【作者】 张涛；

【导师】 李英；

【作者基本信息】 河北医科大学 ， 内科学， 2014， 博士

【摘要】 目的：糖尿病肾病（diabetic nephropathy，DN）由于其高发率、高致残率以及高死亡率，多年来一直是肾病领域研究的热点。研究发现，足细胞在保持肾小球滤过屏障的完整性及预防蛋白尿的产生中具有重要作用，足细胞损伤可影响DN的发生、发展和预后。足细胞是一种高度分化的终末细胞，再生能力差，其多级足突结构功能的正常维持需要大量的能量消耗，对氧化应激反应相对敏感。正是这些特性导致了足细胞的易损。有研究报道，足细胞是DN最早受损细胞，在DN早期即可出现凋亡、脱落，加重蛋白尿和肾小球硬化的进程。DN的发病机制非常复杂，既往大量研究表明，高糖可通过4条经典生化途径诱导肾脏损害，包括糖基化终末产物（advanced glycation endproducts, AGE）增加，蛋白激酶C（PKC）激活，多元醇通路与已糖胺途径的激活。但其之间的相互关系尚未明确。2001年，Brownlee教授首次提出了“统一机制理论”：认为高糖诱导细胞线粒体呼吸链产生过量活性氧（reactive oxygen species，ROS）是糖尿病并发症发生的启动因素。ROS的过度合成可激活上述途径，进一步促进ROS的积聚，通过直接过氧化生物大分子和激活相关信号通路等方式，导致细胞的损伤和凋亡，造成糖尿病多器官并发症的发生。通过多年的实验研究，这一统一理论已被多数学者认可。由线粒体诱导的ROS可能在DN发病中发挥着起始和关键性作用。线粒体内产生的ROS增多可将自身作为首要靶目标导致线粒体损伤，包括对呼吸链及内外膜及基质蛋白的影响，导致ROS产生更多，ATP合成减少；进而线粒体膜通透性改变，细胞色素C、AIF和Smac-DIABLO等一系列促凋亡因子外溢，启动线粒体凋亡途径诱导细胞凋亡的发生。由此可见，线粒体氧化损伤和诱发细胞凋亡可能是DN的重要致病机制，也有望成为DN治疗的新靶点。线粒体生物合成(Mitochondrial biogenesis)是维护和修复线粒体结构的生理活动，是介导核基因对线粒体基因及蛋白表达调控的重要桥梁。研究发现，PGC-1α是线粒体生物合成的中枢调节因子。其可促进线粒体生物合成，增强不同组织细胞有氧呼吸功能。PGC-1α最初是作为核受体过氧化物酶体增殖物激活受体γ（PPARγ）的转录辅激活因子被发现的，此后一系列的研究表明它在能量平衡、线粒体氧化代谢、热量控制及糖、脂代谢中发挥重要作用。作为辅激活因子通过激活和并上调转录因子NRFl和TFAM的表达，对线粒体DNA的复制、转录和蛋白合成发挥重要的调控作用。近年来，随着研究的深入，在多种糖尿病并发症中发现了PGC-1α表达的下调，提示由其调控线粒体生物合成受损导致的线粒体功能障碍可能是糖尿病并发症发病的重要机制之一。更进一步的研究发现，PGC-1α可被NAD+依赖性去乙酰化酶沉默信息调节因子2（sir2）相关酶1（SIRT1）通过去乙酰化作用而激活。白藜芦醇（resveratrol）是目前发现的SIRT1最强的激活剂。具有抗炎、抗氧化、抗癌、调节糖脂代谢等重要生物学功效。多数研究认为，其药理作用的发挥有赖于SIRT1的激活。近来，白藜芦醇在肾脏疾病治疗中的作用也越来越受到学者们的关注。在DN动物模型中发现，白藜芦醇具有降低蛋白尿；减少细胞外基质沉积，抑制氧化应激反应和细胞凋亡等众多肾脏保护作用。在心肌和骨骼肌的研究中，有学者指出，白藜芦醇可通过激活SIRT1进一步调节其下游基因PGC-1α和FOXO来改善线粒体功能。糖尿病肾病时，白藜芦醇的肾脏保护作用是否也与其激活SIRT1/PGC-1α通路改善线粒体功能，进而减少氧化应激和凋亡有关尚需进一步研究证实。本研究通过建立1型糖尿病小鼠模型和高糖刺激体外培养足细胞，同时应用白藜芦醇以及PGC-1α siRNA转染技术干预，深入探讨线粒体氧化损伤和凋亡在糖尿病足细胞病变中的可能作用机制，为进一步阐明DN的发病机制及其早期防治提供理论依据。方法：1SIRT1/PGC-1α在糖尿病小鼠肾组织的表达及白藜芦醇对其的干预研究将健康雄性CD-1小鼠（4-6周）随机分为3组：正常对照组（normalcontrol group NC组）、糖尿病组（diabetic group DM组）、糖尿病+白藜芦醇干预组（diabetic+Resveratrol group DR组）。应用单次腹腔注射链脲佐菌素（streptozotocin, STZ，140mg/kg）法制备糖尿病小鼠模型，72小时后检测尾尖血糖≥16.7mmol/L，尿糖+++~++++确定为糖尿病模型制备成功。DR组给予白藜芦醇30mg·kg-1·d-1灌胃，每日一次，NC组与DM组仅给予等量溶剂(羧甲基纤维素)灌胃。分别于成模后4、8、12周留取各组小鼠血液及24小时尿液标本，用于血糖(blood glucose, BG)、血尿素氮(Blood urea nitrogen,BUN)、血清总胆固醇(total cholesterol, TC)和尿蛋白定量(urinary protein, UP)的检测。切取部分肾皮质组织于4%中性甲醛固定制备石蜡切片用于PAS、HE病理染色，TUNEL染色以及SIRT1、PGC-1α、NRF1和TFAM免疫组织化学染色；部分肾皮质组织于4%戊二醛固定后用于电镜观察；部分肾皮质用于提取总蛋白行Western blot检测SIRT1、PGC-1α、NRF1、TFAM和Nephrin，cleaved caspase-3的蛋白表达，比色法检测肾皮质MDA含量和Mn-SOD活力变化。2高糖对足细胞SIRT1/PGC-1α表达及线粒体氧化损伤与凋亡的影响条件性永生化小鼠足细胞在33℃，含有γ-IFN（10U/ml）及10%胎牛血清的RPMI1640培养液条件下培养传代后，置于37℃，不含γ-IFN的RPMI1640培养液条件下培养10-14天，待足细胞分化成熟后开始后续试验。将实验细胞随机分为3组：正常糖组（5.5mmol/L glucose, NG）、高渗对照组（24.5mmol/L mannitol, MG）和高糖组（30mmol/L glucose，HG）。同步化后分组干预，刺激0、12、24、48、72小时后收集细胞，免疫细胞化学法检测SIRT1、PGC-1α、NRF1、TFAM蛋白表达；Real-timePCR检测SIRT1、PGC-1α、NRF1、TFAM mRNA表达；提取细胞总蛋白行Western blot检测SIRT1、PGC-1α、NRF1、TFAM和Nephrin，cleavedcaspase-3的蛋白表达；提取去线粒体胞浆蛋白行western blot检测Cyto C和DIABLO的表达；JC-1染色检测活细胞线粒体膜电位的变化；DCHF-DA染色检测细胞内总ROS含量；MitoSOX染色检测线粒体ROS的含量；比色法检测线粒体呼吸链复合物Ⅰ、Ⅲ的活性变化。