根皮苷对db/db小鼠主动脉的保护作用机制研究
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摘要
第一部分根皮苷对db/db小鼠主动脉的保护作用
研究背景
糖尿病以慢性血糖水平增高为特征,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缺陷而导致的代谢性疾病。糖尿病临床分型主要包括1型和2型糖尿病。其中,2型糖尿病患病率最高,发病机制最为复杂。过去的20年,随着人们生活水平的提高、生活方式的改变以及人口的老龄化,2型糖尿病的发病率在全球范围内逐年增加。目前我国己成为糖尿病第一大国。糖尿病威胁人们健康与生命,严重影响人类的生活质量,已经成为世界性公共卫生问题。
长期的高血糖可使一些组织或器官发生结构改变和功能障碍,导致各种并发症的产生。糖尿病血管病变是糖尿病的主要并发症之一,是糖尿病致死和致残的首要原因。其中大血管病变表现为主动脉、冠状动脉、脑基底动脉等大、中型血管的动脉粥样硬化,这些血管的损害是糖尿病心脑血管疾病的病理基础。糖尿病患者的动脉粥样硬化相对非糖尿病患者患病率高,发病年龄早,病情进展快。几乎超过半数的2型糖尿病患者最终会并发糖尿病的大血管病变。但是迄今为止,针对糖尿病大血管病变的防治手段仍然集中于代谢危险因素的控制(如严格控制血糖、血脂、血压),戒烟,血管紧张素转换酶抑制剂的应用等。然而上述治疗方法并不能完全阻断动脉粥样硬化的发展。因此,深刻理解2型糖尿病及其血管并发症的病理生理,开发研制新型药物,在积极控制血糖的同时,关注并加强对其血管并发症的干预和治疗有着极为重要的意义。
根皮苷是一种天然酚类物质,可由苹果树的根、皮、茎、嫩叶及果实中提取获得。研究资料表明,苹果树体内酚类物质总含量中,根皮苷占到95%。根皮苷具有多种潜在的功效,包括降血糖,抗氧化,抗衰老,抗肿瘤等。根皮苷抑制内皮粘附分子和血小板激活,对血管内皮有保护作用。然而,根皮苷对糖尿病的大血管是否有保护作用,目前国内外尚未见报道。本实验以db/db小鼠作为2型糖尿病动物模型,给予根皮苷干预,观察根皮苷对2型糖尿病大血管并发症的影响,讨论可能的作用机制,以探索根皮苷治疗糖尿病的可能性,为临床治疗糖尿病大血管并发症提供新的思路和途径。
研究目的
1.观察db/db小鼠主动脉组织病理学变化和超微结构变化,以及根皮苷对db/db小鼠主动脉损害的影响。
2.观察db/db小鼠根皮苷干预对血糖、血脂、甘油三酯、胆固醇、糖基化终末产物、丙二醛、谷丙转氨酶、谷草转氨酶、肌酐、尿素氮等指标的影响,探讨根皮苷对db/db小鼠主动脉保护作用的可能的机制及其安全性。
3.研究根皮苷对于STAT3蛋白与P-STAT3蛋白在主动脉组织表达的影响,初步探讨根皮苷对db/db小鼠主动脉保护作用的分子机制,为开辟糖尿病大血管病变治疗的新途径完善理论基础。
研究方法
7周龄雄州C57BLKS/J db/m小鼠8只以及7周龄雄性C57BLKS/J db/db小鼠16只,均饲喂颗粒饲料,饮水不限,观察1周进入实验。16只db/db小鼠随机分为两组：一组8只为糖尿病根皮苷干预组(DMT),每日给予根皮苷(20mg/kg/d)生理盐水溶液灌胃;另一组8只为糖尿病组(DM),给予相同体积的生理盐水灌胃；db/m小鼠8只作为正常对照组(CC),给予相同体积的生理盐水灌胃10周。实验期间每周定期测量体重(BW)并记录。于10周末,所有小鼠空腹过夜,10%水合氯醛麻醉后(350mg/kg),腹主动脉取血处死。留取血清检测空腹血糖(FBG),甘油三酯(TG)、胆固醇(TC)、糖基化终末产物(AGEs)、(?)丙二醛(MDA)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、尿素氮(BUN)、肌酐(Cr)等生化指标。留取的主动脉用4%多聚甲醛固定后,石蜡包埋、切片,HE染色,进行光学显微镜及电子显微镜检查。部分主动脉组织分离后速存于液氮,速冻后-80℃保存,用作蛋白质组学实验。
