根皮苷对db/db小鼠视网膜保护机制的研究
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摘要
第一部分根皮苷对db/db小鼠视网膜保护作用的研究
研究背景
糖尿病(diabetes mellitus, DM)是一种由于胰岛素抵抗伴有相对胰岛素不足或胰岛素分泌的缺陷而导致的以慢性血葡萄糖水平增高为特征的代谢性疾病。近年来,随着生活水平的提高、生活方式的改变以及人口的老龄化,DM的患病率在全球范围内呈现逐步增高的趋势,业已成为严重威胁人类生命健康与生活质量的世界性公共卫生问题。
DM不仅仅表现为持续的血糖代谢紊乱,更重要的是其可以导致机体大血管与微血管的严重损害。DM视网膜病变是DM微血管病变最常见的并发症之一,也是最为严重的DM眼部并发症。几乎所有的1型DM与超过半数的2型DM患者最终均会发展并导致DM视网膜病变。在发达的工业化国家,DM的视网膜病变是成人中获得性视力损害与失明的最重要的病因。与非DM人群比较,DM患者致盲的危险性升高25倍。由于DM流行趋势的影响,近年来与DM伴行的DM视网膜病变同样严重并备受重视。迄今为止,针对DM视网膜病变的防治手段主要集中于代谢危险因素的控制(如)严格控制血糖、血压等)以及眼的局部治疗(如药物、激光与手术治疗等)。然而,上述治疗方法均受限于局部并发症与治疗效果的困扰,并不能完全阻断视网膜病变的发展。因此,在临床实践中迫切需要寻找防治DM视网膜病变的新型药物与途径,旨在进一步完善与充实DM视网膜病变的治疗策略。
根皮苷(phlorizn,PHL)是根皮素(phloretin)的2‘-β-D-葡萄糖苷,属于植物黄酮类中的二氢查尔酮苷。PHL作为苹果多酚中的重要组分,主要有在于苹果属的多种植物中,在植物的根、茎、叶与果实中均有分布。大量研究结果表明,PHL具有多种生物活性与药理作用,如抗炎,抗肿瘤,抗氧化,以及降低血糖与改善认知等。近年来,PHL已经作为新型的候选药物应用于DM血管并发症的基础研究中。动物实验发现,PHL以显著改善InsAkitaDM小鼠的心室肥厚与心室重塑。PHL还可以明显抑制db/db小鼠的主动脉病变与肝损害。还有证据表明,在STD诱发的DM大鼠中,PHL可以显著减少其蛋白尿,改善其肾小球的高滤过状态。PHL的衍生物T-1095能够明显降低db/db小鼠的蛋白尿,提示PHL具有DM肾脏病变的保护作用。但是,PHL是否对于db/db小鼠视网膜病变具有保护作用,目前国内外未见报道。本研究应用目前较为普遍认可的db/db小鼠作为2型DM的动物模型,采用脱氧核桃核苷酸末端转移酶介导的的dUTP缺口末端标记法(TUNEL)检测db/db上鼠视网膜中审经元药细胞与血管内皮细胞与血管内皮细胞的凋亡特征,并检测视网膜神经胶质细胞胶质纤维酸性蛋白(GFAP)的表达变化以评价db/db小鼠视网膜的病变损伤。同时检测糖原合成酶激酶3β(GSK3β)蛋白以及磷酸化糖原合成酶激酶3β(phospho-GSK3β)蛋白在视网膜组织的表达。本研究旨在观察天然药物PHL.对于db/db小鼠视网膜病变的影响,初步并探讨PHL实现其保护作用的可能分子机制,从为临床上治疗DM慢性血秘并发症提供新的治疗思路与途径。
研究目的
1.研究db/db小鼠视网膜组细胞的凋亡以及视网膜GFAP的表达等视网膜病变早.期阶段的特点：
2.研究PHL对于db/db小鼠视网膜强胞凋亡视网膜GFAP表达的影响，评价PHL对于db/db小鼠视网膜的保护作用；
3.研究PHL对于GSK3β蛋白与phospho-GSK3β蛋白在视网膜组织表达的影响。初步探讨PHL对db/db小鼠视网膜保护作用的机制，为开辟DM视网膜病变治疗的新途径完善理论基础。
研究方法
7周龄性C57BLKS/J db/db小鼠16只,以及7周龄雄性C57BLKS/J db/m小鼠8只,均由南京大学模式动物研究所提供。正式实验开始前所有小鼠均予观察1周。实验开始后分组,C57BLKS/J db/m小鼠8只作为正常对照组(CC), C57BLKS/J db/db小鼠随机分为两组：一组随机8只小鼠为DM模型组(DM),每日予生理盐水灌胃；另一组随机8只为PHL干预组(DMT), PHL20mg/kg/d溶液灌胃(与DM组生理盐水体积相同),共进行10周。实验期间每周定期测量体重(BW)并记录。实验周期结束,所有小鼠空腹过夜并处死。采血检测空腹血糖(FBG),糖基化终末代谢产物(AGEs)等指标。并迅速摘除眼球,多聚甲醛固定后并常规分离视网膜,按照病理取材常规制作视网膜石蜡切片。然后应用TUNEL法检测视网膜细胞凋亡情况,并应用蛋白质免疫印迹(western blotting)技术分析GFAP蛋白、GSK3β蛋白与phospho-GSK3β蛋白在视网膜组织的表达。部分视网膜组织分离后立即于液氮速冻后-80°保存留待进一步蛋白质组学实验用。
研究结果
1.一般观察
CC组小鼠生长以及精神状况良好,活跃,毛发光顺。DM组小鼠实验进程中,逐渐表现为污秽无泽,被毛蓬松,少动,明显多饮、多食、多尿,体重快速增加。DMT组小鼠上述表现有所改善。
2.PHL对db/db小鼠体重、FBG与AGEs的影响
实验开始DM组与DMT组的体重差异并无统计学意义(P>0.05),但均显著高于CC组(P<0.01)。自实验第2周开始, DM组小鼠体重逐渐增加,该趋势一直持续至实验结束(即实验第10周)。然而,DMT组小鼠与DM组相比,体重表现为明显下降(P<0.01)。
实验开始时DM组与DMT组间的FBG与AGEs水平无显著性差异(P>0.05),但与CC组比较,两组FBG与AGEs水平均明显升高(P<0.05)。给予PHL干预10周后,DMT组小鼠的FBG与AGEs水平与DM组比较则明显降低(P 3.PHL对db/db小鼠视网膜细胞凋亡的影响
实验结束后,于TUNEL法以及苏木素-伊红(HE)染色复染检测小鼠视网膜细胞的凋亡,以细胞核棕黄(褐)色染色结果判定为阳性细胞。三组实验小组同样条件下至少各计数10个视野的凋亡细胞,以凋亡细胞与细胞总数的比值表示。结果发现,CC组小鼠视网膜未见明显细胞凋亡,正常细胞核在HE复染后呈蓝色,细胞核形态、大小较一致。与CC组比较,DM组小鼠视网膜TUNEL-阳性细胞的数量显著增加(P<0.01),凋亡细胞的细胞核中出现棕黄色或者棕褐色颗粒，细胞核形态不规则,大小不一,且多位于视网膜神经节细胞与血管内皮细胞。DMT组小鼠视网膜与DM组小鼠相比较,PHL治疗后,其视网膜凋亡细胞数量明显减少(P<0.01)。
4．PHL对db/db小鼠视网膜GFAP表达的影响
Western blotting染色显示,实验结束时,CC组小鼠视网膜组织GFAP显色反应最弱,含量最低。DM组视网膜组织GFAP表达明显增高,PHL治疗后,GFAP表达则显著降低,但仍明显强于对照组。
5.PHL对db/db小鼠视网膜GSK3β蛋白与phospho-GSK3β蛋白表达的影响
Western blotting染色显示,实验结束时,与CC组小鼠视网膜组织phospho-GSK3β蛋白表达相比较DM组视网膜组织phospho-GSK3β蛋白表达明显减少。经PHL治疗后,phospho-GSK3β蛋白表达则显著增高。
结论
1.与正常对照组比较,db/db小鼠随年龄增长其体重显著增加,PHL干预可以显著改善db/db小鼠的肥胖。
2.db/db小鼠的FBG与AGI水平较正常对照组显著升高,经PHL治疗后能够显著降低其FBG与AGEs水平。
3.PHL治疗可以显著减少db/db小鼠视网膜凋亡细胞的数量,抑制db/db小鼠视网膜组织GEAP的过量表达,提示PHL对于db/db小鼠视网膜的保护作用。
4.PHL可以通过减少phospho-GSK3β蛋白表达,抑制GSK3β蛋白活性的途，减轻db/db小鼠视网膜的细胞凋亡,起到保护其视网膜病变的作用。
第二部分基于iTRAQ定量蛋白质组学技术探讨根皮苷对db/db小鼠视网膜的保护机制
研究背景
人类基因组计划的重大研究成果——人类基因组序列图谱的完成,宣告了生命科学研究经步入了一个新的纪元——“后基因组时代(postgenome era)"的到来。人们认识到,基因仅仅只是遗传信息的源头与载体,而功能性蛋白才是基因功能的执行者,是生命现象的直接体现者。对蛋白质结构与功能的研究将直接阐明生命在生理或病理状态下的变化机制。然而,传统的对单个蛋白质进行研究的方式已经无法满足功能基因组时代的要求。因此,必须需要在整体、动态、网络的水平上进行探讨,才能揭示生命现象的本质。由此,蛋白质组学应运而生,它是以细胞内或是一种基因组的全部蛋白质及其活动方式为研究对象。可以说,蛋白质组学研究的开展不仅是生命科学研究进入后基因组时代的里程碑,也是后基因组时代生命科学研究的重点内容之一。
随着蛋白质组学研究的不断深入与发展,人们已经不再满足于对一个混合体系中的蛋白质进行简单的定性分析。蛋白质组从静态的定性分析发展到动态变化的定量研究,于是有学者提出“定量蛋白质组学(quantitative proteomics)"的概念。定量蛋白质组学是功能蛋白质组学中的重要内容之一,就是把一个基因组表达的全部蛋白质或一个复杂的混合体系中所有蛋白质进行精确地定量与鉴定。定量蛋白质组学着重于定量解析细胞蛋白质的动态变化与动态行为,进而真实的反映了细胞功能、过程机制等综合信息。因此,定量蛋白质组学常被用于分析疾病与健康、药物处理前后蛋白质中的差异蛋白的确定,并有助于发现新的生物功能、可用于鉴定诊断或预警的疾病标志物和发现用于疾病治疗的靶标蛋白质。近年来该领域发展非常迅速,全新的应用和方法设计正在不断推进着技术创新。近年来出现的一种基于质谱的蛋白质组学定量分析技术即同位素标记的相对和绝对定量技术(isobaric tag for relative and absolute quantification, iTRAQ)以其独特的优越性,正逐渐成为定量研究中的主要方法之一。iTRAQ相对定量技术为开展针对特定细胞或状态机制等的研究提供了一种差减分析的工具,最终可以提供上调或者下调蛋白质的功能信息,其将在探讨疾病的发生机制、疾病标志物的寻找、药物靶标的鉴定以及新药的研发与利用等研究领域得到很好的应用
PHL是一种天然多酚类化合物,广泛存在于苹果属的多种植物中。大量研究发现,PHL具有多种生物活性、药理作用与临床功效,如抗氧化损伤、清除自由基、调节血糖与血压、保护心脏、抗肿瘤以及改善认知等等。已有研究结果提示,PHL对于DM心肌肥厚、主动脉病变、肾脏蛋白尿、视网膜早期周细胞损伤等DM大血管与微血管病变具有改善作用。本课题第一部分研究也发现：PHL于db/db小鼠视网膜具有明显的保护作用。然而,目前尚未明确PHL保护DM视网膜病变的作用机制以及分子靶点。
药物研发的过程中需要特别重视药物治疗、蛋白质表达和生理反应之间存在的密切关系。绝大多数情况下,药物治疗会导致基因产物表达的调控与修饰。同样,复杂的疾病过程如DM的代谢紊乱也会引起蛋白质表达的改变。为此,可以推断理想的药物有能力将受扰系统的整体蛋白质表达恢复至正常状态的水平。传统意义上的药物研究往往注重药物表型的观察与单个大分子或酶解的活性,无法实现对于药物的药理活检的检测以及药物作用的靶点的全面分析。随着定量蛋白质组学与药学研究的共同发展与结合,为药物高效率开发与利用带来了新的希望。在实际应用中,定量蛋白质组学可以实现高高通量的对比分析药物作用前后蛋白质表达谱或者蛋白质表达丰度的改变,从而详尽的阐明药物的作用机制并可以发现与确认药物作用的靶点,揭示药物作用环节与作用过程。这些无疑将大大有助于理性高效的的开展药物研发工作,为药物研制提供新的思路与途径。
本研究第二部分将应用iTRAQ蛋白质组学相对定量技术,并利用Turbo SEQUEST欠件与国际蛋白质索引(international protein index, IPI)数据库检索等生物信息学方法,分离与鉴定正常对照组(CC组),db/db小鼠组(DM组)与db/db小鼠PHL治疗组(DMT组)等小鼠视网膜组织的差异农达蛋白,旨在探讨PHL对于db/db小鼠视网膜病变的保护机制与作用靶点,为临床上DM视网膜病变的治疗寻找和研发展有效的药物而提供蛋白质靶标,希望有助于理性高效的为治疗DM视网膜病变而开展药物研发工作。
研究目的
1.初步探讨db/db小鼠视网膜病变的差异表达蛋白的特征,进一步了解DM视网膜病变的发病机制：
2.探讨db/db小鼠经PHL治疗后视网膜组织的差异表达蛋白，并筛选以及初步验证药物作用的关键蛋白与侯选靶标,基于蛋白质组学角度进一步揭示PHL对于DM视网膜的保护机制,从而为开辟DM视网膜病变的治疗的新途径奠定理论基础。
研究方法
从正常对照组、db/db小鼠与PHL干预db/db三组小鼠中各选取4只,分离视网膜组织。按照蛋白质组学样本制备标准技术流程,包括视网膜组织的研磨、切碎、超声破碎、裂解等流程来提取视网膜总蛋白,并以Bradford蛋白质定量法测定蛋白质浓度,分装提取样品,-80°保存备用。提取各组视网膜总蛋白后,各组上样20ug样品进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE),观察各组之间电泳后蛋白点迁移平行度较好后,再采用iTRAQ定量蛋白质组学技术。各组视网膜总蛋白采用FASP酶解得到相应各组肽段。各组取60ug肽段,按照ABI公司说明书操作与标记消化后的各组肽段,依次分别为114标记正常对照组,116标记PHL干预组,117则标记db/db组。标记结束后,各组均随机选取一定数量肽段,利用ABI4800MALDI-TOF/TOF质谱仪检测其二级质谱图中低分子量端是否存在相应质量标记试剂,确定标记成功后将各组已标记的肽段混合,然后用AgilentHPLC1200强阳离子交换柱(strong cation exchange, SCX)进行分离分级,收集穿流以及洗脱部分合并成10组,之后予C18Cartridge脱盐进行下一步质谱鉴定。本研究第二部分使用Thermo Finnigan LTQ Velos质谱仪进行液相质谱-串联质谱分析(LC-ESI-MS/MS)。质谱仪在线配备0.15mm*150mm C-18反相色谱柱,液相A液为0.1%甲酸水溶液,B液为0.1%甲酸乙腈水溶液(乙腈为84%)。质谱的进样方式为Microspray,毛细管温度为200度,检测方式为正离子。多肽和多肽的碎片的质量电荷比按照下列方法采集：每次全扫描(full scan)后采集5个碎片图谱(MS2scan)。原始文件(raw file)用iTRAQ Result Multiple File Distiller分析定量数据,并用SEQUEST软件鉴定多肽分子。最后,使用相关软件将定量及鉴定结果进行合并处理,得到定量和鉴定结果。定量方法采用Identified iTRAQ Statistic Builder软件分析,采用软件计算的ratio_biweight值作为蛋白质定量结果,以114标记为内参。搜索使用的数据库为ipi. MOUSE. v3.72. REVERSED. fasta蛋白库(SEQUEST结果过滤参数为：Protein FDR≤0.01; Peptide FDR≤0.01)。应用EXPASY蛋白质组学工具分析等电点、分子量等。并采用基因本体论GO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi,Version1.8)对经鉴定的视网膜蛋白质功能进行分类,从分子功能(Molecular function)、生物过程(Biological process)与细胞组成(Cellular component)三个方面对蛋白质表达谱进行定位分类与功能分析。并将筛选出的部分差异蛋白用western blotting方法给予进一步验证其在视网膜组织中的表达。
研究结果
1.质潜鉴定结果
经LC-ESI-MS/MS鉴定并予软件分析、数据库检索,共鉴定出蛋白1651种,符合鉴定条件者可信蛋白636中,具有唯一肽段8972个。其中鉴定db/db小鼠组与正常对照组视网膜的差异表达蛋白348个,db/db小鼠视网膜组织表达上调的点177个,下调的点171个。另外,经PHL.治疗后有60个回调的差异农达蛋白,其中PHL治疗后下调33个点,上调27个点。
2.PHL干预后差异蛋白的定位分析
对于PHL干预后的60个差异蛋白点,予采用基因本体论GO进行定位分析。结果发现，PHL干预后的差异蛋白包括胞浆蛋白、核蛋白、膜蛋白、内质网蛋白与线粒粒体蛋白等,其中以胞浆蛋白与核蛋白为主,均占33.87%其中γ晶体蛋白(γ-crystallin)位于胞浆以及胞核内,谷氧还蛋白(glutaredoxin-3)位于胞浆中。
3.PHL干预后差异蛋白的功能分析
对于PHL干预后的60个差异蛋白点,根据有知识、数据库注释与文献资料,采用基因本体论GO进行功能分析。结果发现,PHL干预后的60个差异蛋白点,其功能涉及到信号转导、细胞骨架、细胞代谢、凋亡、氧化应激等等,其中参与细胞代谢占55%,比例相对最大。
4.筛选的部分差异蛋白在宙网膜组织的表达改变以验证
为验证蛋白质组学鉴定出的差异蛋白在视网膜组织的表达,应用western blotting方法检检其中部分差异蛋白如γ晶体蛋白(γ=crystallin)与谷氧还蛋白(glutaredoxin-3)在各组小鼠视网膜组织中的表达。结果显示,与正常对照组小鼠视网膜中表达比较,γ晶体蛋白在DM小鼠中表达明显增高。然而，应用PHL治疗后,表达增高的γ晶体蛋白有所下调。另外,谷氧还蛋白在db/db小鼠中表达与正常对照组比较明显降低,经过PHL干预后该蛋白表达回调,因此,γ晶体蛋白与谷氧还蛋白的蛋白免疫印迹。实验结果均与TRAQ鉴定结果是一致的。
结论
1.作为蛋白质组学的新颖技术iTRAQ,具有高灵敏性、高通量的特点,能够对于多组样本同时进行比较分析,可以获得更好的蛋白质组覆盖率。因此,iTRAQ能够用于研究PHL对db/db小鼠视网膜保护作用的分子机制,并助于发现新的药物靶标。
2.应用LC-ESI-MS/MS鉴定得到PHL干预db/db小鼠视网膜差异表达蛋白60个,其中表达下调33个,表达上调27个,均以胞浆蛋白为主。差异蛋白其功能涉及到信号转导、细胞骨架、细胞代谢、凋亡与氧化应激等等,提示这些过程与机制可能参与了DM视网膜的发生与发展。
3.在差异表达蛋白中,以正常对照小鼠视网膜作为对照,蛋白质免疫印迹方法发现,γ晶体蛋白在db/db小鼠组明显上调,在PHL干预组则显著下调；相反,谷氧还蛋白在db/db小鼠明显下调,在PHL干预组则显著上调。该结果与质谱数据解析结果一致,提示Y晶体蛋白与谷氧还蛋白可能是PHL对db/db小鼠视网膜保护作用的候选的关键蛋白。
4.本研究提供了PHL对于db/db小鼠视网膜保护作用的蛋白质表达谱的重要信息,为全面揭示PHL作用机制带来重要手段与思路,同时为寻找DM视网膜病变的治疗提供了可能有意义的药物靶标,从而为临床诊治DM视网膜病变奠定与充实崭新的理论基础。
Part One
Effects of Phlorizin on Retinopathy in db/db Mice
Background
Diabetes is a metabolic disorder characterized by hyperglycemia and insufficiency of secretion or action of endogenous insulin as well as insulin resistance. Nowdays the incidence of diabetes mellitus (DM) has increased worldwild due to various reasons, including the elevation of living standards, the modem life styles and aging. DM has presented a severe social problem, affecting both the health and quality of life among publie.