AnnexinⅤ/PI染色检测足细胞凋亡率。3白藜芦醇及PGC-1α siRNA对高糖诱导足细胞线粒体氧化损伤与凋亡的影响将分化成熟的足细胞随机分为6组：正常糖组（5.5mmol/L glucose，NG组），高糖组（glucose30mmol/L，HG组），高糖+白藜芦醇干预组（glucose30mmol/L+resveratrol10μmol/L，HG+Res组），高糖+EX527干预组（glucose30mmol/L+EX52710μmol/L，HG+EX527组），高糖+PGC1siRNA干预组(glucose30mmol/L+PGC1siRNA，HG+PGC1siRNA组)和高糖+PGC1siRNA+白藜芦醇干预组(glucose30mmol/L+PGC1siRNA+resveratrol10μmol/L，HG+PGC1siRNA+Res组)，各组同步化后干预48h收集细胞，Real-time PCR检测SIRT1、PGC-1α、NRF1、TFAMmRNA表达；提取细胞总蛋白行Western blot检测SIRT1、PGC-1α、NRF1、TFAM和Nephrin，cleaved caspase-3的蛋白表达；提取去线粒体胞浆蛋白行western blot检测Cyto C和DIABLO的表达；JC-1染色检测活细胞线粒体膜电位的变化；DCHF-DA染色检测细胞内总ROS含量；MitoSOX染色检测线粒体ROS的含量；比色法检测线粒体呼吸链复合物Ⅰ、Ⅲ的活性变化。AnnexinⅤ/PI染色检测足细胞凋亡率。结果：1SIRT1/PGC-1α在糖尿病小鼠肾组织的表达及白藜芦醇对其的干预研究①生化结果显示：4周时，DM组小鼠尿蛋白较NC组升高，且随病程延长呈上升趋势；8周时，DM组BUN和TC水平升高，12周时升高更加显著。DR组小鼠蛋白尿水平较同时期DM组降低；12周时，DR组TC和BUN水平均较DM组改善。②光镜下观察：DM组小鼠4周时出现肾小球直径增大，系膜区基质增多；8周时，肾小管上皮细胞出现空泡变性；12周时，肾小球内系膜区增宽更为显著，并可见肾间质水肿，部分肾小管扩张，肾小管上皮细胞脱落再生。③电镜下观察，12周时DM组小鼠肾小球基底膜明显不均匀增厚，足突广泛融合，裂孔隔膜消失，足细胞线粒体数量减少，并出现肿胀、空泡变、内部膜及嵴结构紊乱消失等病理改变；DR组上述病变均有所改善。④比色法检测结果显示，DM组小鼠肾皮质MDA含量较NC组明显升高，且随病程延长呈上升趋势，而Mn-SOD活性则较同时期NC组降低，12周时，降低最为明显。DR组较同时期DM组有所改善，差异有统计学意义。⑤TUNEL染色结果显示：8周时，DM组小鼠肾小球及肾小管间质中均可见凋亡细胞，12周时，凋亡细胞数量较NC组明显增多。DR组凋亡现象较DM组有所减轻。⑥免疫组织化学结果显示，SIRT1、PGC-1α、NRF1和TFAM在NC组肾小球固有细胞及肾小管上皮细胞中均可见阳性表达。其中SIRT1、NRF1主要表达于胞核；PGC-1α在胞核和胞浆中均有表达，以胞浆表达更为明显；TFAM则主要表达于胞浆中。8周时，DM组上述指标的阳性表达较NC组减少，12周时，减少更为显著。⑦Western blot结果显示：DM组肾皮质SIRT1蛋白表达在4周时已较正常组减低，随病程延长，下降更为显著。而PGC-1α、NRF1和TFAM的表达在8周时表达下调，12周时下调更加明显。DR组上述4种蛋白表达均较DM组增多，但仍低于同时期NC组。12周时，DM组小鼠肾皮质Nephrin表达较NC组明显减低，cleavedcaspase3表达较NC组明显升高，DR组上述两指标均较DM组改善。2高糖对足细胞SIRT1/PGC-1α表达及线粒体氧化损伤与凋亡的影响①免疫荧光化学结果显示，SIRT1、PGC-1α、NRF1、TFAM在NG组均可见阳性表达，SIRT1、NRF1主要表达于细胞核；PGC-1α胞浆、胞核均有阳性表达，以胞浆表达更为明显；TFAM主要表达于胞浆中。HG组上述蛋白均较NG组阳性表达减少。②Western blot显示，高糖干预12h，SIRT1表达较0h降低，随干预时间延长，表达呈递减趋势；PGC-1α表达在12h略有增加，NRF1和TFAM则较0h无明显变化，三者均从高糖干预24h开始，呈时间依赖性递减，72h降低最为显著。③r eal time PCR结果显示，高糖刺激呈时间依赖性引起SIRT1mRNA表达降低，以48h减低最显著。PGC-1α、NRF1、TFAM mRNA减低发生在高糖刺激24h，此后随干预时间延长表达逐渐下调。④W estern blot结果显示，高糖干预下，足细胞Nephrin蛋白呈时间依赖性下调，cleaved caspase3则呈逐渐上调趋势。去除线粒体的胞质蛋白中Cyto C和Diablo蛋白也随高糖干预时间延长表达升高。⑤流式细胞术检测结果显示，高糖干预12h，细胞内总ROS含量较0h升高，且随时间延长呈递增趋势。⑥荧光显微镜下显示，高糖干预12h时，足细胞线粒体中ROS含量较未干预时明显增多，且随干预时间延长荧光强度逐渐增强。⑦比色法结果显示，高糖干预24h，足细胞线粒体呼吸链复合物Ⅰ、Ⅲ活性下降，48h、72h降低更为明显，⑧足细胞线粒体膜电位随高糖干预时间延长呈降低趋势，共聚焦显微镜下可见由红色荧光向绿色荧光的转变过程。⑨流式细胞仪检测显示，足细胞凋亡率随高糖干预时间延长逐渐上调。3白藜芦醇及PGC-1α siRNA对高糖诱导足细胞线粒体氧化损伤与凋亡的影响①W estern blot与Real time PCR检测结果显示，干预48h，HG组SIRT1、PGC-1α、NRF1、TFAM蛋白及mRNA表达量均较NG组明显降低，HG+Res组上述4种蛋白及mRNA表达较HG组升高，而HG+EX527组则均较HG组进一步降低，差异有统计学意义。②Western blot检测结果显示，干预48h，HG组Nephrin蛋白表达量较NG组降低，HG+EX527组降低更加显著；HG+Res组其表达量较HG组升高。HG组cleaved caspase3的表达较NG组升高，HG+EX527组上升更为显著；HG+Res组其表达量较HG组降低。去线粒体胞质蛋白中Cyto C、DIABLO的表达在HG组也较NG组明显升高，HG+Res组其表达量较HG组有所降低，HG+EX527组升高较HG组更为明显。③HG+PGC1siRNA组SIRT1表达较HG组无明显变化，HG+PGC1siRNA+Res组SIRT1表达较HG组升高，与HG+Res组表达无明显差异。HG+PGC1siRNA组PGC-1α、NRF1和TFAM表达均较HG组明显降低，HG+PGC1siRNA+Res组上述3种蛋白较HG+Res组表达降低，同HG+PGC1siRNA组无明显差异。