【作者】 李航；

【导师】 段惠军；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的:糖尿病肾病(diabetic nephropathy , DN)是糖尿病常见的微血管并发症,也是糖尿病致死、致残的重要原因。DN早期病理特征是肾小球肥大、基底膜增厚和系膜扩张,晚期则表现为肾小球硬化和间质纤维化。细胞外基质(extracellular matrix ,ECM)过度积聚是DN发生的病理生理基础。研究表明,氧化应激(Oxidative stress)在DN发病中起重要作用,高糖状态下产生过多的活性氧(Reactive oxygen species, ROS)不但激活了几乎所有已知的与糖尿病微血管并发症发生发展有关的信号传导通路,如PKC通路,多元醇通路,氨基己糖通路及AGEs形成,还可激活NF-κB上调黏附分子及炎性因子的基因转录,这是包括DN在内的糖尿病微血管并发症发病的共同机制。以上异常情况的长期存在使肾小球系膜基质及基底膜合成增加、降解减少,导致DN的发生发展。机体在应对ROS损害时形成了一套复杂的氧化应激应答系统,当暴露于ROS时,机体自身能诱导出一系列保护性蛋白,以缓解细胞所受的损害。这一协调反应是由这些保护性基因上游调节区的抗氧化反应元件(antioxidant responsive element ARE)来调控的。而近年来的研究发现,核因子NF-E2相关因子(Nuclear factor erythroid 2-related factor 2, Nrf2)是ARE的激活因子。Nrf2是外源性有毒物质和氧化应激的感受器,在参与细胞抗氧化应激和外源性有毒物质诱导的主要防御机制中发挥重要的作用。Nrf2-ARE通路是迄今为止发现的最为重要的内源性抗氧化应激通路。国内外对于Nrf2/ARE通路的研究主要集中在抗环境应激、抗老化、抗凋亡、神经保护和抗肿瘤等方面,而此通路在糖尿病氧化应激中的作用的研究未见到报道。机体内氧化还原水平失衡是糖尿病肾病的病理生理基础,调节体内抗氧化蛋白水平是改善其病理变化的策略之一,不同于以往的外源性抗氧化剂, Nrf2--ARE抗氧化系统是调控多种抗氧化酶转录表达的关键内源性通路,对于此通路的调控为实现有效、适度抗氧化治疗提供了新思路。本实验拟在整体与细胞水平,研究①Nrf2/ARE通路是否参与DN中的氧化应激反应;②通过增强Nrf2蛋白表达,是否可以减轻DN的氧化应激损伤,从而延缓DN的发生发展,试图为糖尿病肾病的防治提供可靠的实验依据。方法:1糖尿病动物模型的建立及相关指标检测右肾切除的雄性CD-1小鼠随机分为二组:对照组(C组),糖尿病组(DM组)。糖尿病组小鼠按130mg/kg腹腔注射STZ(溶于0.1 mol·L-1枸橼酸缓冲液中,pH为4.5),对照组注射等体积枸橼酸缓冲液。72小时后尾尖取血测定血糖,留尿测尿糖,以血糖≥16.7 mol/L、尿糖(+++)～(++++)作为糖尿病模型成功的标志。分别于注射STZ后4周、12周每组取6只小鼠称重后分别用代谢笼收集24h尿,用于测定尿白蛋白(Ualb,于四周时)和尿蛋白(Upro,于12周时);股动脉取血,分离血清,用于测定血生化和血清丙二醛(malondialdehyde,MDA)浓度;切取肾脏用于常规病理及免疫组化检测Nrf2、HO-1、γ-GCS在肾脏的定位和表达水平;取部分肾皮质匀浆测定MDA浓度;取部分皮质提取总蛋白及核蛋白,用于Western blot检测;2瞬时转染pcDNA3/mNrf2质粒于高糖培养的小鼠肾小球系膜细胞株(Mouse mesangial cells,MMC)及相关指标检测小鼠肾小球系膜细胞株培养于含10%胎牛血清及100KU/L青霉素、100mg/L链霉素的DME-F12培养基中,葡萄糖浓度为5.5mM。收获处于指数生长期细胞接种于6孔细胞培养板中,37℃、5%CO2培养箱培养,使细胞融合到70-80%;每孔中加入DME-F12无血清培养液培养12h后转染。瞬时转染步骤按Lipofectamine 2000说明书进行。转染6 h后换含10%胎牛血清的DME-F12培养液,,12h后将细胞分成5组①对照组(5.5mM葡萄糖),甘露醇对照组:(5.5 mM葡萄糖+24.5 mM甘露醇)、③高糖组:(30 mM葡萄糖),④高糖+空质粒转染对照组(转染pcDNA3+30 mM葡萄糖),⑤高糖+质粒转染组(转染pcDNA3/mNrf2+30 mM葡萄糖)分别刺激48h ,收取细胞。免疫细胞化学检测Nrf2和γ-GCS在细胞内的表达部位;Western-blot检测Nrf2、HO-1、γ-GCS总蛋白和Nrf2核蛋白表达;反转录-多聚酶链反应(Reverse transcription polymerse chain reaction,RT-PCR)检测Nrf2、HO-1、γ-GCS mRNA表达; 2’7’-二氯荧光素探针(2’7’-Dichlorofluorescein diacetate,2’7’-DCFH-DA)检测细胞内ROS水平;细胞裂解液MDA检测用于衡量细胞脂质氧化损伤程度。酶联免疫吸附实验(ELISA)检测细胞上清液TGF-β1分泌水平;流式细胞仪检测系膜细胞增殖。3瞬时转染Nrf2 siRNA于高糖培养的MMC细胞及相关指标检测。siRNA瞬时转染步骤按Lipofectamine 2000说明书进行,优化转染条件后,细胞分为以下五组:①对照组(5.5 mM葡萄糖)②甘露醇对照组:(5.5 mM葡萄糖+24.5 mM甘露醇)、③高糖组:(30 mM葡萄糖),④siRNA对照+高糖组:(转染Control siRNA +30 mM葡萄糖),⑤Nrf2 siRNA(m)+高糖组:(转染Nrf2 siRNA(m)+ 30mM葡萄糖),分别刺激48h,收取细胞。小鼠肾小球系膜细胞Nrf2、HO-1、γ-GCS表达、ROS和MDA含量、TGF-β1分泌、细胞增殖的检测方法同前。4 1%tBHQ干预糖尿病小鼠及相关指标检测。右肾切除的雄性CD-1小鼠随机分为3组,对照组(C组);糖尿病组(DM组); tBHQ干预组(DM+tBHQ组)。腹腔注射STZ诱发糖尿病小鼠模型,DM+tBHQ组在成模3日后给予添加了1%tBHQ(w/w)的饲料喂养,于4周、12周时收取标本,筛网法分离肾小球。检测3组动物的肾重/体重、24 h尿白蛋白定量(4周时)、24h尿蛋白定量(12周时);血清及肾小球组织匀浆MDA含量; Western印迹检测Nrf2,HO-1和γ-GCS蛋白在肾小球表达水平;RT-PCR检测12周时肾小球Nrf2、HO-1、γ-GCS mRNA的表达;透射电镜、HE及PAS染色,FN免疫组化半定量检测肾小球细胞外基质沉积水平。