DM not only develops impaired glucose metabolism, but attacks both macrovascular and microvascular damages throughout the body. Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes and the worst ocular complications of diabetes. Nearly all people with type1and more than half with type2diabetes develop retinopathy. DR remains the leading cause of visual loss and acquired blindness among working-age adults in the industrialized world. Person with DR are25times more likely to become blind than individuals without diabetes. With an ongoing pandemic of DM. more concerns have been focused on the DR expressing an consistanl increase of incidence. The present measures to treat DR are systemic therapy (tight control of glucose and blood pressure) in conjuction with ocular approaches (laser photocoagulation, surgical intervention) as needed. However, current therapeutic options for treating DR, as described above, are limited by considerable side effects and are far from satisfactory. In spite of present managements, DR has remained difficult to prevent or arrest. Thus, there clearly is a strong incentive to search for potential candidates combating DR.
Phlorizin, a phloretin glucoside, is a dihydrochalcones and is maily distributed in the root bark, stem, leaves and fruit of apple trees. Phlorizin makes the major sources of flavonoid. Phlorizin has been reported to possess various properties, including being anti-inflammatory, anti-tumorigenic, antioxidative, and having the ability to lower glucose concentration and improve memory. Recently a series of studies were conducted using phlorizin to curb diabetic complications. In Ins2Akita mice, chronic phlorizin treatment further normalized cardie dysfunction and alleviated ventricular hypertrophy. Also, phlorizin has been reported to inhibit aorta lesion and hepatic damage in db/db mice. In streptozotocin-induced diabetic rats, phlorizin prevented proteinuria, hyperfiltration and kidney hypertrophy, alleviating the early renal functional and preventing some structural changes in diabetes. T-1095, a derivative of phlorizin, suppressed the development of albuminuria and the expansion of the glomerular mesangial area in db/db mice, indicating that the progression of diabetic nephropathy was prevented. Though having evidence that phlorizin treated vascular comlications of DM, no reports have shown the effect of phlorizin on retinopathy in db/db mice. Therefore, We took db/db mice, generally popular applied, as an animal model for type2DM to observe the retina cells apoptosis, detected by Terminal transferase dUTP nick end labeling (TUNEL) and to measure glial fibrillary acidic protein (GFAP) expression and in retinas using western blotting analysis among the control, db/db mice, and phlorizin treated db/db mice. Meanwhile, the expression of glycogen synthase kinase-3beta (GSK-3β) and phosphorylation GSK-3β were also monitored in retinas. The purpose of this study was to examine the effects of phlorizin on DR and to explore the mechanisms underlying the phlorizin therapy and the onset and progression of DR, thus providing new approaches for the management of DR.
Objectives
1. The aim of the study was to characterize the early pathological changes of retina in db/db mice, including retinal cells apoptosis and the GFAP expression in db/db mice.
2. The aim of the study was to examine the effects of phlorizin on the retinal cells apoptosis and the GFAP expression in db/db mice.
3. The aim of the study was to examine the effects of phlorizin on the expression of GSK-3β and phosphor-GSK-3β in db/db mice, and to explore the mechanism by which phlorizin treated DR, further to provide new approaches for the management of DR.
Methods
Male C57BLKS/J db/db(n=16, seven weeks old) and db/m mice (n=8, seven weeks old) were purchased from the Model Animal Research Center of Nanjing University (Jiangsu, China). The mice were kept under observation for one week before the experiments started. C57BLKS/J db/m mice were selected as the control group (CC, n=8). The db/db mice were divided into2groups:an untreated diabetic group (DM, n=8) administered normal saline solution by intragastric gavage and another diabetic group treated with a dosage of20mg/kg of phlorizin (DMT, n=8) for ten weeks. All mice were weighed regularly during the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured. Afterwards, the whole eyes from the controls, db/db mice and db/db mice treated with phlorizin were immediately enucleated and fixed in4%paraformaldehyde and embedded in paraffin. Then the retinas were dissected. The retina sections were then isolated and cut using standard histological procedures. To determine whether retinal cells apoptosis was influenced by diabetic state and treatment of phlorizin. the TUNKL method for detecting DNA breaks in situ was applied to retinal tissue. Meanwhie, the expressions of GFAP, GSK-3β and phosphor-GSK-3β in retinas were also monitored. Moreover, some retina tissue were kept at-80℃until further protcomic analysis.
Results
1. General characteristics
During the study, the CC group mice showed good condition and grew up sound with smooth furs. No diabetic related symptoms were shown in this group. The db/db mice featured polydipsia, polyphagia and polyuris with shaggy furs and a rapid weight gain. Phlorizin treated db/db mice also displayed abnormal, much better than the db/db mice.
2. Effects of phlorizin on body weight, FBG and AGEs
Baseline body weights were similar between DM group and DMT group (P>0.05). The elevated body weights in DM group and DMT group were found comparing with the CC group (P<0.01). At as early as week2of the study, the body weights in DM group and DMT group showed signs of increase. This trend did not change until the end of the experiment. However, the body weight was significantly inhibited at10weeks,12weeks,14weeks,16weeks, and18weeks after phlorizin administration in the DMT group compared to the DM group (P<0.01).
Baseline FBG and AGEs were similar between DM group and DMT group (P>0.05). The elevated FBG and AGEs in DM group and DMT group were found comparing with the CC group (P<0.01). Phlorizin administration in the DMT group for week10caused a reduction in FBG and AGEs, as compared wth DM group (P<0.05).
3. Effect of phlorizin on diabetes-induced retinal cells apoptosis
TUNEL array was performed to detect the retinal cells apoptosis following counterstained with hematoxylin. TUNEL-positive cell was defined as cell with brown-coloured in nucleus after staining. Almost no TUNEL-positive cell staining was detected anywhere in the retina in the CC group. The nucleuses of the cell were in the state of normal structure, showing blue under the hematoxylin staining. The number of TUNEL-positive cells in db/db mice was significantly higher than those of the CC group (P<0.05). The nucleuses of the apoptosis cells were in the state of irregular structure, showing brown staining. TUNEL-positive cells were predominantly located in the ganglion cell layer and in the vascular endothelium. However, Treating db/db mice with phlorizin significantly reduced the number of TUNEL-positive cells(P<0.05).
4. Effect of phlorizin on GFAP expression with western blotting
GFAP expression, monitored with western blotting analysis, increased in retinas of db/db mice compared with the control group. In contrast, phlorizin treatment downregulated the retinal GFAP expression in db/db mice.
5. Effects of phlorizin on the expressions of GSK-3β and phospho-GSK-3β
The expression of phospho-GSK-3β with western blotting decreased in db/db mice when comparing with the control, however, this effect was partially ameliorated with phlorizin treatment.
Conclusion
1. Phlorizin-treated db/db mice experienced total body weight reduction.
2. Phlorizin caused significant decrease both in FBG and in AGEs of db/db mice.
3. Phlorizin remarkably inhibited diabetic retina cells apoptosis and downregulated GFAP expression in retinas, suggesting that phlorizin could be of potential benefit in preventing diabetic retinal damage.
4. Phlorizin could ameliorate phospho-GSK-3β expression in retinas and suppress the GSK-3β activity, which led to the inbibition of retinal cells apoptosis and the protection of DR.
Part Two
iTRAQ-based Proteomic Analysis on the Protective Mechanism of Phlorizin on Retinopathy in db/db Mice
Background
The completion of the draft version of human sequence, as an important part of the Human Genome Project, marked the start of a novel era-the postgenone era. It has been recognized that the gene is the only carrier for genetic information and that the proteins facilitate all biological function and carry out the duties specified by the information encoded in genes. Thus, researches concerning the structure and function of proteins help to explore the mechanisms underline the life response to psychology and pathology. However, traditional research methods for one-by-one protein makes it hard to meet the needs of the functional genome, therefore, exploring the nature of life depends on the research of proteins focusing on the global view, dynamic level and network information, hence the advent of proteomics. Proteomics refers to the global analysis of entire protein complement of an organism at a given time. Thus, proteomics remains a milestone of life science in the postgenome era.
With the continuing improvement of proteomic research, it is hard for the proteomes of qualitative analysis to meet the needs for the rapidly growth. Thus, more attention has been paid to the proteomes of dynamic quantitative analysis, rather than that of stationary qualitative analysis. Hence quantitative proteomics was approaching. Quantitative proteomics is one of the major components of functional proteomics that identifies systematically the proteins in complex samples and determines their quantity or quantitative change. Quantitative proteomics pays more attention to the dynamic alterations of proteins in cell, suggesting the informations on the structure and function of given cell. Therefore, quantitative proteomics profiling is often applied to study and to identify the differentially protein expression occurring with state of disease as well as drug intervention. Thus, quantitative proteomics is anticipated to provide insights into the function of biological processes, facilitate the identification of diagnostic or prognostic disease markers, and contribute to the discovery of proteins as therapeutic targets. This field is growing rapidly and new applications and protocol design are driving technological innovations. Recently a novel, MS-based approach for the relative quantification of proteins, using isobaric tag for relative and absolute quantitation (iTRAQ) is presented, which is regarded as one of the important array for quantitation in proteomics due to its advances. iTRAQ technology, a popular relative quantitative measurement, has provided a means for subtractive analysis where one can target a particular cell mechanism, which in turn provides information about the function of those proteins that are upregulated or downregulated. iTRAQ technology has many roles to play in proteomics research, including exploring the onset of diseases, uncovering the new biomarkers, drug targets validation and new drug development.
As a kind of natural flavonoid compound, phlorizin is widely distributed in the root bark, stem, leaves and fruit of apple trees. It has been demonstrated that phlori/.in possesses a variety of potent properties, pharmacological activities, and clinical benefits, including anti-oxidation, free radical scavenging, regularing glucose concentrations and blood pressure, cardioprotection, anti-tumor, and memory improvement. Recently a series of studies were conducted using phlorizin to curb diabetic complications, including diabetes-induced myocardial hypertrophy, aorta lesion, albuminuria and diabetic retinal pericytes lesions. Our previous study addressed in the part one showed that phlorizin had a protective effect on retinopathy in db/db mice. But the mechanism underlying the protection against retinopathy lesions remains unclear, and so does the molecular targets by which phlorizin treated diabetic retinopathy.
There is a close relationship between drug treatment, protein expression, and resulting physiological effects. Most of the time, pharmacological intervention results in the regulation or modulation of gene-product expression, in a similar way that complex disease processes, e,g, diabetes induced metabolic dysfunction, alter global protein expression. From this, it could be ascertained that an ideal drug is one that restores global protein expression of a disturbed system to a normal state. Drug researches traditionally focuses on the phenotypic observation only, which make it hard to detect the pharmacological activity and to identify the targets of drug. However, quantitative proteomics, coupling with pharmacy study, provides a means for the potential development of new pharmacological molecules. In practice, it is desirable to analyse proteins expression alterations after drugs administration using quantitative proteomics with high throughout. Thus, quantitative proteomics could help to explore the mechanisms behind the drug, uncover the therapeutic targets, and contribute to the processing of drug actions. From this, quantitative proteomics could provide new approaches for researching and developing drugs.
In the part2of this study, We separated and identified differently expressed proteins among the control db/m mice, db/db mice and phlorizin treated db/db mice using iTRAQ technology, followed by urbo SEQUEST program software and international protein index (IPI) mouse protein database. The purpose of this experiment was to explore the mechanisms by which phlorizin curbed diabetic retinopathy, to uncover discovery of proteins as therapeutic targets, and to develop potential for treating diabetes induced retinal lesion.
Objective
1. The aim of the study was to characterize the differently expressed proteins, further to dermine the mechanism of the onset of diabetic retinopathy.
2. The objective of this study was to identify retinal proteomic alterations associated with db/db mice and phlorizin treatment and to uncover discovery of proteins as promising drug target. Also, this study was performed on purpose of determining the mechaniam underlying the protection of phlorizin against diabetic retinopathy and in order to provide novel approaches for the treatment of diabetic retinopathy.
Methods
The retinas tissue was dissected among the control, db/db mice, phlorizin treated db/db mice,4from each group, respectively. The processing of proteomic analysis is as follows. Firstly, the separated retinas tissue sample were prepared for following array using universal procedure, including tissue disruption, sample clean-up, and protein solubilization. After that, the concentrations of the proteins can be estimated using Bradford array. Then the retinas tissue were kept at-80°until further analysis. About20ug of peptides of each group were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to detect the similarities of protein bands loaded on the gels among the three groups for further study. Then, the proteins were digested followed by iTRAQ labeling, according to ABI manufacturer's instructions (Applied Biosystems). About60ug peptides of each group were labeled with iTRAQ reagents (114for the peptides of C group,116for the peptides of DMT group, and117for the peptides of DM group, respectively). All three labeled samples were finally combined together following by separated using Strong Cation Exchange (SCX) chromatography into10fractions and by desalted by an offline fraction collector and C18cartridges (Sigma). Of note, After labeling, an amount of peptides among each group were performed on mass spectrometric analysis using MALDI-TOF/TOF (ABI4800, USA) to evaluate the effectiveness of reagents labeling base on the observation that the peak for reporter ions are encoded in the low mass-to-charge ratio portion of the MS/MS spectrum. In part2of present study, Mass spectrometric analysis was performed using a micro liquid chromatography system (MDLC, GE Healthcare) and a LTQ-Velos ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). The separation column was a0.15mm×150mm capillary packed with Zorbax300SB-C18particles (Agilent Technologies). Mobile phase A (0.1%formic acid in water) and the mobile phase B (0.1%formic acid in ACN) were selected. MS data were acquired using data-dependent acquisition conditions:each MS event was followed by zoom/MS2scans on the five top-most intense peaks. For protein identification and statistical validation, the acquired MS/MS spectra were automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version3.72) using the Turbo SEQUEST program in the BioWorksTM3.1software suite. Data filtering parameters were chosen to generate false positive protein identification rates of<1%, as calculated by searching the MS2scans against a forward reversed database of proteins. A threshold was set to1.5with a P-value<0.05yielding at least a50%change in abundance compared to the reference (114, control group). All identified proteins were classified by their molecular function, biologicai process, and cellular component using AmiGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi Version1.8). Finally, some candidated differentially expressed proteins in the retinal were validated with Western blotting analysis.
Results
1. Mass spectrometry identify the differently expressed proteins
Protein profiling was analyzed using iTRAQ approach followed by LC-MS/MS identification and IPI database searching. A total of1,651proteins were identified. Of them,1636proteins meet the standard. Also,8972unique peptides were observed by mass analysis. Among them,348proteins were differentially expressed in diabetic retina in comparison to control, comprised of177proteins that were increased and171proteins that were decreased. Moreover, in order to examine the effect of phlorizin on the proteome change, proteome analysis was also conducted on the phlorizin treated diabetic retina. Of the significantly changed proteins between DMT group and DM group,33proteins were down-regulated with the treatment of phlorizin, while27proteins up-regulated.
2. Subcellular localization analysis phlorizin associated retina proteins in db/db mice
The localization analysis of the identified proteins in retinas using AmiGO (Version1.8) is shown in our study. The proteins are associated with many subcellular locations, including cytoplasm, nucleus, plasma membrane, mitochondrion and endoplasmic reticulum. The two largest proportion of changed proteins were locates in cytoplasm(33.87%) and nucleus(33.87%). Of note, γ-crystallin was in cytoplasm and in nucleus, while glutaredoxin-3in cytoplasm only.
3. Subcellular bioinformatic functional analysis phlorizin associated retina proteins in db/db mice
Most of these back-regulated proteins were involved in oxidative stress, apoptosis, energy metabolism and signaling transduction. Among the functional assignment of the proteins,55.00%were in metabolic processes,16.67%in cytoskeleton,6.67%were in stress response,6.67%were in immune response,6.67%were in transport,3.33%in extracellular matrix. Obviously, proteins involved in metabolic processes constituted the largest functional group.
