本文選題：糖尿病腎病 + 核因子相關(guān)因子2��； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：氧化應(yīng)激為糖尿病腎病(DN)的重要發(fā)病機(jī)制之一，它可以促進(jìn)DN的發(fā)生和發(fā)展。核因子相關(guān)因子2(Nrf2)作為轉(zhuǎn)錄因子，可以增加多種內(nèi)源性抗氧化蛋白的轉(zhuǎn)錄及表達(dá)，如血紅素加氧酶1(HO-1)、醌氧化還原酶-1(NQO1)、超氧化物歧化酶(SOD)、過氧化氫酶(CAT)和谷氨酸半胱氨酸連接酶(GCL)等。有研究證明蘿卜硫素(SFN)可以通過上調(diào)Nrf2改善DN，但是Nrf2的哪個(gè)下游抗氧化蛋白是SFN的主要保護(hù)途徑尚不清楚。另外，實(shí)驗(yàn)證明給予大鼠SFN干預(yù)后，肝臟基因表達(dá)上調(diào)最顯著的是金屬硫蛋白(MT)，上調(diào)了10倍以上；MT作為強(qiáng)效內(nèi)源性抗氧化蛋白，可以顯著減輕氧化應(yīng)激，延緩DN的病情進(jìn)展。然而，至今沒有研究顯示SFN是否可以通過上調(diào)腎臟MT表達(dá)改善DN，也沒有研究顯示MT是否為Nrf2的下游抗氧化因子。 為探討SFN是否能夠增加腎臟MT的表達(dá)，我們采用高脂飲食聯(lián)合鏈脲佐菌素腹腔注射的方法在C57BL/6J野生型小鼠中建立2型糖尿病(T2DM)小鼠模型，在糖尿病模型建立的同時(shí)給予SFN干預(yù)4個(gè)月，即小鼠被分為對(duì)照組、SFN干預(yù)組、糖尿病組(DM)、糖尿病+SFN干預(yù)組(DM/SFN)。4個(gè)月后，處死小鼠并收集血液、尿液及腎臟組織。實(shí)驗(yàn)結(jié)果顯示，與對(duì)照組小鼠相比，DM小鼠尿白蛋白/肌酐(UACR)顯著增加，腎臟病理改變、炎癥、纖維化和氧化損傷均增加，并伴隨著腎臟組蛋白去乙�；�2(HDAC2)和HDAC4表達(dá)增加。SFN干預(yù)4個(gè)月顯著改善了上述病變、降低了HDAC2和HDAC4的表達(dá)，這些過程伴隨著Nrf2和MT的上調(diào)。機(jī)制上，SFN抑制HDAC2和HDAC4的表達(dá)，導(dǎo)致MT1基因啟動(dòng)子區(qū)組蛋白高度乙�；�。另外，SFN顯著上調(diào)轉(zhuǎn)錄因子Nrf2，并增加MT1基因啟動(dòng)子區(qū)Nrf2的結(jié)合而促進(jìn)MT1的轉(zhuǎn)錄。 為探討SFN對(duì)DN的保護(hù)作用是否絕對(duì)依賴于Nrf2，并進(jìn)一步證實(shí)MT為Nrf2的下游抗氧化因子，我們分別用C57BL/6J野生型小鼠和C57BL/6J Nrf2敲除小鼠誘導(dǎo)T2DM小鼠模型，并于糖尿病模型建立成功的同時(shí)給予SFN干預(yù)，持續(xù)4個(gè)月，即C57BL/6J野生型小鼠和C57BL/6J Nrf2敲除小鼠分別被分為對(duì)照組、SFN干預(yù)組、糖尿病組(DM)、糖尿病+SFN干預(yù)組(DM/SFN)。實(shí)驗(yàn)結(jié)果顯示，在野生型小鼠中，SFN顯著降低糖尿病小鼠的UACR、腎臟炎癥、纖維化，，這個(gè)過程伴隨Nrf2和MT的上調(diào)。在Nrf2敲除小鼠中，SFN對(duì)DN無任何保護(hù)作用，且SFN不能上調(diào)MT的表達(dá)。 為探討MT是否在SFN的腎臟保護(hù)作用中起作用，我們分別用129S1野生型小鼠和129S1MT敲除小鼠誘導(dǎo)T2DM小鼠模型，并于糖尿病模型建立成功的同時(shí)給予SFN干預(yù)，持續(xù)4個(gè)月，即129S1野生型小鼠和129S1MT敲除小鼠分別被分為對(duì)照組、SFN干預(yù)組、糖尿病組(DM)、糖尿病+SFN干預(yù)組(DM/SFN)。實(shí)驗(yàn)結(jié)果顯示，在野生型小鼠中，SFN顯著降低糖尿病小鼠的UACR，約降低了55%；在MT敲除小鼠中，SFN部分降低了糖尿病小鼠的UACR，約降低了27%，這兩個(gè)降低百分比之間存在顯著性差異。另外，為明確MT是否影響Nrf2的表達(dá)，我們檢測(cè)了129S1野生型小鼠和129S1MT敲除小鼠腎臟Nrf2的表達(dá)。