结果:1 Nrf2及其下游调控的抗氧化蛋白在糖尿病小鼠肾脏的表达及其与氧化应激损伤的关系。①由HE、PAS染色可见,糖尿病组于4周开始肾小球体积增大,12周时系膜细胞增生明显,系膜区增宽,肾小球肥大,基底膜增厚,基质增多。②与对照组相比,糖尿病组肾重/体重、24小时尿白蛋白定量(4周时)、24小时尿蛋白定量(12周时)明显增高。③糖尿病组血清及肾皮质匀浆MDA浓度较同期对照组明显增高;④免疫组化显示: Nrf2蛋白阳性表达定位于肾小球固有细胞的胞浆和胞核;与对照组相比,糖尿病组Nrf2蛋白表达在胞核明显增强,并且随病程的延长可见胞核表达有升高的趋势。对照组肾小球胞浆可见HO-1阳性表达,而糖尿病组表达在4周时较对照组降低,至12周时糖尿病组HO-1表达较对照组增高。γ-GCS蛋白的阳性表达主要分布于肾小球固有细胞、肾小管上皮细胞的胞浆中。糖尿病组γ-GCS蛋白的表达均高于同期对照组。⑤Western印记结果显示: 4周时,糖尿病组肾皮质Nrf2总蛋白表达与对照组无差异; HO-1蛋白表达明显低于对照组;γ-GCS蛋白及核蛋白Nrf2表达明显高于对照组;12周时,糖尿病组肾皮质Nrf2总蛋白表达与对照组无差异; HO-1蛋白、γ-GCS、核蛋白Nrf2表达均明显高于对照组。2 Nrf2、HO-1、γ-GCS在高糖培养的MMC细胞的表达及Nrf2-ARE信号通路高糖培养的MMC细胞ROS生成,脂质损伤、TGF-β1分泌、细胞增殖的影响。①与同期正常对照组相比,高糖培养组小鼠系膜细胞内ROS、MDA生成明显升高,并且随着刺激时间的延长有逐渐增高的趋势。②瞬时转染pcDNA3/mNrf2的MMC细胞在高糖刺激48小时后Nrf2蛋白被激活,细胞核Nrf2蛋白表达较高糖对照组明显增强,受其转录调控的抗氧化蛋白HO-1、γ-GCS mRNA及蛋白表达较高糖对照组均明显增强。而给予Nrf2siRNA(m)转染的MMC细胞细Nrf2核蛋白表达、Nrf2、HO-1、γ-GCS mRNA及蛋白表达较高糖对照组明显降低。③免疫细胞化学染色结果显示:转染pcDNA3/mNrf2后再给予高糖刺激48小时,Nrf2蛋白在MMC细胞浆及细胞核内表达较高糖刺激组均明显增强;γ-GCS蛋白在细胞浆内的表达较高糖刺激组明显增强,而转染Nrf2siRNA(m)则出现相反结果。④高糖刺激48小时, pcDNA3/mNrf2质粒转染组MMC细胞ROS,MDA生成,细胞增殖和TGF-β1分泌明显低于高糖刺激组,而给予Nrf2siRNA(m)特异敲低Nrf2的表达后以上保护作用消失。3 tBHQ通过激活Nrf2-ARE信号通路减轻糖尿病小鼠肾小球的氧化应激损伤。①糖尿病组肾重/体重、24 h尿白蛋白定量、24h尿蛋白定量较同期对照组显著升高;tBHQ干预组以上指标较糖尿病组明显改善。②糖尿病组血清及肾小球MDA含量较同期对照组显著升高。tBHQ干预组上述指标均降低。③与同期糖尿病组相比,tBHQ干预组肾小球总蛋白Nrf2、HO-1、γ-GCS、核蛋白Nrf2和Nrf2、HO-1、γ-GCS mRNA表达水平明显增高;④4周电镜、12周HE及PAS染色,FN免疫组化半定量结果均表明:tBHQ干预可以减少糖尿病小鼠肾小球ECM的沉积。结论:我们的研究结果表明,STZ诱导的糖尿病小鼠肾脏存在明显的氧化应激损伤,Nrf2和其转录调控的下游抗氧化蛋白HO-1和γ-GCS参与了糖尿病肾病的抗氧化防御机制。tBHQ特异激活糖尿病小鼠Nrf2蛋白可以启动抗氧化蛋白HO-1、γ-GCS的转录和表达进而减轻ROS引起的肾脏损害,减少肾小球细胞外基质沉积。高糖培养的小鼠肾小球系膜细胞可以产生过多的ROS,通过Nrf2表达质粒转染小鼠系膜细胞可以激活Nrf2和其转录调控的抗氧化蛋白HO-1、γ-GCS,减轻由高糖引起的ROS生成,脂质过氧化损伤,系膜细胞增殖和TGF-β1分泌,而转染siRNA特异敲低Nrf2表达则具有相反作用。本实验从体内和体外两方面证实,Nrf2是糖尿病肾病发病中抗氧化基因表达的关键转录调控因子,通过激活机体内源性抗氧化应激通路Nrf2-ARE,可以保护糖尿病肾脏免受氧化应激损害,减轻细胞外基质沉积,保护肾脏功能。更多还原

【Abstract】 Objective: Diabetic nephropathy (DN) is one of the most common complications and the leading cause of mortality and morbidity of diabetes mellitus. The pathologic manifestations of DN include glomerular hypertrophy, basement membrane thickening, mesangial dilation, and glomerularsclerosis and interstitial fibrosis in the terminal stage. The excessive accumulation of extracellular matrix (ECM) is the common pathophysiological foundation. Recent studies showde that the oxidative stress plays an important role in the pathogenesis of DN. The excessive production of reactive oxygen species (ROS) not only stimulate all signaling pathways known to be associated with diabetic microvascular complications including DN, such as PKC pathway, polyol pathway, hexosamine pathway and AGEs formation, but also activate NF-κB and upregulate the transcription of adhesive molecule and inflammatory factors. That is the common mechanism of diabetic microvascular complications including DN. Persistent existence of those abnormal changes will increase the synthesis of mesangial matrix and basement