4. Validation ofiTRAQ data on selected candidate proteins
To provide confirmation of differentially expressed protein, two candidate proteins were validated using Western blotting analysis, y-crystallin was found to be inhibited whereas Glrx-3was enhanced in the DMT group compared to the DM group. This result verified the reliability of the iTRAQ results.
Conclusion
1. As a novel technology of quantitative proteomics, iTRAQ enables an analysis of up to four samples simultaneously in one experiment with more advantages, including high-throughout, accuracy, and high-sensitivity. Thus, in our study with the help ofiTRAQ, it is possible to explore the mechanism underline the protection of phlorizin against diabetic retinopathy and to uncover discovery of proteins as drug tagets.
2. A total of60proteins differentially changed occurring with phlorizin treatment in retinas of db/db mice were identified using LC-ESI-MS/MS methods. Of these,33proteins were downregulated while27were upregulated with phlorizin administration. Most of these differentially changed proteins were located in cytoplasm and were related to special functions, including signal transduction, cytoskeleton, metabolic process, apoptosis and oxidative stress. Our finding indicted the above-mentioned mechanisms and processes are linked to the onset and pathogenesis of diabetic retinopathy.
3. Phlorizin treatment significantly down-regulated gamma-crystallin in retina of db/db mice while up-regulated glutaredoxin-3. The findings confirmed using Western blotting analysis were consistent with the results from iTRAQ array. Therefore, it could be supposed that gamma-crystallin and glutaredoxin-3might be the critical proteins related to the protection of phlorizin against diabetic retinal lesions.
4. Our present study has provided valuable results in highlighting the protection of phlorizin against diabetic retinapathy to better understand the diabetic retinal damage and phlorizin treatment processes and to discover potential therapeutic targets for curbing diabetic-induced retinal lesions.
研究背景
糖尿病(diabetes mellitus, DM)是一种由于胰岛素抵抗伴有相对胰岛素不足或胰岛素分泌的缺陷而导致的以慢性血葡萄糖水平增高为特征的代谢性疾病。近年来,随着生活水平的提高、生活方式的改变以及人口的老龄化,DM的患病率在全球范围内呈现逐步增高的趋势,业已成为严重威胁人类生命健康与生活质量的世界性公共卫生问题。
DM不仅仅表现为持续的血糖代谢紊乱,更重要的是其可以导致机体大血管与微血管的严重损害。DM视网膜病变是DM微血管病变最常见的并发症之一,也是最为严重的DM眼部并发症。几乎所有的1型DM与超过半数的2型DM患者最终均会发展并导致DM视网膜病变。在发达的工业化国家,DM的视网膜病变是成人中获得性视力损害与失明的最重要的病因。与非DM人群比较,DM患者致盲的危险性升高25倍。由于DM流行趋势的影响,近年来与DM伴行的DM视网膜病变同样严重并备受重视。迄今为止,针对DM视网膜病变的防治手段主要集中于代谢危险因素的控制(如)严格控制血糖、血压等)以及眼的局部治疗(如药物、激光与手术治疗等)。然而,上述治疗方法均受限于局部并发症与治疗效果的困扰,并不能完全阻断视网膜病变的发展。因此,在临床实践中迫切需要寻找防治DM视网膜病变的新型药物与途径,旨在进一步完善与充实DM视网膜病变的治疗策略。
根皮苷(phlorizn,PHL)是根皮素(phloretin)的2‘-β-D-葡萄糖苷,属于植物黄酮类中的二氢查尔酮苷。PHL作为苹果多酚中的重要组分,主要有在于苹果属的多种植物中,在植物的根、茎、叶与果实中均有分布。大量研究结果表明,PHL具有多种生物活性与药理作用,如抗炎,抗肿瘤,抗氧化,以及降低血糖与改善认知等。近年来,PHL已经作为新型的候选药物应用于DM血管并发症的基础研究中。动物实验发现,PHL以显著改善InsAkitaDM小鼠的心室肥厚与心室重塑。PHL还可以明显抑制db/db小鼠的主动脉病变与肝损害。还有证据表明,在STD诱发的DM大鼠中,PHL可以显著减少其蛋白尿,改善其肾小球的高滤过状态。PHL的衍生物T-1095能够明显降低db/db小鼠的蛋白尿,提示PHL具有DM肾脏病变的保护作用。但是,PHL是否对于db/db小鼠视网膜病变具有保护作用,目前国内外未见报道。本研究应用目前较为普遍认可的db/db小鼠作为2型DM的动物模型,采用脱氧核桃核苷酸末端转移酶介导的的dUTP缺口末端标记法(TUNEL)检测db/db上鼠视网膜中审经元药细胞与血管内皮细胞与血管内皮细胞的凋亡特征,并检测视网膜神经胶质细胞胶质纤维酸性蛋白(GFAP)的表达变化以评价db/db小鼠视网膜的病变损伤。同时检测糖原合成酶激酶3β(GSK3β)蛋白以及磷酸化糖原合成酶激酶3β(phospho-GSK3β)蛋白在视网膜组织的表达。本研究旨在观察天然药物PHL.对于db/db小鼠视网膜病变的影响,初步并探讨PHL实现其保护作用的可能分子机制,从为临床上治疗DM慢性血秘并发症提供新的治疗思路与途径。
研究目的
1.研究db/db小鼠视网膜组细胞的凋亡以及视网膜GFAP的表达等视网膜病变早.期阶段的特点：
2.研究PHL对于db/db小鼠视网膜强胞凋亡视网膜GFAP表达的影响，评价PHL对于db/db小鼠视网膜的保护作用；
3.研究PHL对于GSK3β蛋白与phospho-GSK3β蛋白在视网膜组织表达的影响。初步探讨PHL对db/db小鼠视网膜保护作用的机制，为开辟DM视网膜病变治疗的新途径完善理论基础。
研究方法
7周龄性C57BLKS/J db/db小鼠16只,以及7周龄雄性C57BLKS/J db/m小鼠8只,均由南京大学模式动物研究所提供。正式实验开始前所有小鼠均予观察1周。实验开始后分组,C57BLKS/J db/m小鼠8只作为正常对照组(CC), C57BLKS/J db/db小鼠随机分为两组：一组随机8只小鼠为DM模型组(DM),每日予生理盐水灌胃；另一组随机8只为PHL干预组(DMT), PHL20mg/kg/d溶液灌胃(与DM组生理盐水体积相同),共进行10周。实验期间每周定期测量体重(BW)并记录。实验周期结束,所有小鼠空腹过夜并处死。采血检测空腹血糖(FBG),糖基化终末代谢产物(AGEs)等指标。并迅速摘除眼球,多聚甲醛固定后并常规分离视网膜,按照病理取材常规制作视网膜石蜡切片。然后应用TUNEL法检测视网膜细胞凋亡情况,并应用蛋白质免疫印迹(western blotting)技术分析GFAP蛋白、GSK3β蛋白与phospho-GSK3β蛋白在视网膜组织的表达。部分视网膜组织分离后立即于液氮速冻后-80°保存留待进一步蛋白质组学实验用。
研究结果
1.一般观察
CC组小鼠生长以及精神状况良好,活跃,毛发光顺。DM组小鼠实验进程中,逐渐表现为污秽无泽,被毛蓬松,少动,明显多饮、多食、多尿,体重快速增加。DMT组小鼠上述表现有所改善。
2.PHL对db/db小鼠体重、FBG与AGEs的影响
实验开始DM组与DMT组的体重差异并无统计学意义(P>0.05),但均显著高于CC组(P<0.01)。自实验第2周开始, DM组小鼠体重逐渐增加,该趋势一直持续至实验结束(即实验第10周)。然而,DMT组小鼠与DM组相比,体重表现为明显下降(P<0.01)。
实验开始时DM组与DMT组间的FBG与AGEs水平无显著性差异(P>0.05),但与CC组比较,两组FBG与AGEs水平均明显升高(P<0.05)。给予PHL干预10周后,DMT组小鼠的FBG与AGEs水平与DM组比较则明显降低(P
实验结束后,于TUNEL法以及苏木素-伊红(HE)染色复染检测小鼠视网膜细胞的凋亡,以细胞核棕黄(褐)色染色结果判定为阳性细胞。三组实验小组同样条件下至少各计数10个视野的凋亡细胞,以凋亡细胞与细胞总数的比值表示。结果发现,CC组小鼠视网膜未见明显细胞凋亡,正常细胞核在HE复染后呈蓝色,细胞核形态、大小较一致。与CC组比较,DM组小鼠视网膜TUNEL-阳性细胞的数量显著增加(P<0.01),凋亡细胞的细胞核中出现棕黄色或者棕褐色颗粒，细胞核形态不规则,大小不一,且多位于视网膜神经节细胞与血管内皮细胞。DMT组小鼠视网膜与DM组小鼠相比较,PHL治疗后,其视网膜凋亡细胞数量明显减少(P<0.01)。
4．PHL对db/db小鼠视网膜GFAP表达的影响
Western blotting染色显示,实验结束时,CC组小鼠视网膜组织GFAP显色反应最弱,含量最低。DM组视网膜组织GFAP表达明显增高,PHL治疗后,GFAP表达则显著降低,但仍明显强于对照组。
5.PHL对db/db小鼠视网膜GSK3β蛋白与phospho-GSK3β蛋白表达的影响
Western blotting染色显示,实验结束时,与CC组小鼠视网膜组织phospho-GSK3β蛋白表达相比较DM组视网膜组织phospho-GSK3β蛋白表达明显减少。经PHL治疗后,phospho-GSK3β蛋白表达则显著增高。
结论
1.与正常对照组比较,db/db小鼠随年龄增长其体重显著增加,PHL干预可以显著改善db/db小鼠的肥胖。
2.db/db小鼠的FBG与AGI水平较正常对照组显著升高,经PHL治疗后能够显著降低其FBG与AGEs水平。
3.PHL治疗可以显著减少db/db小鼠视网膜凋亡细胞的数量,抑制db/db小鼠视网膜组织GEAP的过量表达,提示PHL对于db/db小鼠视网膜的保护作用。
4.PHL可以通过减少phospho-GSK3β蛋白表达,抑制GSK3β蛋白活性的途，减轻db/db小鼠视网膜的细胞凋亡,起到保护其视网膜病变的作用。
第二部分基于iTRAQ定量蛋白质组学技术探讨根皮苷对db/db小鼠视网膜的保护机制
研究背景
人类基因组计划的重大研究成果——人类基因组序列图谱的完成,宣告了生命科学研究经步入了一个新的纪元——“后基因组时代(postgenome era)"的到来。人们认识到,基因仅仅只是遗传信息的源头与载体,而功能性蛋白才是基因功能的执行者,是生命现象的直接体现者。对蛋白质结构与功能的研究将直接阐明生命在生理或病理状态下的变化机制。然而,传统的对单个蛋白质进行研究的方式已经无法满足功能基因组时代的要求。因此,必须需要在整体、动态、网络的水平上进行探讨,才能揭示生命现象的本质。由此,蛋白质组学应运而生,它是以细胞内或是一种基因组的全部蛋白质及其活动方式为研究对象。可以说,蛋白质组学研究的开展不仅是生命科学研究进入后基因组时代的里程碑,也是后基因组时代生命科学研究的重点内容之一。
随着蛋白质组学研究的不断深入与发展,人们已经不再满足于对一个混合体系中的蛋白质进行简单的定性分析。蛋白质组从静态的定性分析发展到动态变化的定量研究,于是有学者提出“定量蛋白质组学(quantitative proteomics)"的概念。定量蛋白质组学是功能蛋白质组学中的重要内容之一,就是把一个基因组表达的全部蛋白质或一个复杂的混合体系中所有蛋白质进行精确地定量与鉴定。定量蛋白质组学着重于定量解析细胞蛋白质的动态变化与动态行为,进而真实的反映了细胞功能、过程机制等综合信息。因此,定量蛋白质组学常被用于分析疾病与健康、药物处理前后蛋白质中的差异蛋白的确定,并有助于发现新的生物功能、可用于鉴定诊断或预警的疾病标志物和发现用于疾病治疗的靶标蛋白质。近年来该领域发展非常迅速,全新的应用和方法设计正在不断推进着技术创新。近年来出现的一种基于质谱的蛋白质组学定量分析技术即同位素标记的相对和绝对定量技术(isobaric tag for relative and absolute quantification, iTRAQ)以其独特的优越性,正逐渐成为定量研究中的主要方法之一。iTRAQ相对定量技术为开展针对特定细胞或状态机制等的研究提供了一种差减分析的工具,最终可以提供上调或者下调蛋白质的功能信息,其将在探讨疾病的发生机制、疾病标志物的寻找、药物靶标的鉴定以及新药的研发与利用等研究领域得到很好的应用
PHL是一种天然多酚类化合物,广泛存在于苹果属的多种植物中。大量研究发现,PHL具有多种生物活性、药理作用与临床功效,如抗氧化损伤、清除自由基、调节血糖与血压、保护心脏、抗肿瘤以及改善认知等等。已有研究结果提示,PHL对于DM心肌肥厚、主动脉病变、肾脏蛋白尿、视网膜早期周细胞损伤等DM大血管与微血管病变具有改善作用。本课题第一部分研究也发现：PHL于db/db小鼠视网膜具有明显的保护作用。然而,目前尚未明确PHL保护DM视网膜病变的作用机制以及分子靶点。
药物研发的过程中需要特别重视药物治疗、蛋白质表达和生理反应之间存在的密切关系。绝大多数情况下,药物治疗会导致基因产物表达的调控与修饰。同样,复杂的疾病过程如DM的代谢紊乱也会引起蛋白质表达的改变。为此,可以推断理想的药物有能力将受扰系统的整体蛋白质表达恢复至正常状态的水平。传统意义上的药物研究往往注重药物表型的观察与单个大分子或酶解的活性,无法实现对于药物的药理活检的检测以及药物作用的靶点的全面分析。随着定量蛋白质组学与药学研究的共同发展与结合,为药物高效率开发与利用带来了新的希望。在实际应用中,定量蛋白质组学可以实现高高通量的对比分析药物作用前后蛋白质表达谱或者蛋白质表达丰度的改变,从而详尽的阐明药物的作用机制并可以发现与确认药物作用的靶点,揭示药物作用环节与作用过程。这些无疑将大大有助于理性高效的的开展药物研发工作,为药物研制提供新的思路与途径。
本研究第二部分将应用iTRAQ蛋白质组学相对定量技术,并利用Turbo SEQUEST欠件与国际蛋白质索引(international protein index, IPI)数据库检索等生物信息学方法,分离与鉴定正常对照组(CC组),db/db小鼠组(DM组)与db/db小鼠PHL治疗组(DMT组)等小鼠视网膜组织的差异农达蛋白,旨在探讨PHL对于db/db小鼠视网膜病变的保护机制与作用靶点,为临床上DM视网膜病变的治疗寻找和研发展有效的药物而提供蛋白质靶标,希望有助于理性高效的为治疗DM视网膜病变而开展药物研发工作。
研究目的
1.初步探讨db/db小鼠视网膜病变的差异表达蛋白的特征,进一步了解DM视网膜病变的发病机制：
2.探讨db/db小鼠经PHL治疗后视网膜组织的差异表达蛋白，并筛选以及初步验证药物作用的关键蛋白与侯选靶标,基于蛋白质组学角度进一步揭示PHL对于DM视网膜的保护机制,从而为开辟DM视网膜病变的治疗的新途径奠定理论基础。
研究方法
从正常对照组、db/db小鼠与PHL干预db/db三组小鼠中各选取4只,分离视网膜组织。按照蛋白质组学样本制备标准技术流程,包括视网膜组织的研磨、切碎、超声破碎、裂解等流程来提取视网膜总蛋白,并以Bradford蛋白质定量法测定蛋白质浓度,分装提取样品,-80°保存备用。提取各组视网膜总蛋白后,各组上样20ug样品进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE),观察各组之间电泳后蛋白点迁移平行度较好后,再采用iTRAQ定量蛋白质组学技术。各组视网膜总蛋白采用FASP酶解得到相应各组肽段。各组取60ug肽段,按照ABI公司说明书操作与标记消化后的各组肽段,依次分别为114标记正常对照组,116标记PHL干预组,117则标记db/db组。标记结束后,各组均随机选取一定数量肽段,利用ABI4800MALDI-TOF/TOF质谱仪检测其二级质谱图中低分子量端是否存在相应质量标记试剂,确定标记成功后将各组已标记的肽段混合,然后用AgilentHPLC1200强阳离子交换柱(strong cation exchange, SCX)进行分离分级,收集穿流以及洗脱部分合并成10组,之后予C18Cartridge脱盐进行下一步质谱鉴定。本研究第二部分使用Thermo Finnigan LTQ Velos质谱仪进行液相质谱-串联质谱分析(LC-ESI-MS/MS)。质谱仪在线配备0.15mm*150mm C-18反相色谱柱,液相A液为0.1%甲酸水溶液,B液为0.1%甲酸乙腈水溶液(乙腈为84%)。质谱的进样方式为Microspray,毛细管温度为200度,检测方式为正离子。多肽和多肽的碎片的质量电荷比按照下列方法采集：每次全扫描(full scan)后采集5个碎片图谱(MS2scan)。原始文件(raw file)用iTRAQ Result Multiple File Distiller分析定量数据,并用SEQUEST软件鉴定多肽分子。最后,使用相关软件将定量及鉴定结果进行合并处理,得到定量和鉴定结果。定量方法采用Identified iTRAQ Statistic Builder软件分析,采用软件计算的ratio_biweight值作为蛋白质定量结果,以114标记为内参。搜索使用的数据库为ipi. MOUSE. v3.72. REVERSED. fasta蛋白库(SEQUEST结果过滤参数为：Protein FDR≤0.01; Peptide FDR≤0.01)。应用EXPASY蛋白质组学工具分析等电点、分子量等。并采用基因本体论GO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi,Version1.8)对经鉴定的视网膜蛋白质功能进行分类,从分子功能(Molecular function)、生物过程(Biological process)与细胞组成(Cellular component)三个方面对蛋白质表达谱进行定位分类与功能分析。并将筛选出的部分差异蛋白用western blotting方法给予进一步验证其在视网膜组织中的表达。
研究结果
1.质潜鉴定结果
经LC-ESI-MS/MS鉴定并予软件分析、数据库检索,共鉴定出蛋白1651种,符合鉴定条件者可信蛋白636中,具有唯一肽段8972个。其中鉴定db/db小鼠组与正常对照组视网膜的差异表达蛋白348个,db/db小鼠视网膜组织表达上调的点177个,下调的点171个。另外,经PHL.治疗后有60个回调的差异农达蛋白,其中PHL治疗后下调33个点,上调27个点。
2.PHL干预后差异蛋白的定位分析
对于PHL干预后的60个差异蛋白点,予采用基因本体论GO进行定位分析。结果发现，PHL干预后的差异蛋白包括胞浆蛋白、核蛋白、膜蛋白、内质网蛋白与线粒粒体蛋白等,其中以胞浆蛋白与核蛋白为主,均占33.87%其中γ晶体蛋白(γ-crystallin)位于胞浆以及胞核内,谷氧还蛋白(glutaredoxin-3)位于胞浆中。
3.PHL干预后差异蛋白的功能分析
对于PHL干预后的60个差异蛋白点,根据有知识、数据库注释与文献资料,采用基因本体论GO进行功能分析。结果发现,PHL干预后的60个差异蛋白点,其功能涉及到信号转导、细胞骨架、细胞代谢、凋亡、氧化应激等等,其中参与细胞代谢占55%,比例相对最大。
4.筛选的部分差异蛋白在宙网膜组织的表达改变以验证
为验证蛋白质组学鉴定出的差异蛋白在视网膜组织的表达,应用western blotting方法检检其中部分差异蛋白如γ晶体蛋白(γ=crystallin)与谷氧还蛋白(glutaredoxin-3)在各组小鼠视网膜组织中的表达。结果显示,与正常对照组小鼠视网膜中表达比较,γ晶体蛋白在DM小鼠中表达明显增高。然而，应用PHL治疗后,表达增高的γ晶体蛋白有所下调。另外,谷氧还蛋白在db/db小鼠中表达与正常对照组比较明显降低,经过PHL干预后该蛋白表达回调,因此,γ晶体蛋白与谷氧还蛋白的蛋白免疫印迹。实验结果均与TRAQ鉴定结果是一致的。
结论
1.作为蛋白质组学的新颖技术iTRAQ,具有高灵敏性、高通量的特点,能够对于多组样本同时进行比较分析,可以获得更好的蛋白质组覆盖率。因此,iTRAQ能够用于研究PHL对db/db小鼠视网膜保护作用的分子机制,并助于发现新的药物靶标。
2.应用LC-ESI-MS/MS鉴定得到PHL干预db/db小鼠视网膜差异表达蛋白60个,其中表达下调33个,表达上调27个,均以胞浆蛋白为主。差异蛋白其功能涉及到信号转导、细胞骨架、细胞代谢、凋亡与氧化应激等等,提示这些过程与机制可能参与了DM视网膜的发生与发展。
3.在差异表达蛋白中,以正常对照小鼠视网膜作为对照,蛋白质免疫印迹方法发现,γ晶体蛋白在db/db小鼠组明显上调,在PHL干预组则显著下调；相反,谷氧还蛋白在db/db小鼠明显下调,在PHL干预组则显著上调。该结果与质谱数据解析结果一致,提示Y晶体蛋白与谷氧还蛋白可能是PHL对db/db小鼠视网膜保护作用的候选的关键蛋白。
4.本研究提供了PHL对于db/db小鼠视网膜保护作用的蛋白质表达谱的重要信息,为全面揭示PHL作用机制带来重要手段与思路,同时为寻找DM视网膜病变的治疗提供了可能有意义的药物靶标,从而为临床诊治DM视网膜病变奠定与充实崭新的理论基础。
Part One
Effects of Phlorizin on Retinopathy in db/db Mice
Background
Diabetes is a metabolic disorder characterized by hyperglycemia and insufficiency of secretion or action of endogenous insulin as well as insulin resistance. Nowdays the incidence of diabetes mellitus (DM) has increased worldwild due to various reasons, including the elevation of living standards, the modem life styles and aging. DM has presented a severe social problem, affecting both the health and quality of life among publie.