結(jié)果顯示，無論在野生型小鼠亦或MT敲除小鼠中，糖尿病均顯著降低了腎臟Nrf2的表達(dá)，SFN干預(yù)顯著增加了Nrf2的表達(dá)（包括mRNA水平和蛋白水平）。 以上結(jié)果表明，SFN可以預(yù)防T2DM所致的腎臟損傷，其腎臟保護(hù)作用完全依賴Nrf2，部分依賴于MT。MT作為Nrf2的下游因子之一，在SFN保護(hù)DN的過程中起重要作用。SFN保護(hù)DN的可能機(jī)制為：SFN抑制腎臟HDAC2和HDAC4的表達(dá)，導(dǎo)致MT1基因啟動(dòng)子區(qū)組蛋白高度乙�；�，染色質(zhì)結(jié)構(gòu)打開，有利于轉(zhuǎn)錄因子的結(jié)合而易化轉(zhuǎn)錄。另外，SFN顯著上調(diào)轉(zhuǎn)錄因子Nrf2，并增加MT1基因啟動(dòng)子區(qū)Nrf2的結(jié)合而促進(jìn)MT1的轉(zhuǎn)錄，降低糖尿病導(dǎo)致的腎臟氧化損傷，改善DN。本研究主要?jiǎng)?chuàng)新點(diǎn)： （1）本研究應(yīng)用T2DM小鼠模型，首次探討了SFN作為HDAC抑制劑和Nrf2激動(dòng)劑通過上調(diào)MT保護(hù)DN的機(jī)制。 （2）以前的研究顯示Nrf2和MT呈同向性變化，即在某些條件下同時(shí)被上調(diào)或下調(diào)，而我們的研究首次用染色質(zhì)免疫共沉淀(ChIP)的方法和Nrf2敲除小鼠、MT敲除小鼠證明了MT是Nrf2的下游抗氧化因子。 （3）本研究首次比較了SFN在129S1野生型DN小鼠和129S1MT敲除DN小鼠中的作用，證明了MT在SFN保護(hù)DN過程中的重要作用。 （4）本研究首次探討了HDAC抑制劑通過表觀遺傳學(xué)機(jī)制上調(diào)MT從而保護(hù)DN的機(jī)制。
[Abstract]:Oxidative stress is one of the important pathogenesis of diabetic nephropathy (DN). It can promote the development and development of DN. As a transcription factor, nuclear factor related factor 2 (Nrf2) can increase the transcription and expression of a variety of endogenous antioxidant proteins, such as heme oxygenase 1 (HO-1), quinone oxidoreductase -1 (NQO1), superoxide dismutase (SOD), and peroxidation Hydrogen enzyme (CAT) and glutamate cysteine ligase (GCL). Some studies have shown that sulforaphane (SFN) can improve DN by up regulation of Nrf2, but which downstream antioxidant protein of Nrf2 is the main protective way of SFN is not clear. In addition, the experiment showed that the prognosis of SFN dry in rats was given, the most significant up regulation of liver gene expression was metallothionein (MT). Up to 10 times up; MT, as a powerful endogenous antioxidant protein, could significantly reduce oxidative stress and delay the progression of DN. However, no study has shown whether SFN can improve DN by up - regulation of renal MT expression, and whether MT is a downstream anti oxidant factor of Nrf2.