membrane, decrease degradation of them, thus leading to the development and progression of DNOrganism has developed a complex response system to the oxidative stress induced by ROS damage. When exposed to ROS, cells can produce a series of protective proteins to mitigate cellular injury. This coordinated response is controlled by antioxidant responsive element (ARE) in the upstream of protective genes. More recently, nuclear factor erythroid 2-related factor 2 (Nrf2) had been found to be the activator of ARE. The Nrf2-ARE signaling pathway has been proved to be the key regulator of antioxidant and cytoprotective proteins. Protective effect of Nrf2-ARE signal pathway to anti- environment stress, anti-apoptosis, neuro-protection, anti-carcinoma and antii-ageing in vivo and in vitro have been reported, but little is known about the protective role of Nrf2 in DN.Disequilibrium between oxidation and reduction systems is pathophysiological foundation of DN. Some studies indicated that activation of antioxidant protein is effective strategies for the management of DN. The present study try to clarify whether Nrf2-ARE is related to the oxidative stress in DN, and whether diabetic renal injury can be ameliorated by stimulating Nrf2-ARE, in order to provide experimental basis for prevention and trentment of DN.Methods:1 Induction of diabetic model and examination of relative parameters.Uninephrectomized male CD1 mice were randomly divided into two groups: Control group, DM group. The mice of DM group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L citrate buffer (pH 4.5) at a dose of 130mg/kg. The mice of control group only received an injection of the same volume of 0.1mol/L sodium citrate. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L and the glucose in urine was+++～++++after 72 hours of the injection. Six mice of control or DM group were respectively sacrificed at the 4th and 12th weeks after STZ injection. At the end of the study, 24 h urines were collected in metabolic cage for the measurement of urine albumin (Ualb, at the 4th week ) and urine protein (Upro, at the 12th week); parameters of kidney function and lipid peroxidation marker malondialdehyde (MDA) were evaluated in serum samples .The renal cortex were removed and used for MDA content measurement, histopathology examination, immohistochemistry to detected the location and expression of Nrf2, HO-1,γ-GCS in renal cortex, and western blot to detected the expression of Nrf2(total and nucleus)、HO-1、γ-GCS protein .2 Transient transfection of pcDNA3/mNrf2 plasmid into MMC cells cultured in high glucose medium and and examination of relative parameters.Mouse mesangial cells (MMC) were maintained at 37℃in a humidified atmosphere of 5% CO2 in DME-F12 medium containing penicillin/ streptomycin (100 U/ml and 100μg/ml, respectively) and 10% fetal bovine