④HG+PGC siRNA组Nephrin表达较HG组进一步减低，HG+PGC siRNA+Res组表达低于HG+Res组。HG+PGC1siRNA组cleaved caspase3与HG组相比表达升高，HG+PGC1siRNA+Res组表达量高于HG+Res组。⑤流式细胞术检测结果显示，干预48h，HG+Res组线粒体内ROS合成量较HG组明显降低，而HG+EX527组较HG组进一步升高，差异均有统计学意义。⑥H G+Res组线粒体呼吸链复合物Ⅰ、Ⅲ活性及线粒体膜电位均较HG组有所上升，而HG+EX527组较HG组减低更加显著。⑦H G组足细胞内总ROS含量较NG组明显升高，HG+EX527组升高更加明显，HG+Res组足细胞内总ROS含量较HG组明显降低。HG+PGC1siRNA组则较HG组含量更高，HG+PGC1siRNA+Res组较HG+PGC1siRNA组有所降低，但明显高于HG+Res组。⑧HG组足细胞凋亡率明显高于NG组，HG+EX527组较HG组升高更加显著，HG+Res组与HG组比较，凋亡率明显降低。HG+PGC1siRNA组凋亡率高于HG组，HG+PGC1siRNA+Res组较HG+PGC1siRNA组有所降低，但明显高于HG+Res组。结论：1糖尿病小鼠肾组织、高糖培养的小鼠足细胞均出现氧化应激反应增强，细胞凋亡率升高；并伴有肾组织与足细胞内SIRT1、PGC-1α的表达下调。2糖尿病小鼠肾小球足细胞出现线粒体形态异常，裂孔隔膜消失，足突融合，凋亡等病理改变，高糖诱导足细胞内线粒体ROS合成增多，呼吸链复合物活性下降，线粒体膜电位降低，线粒体凋亡途径被激活。提示线粒体功能障碍参与了糖尿病足细胞的氧化损伤与凋亡。3白藜芦醇可上调糖尿病小鼠肾组织SIRT1、PGC-1α表达，改善糖尿病小鼠足细胞内线粒体形态结构异常及足突融合，上调裂孔隔膜蛋白nephrin的表达，减少足细胞凋亡的发生。其减少尿蛋白的机制可能与足细胞保护作用有关。4白藜芦醇可上调高糖环境中足细胞SIRT1、PGC-1α及其下游基因的表达；减少高糖环境下足细胞线粒体ROS合成增多，改善呼吸链复合物活性，提高线粒体膜电位，抑制线粒体凋亡通路的激活。5白藜芦醇可能通过SIRT1/PGC-1α介导的线粒体保护机制减轻了糖尿病小鼠足细胞的氧化应激与凋亡。更多还原

【Abstract】 Objectives:Diabetic nephropathy (DN) has been the focus in researchesof kidney disease for years due to its high morbidity,high disability and highmortality.Studies found that, podocyte plays an important role in maintainingthe integrity of the glomerular filtration barrier and preventing proteinuria.Thepodocyte damage affect the development and prognosis of diabeticnephropathy. Podocyte is a kind of highly specialized cell,whose regeneratedability is poor. Podocyte need large amount of energy consumption tomaintain its normal function of multi-level structure of processes,which issensitive to oxidative stress. All of these characteristics led to the vulnerabilityof podocyte. Some researches reported that podocyte was the earliest damagedcell in diabetic nephropathy, which appeared apoptosis and fall off in the earlystage of DN, then aggravated the process of proteinuria and glomerularsclerosis.The pathogenesis of diabetic nephropathy is very complicated, Previousstudies have shown that, four major molecular mechanisms were implicated inhyperglycemia-induced tissue damage：activation of protein kinase C(PKC)isoforms via de novo synthesis of the lipid second messengerdiaeylglycerol(DAG)， increased hexosamine pathway flux， increasedadvanced glycation endproduct(AGE)formation, and increased polyol pathwayflux． However, there was no apparent common element linking thesemechanisms. In2002， Professor Michael Brownlee pointed out thathyperglycemia-induced mitochondrial overproduction of reactive oxygenspecies(ROS) is the primary initial mechaniam of diatbeticcomplications．