DM not only develops impaired glucose metabolism, but attacks both macrovascular and microvascular damages throughout the body. Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes and the worst ocular complications of diabetes. Nearly all people with type1and more than half with type2diabetes develop retinopathy. DR remains the leading cause of visual loss and acquired blindness among working-age adults in the industrialized world. Person with DR are25times more likely to become blind than individuals without diabetes. With an ongoing pandemic of DM. more concerns have been focused on the DR expressing an consistanl increase of incidence. The present measures to treat DR are systemic therapy (tight control of glucose and blood pressure) in conjuction with ocular approaches (laser photocoagulation, surgical intervention) as needed. However, current therapeutic options for treating DR, as described above, are limited by considerable side effects and are far from satisfactory. In spite of present managements, DR has remained difficult to prevent or arrest. Thus, there clearly is a strong incentive to search for potential candidates combating DR.
Phlorizin, a phloretin glucoside, is a dihydrochalcones and is maily distributed in the root bark, stem, leaves and fruit of apple trees. Phlorizin makes the major sources of flavonoid. Phlorizin has been reported to possess various properties, including being anti-inflammatory, anti-tumorigenic, antioxidative, and having the ability to lower glucose concentration and improve memory. Recently a series of studies were conducted using phlorizin to curb diabetic complications. In Ins2Akita mice, chronic phlorizin treatment further normalized cardie dysfunction and alleviated ventricular hypertrophy. Also, phlorizin has been reported to inhibit aorta lesion and hepatic damage in db/db mice. In streptozotocin-induced diabetic rats, phlorizin prevented proteinuria, hyperfiltration and kidney hypertrophy, alleviating the early renal functional and preventing some structural changes in diabetes. T-1095, a derivative of phlorizin, suppressed the development of albuminuria and the expansion of the glomerular mesangial area in db/db mice, indicating that the progression of diabetic nephropathy was prevented. Though having evidence that phlorizin treated vascular comlications of DM, no reports have shown the effect of phlorizin on retinopathy in db/db mice. Therefore, We took db/db mice, generally popular applied, as an animal model for type2DM to observe the retina cells apoptosis, detected by Terminal transferase dUTP nick end labeling (TUNEL) and to measure glial fibrillary acidic protein (GFAP) expression and in retinas using western blotting analysis among the control, db/db mice, and phlorizin treated db/db mice. Meanwhile, the expression of glycogen synthase kinase-3beta (GSK-3β) and phosphorylation GSK-3β were also monitored in retinas. The purpose of this study was to examine the effects of phlorizin on DR and to explore the mechanisms underlying the phlorizin therapy and the onset and progression of DR, thus providing new approaches for the management of DR.
Objectives
1. The aim of the study was to characterize the early pathological changes of retina in db/db mice, including retinal cells apoptosis and the GFAP expression in db/db mice.
2. The aim of the study was to examine the effects of phlorizin on the retinal cells apoptosis and the GFAP expression in db/db mice.
3. The aim of the study was to examine the effects of phlorizin on the expression of GSK-3β and phosphor-GSK-3β in db/db mice, and to explore the mechanism by which phlorizin treated DR, further to provide new approaches for the management of DR.
Methods
Male C57BLKS/J db/db(n=16, seven weeks old) and db/m mice (n=8, seven weeks old) were purchased from the Model Animal Research Center of Nanjing University (Jiangsu, China). The mice were kept under observation for one week before the experiments started. C57BLKS/J db/m mice were selected as the control group (CC, n=8). The db/db mice were divided into2groups:an untreated diabetic group (DM, n=8) administered normal saline solution by intragastric gavage and another diabetic group treated with a dosage of20mg/kg of phlorizin (DMT, n=8) for ten weeks. All mice were weighed regularly during the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured. Afterwards, the whole eyes from the controls, db/db mice and db/db mice treated with phlorizin were immediately enucleated and fixed in4%paraformaldehyde and embedded in paraffin. Then the retinas were dissected. The retina sections were then isolated and cut using standard histological procedures. To determine whether retinal cells apoptosis was influenced by diabetic state and treatment of phlorizin. the TUNKL method for detecting DNA breaks in situ was applied to retinal tissue. Meanwhie, the expressions of GFAP, GSK-3β and phosphor-GSK-3β in retinas were also monitored. Moreover, some retina tissue were kept at-80℃until further protcomic analysis.
Results
1. General characteristics
During the study, the CC group mice showed good condition and grew up sound with smooth furs. No diabetic related symptoms were shown in this group. The db/db mice featured polydipsia, polyphagia and polyuris with shaggy furs and a rapid weight gain. Phlorizin treated db/db mice also displayed abnormal, much better than the db/db mice.
2. Effects of phlorizin on body weight, FBG and AGEs
Baseline body weights were similar between DM group and DMT group (P>0.05). The elevated body weights in DM group and DMT group were found comparing with the CC group (P<0.01). At as early as week2of the study, the body weights in DM group and DMT group showed signs of increase. This trend did not change until the end of the experiment. However, the body weight was significantly inhibited at10weeks,12weeks,14weeks,16weeks, and18weeks after phlorizin administration in the DMT group compared to the DM group (P<0.01).
Baseline FBG and AGEs were similar between DM group and DMT group (P>0.05). The elevated FBG and AGEs in DM group and DMT group were found comparing with the CC group (P<0.01). Phlorizin administration in the DMT group for week10caused a reduction in FBG and AGEs, as compared wth DM group (P<0.05).
3. Effect of phlorizin on diabetes-induced retinal cells apoptosis
TUNEL array was performed to detect the retinal cells apoptosis following counterstained with hematoxylin. TUNEL-positive cell was defined as cell with brown-coloured in nucleus after staining. Almost no TUNEL-positive cell staining was detected anywhere in the retina in the CC group. The nucleuses of the cell were in the state of normal structure, showing blue under the hematoxylin staining. The number of TUNEL-positive cells in db/db mice was significantly higher than those of the CC group (P<0.05). The nucleuses of the apoptosis cells were in the state of irregular structure, showing brown staining. TUNEL-positive cells were predominantly located in the ganglion cell layer and in the vascular endothelium. However, Treating db/db mice with phlorizin significantly reduced the number of TUNEL-positive cells(P<0.05).
4. Effect of phlorizin on GFAP expression with western blotting
GFAP expression, monitored with western blotting analysis, increased in retinas of db/db mice compared with the control group. In contrast, phlorizin treatment downregulated the retinal GFAP expression in db/db mice.
5. Effects of phlorizin on the expressions of GSK-3β and phospho-GSK-3β
The expression of phospho-GSK-3β with western blotting decreased in db/db mice when comparing with the control, however, this effect was partially ameliorated with phlorizin treatment.
Conclusion
1. Phlorizin-treated db/db mice experienced total body weight reduction.
2. Phlorizin caused significant decrease both in FBG and in AGEs of db/db mice.
3. Phlorizin remarkably inhibited diabetic retina cells apoptosis and downregulated GFAP expression in retinas, suggesting that phlorizin could be of potential benefit in preventing diabetic retinal damage.
4. Phlorizin could ameliorate phospho-GSK-3β expression in retinas and suppress the GSK-3β activity, which led to the inbibition of retinal cells apoptosis and the protection of DR.
Part Two
iTRAQ-based Proteomic Analysis on the Protective Mechanism of Phlorizin on Retinopathy in db/db Mice
Background
The completion of the draft version of human sequence, as an important part of the Human Genome Project, marked the start of a novel era-the postgenone era. It has been recognized that the gene is the only carrier for genetic information and that the proteins facilitate all biological function and carry out the duties specified by the information encoded in genes. Thus, researches concerning the structure and function of proteins help to explore the mechanisms underline the life response to psychology and pathology. However, traditional research methods for one-by-one protein makes it hard to meet the needs of the functional genome, therefore, exploring the nature of life depends on the research of proteins focusing on the global view, dynamic level and network information, hence the advent of proteomics. Proteomics refers to the global analysis of entire protein complement of an organism at a given time. Thus, proteomics remains a milestone of life science in the postgenome era.
With the continuing improvement of proteomic research, it is hard for the proteomes of qualitative analysis to meet the needs for the rapidly growth. Thus, more attention has been paid to the proteomes of dynamic quantitative analysis, rather than that of stationary qualitative analysis. Hence quantitative proteomics was approaching. Quantitative proteomics is one of the major components of functional proteomics that identifies systematically the proteins in complex samples and determines their quantity or quantitative change. Quantitative proteomics pays more attention to the dynamic alterations of proteins in cell, suggesting the informations on the structure and function of given cell. Therefore, quantitative proteomics profiling is often applied to study and to identify the differentially protein expression occurring with state of disease as well as drug intervention. Thus, quantitative proteomics is anticipated to provide insights into the function of biological processes, facilitate the identification of diagnostic or prognostic disease markers, and contribute to the discovery of proteins as therapeutic targets. This field is growing rapidly and new applications and protocol design are driving technological innovations. Recently a novel, MS-based approach for the relative quantification of proteins, using isobaric tag for relative and absolute quantitation (iTRAQ) is presented, which is regarded as one of the important array for quantitation in proteomics due to its advances. iTRAQ technology, a popular relative quantitative measurement, has provided a means for subtractive analysis where one can target a particular cell mechanism, which in turn provides information about the function of those proteins that are upregulated or downregulated. iTRAQ technology has many roles to play in proteomics research, including exploring the onset of diseases, uncovering the new biomarkers, drug targets validation and new drug development.
As a kind of natural flavonoid compound, phlorizin is widely distributed in the root bark, stem, leaves and fruit of apple trees. It has been demonstrated that phlori/.in possesses a variety of potent properties, pharmacological activities, and clinical benefits, including anti-oxidation, free radical scavenging, regularing glucose concentrations and blood pressure, cardioprotection, anti-tumor, and memory improvement. Recently a series of studies were conducted using phlorizin to curb diabetic complications, including diabetes-induced myocardial hypertrophy, aorta lesion, albuminuria and diabetic retinal pericytes lesions. Our previous study addressed in the part one showed that phlorizin had a protective effect on retinopathy in db/db mice. But the mechanism underlying the protection against retinopathy lesions remains unclear, and so does the molecular targets by which phlorizin treated diabetic retinopathy.
There is a close relationship between drug treatment, protein expression, and resulting physiological effects. Most of the time, pharmacological intervention results in the regulation or modulation of gene-product expression, in a similar way that complex disease processes, e,g, diabetes induced metabolic dysfunction, alter global protein expression. From this, it could be ascertained that an ideal drug is one that restores global protein expression of a disturbed system to a normal state. Drug researches traditionally focuses on the phenotypic observation only, which make it hard to detect the pharmacological activity and to identify the targets of drug. However, quantitative proteomics, coupling with pharmacy study, provides a means for the potential development of new pharmacological molecules. In practice, it is desirable to analyse proteins expression alterations after drugs administration using quantitative proteomics with high throughout. Thus, quantitative proteomics could help to explore the mechanisms behind the drug, uncover the therapeutic targets, and contribute to the processing of drug actions. From this, quantitative proteomics could provide new approaches for researching and developing drugs.
In the part2of this study, We separated and identified differently expressed proteins among the control db/m mice, db/db mice and phlorizin treated db/db mice using iTRAQ technology, followed by urbo SEQUEST program software and international protein index (IPI) mouse protein database. The purpose of this experiment was to explore the mechanisms by which phlorizin curbed diabetic retinopathy, to uncover discovery of proteins as therapeutic targets, and to develop potential for treating diabetes induced retinal lesion.
Objective
1. The aim of the study was to characterize the differently expressed proteins, further to dermine the mechanism of the onset of diabetic retinopathy.
2. The objective of this study was to identify retinal proteomic alterations associated with db/db mice and phlorizin treatment and to uncover discovery of proteins as promising drug target. Also, this study was performed on purpose of determining the mechaniam underlying the protection of phlorizin against diabetic retinopathy and in order to provide novel approaches for the treatment of diabetic retinopathy.
Methods
The retinas tissue was dissected among the control, db/db mice, phlorizin treated db/db mice,4from each group, respectively. The processing of proteomic analysis is as follows. Firstly, the separated retinas tissue sample were prepared for following array using universal procedure, including tissue disruption, sample clean-up, and protein solubilization. After that, the concentrations of the proteins can be estimated using Bradford array. Then the retinas tissue were kept at-80°until further analysis. About20ug of peptides of each group were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to detect the similarities of protein bands loaded on the gels among the three groups for further study. Then, the proteins were digested followed by iTRAQ labeling, according to ABI manufacturer's instructions (Applied Biosystems). About60ug peptides of each group were labeled with iTRAQ reagents (114for the peptides of C group,116for the peptides of DMT group, and117for the peptides of DM group, respectively). All three labeled samples were finally combined together following by separated using Strong Cation Exchange (SCX) chromatography into10fractions and by desalted by an offline fraction collector and C18cartridges (Sigma). Of note, After labeling, an amount of peptides among each group were performed on mass spectrometric analysis using MALDI-TOF/TOF (ABI4800, USA) to evaluate the effectiveness of reagents labeling base on the observation that the peak for reporter ions are encoded in the low mass-to-charge ratio portion of the MS/MS spectrum. In part2of present study, Mass spectrometric analysis was performed using a micro liquid chromatography system (MDLC, GE Healthcare) and a LTQ-Velos ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). The separation column was a0.15mm×150mm capillary packed with Zorbax300SB-C18particles (Agilent Technologies). Mobile phase A (0.1%formic acid in water) and the mobile phase B (0.1%formic acid in ACN) were selected. MS data were acquired using data-dependent acquisition conditions:each MS event was followed by zoom/MS2scans on the five top-most intense peaks. For protein identification and statistical validation, the acquired MS/MS spectra were automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version3.72) using the Turbo SEQUEST program in the BioWorksTM3.1software suite. Data filtering parameters were chosen to generate false positive protein identification rates of<1%, as calculated by searching the MS2scans against a forward reversed database of proteins. A threshold was set to1.5with a P-value<0.05yielding at least a50%change in abundance compared to the reference (114, control group). All identified proteins were classified by their molecular function, biologicai process, and cellular component using AmiGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi Version1.8). Finally, some candidated differentially expressed proteins in the retinal were validated with Western blotting analysis.