serum (FBS), with a normal D-glucose concentration of 5.5mM. Prior to use, cells at 70～80% confluence were incubated in serum-free medium for 12 hours. Transient transfection of MMC cells was carried out according to the Lipofectamine 2000 manufacturer’s instruction. Six hours after transfection, the medium was replaced by normal DME-F12 medium with 10% FBS for 12 hours. MMC cells were randomly divided into 5 groups: Control group (media containing 5.5mM glucose), Mannitol control group (media containing 5.5mM glucose and 24.5mM mannitol), HG group (media containing 30mM glucose), HG+ pcDNA3 group (media containing 30mM glucose and transfected pcDNA3). HG+pcDNA3/mNrf2 group (media containing 30mM glucose and transfected pcDNA3/Nrf2).Cells were collected at 48 hours after high glucose stimulation. Immunocytochemistry were used to analyze expressed location of Nrf2 andγ-GCS. Western-blot were used to detected the expression of Nrf2(total and nucleus)、HO-1、γ-GCS protein. Reverse transcription polymerse chain reaction (RT-PCR) were used to detected the expression of Nrf2、HO-1、γ-GCS mRNA. 2’7’-Dichloro-fluorescein diacetate (2’7’-DCFH-DA) and MDA were assessed as cellular ROS generation and lipid peroxidation. The contents of TGF-β1 in the supernatants were measured by Enzyme linked immunosorben assay (ELISA). Proliferation activity of mesangial cells were determined by flow cytometry and analyzed by DNA analysis software modeling program.3 Transient transfection of siRNA Nrf2(m) into MMC cells cultured in high glucose medium and examination of relative parameters.siRNA targeting mouse Nrf2 mRNA was used to silence Nrf2 gene expression. Transient transfection of MMC cells was carried out according to the Lipofectamine 2000 manufacturer’s instruction. After optimizing transfection conditions, MMC cells were divided into 5 groups: Control group (media containing 5.5mM glucose), Mannitol control group (media containing 5.5mM glucose and 24.5mM mannitol), HG group (media containing 30mM glucose), HG+ siRNA control group (media containing 30mM glucose and transfected non-specific siRNA). HG+ siRNANrf2(m)group(media containing 30mM glucose and transfected siRNA Nrf2(m)). Cells were collected at 48 hours after high glucose stimulation. Exam methods of Nrf2、HO-1、γ-GCS expression and ROS generation ,lipid peroxidation , cell proliferation , TGF-β1 secretion in different groups were described as former.4 Treatment of diabetic mice with 1%tBHQ food and examination of relative parameters.Uninephrectomized male CD-1 mice were randomly assigned to three groups: Control group(C group); Diabetes mellitus group (DM group); tBHQ intervention group (DM+tBHQ group).The mice of DM group and DM+tBHQ group were induced by single-dose intraperitoneal injection of STZ.Mice of DM+tBHQ group were fed with food containing 1%tBHQ(w/w)three