ROS overexpression can activate the above four biochemicalpathways, and these pathways can also cause the accumulation of ROS. Through direct peroxidation and activation of related signaling pathways,ROS can lead to cellular damage and apoptosis, then cause multiple organcomplications of diabetes. Through experimental researches for many years,this unified theory has been accepted by most scholars. Mitochondrial ROSoverexpression may play a beginning and key role in the pathogenesis ofdiabetic nephropathy.The overexpression of mitochondrial ROS can target itself and lead tomitochondrial damage, including the respiratory chains, inner and outermembranes and matrix proteins. That lead to produce more ROS, while ATPsynthesis is reduced, and then the mitochondrial membrane permeabilitychanges. A series of pro-apoptotic factors such as cytochrome C, AIF andsmac-diablo outflowing into cytoplasm induces the activation ofmitochondrial apoptosis pathway. Therefore, mitochondrial oxidative damageand cell apoptosis may be the important pathogenic mechanism of diabetes,which is also expected to become the new target for treatment of diabeticnephropathy.Mitochondrial biogenesis is the physiological activities for maintainingand repairing the Mitochondrial structure. It is the important bridge to mediatregulation of nuclear genes to Mitochondrial genes. The study found that,PGC-1α had been identified as a master regulator of mitochondrial biogenesis.Its main biological characteristics was to stimulate mitochondrial biosynthesisand enhance the capacity of oxidative metabolism in different organizations.PGC-1α was found initially as a transcriptional coactivator of nuclearhormone receptor Peroxisome Proliferator Activated Receptor gamma (PPARγ) in mice. A series of studies have shown that it plays an important role inenergy balance, mitochondrial oxidative metabolism, glucose and lipidmetabolism. PGC-lα stimulates the expression of nuclear respiratory factor(NRF) and mitochondrial transcriptional factor A (TFAM), and then activatesexpression of nuclear and mitochondrial genes encoding mitochondrialproteins．In recent years, lots of researches have found the down-regulation ofPGC-1α expression in a variety of diabetic complications, with close relationship with mitochondrial damage, which may be one of the importantmechanisms of diabetic complications. Further studies found that PGC-1α wasactivated by deacetylation of NAD+dependented deacetylatise silentinformation factor2(sir2) related enzyme (SIRT1). Resveratrol is thestrongest SIRT1activator found by now, which has many important biologicalefficacies such as anti-inflammation, antioxidant, anticancer, regulatingglucose and lipid metabolism. Most studies suggested that thepharmacological effects of resveratrol were depended on the activation ofSIRT1. Recently, the role of resveratrol in the treatment of kidney disease getsmore and more scholars’ attention. Researches about animal models of diabeticnephropathy have showed that resveratrol has renal protective effects, such asreducing proteinuria and extracellular matrix deposition, inhibiting oxidativestress and apoptosis. In the studies about myocardial and skeletal muscles,resveratrol