Results
1. Mass spectrometry identify the differently expressed proteins
Protein profiling was analyzed using iTRAQ approach followed by LC-MS/MS identification and IPI database searching. A total of1,651proteins were identified. Of them,1636proteins meet the standard. Also,8972unique peptides were observed by mass analysis. Among them,348proteins were differentially expressed in diabetic retina in comparison to control, comprised of177proteins that were increased and171proteins that were decreased. Moreover, in order to examine the effect of phlorizin on the proteome change, proteome analysis was also conducted on the phlorizin treated diabetic retina. Of the significantly changed proteins between DMT group and DM group,33proteins were down-regulated with the treatment of phlorizin, while27proteins up-regulated.
2. Subcellular localization analysis phlorizin associated retina proteins in db/db mice
The localization analysis of the identified proteins in retinas using AmiGO (Version1.8) is shown in our study. The proteins are associated with many subcellular locations, including cytoplasm, nucleus, plasma membrane, mitochondrion and endoplasmic reticulum. The two largest proportion of changed proteins were locates in cytoplasm(33.87%) and nucleus(33.87%). Of note, γ-crystallin was in cytoplasm and in nucleus, while glutaredoxin-3in cytoplasm only.
3. Subcellular bioinformatic functional analysis phlorizin associated retina proteins in db/db mice
Most of these back-regulated proteins were involved in oxidative stress, apoptosis, energy metabolism and signaling transduction. Among the functional assignment of the proteins,55.00%were in metabolic processes,16.67%in cytoskeleton,6.67%were in stress response,6.67%were in immune response,6.67%were in transport,3.33%in extracellular matrix. Obviously, proteins involved in metabolic processes constituted the largest functional group.
4. Validation ofiTRAQ data on selected candidate proteins
To provide confirmation of differentially expressed protein, two candidate proteins were validated using Western blotting analysis, y-crystallin was found to be inhibited whereas Glrx-3was enhanced in the DMT group compared to the DM group. This result verified the reliability of the iTRAQ results.
Conclusion
1. As a novel technology of quantitative proteomics, iTRAQ enables an analysis of up to four samples simultaneously in one experiment with more advantages, including high-throughout, accuracy, and high-sensitivity. Thus, in our study with the help ofiTRAQ, it is possible to explore the mechanism underline the protection of phlorizin against diabetic retinopathy and to uncover discovery of proteins as drug tagets.
2. A total of60proteins differentially changed occurring with phlorizin treatment in retinas of db/db mice were identified using LC-ESI-MS/MS methods. Of these,33proteins were downregulated while27were upregulated with phlorizin administration. Most of these differentially changed proteins were located in cytoplasm and were related to special functions, including signal transduction, cytoskeleton, metabolic process, apoptosis and oxidative stress. Our finding indicted the above-mentioned mechanisms and processes are linked to the onset and pathogenesis of diabetic retinopathy.
3. Phlorizin treatment significantly down-regulated gamma-crystallin in retina of db/db mice while up-regulated glutaredoxin-3. The findings confirmed using Western blotting analysis were consistent with the results from iTRAQ array. Therefore, it could be supposed that gamma-crystallin and glutaredoxin-3might be the critical proteins related to the protection of phlorizin against diabetic retinal lesions.
4. Our present study has provided valuable results in highlighting the protection of phlorizin against diabetic retinapathy to better understand the diabetic retinal damage and phlorizin treatment processes and to discover potential therapeutic targets for curbing diabetic-induced retinal lesions.
引文
1. Unwin N, Gan D, Whiting D. The IDF Diabetes Atlas:providing evidence, raising awareness and promoting action. Diabetes Res Clin Pract.2010;87(1):2-3.
2. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 2004;27(5):1047-1053.
3. Nazimek-Siewniak B, Moczulski D, Grzeszczak W. Risk of macrovascular and microvascular complications in Type 2 diabetes:results of longitudinal study design. J Diabetes Complications.2002; 16(4):271-276.
4. Xie XW, Xu L, Jonas JB, Wang YX. Prevalence of diabetic retinopathy among subjects with known diabetes in China:the Beijing Eye Study, Eur J Ophthalmol. 2009;19(1):91-99.
5. Fong DS, Aiello L, Gardner TW, King GL, Blankenship G, Cavallerano JD, Ferris FL,3rd, Klein R. Retinopathy in diabetes. Diabetes Care.2004;27 Suppl 1:S84-87.
6. Congdon NG, Friedman DS, Lietman T. Important causes of visual impairment in the world today. JAMA.2003;290(15):2057-2060.
7. Cheung N, Wong TY. Diabetic retinopathy and systemic vascular complications. Prog Retin Eye Res.2008:27(2):161-176.
8. Zhang X, Saaddine JB, Chou CF, Cotch MF, Cheng YJ. Geiss LS. Gregg FW, Albright AL, Klein BE, Klein R. Prevalence of diabetic retinopathy in the united states,2005-2008. JAMA.2010;304(6):649-656.
9. Heintz E, Wirehn AB, Peebo BB, Rosenqvist U, Levin LA. Prevalence and healthcare costs of diabetic retinopathy:a population-based register study in Sweden. Diabetologia.2010;53(10):2147-2154.
10.纪立农.对2型糖尿病新的大型临床试验结果的解读和分析.中国糖尿病杂志.2008；16(11)：642-647.
11.彭晓燕,张永鹏.糖尿病视网膜病变的治疗趋势.眼科.2011；11(4)：217-221.
12. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature.2001;414(6865):813-820.
13. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet.2010;376(9735):124-136.
14. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes.1998;47(6):859-866.
15. Hammes HP, Alt A, Niwa T, Clausen JT, Bretzel RG, Brownlee M, Schleicher ED. Differential accumulation of advanced glycation end products in the course of diabetic retinopathy. Diabetologia.1999;42(6):728-736.
16. Brownlee M. The pathobiology of diabetic complications:a unifying mechanism. Diabetes.2005;54(6):1615-1625.
17. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res.2010;107(9):1058-1070.
18. Barber AJ. A new view of diabetic retinopathy:a neurodegenerative disease of the eye. Prog Neuropsychopharmacol Biol Psychiatry.2003;27(2):283-290.
19. Hammes HP, Federoff HJ, Brownlee M. Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes. Mol Med.1995;1(5):527-534.
20. Antonetti DA, Barber AJ, Bronson SK, Freeman WM, Gardner TW, Jefferson LS, Kester M, Kimball SR, Krady JK, LaNoue KF, Norbury CC, Quinn PG,Sandirasegarane L, Simpson IA; JDRF Diabetic Retinopathy Center Group. Diabetic retinopathy:seeing beyond glucose-induced microvascular disease. Diabetes.2006;55(9):2401-2411.
21. Barber AJ, Gardner TW, Abcouwer SF.The significance of vascular and neural apoptosis to the pathology of diabetic retinopathy. Invest Ophthalmol Vis Sci.2011;52(2):1156-1163.
22. Kowluru RA, Kanwar M. Oxidative stress and the development of diabetic retinopathy:contributory role of matrix metalloproteinase-2. Free Radic Biol Med.2009;46(12):1677-1685.
23. Silva KC, Rosales MA, Biswas SK, Lopes de Faria JB, Lopes de Faria JM. Diabetic retinal neurodegeneration is associated with mitochondrial oxidative stress and is improved by an angiotensin receptor blocker in a model combining hypertension and diabetes. Diabetes.2009;58(6):1382-1390.
24. Kowluru RA, Koppolu P. Diabetes-induced activation of caspase-3 in retina: effect of antioxidant therapy. Free Radic Res.2002;36(9):993-999.
25. Kowluru RA, Odenbach S. Effect of long-term administration of alpha-lipoic acid on retinal capillary cell death and the development of retinopathy in diabetic rats. Diabetes.2004;53(12):3233-3238.
26. Sasaki M, Ozawa Y, Kurihara T, Kubota S, Yuki K, Noda K, Kobayashi S, Ishida S, Tsubota K. Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes. Diabetologia.2010;53(5):971-979.
27. Arnal E, Miranda M, Johnsen-Soriano S, Alvarez-Nolting R, Diaz-Llopis M, Araiz J, Cervera E, Bosch-Morcll F, Romero FJ. Beneficial effect of docosahexanoic acid and lutein on retinal structural, metabolic, and functional abnormalities in diabetic rats. Curr Eye Res.2009;34(11):928-938.
28. Millen AE, Gruber M, Klein R, Klein BE, Palta M, Mares JA. Relations of serum ascorbic acid and alpha-tocopherol to diabetic retinopathy in the Third National Health and Nutrition Examination Survey. Am J Epidemiol.2003; 158(3):225-233.
29. Millen AE, Klein R, Folsom AR, Stevens J, Palta M, Mares JA. Relation between intake of vitamins C and E and risk of diabetic retinopathy in the Atherosclerosis Risk in Communities Study. Am J Clin Nutr.2004:79(5):865-873.
30. Madsen-Bouterse SA, Kowluru RA. Oxidative stress and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Rev Endoer Metab Disord.2008;9(4):315-327.
31. Gosch C, Halbwirth H, Stich K. Phloridzin:biosynthesis, distribution and physiological relevance in plant. Phytochemistry.2010:71(8-9):838-843.
32. Ridgway T, O'Reilly J, West G, Tucker G, Wiseman H. Antixoxidant action of novel derivatives of the apple-derived flavonoid phloridzin compared to oestrogen; relevance to potential cardioprotective action. Biochen Soc Trans. 1997; 25(1):106S.
33. Lee KW, Kim YJ, Kim DO, Lee HJ, Lee CY. Major phenolics in apple and their contribution to the total antioxidant capacity. J Agric Food Chem.2003;51(22): 6516-6520.
34. Rezk BM, Haenen GR, van der Vijgh WJ, Bast A. The antioxidant actitivity of phloretin:the disclosure of new antioxidant pharmacophore in flavonoids. Biochem Biophys Res Commun.2002; 295(1):9-13.
35. Hong EG, Jung DY, Ko HJ, Zhang Z, Ma Z, Jun JY, Kim JH, Sumner AD, Vary TC, Gardner TW, Bronson SK, Kim JK. Nonobese, insulin-deficient ins2Akita mice develop type 2 diabetes phenotypes including insulin resistance and cardiac remodeling. Am J Physiol Endocrinol Metab.2007;293(6):E 1687-1696.
36. Shen L, You BA, Gao HQ, Li BY. Yu F. Pei F. Effects of phlorizin on vascular complications in diabetes db/db mice. Chin Med J (Engl). 2012;125(20):3692-3696.
37. Malatiali S, Francis I, Barac-Nieto M. Phlorizin prevents glomerular hyperfiltration but not hypertrophy in diabetic rats. Exp Diabetes Res. 2008;2008:305403.
38. Arakawa K, Ishihara T, Oku A, Nawano M, Ueta K, Kitamura K, Matsumoto M, Saito A. Improved diabetic syndrome in c57bl/ksj-db/db mice by oral administration of the Na(+)-glucose cotransporter inhibitor t-1095. Br J Pharmacol.2001;132(2):578-586.
39. Wakisaka M, Yoshinari M, Yamamoto M, Nakamura S, Asano T, Himeno T, Ichikawa K, Doi Y, Fujishima M. Na+-dependent glucose uptake and collagen synthesis by cultured bovine retinal pericytes. Biochim Biophys Acta. 1997; 1362(1):87-96.
40. Wakisaka M, Yoshinari M, Asano T, Iino K, Nakamura S, Takata Y, Fujishima M. Normalization of glucose entry under the high glucose condition by phlorizin attenuates the high glucose-induced morphological and functional changes of cultured bovine retinal pericytes. Biochim Biophys Acta.1999; 1453(1):83-91.
41. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP. Evidence that the diabetes gene encodes the leptin receptor:identification of a mutation in the leptin receptor gene in db/db mice. Cell.1996;84(3):491-495.
42.唐慧青,陈立新,染磊,等.Ⅱ型糖尿病模型C57BL/KsJ-db/db小鼠的生物学特征研究.实验动物与比较医学.2011；31(1)：62-65.
43. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol.1992;119(3):493-501.
44. Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, Jerums G. Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes.1991;40(10):1328-1334.
45. Joussen AM, Doehmen S, Le ML, Koizumi K, Radetzky S, Krohne TU, Poulaki V, Semkova I, Kociok N. TNF-alpha mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations. Mol Vis.2009; 15:1418-1428.
46. Xie XW, Xu L, Wang YX, Jonas JB. Prevalence and associated factors of diabetic retinopathy. The Beijing Eye Study 2006. Grades Arch Clin Exp Ophthalmol.2008;246(11):1519-1526.
47. Wang FH, Liang YB, Zhang F,Wang JJ, Wei WB, Tao QS, Sun LP, Friedman DS, Wang NL, Wong TY. Prevalence of diabetic retinopathy in rural China:the Handan Eye Study. Ophthalmology.2009;116(3):461-467.
48. White NH, Sun W, Cleary PA, Tamborlane WV, Danis RP, Hainsworth DP, Davis MD; DCCT-EDIC Research Group. Effect of prior intensive therapy in type 1 diabetes on 10-year progression of retinopathy in the DCCT/EDIC:comparison of adults and adolescents. Diabetes.2010;59(5):1244-1253.
49. Wong TY, Liew G, Tapp RJ, Schmidt Mi, Wang JJ, Mitchell P,Klein R,Klein BE, Zimmet P, Shaw J. Relation between fasting glucose and retinopathy for diagnosis of diabetes:three population-based cross-sectional studies. Lancet.2008;371(9614):736-743.
50.许迅,刘堃.统一机制学说和慢性炎症学说：糖尿病视网膜病变基础研究的新方向.中华眼底病杂志.2008；24(4)：237-239.
51. Gardner TW, Abcouwer SF, Barber AJ, Jackson GR. An integrated approach to diabetic retinopathy research. Arch Ophthalmol.2011;129(2):230-235.
52. Kowluru RA, Chan PS. Oxidative stress and diabetic retinopathy. Exp Diabetes Res.2007;2007:43603.
53.王泓,许迅.氧化应激反应与糖尿病视网膜病变.临床眼科杂志.2009；17(5)：474-477.
54. Hummel KP, Dickie MM, Coleman DL. Diabetes, a new mutation in the mouse. Science.1966;153(3740):1127-1128.
55. Sharma K, McCue P, Dunn SR. Diabetic kidney disease in the db/db mouse. Am J Physiol Renal Physiol.2003;284(6):F1138-1144.
56. Shoham S, Bejar C, Kovalev E, Schorer-Apelbaum D, Weinstock M. Ladostigil prevents gliosis, oxidative-nitrative stress and memory deficits induced by intracerebroventricular injection of streptozotocin in rats. Neuropharmacology.2007;52(3):836-843.
57. Barber AJ, Antonetti DA, Gardner TW. Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes. The Penn State Retina Research Group. Invest Ophthalmol Vis Sci 2000;41(11):3561-3568.
58. Ehrenkranz JR, Lewis NG, Kahn CR, Roth J. Pholorizin:a review. Diabetes Metab Res Rev,2005;21(1):31-38.
59. Hertog MG, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F, Giampaoli S, Jansen A, Menotti A, Nedeljkovic S, et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch Intern Med.1995;155(4):381-386.
60. Vasantha Rupasinghe HP, Yasmin A. Inhibition of oxidation of aqueous emulsions of omega-3 fatty acids and fish oil by phloretin and phloridzin. Molecules.2010;15(1):251-257.
61. Xiang L, Sun K, Lu J, Weng Y, Taoka A, Sakagami Y, Qi J. Anti-aging effects of phloridzin, an apple polyphenol, on yeast via the SOD and Sir2 genes. Biosci Biotech Bioch,2011;75(5):854-858.
62. Oresajo C, Stephens T, Hino PD, Law RM, Yatskayer M, Foltis P, Pillai S, Pinnell SR. Protective effects of a topical antioxidant mixture containing vitamin C ferulic acid, and phloretin against ultraviolet-induced photodamage in human skin. J Cosmet Dermatol.2008;7(4):290-297.
63. Abdul-Ghahi MA, DeFronzo R A. Inhibition of renal glucose absorption:a novel strategy for achieving glucose control in type 2 diabetes mellitus. Endocr Pract. 2008;14(6):782-790.
64. Lu WD, Li BY, Yu F, Cai Q, Zhang Z, Yin M, Gao HQ. Quantitative proteomics study on the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice. Mol Med Report.2012；5(5):1285-1294.
65. Bradford BJ, Allen MS. Phlorizin induces lipolysis and alters meal patterns in both early- and late-lactation dairy cows. J Dairy Sci.2007;90(4):1810-1815.
66. Thomas MC. Advanced glycation end products. Contrib Nephrol.2011；170:66-74.
67. Yamagishi S, Maeda S, Matsui T, Ueda S, Fukami K, Okuda S. Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications indiabetes. Biochim Biophys Acta.2012;1820(5):663-671.
68. Hernandez C, Simo R. Neuroprotection in diabetic retinopathy. Curr Diab Rep.2012; 12(4):329-337.
69. Carrasco E, Hernandez C, de Torres I, Farres J, Simo R. Lowered eortistatin expression is an early event in the human diabetic retina and is associated with apoptosis and glial activation. Mol Vis.2008;14:1496-1502.
70. Kmbi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem.1980; 107(2):519-27.
71. Jacobs KM. Bhave SR, Ferraro DJ, Jaboin JJ, Hallahan DE, Thotala D. GSK-3β: A Bifunctional Role in Cell Death Pathways. Int J Cell Biol.2012;2012:930710.
72..lope RS, Roh MS. Glycogen synthase kinase-3 (GSK3) in psychiatric diseases and therapeutic interventions. Curr Drug Targets.2006;7(11):1421-1434.
73. Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci.2004;29(2):95-102.
74. Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A. Glycogen synthase kinase 3: more than a namesake. Br J Pharmacol.2009;156(6):885-898.
75. Frame S, Cohen P, Biondi RM. A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell.2001;7(6):1321-1327.
76. Frame S, Zheleva D. Targeting glycogen synthase kinase-3 in insulin signalling. Expert Opin Ther Targets.2006;10(3):429-444.
