略。研究表明，微小RNA-200a (miR-200a)可结合Keap1mRNA的3’非翻译区，导致Keap1mRNA降解，最终引起Keap1蛋白水平降低。另有研究表明，姜黄素可上调miR-200a。因此，我们假设C66通过上调miR-200a，降低Keap1水平，进而上调Nrf2。为了验证该假设是否成立，我们在野生型糖尿病小鼠中同时应用C66和miR-200a的特异性抑制剂-锁核苷酸修饰的抗miR-200a (LNA-200a)干预6个月。结果显示C66可显著上调肾脏miR-200a、降低Keap1mRNA及蛋白水平、上调Nrf2蛋白水平及其下游抗氧化基因血红素加氧酶1(HO1)、醌氧化还原酶-1(NQO1)的mRNA水平；而LNA-200a的应用完全阻断了C66的这些作用。这些结果证实C66通过上调miR-200a进而激活Nrf2。 本研究的第三个目的是寻找并探讨除上调Nrf2外，C66保护DN的其他机制。在DN相关的microRNA中，姜黄素除上调miR-200a外，尚可下调miR-21水平；且miR-21在DN的发病机制中起重要作用。miR-21可结合并降解Smad7的mRNA，导致Smad7蛋白水平下调，进而引起其下游致纤维化转录因子Smad3活化，诱导糖尿病肾脏纤维化。因此，我们假设C66通过下调miR-21减轻DN。我们首先在野生型小鼠中观察C66对miR-21和miR-21基因转录初级产物pri-miR-21的影响，结果显示C66在非糖尿病及糖尿病肾脏中均可显著下调miR-21及pri-miR-21的水平。这些结果肯定了C66对miR-21的下调作用，并揭示了C66对miR-21的下调作用发生在转录水平，而非miR-21的转录后加工过程。为明确C66对Smad7/Smad3信号通路的影响，，我们进一步检测了Smad7的mRNA和蛋白水平，以及磷酸化Smad3(p-Smad3)的蛋白水平。结果提示C66在非糖尿病与糖尿病肾脏中均可上调Smad7表达、下调p-Smad3的蛋白水平。为进一步排除Nrf2的影响，我们在Nrf2敲除糖尿病小鼠中分别给予C66和miR-21的特异性抑制剂LNA-21（阳性对照）处理6个月，结果显示C66和LNA-21相似程度地降低了肾脏miR-21的表达，减轻了糖尿病引起的蛋白尿及肾脏损伤。这些结果证实除上调Nrf2以外，C66可通过抑制miR-21减轻DN。 综上所述，本研究首次阐明了新型姜黄素同源物C66减轻DN的双重作用机制：上调Nrf2和抑制miR-21。此外，本研究进一步阐明了C66通过上调miR-200a从而活化Nrf2的机制。
[Abstract]:With the increase of the incidence of diabetes, diabetic nephropathy (DN) incidence increased year by year.DN is an important cause of end-stage renal disease, causing high mortality and disability rate, but there is still a lack of effective means of control. Therefore, it is very important to establish a new method for effective prevention and treatment of DN. Oxidative stress is an important pathogenesis the mechanism of DN. Under the condition of high blood glucose, kidney of reactive oxygen species (ROS) have increased more than the antioxidant defense system scavenging ability, induced the occurrence of DN, through a variety of ways. The development of nuclear factor related factor 2 (Nrf2) plays a central role in the antioxidant system. As a transcription factor, Nrf2 can enter the nucleus, binds the antioxidant response element downstream antioxidant gene promoter (ARE), transcription of these genes, thereby reducing oxidative damage caused by diabetes, slowed the progression of DN disease. Curcumin Is a natural plant extract, has a good protective effect on various diseases model, but its bioavailability is low, which greatly restricts its clinical application. The effective dose of curcumin in animal models of DN is 50-200mg/kg, once a day; and the effective dose of novel curcumin homologue of C66 is only 5mg/kg, once every other day, compared with the natural curcumin bioavailability significantly improved, with good prospects for clinical application. However, the mechanism of protective effect of C66 on DN is not clear.
Our previous study showed that C66 could upregulate the expression of Nrf2 in aorta, reduce macrovascular disease caused by diabetes. In order to investigate whether C66 through upregulation of renal Nrf2 reduce DN, we in the C57BL/6J wild-type and C57BL/6J Nrf2 knockout mice by repeated low-dose intraperitoneal injection of streptozotocin induced diabetic model method, at the same time. The C66, 5mg/kg, once every other day, lasted for 6 months. The results showed that in wild-type mice, C66 can significantly reduce the proteinuria and renal fibrosis in diabetic mice; while Nrf2 knockdown attenuated the protective effect of C66 on DN. These results confirmed that C66 plays an important protective effect on DN through the up regulation of Nrf2; on the other hand, suggesting that in addition to the upregulation of Nrf2, there are other mechanisms involved in the protective effect of C66 on DN.