was reported to promote mitochondrial biogenesis and protect cellsfrom metabolic disease through SIRT1-dependented deacetylation of PGC-1αand FOXO. In diabetic nephropathy, whether resveratrol has renal protectiveeffect by the improvement of mitochondrial function through its activation ofSIRT1/PGC-1α, and then reducing oxidative stress and apoptosis still needmore studies to confirm.The present research will discuss possible mechanisms of mitochondrialoxidative damage and cell apoptosis in diabetic podocyte lesions throughestablishing mouse models of type1diabetes and stimulating podocytes withhigh glucose, and interventing with resveratrol and PGC-1α siRNAtransfection, in order to provide theoretical basis for early prevention ofdiabetic nephropathy.Methods：1The expression of SIRT1/PGC-1α in the renal tissues of STZ-induceddiabetic mice and the effects of resveratrolMale CD-1mice were randomly divided into three groups: normal controlgroup(NC), diabetic group (DM) and diabetic+Resveratrol group(DR).Diabetes were induced by intraperitoneal injection of streptozotocin (STZ, 150mg/kg). Individual animals with blood glucose concentrations more than16.7mM and the glucose in urine positive for3consecutive days afterinjection were confirmed as diabetes. The control mice were received0.1Mcitrate buffer solution only. After the diabetic model was affirmed to besuccessful, the mice of diabetic+resveratrol group were administered dailywith resveratrol (30mg/kg) by gavage. The mice of control group and diabeticgroup were only administered daily with the same volume of solvent bygavage. At4,8and12weeks after treatment, mice from every group wererespectively sacrificed. Blood and24h urine samples were collected forbiochemical indicators such as blood glucose, serum creatinine, totalcholesterol and urinary protein. Partial renal tissures were fixed in4%neutralformalin for histochemical, TUNEL and immunohistochemical staining.Partial renal cortices were fixed in4%glutaraldehyde for electron microscopicobservation. Protein extracted from partial renal cortices were used to detectedthe expression of SIRT1, PGC-1α, NRF1, TFAM, nephrin and cleave caspase3by Western blot.The other Protein was used to evaluated the MDA contentand Mn-SOD activity in renal cortex by colorimetry.2The effects of high glucose on SIRT1/PGC-1αexpression, mitochondrial oxidative damage and apoptosis in podocytesTo induce proliferation, the conditionally immortalized mouse podocyteswere cultured at33℃with γ-INF under growth permissive conditions, andthen cells were cultured at37℃without γ-INF under growth restrictiveconditions for10-14days to induce quiescence and the differentiatedphenotype. The podocytes were divided into3groups: Normal glucose group(NG), mannitol group (MG), high glucose group (HG). The three groups werecultured for12,24,48and72h respectively, and then podocytes