77. Pearce NJ, Arch JR, Clapham JC, Coghlan MP, Corcoran SL, Lister CA, Llano A, Moore GB, Murphy GJ, Smith SA, Taylor CM, Yates JW, Morrison AD, Harper AJ, Roxbee-Cox L, Abuin A, Wargent E, Holder JC. Development of glucose intolerance in male transgenic mice overexpressing human glycogen synthase kinase-3beta on a muscle-specific promoter. Metabolism.2004;53(10):1322-1330.
78. Dokken BB, Henriksen EJ. Chronic selective glycogen synthase kinase-3 inhibition enhances glucose disposal and muscle insulin action in prediabetic obese Zucker rats. Am J Physiol Endocrinol Metab.2006;291(2):E207-213.
79. Lajoie C, Calderone A, Trudeau F, Lavoie N, Massicotte G, Gagnon S, Beliveau L. Exercise training attenuated the PKB and GSK-3 dephosphorylation in the myocardium of ZDF rats. J Appl Physiol.2004;96(5):1606-1612.
80. Dokken BB, Sloniger JA, Henriksen EJ. Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle. Am J Physiol Endocrinol Metab.2005;288(6):E1188-1194.
81. Stukenbrock H, Mussmann R, Geese M, Ferandin Y, Lozach O, Lemcke T, Kegel S, Lomow A, Burk U, Dohrmann C, Meijer L, Austen M, Kunick C. 9-cyano-l-azapaullone (cazpaullone), a glycogen synthase kinase-3 (GSK-3) inhibitor activating pancreatic beta cell protection and replication. J Med Chem.2008;51(7):2196-2207.
82.吴薇,罗小平.糖原合成酶激酶3抑制剂降低胰岛β细胞凋亡.中华内分泌代谢杂志.2008；24(1)：48-49.
83. Watcharasit P, Bijur GN, Zmijewski JW, Song L, Zmijewska A, Chen X, Johnson GV, Jope RS. Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage. Proc Natl Acad Sci U S A.2002;99(12):7951-7955.
84. Reiter CE, Wu X, Sandirasegarane L, Nakamura M, Gilbert KA, Singh RS, Fort PE, Antonetti DA, Gardner TW. Diabetes reduces basal retinal insulin receptor signaling:reversal with systemic and local insulin. Diabetes.2006;55(4):1148-1156.
1. Abbott A. And now for the proteome. Nature.2001;409(6822):747.
2. Fields S. Proteomics. Proteomies in genomeland. Science.2001;291 (5507):1221-1224.
3. Wasinger VC, Cordwell SJ, Ccrpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humph cry-Smith I. Progress with gene-product mapping of the Mollicutes:Mycoplasma genitalium. Electrophoresis.1995; 16(7):1090-1094.
4. Wilkins MR, Appel RD, Van Eyk JE, Chung MC, Gorg A, Hecker M, Huber LA, Langen H, Link AJ, Paik YK, Patterson SD, Pennington SR, Rabilloud T, Simpson RJ, Weiss W, Dunn MJ. Guidelines for the next 10 years of proteomics. Proteomics.2006;6(1):4-8.
5. Smith R. Two-dimensional electrophoresis:an overview. Methods Mol Biol.2009;519:1-16.
6. Van Riper SK, de Jong EP, Carlis JV, Griffin TJ. Mass spectrometry-based proteomics:basic principles and emerging technologies and directions. Adv Exp Med Biol.2013;99():1-35.
7. Roepstorff P. Mass spectrometry based proteomics, background, status and future needs. Protein Cell.2012;3(9):641-647.
8.邱洁,高海青.蛋白质组学技术在心血管疾病中应用.中华老年医学杂志2005；24(2)：152-155.
9. Becker CH, Bern M. Recent developments in quantitative proteomics. Mutat Res.2011;722(2):171-182.
10. Foth BJ, Zhang N, Mok S, Preiser PR, Bozdech Z. Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites. Genome Biol.2008;9(12):R177.
11.曹冬,张养军,钱小红.基于生物质谱的蛋白质组学绝对定量方法研究进展. 质谱学报.2008；29(3)：185-191.
12. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ. Multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics.2004;3(12):1154-1169.
13. Karp NA, Huber W, Sadowski PG, Charles PD, Hester SV, Lilley KS. Addressing accuracy and precision issues in iTRAQ quantitation. Mol Cell Proteomics.2010;9(9):1885-1897.
14. Evans C, Noirel J, Ow SY, Salim M, Pereira-Medrano AG, Couto N, Pandhal J, Smith D, Pham TK, Karunakaran E, Zou X, Biggs CA, Wright PC. An insight into iTRAQ:where do we stand now? Anal Bioanal Chem.2012;404(4):1011-1027.
15. Yu F, Li BY, Li XL, Cai Q, Zhang Z, Cheng M, Yin M, Wang JF, Zhang JH, Lu WD, Zhou RH, Gao HQ. Proteomic analysis of aorta and protective effects of grape seed procyanidin B2 in db/db mice reveal a critical role of milk fat globule epidermal growth factor-8 in diabetic arterial damage. PLoS One.2012;7(12):e52541.
16. Magharious M, D'Onofrio PM, Hollander A, Zhu P, Chen J, Koeberle PD. Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush. J Proteome Res.2011;10(8):3344-3362.
17. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet.2010;376(9735):124-136.
18. Cheung N, Wong TY. Diabetic retinopathy and systemic vascular complications. Prog Retin Eye Res.2008;27(2):161-176.
19. Ehrenkranz JR, Lewis NG, Kahn CR, Roth J. Pholorizin:a review. Diabetes Metab Res Rev,2005;21(1):31-38.
20. Shen L, You BA, Gao HQ, Li BY, Yu F, Pei F. Effects of phlorizin on vascular complications in diabetes db/db mice. Chin Med J (Engl). 2012;125(20):3692-3696.
21. Lu WD, Li BY, Yu F, Cai Q, Zhang Z, Yin M, Gao HQ.Quantitative proteomics study on the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice.Mol Med Rep.2012;5(5):1285-1294.
22. Chakravarti B, Mallik B, Chakravarti DN. Proteomics and systems biology: application in drug discovery and development. Methods Mol Biol.2010;662:3-28.
23. Sanchez JC, Converset V, Nolan A, Schmid G, Wang S, Heller M, Sennitt MV, Hochstrasser DF, Cawthorne MA. Effect of rosiglitazone on the differential expression of obesity and insulin resistance associated proteins in lep/lep mice. Proteomics.2003;3(8):1500-1520.
24. Sanchez JC, Converset V, Nolan A, Schmid G, Wang S, Heller M, Sennitt MV, Hochstrasser DF, Cawthorne MA. Effect of rosiglitazone on the differential expression of diabetes-associated proteins in pancreatic islets of C57B1/6 lep/lep mice. Mol Cell Proteomics.2002;1(7):509-516.
25. Schirle M, Bantscheff M, Kuster B. Mass spectrometry-based proteomics in preclinical drug discovery. Chem Biol.2012;19(1):72-84.
26. Drewes G. Chemical proteomics in drug discovery. Methods Mol Biol.2012;803:15-21.
27. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods.2009;6(5):359-362.
28. Gene Ontology Consortium.The Gene Ontology:enhancements for 2011. Nucleic Acids Res.2012;40(Database issue):D559-564.
29. Becker CH. Bern M. Recent developments in quantitative proteomics. Mutat Res.2011；722(2):171-182.
30. Elliott MH, Smith DS， Parker CE， Borchers C. Current trends in quantitative proteomics. J Mass Spectrom.2009；44(12):1637-1660.
31. Kolla V, Jeno P, Moes S, Tercanli S, Lapaire O, Choolani M,Hahn S. Quantitative proteomics analysis of maternal plasma in Down syndrome pregnancies using isobaric tagging reagent (iTRAQ). J Biomed Biotechnol.2010;2010:952047.
32. Bantscheff M, Kuster B. Quantitative mass spectrometry in proteomics. Anal Bioanal Chem.2012;404(4):937-938.
33.李伟.iTRAQ多重化学标记串联质谱技术在比较蛋白质组学中的应用.生命的化学.2006；26(5)：453-456.
34. Xi J, Farjo R, Yoshida S, Kern TS, Swaroop A, Andley UP. A comprehensive analysis of the expression of crystallins in mouse retina. Mol Vis. 2003;9:410-419.
35.叶鸿飞,缪爱珠,卢奕.蛋白质组学技术用于晶状体蛋白的研究进展.中华眼科杂志.2012；48(10)：952-955.
36. Andley UP. Crystallins in the eye:Function and pathology. Prog Retin Eye Res.2007;26(1):78-98.
37.张民英,吴晓燕,王亚东,等.αA晶体蛋白在2型糖尿病大鼠神经视网膜的异常表达.中华眼底病杂志.2010；26(2)：160-164.
38. Fort PE, Lampi KJ. New focus on alpha-crystallins in retinal neurodegenerative diseases. Exp Eye Res.2011;92(2):98-103.
39. Whiston EA, Sugi N, Kamradt MC, Sack C, Heimer SR, Engelbert M, Wawrousek EF, Gilmore MS, Ksander BR, Gregory MS. AlphaB-crystallin protects retinal tissue during Staphylococcus aureus-induced endophthalmitis. Infect Immun.2008;76(4):1781-1790.
40. Yaung J, Kannan R, Wawrousek EF, Spee C, Sreekumar PG, Hinton DR. Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia. Exp Eye Res.2008;86(2):355-365.
41. VanGuilder HD, Bixler GV, Kutzler L, Brucklacher RM, Bronson SK, Kimball SR, Freeman.Multi-modal proteomic analysis of retinal protein expression alterati ons in a rat model of diabetic retinopathy. WM. PLoS One.2011;6(1):e 16271.
42. Li M, Ma YB, Gao HQ, Li BY, Cheng M, Xu L, Li XL, Li XH. A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats. Chin Med J (Engl). 2008;121(24):2544-2552.
43. Wang K, Cheng C. Li L, Liu H, Huang Q, Xia CH, Yao K, Sun P, Horwitz J, Gong X. Gammad-crystallin associated protein aggregation and lens fiber cell denucleation. Invest Ophthalmol Vis Sci.2007;48(8):3719-3728.
44. Zhang C, Gehlbach P, Gongora C, Cano M, Fariss R, Hose S, Nath A, Green WR, Goldberg MF, Zigler JS, Jr., Sinha D. A potential role For beta-and gamma-crystallins in the vascular remodeling of the eye. Dev Dyn.2005;234(1):36-47.
45. Zhan X, Du Y, Crabb JS, Gu X, Kern TS, Crabb JW. Targets of tyrosine nitration in diabetic rat retina. Mol Cell Proteomics.2008;7(5):864-874.
46. Fort PE, Freeman WM, Losiewicz MK, Singh RS, Gardner TW. The retinal proteome in experimental diabetic retinopathy:Up-regulation of crystallins and reversal by systemic and periocular insulin. Mol Cell Proteomics. 2009;8(4):767-779.
47. Lillig CH, Berndt C, I lolmgren A. Glutaredoxin systems. Biochim Biophys Acta. 2008; 1780(11):1304-1317.
48.李民,冯银刚,高杨.谷氧化蛋白系统及其对细胞氧化还原态势的调控.生物物理学报.2007；23(5)：343-350.
49.张春景,于海涛,邹朝霞,等.谷氧化蛋白的生物学活性及其与人类病的关系.生命的化学.2006；26(2)：163-165.
50. Malik G, Nagy N, Ho YS, Maulik N, Das DK. Role of glutaredoxin-1 in cardioprotection:An insight with glrxl transgenic and knockout animals. J Mol Cell Cardiol.2008;44(2):261-269.
51. Murata H, Ihara Y, Nakamura H, Yodoi J, Sumikawa K, Kondo T. Glutaredoxin exerts an antiapoptotic effect by regulating the redox slate of akt. J Biol Chem. 2003;278(50):50226-50233.
52. Lekli I. Mukherjee S, Ray 1). Gurusamy N, Kim YH, Tosaki A, Kngelman RM, Ho YS, Das DK. Functional recovery of diabetic mouse hearts by glutaredoxin-1 gene therapy:Role of akt-foxo-signaling network. Gene Ther. 2010;17(4):478-485.
53. Wu H. Xing K, Lou MF. Glutaredoxin 2 prevents h(2)o(2)-induced cell apoptosis by protecting complex i activity in the mitochondria. Biochim Biophys Acta. 2010;1797(10):1705-1715.
54. Takagi Y, Nakamura T, Nishiyama A, Nozaki K, Tanaka T, Hashimoto N, Yodoi J. Localization of glutaredoxin (thioltransferase) in the rat brain and possible functional implications during focal ischemia. Biochem Biophys Res Commun. 1999;258(2):390-394.
55. Peltoniemi MJ, Rytila PH, Harju TH, Soini YM, Salmenkivi KM, Ruddock LW, Kinnula VL. Modulation of glutaredoxin in the lung and sputum of cigarette smokers and chronic obstructive pulmonary disease. Respir Res.2006;7:133.
56. Babichev Y, Isakov N. Tyrosine phosphorylation of PICOT and its translocation to the nucleus in response of human T cells to oxidative stress. Adv Exp Med Biol.2001;495:41-45.
57. Yun N, Kim C, Cha H, Park WJ, Shibayama H. Park IS, Oh YJ. Caspase-3-mediated cleavage of PICOT in apoptosis. Biochem Biophys Res Commun.2013;432(3):533-538.
58. Zhang SX, Garcia-Gras E, Wycuff DR, Marriot SJ, Kadeer N, Yu W, Olson EN, Garry DJ, Parmacek MS, Schwartz RJ. Identification of direct serum-response factor gene targets during me2so-induced p19 cardiac cell differentiation. J Biol Chem.2005;280(19):19115-19126.
59. Jeong D, Cha H, Kim E, Kang M, Yang DK, Kim JM, Yoon PO, Oh JG, Bernecker OY, Sakata S, Le TT, Cui L, Lee YH, Kim do H, Woo SH, Liao R, Hajjar RJ, Park WJ. Picot inhibits cardiac hypertrophy and enhances ventricular function and cardiomyocyte contractility. Circ Res.2006;99(3):307-314.
60. Oh JG, Jeong D, Cha H, Kim JM, Lifirsu E, Kim J, Yang DK, Park CS, Kho C, Park S, Yoo YJ, Kim do H, Kim J, Hajjar RJ, Park WJ. PICOT increases cardiac contractility by inhibiting PKCζ, activity. J Mol Cell Cardiol.2012;53(1):53-63.
61. Cha H, Kim JM, Oh JG, Jeong MH, Park CS, Park J, Jeong HJ, Park BK, Lee YH, Jeong D, Yang DK, Bernecker OY, Kim do H, Hajjar RJ, Park WJ. PICOT is a critical regulator of cardiac hypertrophy and cardiomyocyte contractility. J Mol Cell Cardiol.2008;45(6):796-803.
62. Samarel AM. PICOT:a multidomain scaffolding inhibitor of hypertrophic signal transaction. Circ Res.2008 Mar 28;102(6):625-627.
63. Jeong D, Kim JM, Cha H, Oh JG, Park J, Yun SH, Ju ES, Jeon ES, Hajjar RJ, Park WJ. PICOT attenuates eardiac hypertrophy by disrupting calcineurin-NFAT signaling. Circ Res.2008; 102(6):711-719.
64. Pickart CM. Back to the future with ubiquitin. Cell.2004; 116(2):181-190.
65. Goldberg AL. Protein degradation and protection against misfolded or damaged proteins. Nature.2003;426(6968):895-899.
66. Fernandes R, Ramalho J, Pereira P. Oxidative stress upregulates ubiquitin proteasome pathway in retinal endothelial cells. Mol Vis.2006; 12:1526-1535.
67. Mirza S, Plafker KS, Aston C, Plafker SM. Expression and distribution of the class iii ubiquitin-conjugating enzymes in the retina. Mol Vis. 2010;16:2425-2437.
68. Adachi-Uehara N, Kato M, Nimura Y, Seki N, Ishihara A, Matsumoto E, Iwase K, Ohtsuka S, Kodama H, Mizota A, Yamamoto S, Adachi-Usami E, Takiguchi M.Up-regulation of genes for oxidative phosphorylation and protein turnover in diabetic mouse retina.Exp Eye Res.2006;83(4):849-857.
69.刘源劼,陈国庆,卢辰,周鸿鹰,梅妍,羊惠君.泛素蛋白酶系统在四氧嘧啶诱导的糖尿病大鼠视网膜中的表达.解剖学杂志.2008；31(3)：372-375.
70. Komatsu M, Chiba T, Tatsumi K, Iemura S, Tanida I, Okazaki N, Ucno T， Kominami E, Natsume T, Tanaka K. A novel protein-conjugating system for ufml, a ubiquitin-fold modiiler. EMBO J.2004:23(9):1977-1986.
71. Azfer A, Niu J, Rogers LM, Adamski FM, Kolattukudy PE. Activation of endoplasmic reticulum stress response during the development of ischemic heart disease. Am J Physiol Heart Circ Physiol.2006:291 (3):H1411-1420.
72. Lu H, Yang Y, Allister EM, Wijesekara N, Wheeler MB. The identification of potential factors associated with the development of type 2 diabetes:a quantitative proteomics approach. Mol Cell Proteomics.2008;7(8):1434-1451.
73. Groll M, Ditzel L. Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R. Structure of 20s proteasome from yeast at 2.4 a resolution. Nature. 1997;386(6624):463-471.
74. Pickering AM, Koop AL, Teoh CY, Ermak G, Grune T, Davies KJ. The immunoproteasome, the 20s proteasome and the pa28alphabeta proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem J. 2010;432(2):585-594.
75. Abu El-Asrar AM, Missotten L, Geboes K. Expression of hypoxia-inducible factor-1 alpha and the protein products of its target genes in diabetic fibrovascular epiretinal membranes. Br J Ophthalmol.2007;91(6):822-826.
76. Adamis AP. Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol. 2002;86(4):363-365.
77. Joussen AM, Poulaki V, Le ML, Koizumi K, Esser C, Janicki H, Schraermeyer U, Kociok N, Fauser S, Kirchhof B, Kern TS. Adamis AP. A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J. 2004;18(12):1450-1452.
78. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science.1997;275(5302):964-967.
79. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nature medicine.2002;8(6):607-612.
80. Otani A, Kinder K, Ewalt K, Otero FJ, Schimmel P, Friedlander M. Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis. Nature medicine.2002;8(9):1004-1010.
