本文關(guān)鍵詞： 糖尿病腎病 氧化應(yīng)激 核因子相關(guān)因子2 微小RNA 姜黃素　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：隨著糖尿病發(fā)病率的增加，糖尿病腎病(DN)的發(fā)病率也呈逐年上升趨勢(shì)。DN是引起終末期腎臟病的重要原因，導(dǎo)致極高的致死率和致殘率，然而目前尚缺乏有效防治手段。因此，尋找有效防治DN的新方法至關(guān)重要。氧化應(yīng)激是DN的重要發(fā)病機(jī)制。高血糖狀態(tài)下，腎臟活性氧簇(ROS)產(chǎn)生增多，超過了機(jī)體抗氧化防御系統(tǒng)的清除能力，通過多種途徑誘導(dǎo)DN的發(fā)生、發(fā)展。核因子相關(guān)因子2(Nrf2)在機(jī)體抗氧化系統(tǒng)中起到核心作用。作為轉(zhuǎn)錄因子，Nrf2可進(jìn)入細(xì)胞核，結(jié)合下游抗氧化基因啟動(dòng)子區(qū)的抗氧化反應(yīng)元件(ARE)，啟動(dòng)這些基因的轉(zhuǎn)錄，從而減輕糖尿病引起的氧化應(yīng)激損傷，延緩DN的病情進(jìn)展。姜黃素是天然植物提取物，對(duì)多種疾病模型具有良好的保護(hù)作用，然而其生物利用率低，極大地限制了它的臨床應(yīng)用。在DN動(dòng)物模型中姜黃素的有效劑量為50-200mg/kg，每日一次；而新型姜黃素同源物C66的有效劑量?jī)H為5mg/kg，隔日一次，與天然姜黃素相比生物利用率顯著提高，具有良好的臨床應(yīng)用前景。然而，C66對(duì)DN的保護(hù)作用機(jī)制尚不明確。 我們之前的研究顯示C66可顯著上調(diào)主動(dòng)脈Nrf2表達(dá)，減輕糖尿病引起的大血管病變。為了探討C66是否通過上調(diào)腎臟Nrf2減輕DN，我們?cè)贑57BL/6J野生型及C57BL/6J Nrf2敲除小鼠中采用多次小劑量腹腔注射鏈脲佐菌素的方法誘導(dǎo)糖尿病模型，成模的同時(shí)給予C66處理，5mg/kg，隔日一次，持續(xù)6個(gè)月。結(jié)果顯示，在野生型小鼠中，C66可顯著減輕糖尿病小鼠蛋白尿和腎臟纖維化；而Nrf2敲除部分減弱了C66對(duì)DN的保護(hù)作用。這些結(jié)果一方面證實(shí)了C66通過上調(diào)Nrf2對(duì)DN起重要保護(hù)作用；另一方面提示除上調(diào)Nrf2外，仍有其他作用機(jī)制參與C66對(duì)DN的保護(hù)作用。 本研究的第二個(gè)目的是探討C66上調(diào)Nrf2的機(jī)制。在胞漿中，肌動(dòng)蛋白結(jié)合蛋白Kelch樣環(huán)氧氯丙烷相關(guān)蛋白1(Keap1)結(jié)合Nrf2，阻止Nrf2入核啟動(dòng)下游抗氧化基因的轉(zhuǎn)錄，并介導(dǎo)Nrf2的泛素化降解。因此，下調(diào)Keap1是上調(diào)Nrf2的有效策略。研究表明，微小RNA-200a (miR-200a)可結(jié)合Keap1mRNA的3’非翻譯區(qū)，導(dǎo)致Keap1mRNA降解，最終引起Keap1蛋白水平降低。另有研究表明，姜黃素可上調(diào)miR-200a。因此，我們假設(shè)C66通過上調(diào)miR-200a，降低Keap1水平，進(jìn)而上調(diào)Nrf2。為了驗(yàn)證該假設(shè)是否成立，我們?cè)谝吧吞悄虿⌒∈笾型瑫r(shí)應(yīng)用C66和miR-200a的特異性抑制劑-鎖核苷酸修飾的抗miR-200a (LNA-200a)干預(yù)6個(gè)月。結(jié)果顯示C66可顯著上調(diào)腎臟miR-200a、降低Keap1mRNA及蛋白水平、上調(diào)Nrf2蛋白水平及其下游抗氧化基因血紅素加氧酶1(HO1)、醌氧化還原酶-1(NQO1)的mRNA水平；而LNA-200a的應(yīng)用完全阻斷了C66的這些作用。這些結(jié)果證實(shí)C66通過上調(diào)miR-200a進(jìn)而激活Nrf2。 本研究的第三個(gè)目的是尋找并探討除上調(diào)Nrf2外，C66保護(hù)DN的其他機(jī)制。在DN相關(guān)的microRNA中，姜黃素除上調(diào)miR-200a外，尚可下調(diào)miR-21水平；且miR-21在DN的發(fā)病機(jī)制中起重要作用。miR-21可結(jié)合并降解Smad7的mRNA，導(dǎo)致Smad7蛋白水平下調(diào)，進(jìn)而引起其下游致纖維化轉(zhuǎn)錄因子Smad3活化，誘導(dǎo)糖尿病腎臟纖維化。因此，我們假設(shè)C66通過下調(diào)miR-21減輕DN。我們首先在野生型小鼠中觀察C66對(duì)miR-21和miR-21基因轉(zhuǎn)錄初級(jí)產(chǎn)物pri-miR-21的影響，結(jié)果顯示C66在非糖尿病及糖尿病腎臟中均可顯著下調(diào)miR-21及pri-miR-21的水平。這些結(jié)果肯定了C66對(duì)miR-21的下調(diào)作用，并揭示了C66對(duì)miR-21的下調(diào)作用發(fā)生在轉(zhuǎn)錄水平，而非miR-21的轉(zhuǎn)錄后加工過程。為明確C66對(duì)Smad7/Smad3信號(hào)通路的影響，，我們進(jìn)一步檢測(cè)了Smad7的mRNA和蛋白水平，以及磷酸化Smad3(p-Smad3)的蛋白水平。結(jié)果提示C66在非糖尿病與糖尿病腎臟中均可上調(diào)Smad7表達(dá)、下調(diào)p-Smad3的蛋白水平。為進(jìn)一步排除Nrf2的影響，我們?cè)贜rf2敲除糖尿病小鼠中分別給予C66和miR-21的特異性抑制劑LNA-21（陽性對(duì)照）處理6個(gè)月，結(jié)果顯示C66和LNA-21相似程度地降低了腎臟miR-21的表達(dá)，減輕了糖尿病引起的蛋白尿及腎臟損傷。這些結(jié)果證實(shí)除上調(diào)Nrf2以外，C66可通過抑制miR-21減輕DN。 綜上所述，本研究首次闡明了新型姜黃素同源物C66減輕DN的雙重作用機(jī)制：上調(diào)Nrf2和抑制miR-21。此外，本研究進(jìn)一步闡明了C66通過上調(diào)miR-200a從而活化Nrf2的機(jī)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.2

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