In this study, the second is to explore the mechanism of C66 up regulation of Nrf2. In the cytoplasm, actin binding protein Kelch like ECH associated protein 1 (Keap1) combined with Nrf2, block Nrf2 nuclear transcription downstream antioxidant gene, ubiquitin mediated Nrf2. Therefore, the downregulation of Keap1 is the effective method to increase Nrf2. The results show that micro RNA-200a (miR-200a) can be combined with Keap1mRNA in the 3 'untranslated region, resulting in the degradation of Keap1mRNA, resulting in reduced Keap1 protein levels. Other studies show that curcumin can upregulate miR-200a. therefore, we hypothesized that C66 through upregulation of miR-200a, decrease the level of Keap1, and the up regulation of Nrf2. in order to verify the hypotheses, anti the specific inhibitor of miR-200a - we use both C66 and miR-200a in the wild type diabetic mice lock nucleotide modifications (LNA-200a) intervention for 6 months. The results show that C66 can significantly raise the kidney Dirty miR-200a, reduce the Keap1mRNA and protein levels, increased levels of Nrf2 protein and its downstream antioxidant gene heme oxygenase 1 (HO1), quinone oxidoreductase -1 (NQO1) mRNA level; and the application of LNA-200a completely blocked the effect of C66. These results demonstrated that C66 activates Nrf2. via up regulation of miR-200a
The third objective is to find and study in the up regulation of Nrf2 and other protective mechanism of C66 DN. In DN related microRNA, in addition to curcumin upregulated miR-200a, also lowered the level of miR-21; and miR-21 in the pathogenesis of DN play an important role in the degradation of Smad7 and.MiR-21 can be combined with mRNA, resulting in Smad7 protein levels down, and then cause the downstream transcription factor Smad3 activation induced fibrosis, renal fibrosis induced by diabetes. Therefore, we hypothesized that C66 downregulation by miR-21 attenuated DN. we observed the effect of C66 on miR-21 and miR-21 gene transcription of primary products of pri-miR-21 in wild-type mice, the results showed that C66 can be found in nondiabetic and diabetic kidney significantly down regulated in miR-21 and the level of pri-miR-21. These results confirmed the downregulation of C66 on miR-21, and reveals the effect of C66 on miR-21 down regulated at the transcription level, rather than miR-21 The post transcriptional processing process. Effects of C66 on Smad7/Smad3 signaling pathway, we further examined Smad7 mRNA and protein levels, and phosphorylation of Smad3 (p-Smad3). The results suggest that C66 protein levels in non-diabetic and diabetic kidney can upregulate the expression of Smad7 and down-regulation of p-Smad3 protein level. To further exclude Nrf2 we, in the knockdown of Nrf2 specific inhibitor LNA-21 and C66 miR-21 respectively in diabetic mice (positive control) treatment for 6 months, the results showed that C66 and LNA-21 are similar and decreased the expression of miR-21, reduce the diabetes induced proteinuria and renal damage. These results confirmed that in addition to the upregulation of Nrf2, C66 through inhibition of miR-21 reduced DN.
To sum up, this study first clarifies the dual mechanism of C66, a new curcumin homologue, to reduce DN. Nrf2 and miR-21. inhibit the mechanism of C66 activation through upregulated miR-200a.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2

【参考文献】

相关期刊论文 前2条

1 张丽娜;谢席胜;左川;樊均明;;人参皂甙Rg1对糖尿病肾病大鼠TNF-α、MCP-1表达的影响[J];四川大学学报(医学版);2009年03期

2 袁明霞;唐荣;周巧玲;刘抗寒;肖舟;Veeraragoo Pouranan;;冬虫夏草对糖尿病肾病大鼠肾组织HIF-1α及VEGF表达的影响[J];中南大学学报(医学版);2013年05期

本文编号：1542728