wereharvested.The expression of SIRT1, PGC1α, NRF1and TFAM were detectedby immunocytochemistry and Western blot. The mRNA levels of SIRT1,PGC1α, NRF1and TFAM were evaluated by Real-time PCR. The expressionof SIRT1, PGC-1α, NRF1, TFAM, Nephrin, cleaved caspase-3, Cyto C andDIABLO was respectively evaluated by Western blot. The cell ROS production were detected by DCHF-DA staining.The mitochondrial ROSproduction was detected by MitoSOX staining. The mitochondrial membranepotential was measured by JC-1staining. Complex Ⅰ and Ⅲ enzymeactivities in mitochondria were measured by colorimetry. Podocyte apoptosiswas detected by AnnexinⅤ/PI staining.3The effects of resveratrol and PGC-1α siRNA on mitochondrialoxidative damage and apoptosis in podocytes stimulated by high glucose.Podocytes were randomly divided into six groups: normal glucose group(5.5mmol/L glucose, NG), high glucose group (30mmol/L glucose, HG),high glucose+resveratrol group(30mmol/L glucose+resveratrol10μmol/L，HG+Res), high glucose+EX527group(30mmol/L glucose+EX52710μmol/L,HG+EX527), high glucose+PGC1-siRNA group (30mmol/L glucose+PGC1siRNA, HG+PGC1siRNA) and high glucose+PGC1.siRNA+resveratrolgroup (30mmol/L glucose+PGC1siRNA+resveratrol10μmol/L,HG+PGC1siRNA+Res). The six groups were cultured for48hours, and thenpodocytes were harvested. The expression of SIRT1, PGC1α, NRF1andTFAM were detected by immunocytochemistry and Western blot. The mRNAlevels of them were evaluated by Real-time PCR. The expression of SIRT1,PGC-1α, NRF1, TFAM, Nephrin, cleaved caspase3, Cyto C and DIABLOwas respectively evaluated by Western blot. The cell ROS production weredetected by DCHF-DA staining. The mitochondrial ROS production wasdetected by MitoSOX staining. The mitochondrial membrane potential wasmeasured by JC-1staining. ComplexⅠ and Ⅲ enzyme activities inmitochondria were measured by colorimetry.Podocyte apoptosis was detectedby AnnexinⅤ/PI staining.Results：1The expression of SIRT1/PGC-1α in the renal tissues of STZ-induceddiabetic mice and the effects of resveratrol①Compared with control group,24h urine protein upregulated in atime-dependent manner in diabetic group. The BUN and TC levels wereincreased from week8in diabetic group than control group. Compared with diabetic group,24h urine protein and the BUN,TC levels ofdiabetic+Resveratrol group were significantly decreased.②Diabetic miceshowed significant glomeruli hypertrophy and expansion of the mesangialmatrix in glomeruli by light microscope. Uneven increase in thickness ofglomerular basement membrane, the fusion of podocyte foot processes,andthe decreased number and Morphological abnormalities of mitochondria inpodocytes of diabetic rats were observed by transmission electron microscopy.These lesions were improved in diabetic+Resveratrol group.