days after model establishment. Specimens were harvested at 4th and 12thweek after the model establishment. The glomeruli were separated by sieving method .Renal weight/body index, quantification of 24 hour urine albumin (of 4th week) and protein(of 12th week), MDA contents in the serum and glomerular homogenate of mice were evaluated as indication of ROS injury. The localization and expressions of Nrf2, HO-1 andγ-GCS proteins in the glomerulus were measured by immunohistochemical assay andwestern blot. Expressions of Nrf2, HO-1 andγ-GCS mRNA in the glomerulus were detected by RT-PCR. ECM deposition in the glomerulus were measured by HE and PAS staining, transmission electron microscopy examination, immunohistochemical assay of FN in the glomerulus.Results:1 The expression of Nrf2、HO-1、γ-GC in the renal tissue and relative to oxidative stress in the kidneys of STZ-induced diabetic mice①Compared with C group, renal weight/body index, 24h urine albumin quantification of 4th week and 24h urine protein quantification of 12th week in DM group were significantly higher.②The mice showed slightly enlarged glomeruli at 4th week , and the number of MMC cells was increased with glomeruli hypertrophy, thickened glomerular basement membrane and expanded mesangium matrix were observed in DM group at 12thweek vs C group by HE and PAS staining .③Immunohistochemistry staining indicated that Nrf2 protein was presented in both nuclear and cytoplasm of renal glomeruli cells; Nrf2 nucleus protein expressions were significantly higher than those in C group of the same time; HO-1 protein was mainly expressed in cytoplasm of renal glomeruli cells and the expression was significantly lower than that in C group at the 4th week but higher at the 12th week;γ-GCS protein presented in cytoplasm of renal glomeruli and tubular cells, and the protein expressions were significantly higher than those in C group of the same time.④MDA contents of the serum and renal cortex in DM group were significantly higher than those in C group at the corresponding time .⑤Western blot showed that: At the 4thweek, Nrf2 total protein expression in DM group was similar to that in C group; HO-1 expression was significantly lower than that in C group;γ-GCS and Nrf2 nucleus protein expressions were significantly higher than those in C group; At the 12th week, Nrf2 total protein expression of DM group was similar to that in C group; HO-1,γ-GCS and Nrf2 nucleus protein expressions were significantly higher than those in C group.2 The expression of Nrf2、HO-1、γ-GCS protein and effects of Nrf2-ARE on ROS generation,proliferation,TGF-β1 secretion of MMC cells cultured in high glucose medium.①MMC cells incubated in high-glucose medium resulted higher ROS production and MDA content in time-dependent manner than that incubated in normal-glucose medium.