萝卜硫素在小鼠糖尿病肾病中的肾脏保护机制研究

发布时间：2018-05-01 23:29

本文选题：糖尿病肾病 + 核因子相关因子2　；参考：《吉林大学》2015年博士论文

【摘要】：氧化应激为糖尿病肾病(DN)的重要发病机制之一，它可以促进DN的发生和发展。核因子相关因子2(Nrf2)作为转录因子，可以增加多种内源性抗氧化蛋白的转录及表达，如血红素加氧酶1(HO-1)、醌氧化还原酶-1(NQO1)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷氨酸半胱氨酸连接酶(GCL)等。有研究证明萝卜硫素(SFN)可以通过上调Nrf2改善DN，但是Nrf2的哪个下游抗氧化蛋白是SFN的主要保护途径尚不清楚。另外，实验证明给予大鼠SFN干预后，肝脏基因表达上调最显著的是金属硫蛋白(MT)，上调了10倍以上；MT作为强效内源性抗氧化蛋白，可以显著减轻氧化应激，延缓DN的病情进展。然而，至今没有研究显示SFN是否可以通过上调肾脏MT表达改善DN，也没有研究显示MT是否为Nrf2的下游抗氧化因子。为探讨SFN是否能够增加肾脏MT的表达，我们采用高脂饮食联合链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时给予SFN干预4个月，即小鼠被分为对照组、SFN干预组、糖尿病组(DM)、糖尿病+SFN干预组(DM/SFN)。4个月后，处死小鼠并收集血液、尿液及肾脏组织。实验结果显示，与对照组小鼠相比，DM小鼠尿白蛋白/肌酐(UACR)显著增加，肾脏病理改变、炎症、纤维化和氧化损伤均增加，并伴随着肾脏组蛋白去乙酰化酶2(HDAC2)和HDAC4表达增加。SFN干预4个月显著改善了上述病变、降低了HDAC2和HDAC4的表达，这些过程伴随着Nrf2和MT的上调。机制上，SFN抑制HDAC2和HDAC4的表达，导致MT1基因启动子区组蛋白高度乙酰化。另外，SFN显著上调转录因子Nrf2，并增加MT1基因启动子区Nrf2的结合而促进MT1的转录。为探讨SFN对DN的保护作用是否绝对依赖于Nrf2，并进一步证实MT为Nrf2的下游抗氧化因子，我们分别用C57BL/6J野生型小鼠和C57BL/6J Nrf2敲除小鼠诱导T2DM小鼠模型，并于糖尿病模型建立成功的同时给予SFN干预，持续4个月，即C57BL/6J野生型小鼠和C57BL/6J Nrf2敲除小鼠分别被分为对照组、SFN干预组、糖尿病组(DM)、糖尿病+SFN干预组(DM/SFN)。实验结果显示，在野生型小鼠中，SFN显著降低糖尿病小鼠的UACR、肾脏炎症、纤维化，，这个过程伴随Nrf2和MT的上调。在Nrf2敲除小鼠中，SFN对DN无任何保护作用，且SFN不能上调MT的表达。为探讨MT是否在SFN的肾脏保护作用中起作用，我们分别用129S1野生型小鼠和129S1MT敲除小鼠诱导T2DM小鼠模型，并于糖尿病模型建立成功的同时给予SFN干预，持续4个月，即129S1野生型小鼠和129S1MT敲除小鼠分别被分为对照组、SFN干预组、糖尿病组(DM)、糖尿病+SFN干预组(DM/SFN)。实验结果显示，在野生型小鼠中，SFN显著降低糖尿病小鼠的UACR，约降低了55%；在MT敲除小鼠中，SFN部分降低了糖尿病小鼠的UACR，约降低了27%，这两个降低百分比之间存在显著性差异。另外，为明确MT是否影响Nrf2的表达，我们检测了129S1野生型小鼠和129S1MT敲除小鼠肾脏Nrf2的表达。结果显示，无论在野生型小鼠亦或MT敲除小鼠中，糖尿病均显著降低了肾脏Nrf2的表达，SFN干预显著增加了Nrf2的表达（包括mRNA水平和蛋白水平）。以上结果表明，SFN可以预防T2DM所致的肾脏损伤，其肾脏保护作用完全依赖Nrf2，部分依赖于MT。MT作为Nrf2的下游因子之一，在SFN保护DN的过程中起重要作用。SFN保护DN的可能机制为：SFN抑制肾脏HDAC2和HDAC4的表达，导致MT1基因启动子区组蛋白高度乙酰化，染色质结构打开，有利于转录因子的结合而易化转录。另外，SFN显著上调转录因子Nrf2，并增加MT1基因启动子区Nrf2的结合而促进MT1的转录，降低糖尿病导致的肾脏氧化损伤，改善DN。本研究主要创新点：（1）本研究应用T2DM小鼠模型，首次探讨了SFN作为HDAC抑制剂和Nrf2激动剂通过上调MT保护DN的机制。（2）以前的研究显示Nrf2和MT呈同向性变化，即在某些条件下同时被上调或下调，而我们的研究首次用染色质免疫共沉淀(ChIP)的方法和Nrf2敲除小鼠、MT敲除小鼠证明了MT是Nrf2的下游抗氧化因子。（3）本研究首次比较了SFN在129S1野生型DN小鼠和129S1MT敲除DN小鼠中的作用，证明了MT在SFN保护DN过程中的重要作用。（4）本研究首次探讨了HDAC抑制剂通过表观遗传学机制上调MT从而保护DN的机制。
[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.

【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;R692.9

【参考文献】