81. Lee IG, Chae SL, Kim JC. Involvement of circulating endothelial progenitor cells and vasculogenic factors in the pathogenesis of diabetic retinopathy. Eye (Lond). 2006;20(5):546-552.
82. Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Shimada T, Oike Y, Imaizumi T. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation.2001;103(23):2776-2779.
83. Fadini GP, Sartore S, Baesso I, Lenzi M, Agostini C, Tiengo A, Avogaro A. Endothelial progenitor cells and the diabetic paradox. Diabetes Care. 2006;29(3):714-716.
84. Caballero S, Sengupta N, Aizal A, Chang KH, Li Calzi S, Guberski DL, Kern TS, Grant MB. Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells. Diabetes.2007;56(4):960-967.
85. Taylor CT, Moncada S. Nitric oxide, eytochrome c oxidase, and the cellular response to hypoxia. Arterioscler Thromb Vase Biol.2010;30(4):643-647.
86. Hansson HA. A histochemical study of cellular reactions in rat retina transiently damaged by visible light. Exp Eye Res.1971:12(3):270-274.
87. Caldwell RB, Slapnick SM. Increased eytochrome oxidase activity in the diabetic rat retinal pigment epithelium. Invest Ophthalmol Vis Sci.1989;30(4):591-599.
88. Wang AG, Lee CM, Wang YC, Lin CH, Fann MJ. Up-regulation of cytochrome oxidase in the retina following optic nerve injury. Exp Eye Res. 2002;74(5):651-659.
89.赵欣,蒲小平.蛋白质组学在药物研究中的应用.国药药理学通报.2009；25(8)：988-991.
90. Chan JN, Nislow C, Emili A. Recent advances and method development for drug target identification. Trends Pharmacol Sci.2010;31(2):82-88.
2. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 2004;27(5):1047-1053.
3. Nazimek-Siewniak B, Moczulski D, Grzeszczak W. Risk of macrovascular and microvascular complications in Type 2 diabetes:results of longitudinal study design. J Diabetes Complications.2002; 16(4):271-276.
4. Xie XW, Xu L, Jonas JB, Wang YX. Prevalence of diabetic retinopathy among subjects with known diabetes in China:the Beijing Eye Study, Eur J Ophthalmol. 2009;19(1):91-99.
5. Fong DS, Aiello L, Gardner TW, King GL, Blankenship G, Cavallerano JD, Ferris FL,3rd, Klein R. Retinopathy in diabetes. Diabetes Care.2004;27 Suppl 1:S84-87.
6. Congdon NG, Friedman DS, Lietman T. Important causes of visual impairment in the world today. JAMA.2003;290(15):2057-2060.
7. Cheung N, Wong TY. Diabetic retinopathy and systemic vascular complications. Prog Retin Eye Res.2008:27(2):161-176.
8. Zhang X, Saaddine JB, Chou CF, Cotch MF, Cheng YJ. Geiss LS. Gregg FW, Albright AL, Klein BE, Klein R. Prevalence of diabetic retinopathy in the united states,2005-2008. JAMA.2010;304(6):649-656.
9. Heintz E, Wirehn AB, Peebo BB, Rosenqvist U, Levin LA. Prevalence and healthcare costs of diabetic retinopathy:a population-based register study in Sweden. Diabetologia.2010;53(10):2147-2154.
10.纪立农.对2型糖尿病新的大型临床试验结果的解读和分析.中国糖尿病杂志.2008；16(11)：642-647.
11.彭晓燕,张永鹏.糖尿病视网膜病变的治疗趋势.眼科.2011；11(4)：217-221.
12. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature.2001;414(6865):813-820.
13. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet.2010;376(9735):124-136.
14. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes.1998;47(6):859-866.
15. Hammes HP, Alt A, Niwa T, Clausen JT, Bretzel RG, Brownlee M, Schleicher ED. Differential accumulation of advanced glycation end products in the course of diabetic retinopathy. Diabetologia.1999;42(6):728-736.
16. Brownlee M. The pathobiology of diabetic complications:a unifying mechanism. Diabetes.2005;54(6):1615-1625.
17. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res.2010;107(9):1058-1070.
18. Barber AJ. A new view of diabetic retinopathy:a neurodegenerative disease of the eye. Prog Neuropsychopharmacol Biol Psychiatry.2003;27(2):283-290.
19. Hammes HP, Federoff HJ, Brownlee M. Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes. Mol Med.1995;1(5):527-534.
20. Antonetti DA, Barber AJ, Bronson SK, Freeman WM, Gardner TW, Jefferson LS, Kester M, Kimball SR, Krady JK, LaNoue KF, Norbury CC, Quinn PG,Sandirasegarane L, Simpson IA; JDRF Diabetic Retinopathy Center Group. Diabetic retinopathy:seeing beyond glucose-induced microvascular disease. Diabetes.2006;55(9):2401-2411.
21. Barber AJ, Gardner TW, Abcouwer SF.The significance of vascular and neural apoptosis to the pathology of diabetic retinopathy. Invest Ophthalmol Vis Sci.2011;52(2):1156-1163.
22. Kowluru RA, Kanwar M. Oxidative stress and the development of diabetic retinopathy:contributory role of matrix metalloproteinase-2. Free Radic Biol Med.2009;46(12):1677-1685.
23. Silva KC, Rosales MA, Biswas SK, Lopes de Faria JB, Lopes de Faria JM. Diabetic retinal neurodegeneration is associated with mitochondrial oxidative stress and is improved by an angiotensin receptor blocker in a model combining hypertension and diabetes. Diabetes.2009;58(6):1382-1390.
24. Kowluru RA, Koppolu P. Diabetes-induced activation of caspase-3 in retina: effect of antioxidant therapy. Free Radic Res.2002;36(9):993-999.
25. Kowluru RA, Odenbach S. Effect of long-term administration of alpha-lipoic acid on retinal capillary cell death and the development of retinopathy in diabetic rats. Diabetes.2004;53(12):3233-3238.
26. Sasaki M, Ozawa Y, Kurihara T, Kubota S, Yuki K, Noda K, Kobayashi S, Ishida S, Tsubota K. Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes. Diabetologia.2010;53(5):971-979.
27. Arnal E, Miranda M, Johnsen-Soriano S, Alvarez-Nolting R, Diaz-Llopis M, Araiz J, Cervera E, Bosch-Morcll F, Romero FJ. Beneficial effect of docosahexanoic acid and lutein on retinal structural, metabolic, and functional abnormalities in diabetic rats. Curr Eye Res.2009;34(11):928-938.
28. Millen AE, Gruber M, Klein R, Klein BE, Palta M, Mares JA. Relations of serum ascorbic acid and alpha-tocopherol to diabetic retinopathy in the Third National Health and Nutrition Examination Survey. Am J Epidemiol.2003; 158(3):225-233.
29. Millen AE, Klein R, Folsom AR, Stevens J, Palta M, Mares JA. Relation between intake of vitamins C and E and risk of diabetic retinopathy in the Atherosclerosis Risk in Communities Study. Am J Clin Nutr.2004:79(5):865-873.
30. Madsen-Bouterse SA, Kowluru RA. Oxidative stress and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Rev Endoer Metab Disord.2008;9(4):315-327.
31. Gosch C, Halbwirth H, Stich K. Phloridzin:biosynthesis, distribution and physiological relevance in plant. Phytochemistry.2010:71(8-9):838-843.
32. Ridgway T, O'Reilly J, West G, Tucker G, Wiseman H. Antixoxidant action of novel derivatives of the apple-derived flavonoid phloridzin compared to oestrogen; relevance to potential cardioprotective action. Biochen Soc Trans. 1997; 25(1):106S.
33. Lee KW, Kim YJ, Kim DO, Lee HJ, Lee CY. Major phenolics in apple and their contribution to the total antioxidant capacity. J Agric Food Chem.2003;51(22): 6516-6520.
34. Rezk BM, Haenen GR, van der Vijgh WJ, Bast A. The antioxidant actitivity of phloretin:the disclosure of new antioxidant pharmacophore in flavonoids. Biochem Biophys Res Commun.2002; 295(1):9-13.
35. Hong EG, Jung DY, Ko HJ, Zhang Z, Ma Z, Jun JY, Kim JH, Sumner AD, Vary TC, Gardner TW, Bronson SK, Kim JK. Nonobese, insulin-deficient ins2Akita mice develop type 2 diabetes phenotypes including insulin resistance and cardiac remodeling. Am J Physiol Endocrinol Metab.2007;293(6):E 1687-1696.
36. Shen L, You BA, Gao HQ, Li BY. Yu F. Pei F. Effects of phlorizin on vascular complications in diabetes db/db mice. Chin Med J (Engl). 2012;125(20):3692-3696.
37. Malatiali S, Francis I, Barac-Nieto M. Phlorizin prevents glomerular hyperfiltration but not hypertrophy in diabetic rats. Exp Diabetes Res. 2008;2008:305403.
38. Arakawa K, Ishihara T, Oku A, Nawano M, Ueta K, Kitamura K, Matsumoto M, Saito A. Improved diabetic syndrome in c57bl/ksj-db/db mice by oral administration of the Na(+)-glucose cotransporter inhibitor t-1095. Br J Pharmacol.2001;132(2):578-586.
39. Wakisaka M, Yoshinari M, Yamamoto M, Nakamura S, Asano T, Himeno T, Ichikawa K, Doi Y, Fujishima M. Na+-dependent glucose uptake and collagen synthesis by cultured bovine retinal pericytes. Biochim Biophys Acta. 1997; 1362(1):87-96.
40. Wakisaka M, Yoshinari M, Asano T, Iino K, Nakamura S, Takata Y, Fujishima M. Normalization of glucose entry under the high glucose condition by phlorizin attenuates the high glucose-induced morphological and functional changes of cultured bovine retinal pericytes. Biochim Biophys Acta.1999; 1453(1):83-91.
41. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP. Evidence that the diabetes gene encodes the leptin receptor:identification of a mutation in the leptin receptor gene in db/db mice. Cell.1996;84(3):491-495.
42.唐慧青,陈立新,染磊,等.Ⅱ型糖尿病模型C57BL/KsJ-db/db小鼠的生物学特征研究.实验动物与比较医学.2011；31(1)：62-65.
43. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol.1992;119(3):493-501.
44. Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, Jerums G. Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes.1991;40(10):1328-1334.
45. Joussen AM, Doehmen S, Le ML, Koizumi K, Radetzky S, Krohne TU, Poulaki V, Semkova I, Kociok N. TNF-alpha mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations. Mol Vis.2009; 15:1418-1428.
46. Xie XW, Xu L, Wang YX, Jonas JB. Prevalence and associated factors of diabetic retinopathy. The Beijing Eye Study 2006. Grades Arch Clin Exp Ophthalmol.2008;246(11):1519-1526.
47. Wang FH, Liang YB, Zhang F,Wang JJ, Wei WB, Tao QS, Sun LP, Friedman DS, Wang NL, Wong TY. Prevalence of diabetic retinopathy in rural China:the Handan Eye Study. Ophthalmology.2009;116(3):461-467.
48. White NH, Sun W, Cleary PA, Tamborlane WV, Danis RP, Hainsworth DP, Davis MD; DCCT-EDIC Research Group. Effect of prior intensive therapy in type 1 diabetes on 10-year progression of retinopathy in the DCCT/EDIC:comparison of adults and adolescents. Diabetes.2010;59(5):1244-1253.
49. Wong TY, Liew G, Tapp RJ, Schmidt Mi, Wang JJ, Mitchell P,Klein R,Klein BE, Zimmet P, Shaw J. Relation between fasting glucose and retinopathy for diagnosis of diabetes:three population-based cross-sectional studies. Lancet.2008;371(9614):736-743.
50.许迅,刘堃.统一机制学说和慢性炎症学说：糖尿病视网膜病变基础研究的新方向.中华眼底病杂志.2008；24(4)：237-239.
51. Gardner TW, Abcouwer SF, Barber AJ, Jackson GR. An integrated approach to diabetic retinopathy research. Arch Ophthalmol.2011;129(2):230-235.
52. Kowluru RA, Chan PS. Oxidative stress and diabetic retinopathy. Exp Diabetes Res.2007;2007:43603.
53.王泓,许迅.氧化应激反应与糖尿病视网膜病变.临床眼科杂志.2009；17(5)：474-477.
54. Hummel KP, Dickie MM, Coleman DL. Diabetes, a new mutation in the mouse. Science.1966;153(3740):1127-1128.
55. Sharma K, McCue P, Dunn SR. Diabetic kidney disease in the db/db mouse. Am J Physiol Renal Physiol.2003;284(6):F1138-1144.
56. Shoham S, Bejar C, Kovalev E, Schorer-Apelbaum D, Weinstock M. Ladostigil prevents gliosis, oxidative-nitrative stress and memory deficits induced by intracerebroventricular injection of streptozotocin in rats. Neuropharmacology.2007;52(3):836-843.
57. Barber AJ, Antonetti DA, Gardner TW. Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes. The Penn State Retina Research Group. Invest Ophthalmol Vis Sci 2000;41(11):3561-3568.
58. Ehrenkranz JR, Lewis NG, Kahn CR, Roth J. Pholorizin:a review. Diabetes Metab Res Rev,2005;21(1):31-38.
59. Hertog MG, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F, Giampaoli S, Jansen A, Menotti A, Nedeljkovic S, et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch Intern Med.1995;155(4):381-386.
60. Vasantha Rupasinghe HP, Yasmin A. Inhibition of oxidation of aqueous emulsions of omega-3 fatty acids and fish oil by phloretin and phloridzin. Molecules.2010;15(1):251-257.
61. Xiang L, Sun K, Lu J, Weng Y, Taoka A, Sakagami Y, Qi J. Anti-aging effects of phloridzin, an apple polyphenol, on yeast via the SOD and Sir2 genes. Biosci Biotech Bioch,2011;75(5):854-858.
62. Oresajo C, Stephens T, Hino PD, Law RM, Yatskayer M, Foltis P, Pillai S, Pinnell SR. Protective effects of a topical antioxidant mixture containing vitamin C ferulic acid, and phloretin against ultraviolet-induced photodamage in human skin. J Cosmet Dermatol.2008;7(4):290-297.
63. Abdul-Ghahi MA, DeFronzo R A. Inhibition of renal glucose absorption:a novel strategy for achieving glucose control in type 2 diabetes mellitus. Endocr Pract. 2008;14(6):782-790.
64. Lu WD, Li BY, Yu F, Cai Q, Zhang Z, Yin M, Gao HQ. Quantitative proteomics study on the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice. Mol Med Report.2012；5(5):1285-1294.
65. Bradford BJ, Allen MS. Phlorizin induces lipolysis and alters meal patterns in both early- and late-lactation dairy cows. J Dairy Sci.2007;90(4):1810-1815.
66. Thomas MC. Advanced glycation end products. Contrib Nephrol.2011；170:66-74.
67. Yamagishi S, Maeda S, Matsui T, Ueda S, Fukami K, Okuda S. Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications indiabetes. Biochim Biophys Acta.2012;1820(5):663-671.
68. Hernandez C, Simo R. Neuroprotection in diabetic retinopathy. Curr Diab Rep.2012; 12(4):329-337.
69. Carrasco E, Hernandez C, de Torres I, Farres J, Simo R. Lowered eortistatin expression is an early event in the human diabetic retina and is associated with apoptosis and glial activation. Mol Vis.2008;14:1496-1502.
70. Kmbi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem.1980; 107(2):519-27.
71. Jacobs KM. Bhave SR, Ferraro DJ, Jaboin JJ, Hallahan DE, Thotala D. GSK-3β: A Bifunctional Role in Cell Death Pathways. Int J Cell Biol.2012;2012:930710.
72..lope RS, Roh MS. Glycogen synthase kinase-3 (GSK3) in psychiatric diseases and therapeutic interventions. Curr Drug Targets.2006;7(11):1421-1434.
73. Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci.2004;29(2):95-102.
74. Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A. Glycogen synthase kinase 3: more than a namesake. Br J Pharmacol.2009;156(6):885-898.
75. Frame S, Cohen P, Biondi RM. A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell.2001;7(6):1321-1327.
76. Frame S, Zheleva D. Targeting glycogen synthase kinase-3 in insulin signalling. Expert Opin Ther Targets.2006;10(3):429-444.
77. Pearce NJ, Arch JR, Clapham JC, Coghlan MP, Corcoran SL, Lister CA, Llano A, Moore GB, Murphy GJ, Smith SA, Taylor CM, Yates JW, Morrison AD, Harper AJ, Roxbee-Cox L, Abuin A, Wargent E, Holder JC. Development of glucose intolerance in male transgenic mice overexpressing human glycogen synthase kinase-3beta on a muscle-specific promoter. Metabolism.2004;53(10):1322-1330.
78. Dokken BB, Henriksen EJ. Chronic selective glycogen synthase kinase-3 inhibition enhances glucose disposal and muscle insulin action in prediabetic obese Zucker rats. Am J Physiol Endocrinol Metab.2006;291(2):E207-213.
79. Lajoie C, Calderone A, Trudeau F, Lavoie N, Massicotte G, Gagnon S, Beliveau L. Exercise training attenuated the PKB and GSK-3 dephosphorylation in the myocardium of ZDF rats. J Appl Physiol.2004;96(5):1606-1612.
80. Dokken BB, Sloniger JA, Henriksen EJ. Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle. Am J Physiol Endocrinol Metab.2005;288(6):E1188-1194.
81. Stukenbrock H, Mussmann R, Geese M, Ferandin Y, Lozach O, Lemcke T, Kegel S, Lomow A, Burk U, Dohrmann C, Meijer L, Austen M, Kunick C. 9-cyano-l-azapaullone (cazpaullone), a glycogen synthase kinase-3 (GSK-3) inhibitor activating pancreatic beta cell protection and replication. J Med Chem.2008;51(7):2196-2207.
82.吴薇,罗小平.糖原合成酶激酶3抑制剂降低胰岛β细胞凋亡.中华内分泌代谢杂志.2008；24(1)：48-49.
83. Watcharasit P, Bijur GN, Zmijewski JW, Song L, Zmijewska A, Chen X, Johnson GV, Jope RS. Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage. Proc Natl Acad Sci U S A.2002;99(12):7951-7955.