②Activation of Nrf2 by transfected pcDNA3/Nrf2(m) plasmid induced nuclear translocation of Nrf2 and increased ARE-linked gene HO-1、γ-GCS protein and mRNA expression after 48h-incubation in high-glucose media.This induction was markedly attenuated by pretreatment with siRNA Nrf2(m)③Immunochemistry showed increased Nrf2 andγ-GCS expression in cytoplasm meanwhile increased Nrf2 nuclear expression in transfected pcDNA3-Nrf2 plasmid group vs high-glucose stimulating group. This induction was markedly attenuated by pretreatment with siRNA Nrf2(m)④Hyperglycemia increased the formation of ROS, MDA, proliferation ,TGF-β1 secretion of MMC were markedly prevented by pcDNA3-Nrf2 plasmid transfection while this prevention were attenuated by knockdown of Nrf2 expression through siRNA Nrf2(m) transfection.3 tBHQ attenuates glomerular ROS injury of diabetic mice via activating Nrf2-ARE pathway①Renal weight/body index, 24h urine albumin quantification of the 4thweek and 24h urine protein quantification of the 12thweek in DM group were significantly higher than those in group C; and these parameters were markedly improved in DM+tBHQ group compared to those in DM group.②MDA contents of the serum and renal glomeruli homogenate in DM group were significantly higher than those in C group at the corresponding time, and these parameters decreased in the tBHQ intervention group.③Compared with group DM at corresponding time, expressions of glomerular total protein Nrf2, HO-1,γ-GCS, nuclear protein Nrf2 and Nrf2, HO-1,γ-GCS mRNA in the tBHQ intervention group increased significantly;④Transmission electron microscope at 4th week, HE and PAS staining at 12th week and semi-quantitative FN immunohistochemical assay showed that tBHQ intervention could reduce the ECM deposition in glomerulus of the diabetic mice.Conclusion: Our study showed that the renal tissues of STZ induced diabetic model presented ROS injury and elevation of Nrf2 levels. Nrf2-ARE signaling pathway and the downstream antioxidant protein HO-1 andγ-GCS play an important role in antioxidant mechanism of DN. tBHQ is able to ameliorate renal injury induced by ROS and reduce the ECM deposition in glomerulus of diabetic kidney, The mechanism may be associated with activating Nrf2-ARE signaling pathway and initiating the downstream genes HO-1 andγ-GCS transcription and expression. In the mesangial cells of mice, high glucose induced ROS production, and overexpression of Nrf2 by transient transfection of pcDNA3/Nrf2 plasmid activated expression of Nrf2 and its downstream antioxidant genes HO-1 andγ-GCS, inhibited ROS production, lipid peroxidation , cell proliferation and TGF-β1 secretion whereas knockdown of Nrf2 by siRNA displayed the adverse effects. The above results suggested that activation of Nrf2 could be an effective method to prevent and slow down the progression of DN in vivo and in vitro.更多还原