本文選題：糖尿病腎病　切入點(diǎn)：核因子相關(guān)因子2　出處：《吉林大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：氧化應(yīng)激是導(dǎo)致糖尿病腎病(DN)的一個(gè)重要原因。上調(diào)抗氧化因子核因子相關(guān)因子2(Nrf2)可以延緩DN的病情進(jìn)展。本研究旨在探討Nrf2激動(dòng)劑蘿卜硫素(SFN)和小劑量蛋白酶體抑制劑MG132對(duì)小鼠DN的防治作用。 為探討SFN對(duì)DN的預(yù)防作用，我們采用小劑量多次注射鏈脲佐菌素的方法建立1型糖尿病小鼠模型，并與模型建立成功的同時(shí)給予糖尿病小鼠及同齡對(duì)照小鼠SFN干預(yù)3個(gè)月(0.5mg/kg/day，每周5天)，處死第一批小鼠(3個(gè)月組)，收集血液、尿液及腎臟標(biāo)本；第二批小鼠停止SFN干預(yù)并繼續(xù)喂養(yǎng)3個(gè)月后處死(6個(gè)月組)，收集相應(yīng)標(biāo)本。為探討蛋白酶體抑制劑MG132對(duì)DN的治療作用，我們給予3月齡轉(zhuǎn)基因1型糖尿病(OVE26)小鼠腹腔注射MG132(10μg/kg/day)，此時(shí)OVE26小鼠已表現(xiàn)為腎功能異常(尿蛋白增加)。MG132治療3個(gè)月后，處死小鼠，，收集相應(yīng)標(biāo)本。我們應(yīng)用Real-time PCR、Westernblot、免疫組織化學(xué)以及相關(guān)試劑盒檢測(cè)腎臟功能學(xué)、形態(tài)學(xué)及生物化學(xué)等指標(biāo)。此外，我們尚在體外培養(yǎng)人腎小管上皮細(xì)胞(HK11)，通過給予高糖/高脂和SFN、MG132等處理模擬體內(nèi)實(shí)驗(yàn)，并應(yīng)用Nrf2特異性siRNA沉默HK11細(xì)胞的Nrf2基因，從而探討Nrf2在SFN和MG132防治DN過程中的核心作用。 SFN預(yù)防實(shí)驗(yàn)結(jié)果顯示，與糖尿病(3個(gè)月)組小鼠相比，糖尿病+SFN (3個(gè)月)組小鼠腎臟氧化損傷、炎癥、纖維化以及腎臟結(jié)構(gòu)和功能的改變均顯著減輕，并伴隨Nrf2表達(dá)增加和活性增強(qiáng)；而SFN的這種腎臟保護(hù)作用以及Nrf2的上調(diào)在停止SFN干預(yù)的6個(gè)月組小鼠中未能持續(xù)。MG132治療實(shí)驗(yàn)結(jié)果顯示，與同齡未治療的糖尿病小鼠相比，經(jīng)MG132治療的糖尿病小鼠腎臟病理和功能均明顯改善。MG132的治療作用伴隨著腎臟Nrf2蛋白表達(dá)增加以及Nrf2下游抗氧化基因轉(zhuǎn)錄水平上調(diào)。此外，我們尚發(fā)現(xiàn)糖尿病時(shí)腎臟蛋白酶體活性顯著增加，MG132治療3個(gè)月可以使其降低。體外實(shí)驗(yàn)結(jié)果表明，經(jīng)Nrf2特異性siRNA預(yù)處理，SFN和MG132對(duì)高糖/高脂導(dǎo)致的Nrf2及其下游抗氧化基因的上調(diào)作用均明顯減弱，并且高糖/高脂誘導(dǎo)的HK11細(xì)胞高結(jié)締組織生長(zhǎng)因子表達(dá)被完全逆轉(zhuǎn)。 以上結(jié)果表明，SFN干預(yù)3個(gè)月可以預(yù)防1型糖尿病所致的腎臟損傷，但具有非持續(xù)性；小劑量MG132治療3個(gè)月可以減輕1型糖尿病所致的腎臟氧化損傷、炎癥以及纖維化；Nrf2激動(dòng)劑SFN對(duì)DN預(yù)防作用可能系通過上調(diào)Nrf2的蛋白表達(dá)，增強(qiáng)Nrf2活性；MG132通過上調(diào)Nrf2而發(fā)揮其對(duì)DN的治療作用可能依賴于抑制蛋白酶體活性。
[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.
【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】：2014
【分類號(hào)】：R587.2;R692

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