③Colorimetrytest results showed that the MDA content in renal cortex of DM groupincreased than NC group, and upregulated in a time-dependent manner. TheMnSOD activity were reduced than NC group. Compared with DM group,The two indicators in DR group were improved.④Apoptotic cells byTUNEL were observed in renal tissues of the diabetic kidney. Apoptotic ratewas significantly lower in the diabetic+resveratrol group than that in diabeticgroup.⑤By immunohistochemical staining, the protein expression of SIRT1,PGC-1α, NRF1and TFAM were reduced in glomeruli and tubule cells ofdiabetic group than that in control group. Compared with control group,Western blot indicated that the protein expression of SIRT1in diabetic groupbegan to reduced at week4, and then down-regulated in a time-dependentmanner. The expression of PGC-1α, NRF1and TFAM were reduced fromweek8, while resveratrol increased their protein expression in diabetic mice.The expression of nephrin reduced,while the expression of cleaved caspase3were increased in diabetic group at week12, The two indicators in DR groupwere improved.2The effects of high glucose on SIRT1/PGC-1αexpression, mitochondrial oxidative damage and apoptosis in podocytes①Immunocytochemical staining showed that the expression of SIRT1,PGC-1α, NRF1and TFAM reduced in podocytes induced by high glucose.②By Western blot, the protein expression of SIRT1began to reduce after thestimulation of HG for12h. PGC-1α expression at12h increased slightly than0h, there was no difference between the expression of NRF1and TFAM at 12h with that of0h. They all reduced at24h, and down-regulated intime-dependent manner.③The effects of HG on mRNA levels of SIRT1wererevealed at12h and peaked at48h. HG also reduced PGC-1α, NRF1andTFAM mRNA expression from24h.④Western blot result showed that, theexpression of nephrin was down-regulated with HG stimulated intime-dependent manner.whereas cleaved caspase3, Cyto C and Diablo wereincreased at the same time.⑤Compared with normal glucose group, the ROScontent in podocyte treated with HG for12h was increased, and thenup-regulated over time.⑥The increase of mitochondrial ROS production inpodocyte treated with HG were observed by fluorescence microscope.⑦ComplexⅠ and Ⅲ enzyme activities in mitochondria were reduced by HGtreatment for24h, the reduce was more apparent at48h and72h.⑧Themitochondrial membrane potential was down-regulated in HG groupcompared with NG group.⑨Apoptosis rate of podocyte exposed to HG wasincreased gradually than that of NG group.3The effects of resveratrol and PGC-1α siRNA on mitochondriaoxidative damage and apoptosis of podocytes stimulated by high glucose.①Western blot and Real time PCR resulte showed that, the mRNA andprotein expression of SIRT1, PGC-1α, NRF1and TFAM reduced in HG groupthan those of NG group.They were up-regulated in HG+Res group,while theywere lower in HG+EX527group than HG group.The protein expression ofnephrin in HG group was reduced than NG group.Nephrin expression inHG+EX527group was down-regulated more remarkably than that of HGgroup, while that in HG+Res group was up-regulated than HG group. Theprotein expression of cleaved Caspase3，Cyto C and Diablo in HG group wereall increased than those of NG group.Those in HG+EX527increased morethan HG group，while those in HG+Res group were reduced than HG group.