84. Reiter CE, Wu X, Sandirasegarane L, Nakamura M, Gilbert KA, Singh RS, Fort PE, Antonetti DA, Gardner TW. Diabetes reduces basal retinal insulin receptor signaling:reversal with systemic and local insulin. Diabetes.2006;55(4):1148-1156.
1. Abbott A. And now for the proteome. Nature.2001;409(6822):747.
2. Fields S. Proteomics. Proteomies in genomeland. Science.2001;291 (5507):1221-1224.
3. Wasinger VC, Cordwell SJ, Ccrpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humph cry-Smith I. Progress with gene-product mapping of the Mollicutes:Mycoplasma genitalium. Electrophoresis.1995; 16(7):1090-1094.
4. Wilkins MR, Appel RD, Van Eyk JE, Chung MC, Gorg A, Hecker M, Huber LA, Langen H, Link AJ, Paik YK, Patterson SD, Pennington SR, Rabilloud T, Simpson RJ, Weiss W, Dunn MJ. Guidelines for the next 10 years of proteomics. Proteomics.2006;6(1):4-8.
5. Smith R. Two-dimensional electrophoresis:an overview. Methods Mol Biol.2009;519:1-16.
6. Van Riper SK, de Jong EP, Carlis JV, Griffin TJ. Mass spectrometry-based proteomics:basic principles and emerging technologies and directions. Adv Exp Med Biol.2013;99():1-35.
7. Roepstorff P. Mass spectrometry based proteomics, background, status and future needs. Protein Cell.2012;3(9):641-647.
8.邱洁,高海青.蛋白质组学技术在心血管疾病中应用.中华老年医学杂志2005；24(2)：152-155.
9. Becker CH, Bern M. Recent developments in quantitative proteomics. Mutat Res.2011;722(2):171-182.
10. Foth BJ, Zhang N, Mok S, Preiser PR, Bozdech Z. Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites. Genome Biol.2008;9(12):R177.
11.曹冬,张养军,钱小红.基于生物质谱的蛋白质组学绝对定量方法研究进展. 质谱学报.2008；29(3)：185-191.
12. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ. Multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics.2004;3(12):1154-1169.
13. Karp NA, Huber W, Sadowski PG, Charles PD, Hester SV, Lilley KS. Addressing accuracy and precision issues in iTRAQ quantitation. Mol Cell Proteomics.2010;9(9):1885-1897.
14. Evans C, Noirel J, Ow SY, Salim M, Pereira-Medrano AG, Couto N, Pandhal J, Smith D, Pham TK, Karunakaran E, Zou X, Biggs CA, Wright PC. An insight into iTRAQ:where do we stand now? Anal Bioanal Chem.2012;404(4):1011-1027.
15. Yu F, Li BY, Li XL, Cai Q, Zhang Z, Cheng M, Yin M, Wang JF, Zhang JH, Lu WD, Zhou RH, Gao HQ. Proteomic analysis of aorta and protective effects of grape seed procyanidin B2 in db/db mice reveal a critical role of milk fat globule epidermal growth factor-8 in diabetic arterial damage. PLoS One.2012;7(12):e52541.
16. Magharious M, D'Onofrio PM, Hollander A, Zhu P, Chen J, Koeberle PD. Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush. J Proteome Res.2011;10(8):3344-3362.
17. Cheung N, Mitchell P, Wong TY. Diabetic retinopathy. Lancet.2010;376(9735):124-136.
18. Cheung N, Wong TY. Diabetic retinopathy and systemic vascular complications. Prog Retin Eye Res.2008;27(2):161-176.
19. Ehrenkranz JR, Lewis NG, Kahn CR, Roth J. Pholorizin:a review. Diabetes Metab Res Rev,2005;21(1):31-38.
20. Shen L, You BA, Gao HQ, Li BY, Yu F, Pei F. Effects of phlorizin on vascular complications in diabetes db/db mice. Chin Med J (Engl). 2012;125(20):3692-3696.
21. Lu WD, Li BY, Yu F, Cai Q, Zhang Z, Yin M, Gao HQ.Quantitative proteomics study on the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice.Mol Med Rep.2012;5(5):1285-1294.
22. Chakravarti B, Mallik B, Chakravarti DN. Proteomics and systems biology: application in drug discovery and development. Methods Mol Biol.2010;662:3-28.
23. Sanchez JC, Converset V, Nolan A, Schmid G, Wang S, Heller M, Sennitt MV, Hochstrasser DF, Cawthorne MA. Effect of rosiglitazone on the differential expression of obesity and insulin resistance associated proteins in lep/lep mice. Proteomics.2003;3(8):1500-1520.
24. Sanchez JC, Converset V, Nolan A, Schmid G, Wang S, Heller M, Sennitt MV, Hochstrasser DF, Cawthorne MA. Effect of rosiglitazone on the differential expression of diabetes-associated proteins in pancreatic islets of C57B1/6 lep/lep mice. Mol Cell Proteomics.2002;1(7):509-516.
25. Schirle M, Bantscheff M, Kuster B. Mass spectrometry-based proteomics in preclinical drug discovery. Chem Biol.2012;19(1):72-84.
26. Drewes G. Chemical proteomics in drug discovery. Methods Mol Biol.2012;803:15-21.
27. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods.2009;6(5):359-362.
28. Gene Ontology Consortium.The Gene Ontology:enhancements for 2011. Nucleic Acids Res.2012;40(Database issue):D559-564.
29. Becker CH. Bern M. Recent developments in quantitative proteomics. Mutat Res.2011；722(2):171-182.
30. Elliott MH, Smith DS， Parker CE， Borchers C. Current trends in quantitative proteomics. J Mass Spectrom.2009；44(12):1637-1660.
31. Kolla V, Jeno P, Moes S, Tercanli S, Lapaire O, Choolani M,Hahn S. Quantitative proteomics analysis of maternal plasma in Down syndrome pregnancies using isobaric tagging reagent (iTRAQ). J Biomed Biotechnol.2010;2010:952047.
32. Bantscheff M, Kuster B. Quantitative mass spectrometry in proteomics. Anal Bioanal Chem.2012;404(4):937-938.
33.李伟.iTRAQ多重化学标记串联质谱技术在比较蛋白质组学中的应用.生命的化学.2006；26(5)：453-456.
34. Xi J, Farjo R, Yoshida S, Kern TS, Swaroop A, Andley UP. A comprehensive analysis of the expression of crystallins in mouse retina. Mol Vis. 2003;9:410-419.
35.叶鸿飞,缪爱珠,卢奕.蛋白质组学技术用于晶状体蛋白的研究进展.中华眼科杂志.2012；48(10)：952-955.
36. Andley UP. Crystallins in the eye:Function and pathology. Prog Retin Eye Res.2007;26(1):78-98.
37.张民英,吴晓燕,王亚东,等.αA晶体蛋白在2型糖尿病大鼠神经视网膜的异常表达.中华眼底病杂志.2010；26(2)：160-164.
38. Fort PE, Lampi KJ. New focus on alpha-crystallins in retinal neurodegenerative diseases. Exp Eye Res.2011;92(2):98-103.
39. Whiston EA, Sugi N, Kamradt MC, Sack C, Heimer SR, Engelbert M, Wawrousek EF, Gilmore MS, Ksander BR, Gregory MS. AlphaB-crystallin protects retinal tissue during Staphylococcus aureus-induced endophthalmitis. Infect Immun.2008;76(4):1781-1790.
40. Yaung J, Kannan R, Wawrousek EF, Spee C, Sreekumar PG, Hinton DR. Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia. Exp Eye Res.2008;86(2):355-365.
41. VanGuilder HD, Bixler GV, Kutzler L, Brucklacher RM, Bronson SK, Kimball SR, Freeman.Multi-modal proteomic analysis of retinal protein expression alterati ons in a rat model of diabetic retinopathy. WM. PLoS One.2011;6(1):e 16271.
42. Li M, Ma YB, Gao HQ, Li BY, Cheng M, Xu L, Li XL, Li XH. A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats. Chin Med J (Engl). 2008;121(24):2544-2552.
43. Wang K, Cheng C. Li L, Liu H, Huang Q, Xia CH, Yao K, Sun P, Horwitz J, Gong X. Gammad-crystallin associated protein aggregation and lens fiber cell denucleation. Invest Ophthalmol Vis Sci.2007;48(8):3719-3728.
44. Zhang C, Gehlbach P, Gongora C, Cano M, Fariss R, Hose S, Nath A, Green WR, Goldberg MF, Zigler JS, Jr., Sinha D. A potential role For beta-and gamma-crystallins in the vascular remodeling of the eye. Dev Dyn.2005;234(1):36-47.
45. Zhan X, Du Y, Crabb JS, Gu X, Kern TS, Crabb JW. Targets of tyrosine nitration in diabetic rat retina. Mol Cell Proteomics.2008;7(5):864-874.
46. Fort PE, Freeman WM, Losiewicz MK, Singh RS, Gardner TW. The retinal proteome in experimental diabetic retinopathy:Up-regulation of crystallins and reversal by systemic and periocular insulin. Mol Cell Proteomics. 2009;8(4):767-779.
47. Lillig CH, Berndt C, I lolmgren A. Glutaredoxin systems. Biochim Biophys Acta. 2008; 1780(11):1304-1317.
48.李民,冯银刚,高杨.谷氧化蛋白系统及其对细胞氧化还原态势的调控.生物物理学报.2007；23(5)：343-350.
49.张春景,于海涛,邹朝霞,等.谷氧化蛋白的生物学活性及其与人类病的关系.生命的化学.2006；26(2)：163-165.
50. Malik G, Nagy N, Ho YS, Maulik N, Das DK. Role of glutaredoxin-1 in cardioprotection:An insight with glrxl transgenic and knockout animals. J Mol Cell Cardiol.2008;44(2):261-269.
51. Murata H, Ihara Y, Nakamura H, Yodoi J, Sumikawa K, Kondo T. Glutaredoxin exerts an antiapoptotic effect by regulating the redox slate of akt. J Biol Chem. 2003;278(50):50226-50233.
52. Lekli I. Mukherjee S, Ray 1). Gurusamy N, Kim YH, Tosaki A, Kngelman RM, Ho YS, Das DK. Functional recovery of diabetic mouse hearts by glutaredoxin-1 gene therapy:Role of akt-foxo-signaling network. Gene Ther. 2010;17(4):478-485.
53. Wu H. Xing K, Lou MF. Glutaredoxin 2 prevents h(2)o(2)-induced cell apoptosis by protecting complex i activity in the mitochondria. Biochim Biophys Acta. 2010;1797(10):1705-1715.
54. Takagi Y, Nakamura T, Nishiyama A, Nozaki K, Tanaka T, Hashimoto N, Yodoi J. Localization of glutaredoxin (thioltransferase) in the rat brain and possible functional implications during focal ischemia. Biochem Biophys Res Commun. 1999;258(2):390-394.
55. Peltoniemi MJ, Rytila PH, Harju TH, Soini YM, Salmenkivi KM, Ruddock LW, Kinnula VL. Modulation of glutaredoxin in the lung and sputum of cigarette smokers and chronic obstructive pulmonary disease. Respir Res.2006;7:133.
56. Babichev Y, Isakov N. Tyrosine phosphorylation of PICOT and its translocation to the nucleus in response of human T cells to oxidative stress. Adv Exp Med Biol.2001;495:41-45.
57. Yun N, Kim C, Cha H, Park WJ, Shibayama H. Park IS, Oh YJ. Caspase-3-mediated cleavage of PICOT in apoptosis. Biochem Biophys Res Commun.2013;432(3):533-538.
58. Zhang SX, Garcia-Gras E, Wycuff DR, Marriot SJ, Kadeer N, Yu W, Olson EN, Garry DJ, Parmacek MS, Schwartz RJ. Identification of direct serum-response factor gene targets during me2so-induced p19 cardiac cell differentiation. J Biol Chem.2005;280(19):19115-19126.
59. Jeong D, Cha H, Kim E, Kang M, Yang DK, Kim JM, Yoon PO, Oh JG, Bernecker OY, Sakata S, Le TT, Cui L, Lee YH, Kim do H, Woo SH, Liao R, Hajjar RJ, Park WJ. Picot inhibits cardiac hypertrophy and enhances ventricular function and cardiomyocyte contractility. Circ Res.2006;99(3):307-314.
60. Oh JG, Jeong D, Cha H, Kim JM, Lifirsu E, Kim J, Yang DK, Park CS, Kho C, Park S, Yoo YJ, Kim do H, Kim J, Hajjar RJ, Park WJ. PICOT increases cardiac contractility by inhibiting PKCζ, activity. J Mol Cell Cardiol.2012;53(1):53-63.
61. Cha H, Kim JM, Oh JG, Jeong MH, Park CS, Park J, Jeong HJ, Park BK, Lee YH, Jeong D, Yang DK, Bernecker OY, Kim do H, Hajjar RJ, Park WJ. PICOT is a critical regulator of cardiac hypertrophy and cardiomyocyte contractility. J Mol Cell Cardiol.2008;45(6):796-803.
62. Samarel AM. PICOT:a multidomain scaffolding inhibitor of hypertrophic signal transaction. Circ Res.2008 Mar 28;102(6):625-627.
63. Jeong D, Kim JM, Cha H, Oh JG, Park J, Yun SH, Ju ES, Jeon ES, Hajjar RJ, Park WJ. PICOT attenuates eardiac hypertrophy by disrupting calcineurin-NFAT signaling. Circ Res.2008; 102(6):711-719.
64. Pickart CM. Back to the future with ubiquitin. Cell.2004; 116(2):181-190.
65. Goldberg AL. Protein degradation and protection against misfolded or damaged proteins. Nature.2003;426(6968):895-899.
66. Fernandes R, Ramalho J, Pereira P. Oxidative stress upregulates ubiquitin proteasome pathway in retinal endothelial cells. Mol Vis.2006; 12:1526-1535.
67. Mirza S, Plafker KS, Aston C, Plafker SM. Expression and distribution of the class iii ubiquitin-conjugating enzymes in the retina. Mol Vis. 2010;16:2425-2437.
68. Adachi-Uehara N, Kato M, Nimura Y, Seki N, Ishihara A, Matsumoto E, Iwase K, Ohtsuka S, Kodama H, Mizota A, Yamamoto S, Adachi-Usami E, Takiguchi M.Up-regulation of genes for oxidative phosphorylation and protein turnover in diabetic mouse retina.Exp Eye Res.2006;83(4):849-857.
69.刘源劼,陈国庆,卢辰,周鸿鹰,梅妍,羊惠君.泛素蛋白酶系统在四氧嘧啶诱导的糖尿病大鼠视网膜中的表达.解剖学杂志.2008；31(3)：372-375.
70. Komatsu M, Chiba T, Tatsumi K, Iemura S, Tanida I, Okazaki N, Ucno T， Kominami E, Natsume T, Tanaka K. A novel protein-conjugating system for ufml, a ubiquitin-fold modiiler. EMBO J.2004:23(9):1977-1986.
71. Azfer A, Niu J, Rogers LM, Adamski FM, Kolattukudy PE. Activation of endoplasmic reticulum stress response during the development of ischemic heart disease. Am J Physiol Heart Circ Physiol.2006:291 (3):H1411-1420.
72. Lu H, Yang Y, Allister EM, Wijesekara N, Wheeler MB. The identification of potential factors associated with the development of type 2 diabetes:a quantitative proteomics approach. Mol Cell Proteomics.2008;7(8):1434-1451.
73. Groll M, Ditzel L. Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R. Structure of 20s proteasome from yeast at 2.4 a resolution. Nature. 1997;386(6624):463-471.
74. Pickering AM, Koop AL, Teoh CY, Ermak G, Grune T, Davies KJ. The immunoproteasome, the 20s proteasome and the pa28alphabeta proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem J. 2010;432(2):585-594.
75. Abu El-Asrar AM, Missotten L, Geboes K. Expression of hypoxia-inducible factor-1 alpha and the protein products of its target genes in diabetic fibrovascular epiretinal membranes. Br J Ophthalmol.2007;91(6):822-826.
76. Adamis AP. Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol. 2002;86(4):363-365.
77. Joussen AM, Poulaki V, Le ML, Koizumi K, Esser C, Janicki H, Schraermeyer U, Kociok N, Fauser S, Kirchhof B, Kern TS. Adamis AP. A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J. 2004;18(12):1450-1452.
78. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science.1997;275(5302):964-967.
79. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nature medicine.2002;8(6):607-612.
80. Otani A, Kinder K, Ewalt K, Otero FJ, Schimmel P, Friedlander M. Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis. Nature medicine.2002;8(9):1004-1010.
81. Lee IG, Chae SL, Kim JC. Involvement of circulating endothelial progenitor cells and vasculogenic factors in the pathogenesis of diabetic retinopathy. Eye (Lond). 2006;20(5):546-552.
82. Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Shimada T, Oike Y, Imaizumi T. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation.2001;103(23):2776-2779.
83. Fadini GP, Sartore S, Baesso I, Lenzi M, Agostini C, Tiengo A, Avogaro A. Endothelial progenitor cells and the diabetic paradox. Diabetes Care. 2006;29(3):714-716.
84. Caballero S, Sengupta N, Aizal A, Chang KH, Li Calzi S, Guberski DL, Kern TS, Grant MB. Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells. Diabetes.2007;56(4):960-967.
85. Taylor CT, Moncada S. Nitric oxide, eytochrome c oxidase, and the cellular response to hypoxia. Arterioscler Thromb Vase Biol.2010;30(4):643-647.
86. Hansson HA. A histochemical study of cellular reactions in rat retina transiently damaged by visible light. Exp Eye Res.1971:12(3):270-274.
87. Caldwell RB, Slapnick SM. Increased eytochrome oxidase activity in the diabetic rat retinal pigment epithelium. Invest Ophthalmol Vis Sci.1989;30(4):591-599.
88. Wang AG, Lee CM, Wang YC, Lin CH, Fann MJ. Up-regulation of cytochrome oxidase in the retina following optic nerve injury. Exp Eye Res. 2002;74(5):651-659.
89.赵欣,蒲小平.蛋白质组学在药物研究中的应用.国药药理学通报.2009；25(8)：988-991.
90. Chan JN, Nislow C, Emili A. Recent advances and method development for drug target identification. Trends Pharmacol Sci.2010;31(2):82-88.
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