To investigate whether SFN could increase the expression of MT in the kidney, we used a high fat diet combined with streptozotocin to intraperitoneal injection of type 2 diabetes (T2DM) model in C57BL/6J wild type mice. The diabetic model was established with SFN for 4 months, that is, the rats were divided into the control group, the SFN intervention group, the diabetic group (DM), and the sugar. The results showed that the urinary albumin / creatinine (UACR) increased significantly in the DM mice compared with the control group, and the renal pathological changes, inflammation, fibrosis and oxidative damage were increased, with renal histone deacetylase 2 (HDAC2) 2 (HDAC2) and HDAC4 as compared with the control group. The expression of.SFN intervention for 4 months significantly improved the above lesions and decreased the expression of HDAC2 and HDAC4. These processes were associated with the up regulation of Nrf2 and MT. SFN inhibited the expression of HDAC2 and HDAC4, resulting in the high acetylation of the promoter region histone of the MT1 gene. In addition, SFN significantly increased the Nrf2 of the transcription factor and increased the MT1 gene promoter region. Combined to promote the transcription of MT1.
To investigate whether the protective effect of SFN on DN was absolutely dependent on Nrf2, and further confirmed that MT was a downstream antioxidant factor of Nrf2, we used C57BL/6J wild type and C57BL/6J Nrf2 knockout mice to induce the T2DM mouse model, and the diabetes model was successfully established and the SFN intervention was given at the same time for 4 months, that is, C57BL/6J wild type mice. And C57BL/6J Nrf2 knockout mice were divided into the control group, the SFN intervention group, the diabetes group (DM) and the diabetic +SFN intervention group (DM/SFN). The experimental results showed that in the wild mice, SFN significantly reduced the UACR, renal inflammation and fibrosis in diabetic mice. This process was associated with the up regulation of Nrf2 and MT. In Nrf2 knockout mice, SFN had no protection on the SFN. Effect, and SFN can not increase the expression of MT.
To investigate whether MT plays a role in the renal protection of SFN, we use 129S1 wild type mice and 129S1MT knockout mice to induce T2DM mice model, and the diabetes model was successfully established and the SFN intervention was given for 4 months. The 129S1 wild type mice and 129S1MT knockout mice were divided into the control group, the SFN intervention group, and the diabetes mellitus, respectively. In the disease group (DM), the diabetic +SFN intervention group (DM/SFN). The results showed that in the wild mice, SFN significantly reduced the UACR of diabetic mice by about 55%; in the MT knockout mice, the SFN part reduced the UACR of the diabetic mice, reduced by about 27%, and there was a significant difference between the two reduction percentages. In addition, to determine whether MT affects MT. Nrf2 expression, we detected the expression of Nrf2 in the 129S1 wild type and 129S1MT knockout mice. The results showed that diabetes significantly decreased the expression of Nrf2 in the kidney and MT knockout mice, and the SFN intervention significantly increased the expression of Nrf2 (including the level of mRNA and protein).
These results suggest that SFN can prevent renal injury caused by T2DM, and its renal protection is entirely dependent on Nrf2, partly dependent on MT.MT as one of the downstream factors of Nrf2. The possible mechanism of.SFN to protect DN in the process of SFN protection of DN is that SFN inhibits the expression of HDAC2 and HDAC4, leading to the promoter region histone High acetylation and chromatin structure are open to facilitate the binding of transcription factors and facilitate transcription. In addition, SFN significantly up-regulated the transcription factor Nrf2 and increases the binding of Nrf2 in the promoter region of the MT1 gene to promote the transcription of MT1, reduce the oxidative damage caused by diabetes, and improve the main innovation of the DN. study.
(1) in this study, the T2DM mouse model was used to explore the mechanism of SFN as a HDAC inhibitor and Nrf2 agonist to protect DN through upregulated MT.
(2) previous studies showed that Nrf2 and MT were in the same direction, that is, up or down under certain conditions, and our study was the first to use chromatin immunoprecipitation (ChIP) and Nrf2 knockout mice. MT knockout mice showed that MT was the downstream antioxidant factor of Nrf2.
(3) for the first time, this study compared the role of SFN in 129S1 wild type DN mice and 129S1MT knockout DN mice, demonstrating the important role of MT in SFN protection DN.
(4) in this study, we first explored the mechanism by which HDAC inhibitors can upregulate MT through epigenetic mechanisms to protect DN.

【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;R692.9

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相關(guān)期刊論文 前2條

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本文編號(hào)：1831460