③There was no difference in SIRT1expression of HG+PGC1siRNAgroup and that of HG group.SIRT1expression in HG+PGC1siRNA+Resgroup was increased than HG group, but there was no obvious changes withthat of HG+Res group. The expression of PGC-1α, NRF1and TFAM were reduced than those of HG group, those in HG+PGC1siRNA+Res group werereduced than that of HG+Res group.④The expression of Nephrin inHG+PGC1siRNA group was more lower than that of HG group.That ofHG+PGC1siRNA+Res group was also lower than that of HG+Res group. Theexpression of cleaved caspase3in HG+PGC1siRNA group was higher thanthat of HG group.That of HG+PGC1siRNA+Res group was up-regulated thanthat of HG+Res group.⑤flow cytometry test results showed that, comparedwith HG group, the mitochondrial ROS production in HG+Res group wasdecreased significantly, while that of HG+EX527group was increased.⑥theComplexⅠ and Ⅲ enzyme activities and the mitochondrial membranepotential in HG+Res group were increased,whereas that of HG+EX527groupwere decreased.⑦The total ROS content and apoptosis rate of podocyte inHG+Res group were reduced than that of HG group. Those of HG+PGC1siRNA group were more higher than those of HG group. Those of HG+PGC1siRNA+Res group were higher than HG+Res group.Conclusions：1The expression of SIRT1, PGC-1α in renel tissues from diabetic miceor in the mouse podocytes stimulated by high glucose were all reduced,accompanied with enhanced oxidative stress and increased cell apoptosis.2Abnormal mitochondria morphology, fusion of podocyte foot andapoptosis in the glomerular podocytes were detected in diabetic mice.Mitochondrial ROS synthesis was increased, mitochondrial respiratoryfunction was decreased, mitochondrial apoptotic pathways was activated inpodocyte treated with high glucose, which suggested that mitochondrialdysfunction might participate in the oxidative damage and apoptosis ofpodocyte in diabetic nephropathy.3Resveratrol increased the expression of SIRT1,PGC-1α and nephrin,and improved mitochondrial morphology abnormalities and foot processfusion in podocyte, and reduced cell apoptosis in renal cortex of diabetic mice.4Resveratrol inceased the expression of SIRT1and PGC-1α,andreduced mitochondrial ROS synthesis, improved mitochondrial respiratory function and inhibited the activation of the mitochondrial apoptosis pathwayin podocytes stimulated by high glucose.5Resveratrol could relieve the podocyte oxidative damage and apoptosisin diabetic mice maybe through the mitochondrial protective mechanismsmediated by the activation of SIRT1/PGC-1α pathway.更多还原