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减轻。
2.体重变化
实验开始时,DM组和DMT组基础体重无显著性差异(P>0.05),均明显高于CC组(P<0.01)。自实验第2周开始,DM组小鼠体重逐渐增加,该趋势一直持续至第10周实验结束。然而,DMT组小鼠与DM组相比,在实验的第
2、4、6、8、10周体重均明显降低(P<0.01),表明根皮苷能够显著改善DM小鼠的体重。
3. FBG、TC、TG、AGEs、MDA、ALT、AST、BUN、Cr含量检测结果
实验开始时DM组与DMT组间的FBG、AGEs、MDA水平无显著性差异(P>0.05),但与CC组比较,两组FBG、AGES和MDA水平均明显升高(P<0.05)。10周末,DM组小鼠FBG、AGEs和MDA均明显高于CC组(P<0.01),DMT组小鼠FBG、AGEs和MDA显著低于DM组(P<0.05)。表明根皮苷显著降低了FBG、TC、TG、AGEs和MDA水平。ALT、AST、BUN、Cr在各组间无差异。
4.主动脉组织学观察
正常对照组小鼠主动脉内膜光滑,无异常表现。糖尿病组小鼠主动脉管壁增厚,血管内膜受损,血管平滑肌细胞增殖、移行。根皮苷干预组小鼠比糖尿病小鼠主动脉病变程度减轻。表明根皮苷对糖尿病主动脉有保护作用。
5.根皮苷对db/db小鼠主动脉STAT3蛋白与p-STAT3蛋白表达的影响
蛋白质免疫印迹(Western blotting)染色显示,10周末,与CC组小鼠主动脉组织p-STAT3蛋白表达相比较,DM组主动脉组织p-STAT3蛋白表达明显增多。根皮苷治疗后,p-STAT3蛋白表达则显著减少。表明根皮苷对STAT3传导通路有抑制作用。
结论
根皮苷能够有效减轻db/db小鼠体重,减轻主动脉损害。能够降低小鼠的FBG、改善TC、TG、AGEs、MDA等生化指标,对肝、肾功能无影响。根皮苷对db/db小鼠主动脉的保护作用与其降糖调脂、降低AGEs水平和抗氧化有关。根皮苷抑制db/db小鼠主动脉STAT3的磷酸化,提示根皮苷可以通过抑制STAT3信号转导通路发挥其血管保护作用。
根皮苷为纯天然物质,安全,副作用小,能够减轻2型糖尿病的血管损害,具有降糖调脂、抗氧化等多种功效。可望作为一种新型的功能成分,为2型糖尿病并发症的防治提供新的思路和手段。
第二部分应用TRAQ技术探讨根皮苷对db/db小鼠主动脉的保护机制
研究背景
随着人类基因组工作框架的完成,生命科学的研究进入了后基因组时代。科学家逐渐意识到基因只是遗传信息的载体,而蛋白质才是生命活动的最终执行者,是生命现象的直接体现者,介导几乎所有的生命活动。因此,除了了解蛋白质的数量、结构和性质以外,全面动态的了解蛋白质的生物功能及不同蛋白质相互关系的变化,才能直接阐明生理或病理条件下生命活动的变化机制,从而揭示生命活动的规律和生命现象的本质。因此生命科学的工作重心从获取生命的全部遗传信息而渐渐转移到对生命功能的整体探索上来。对蛋白质组学研究也逐渐成为后基因组研究中的核心内容。
精确鉴定及定量整个细胞基因组所表达的全部蛋白质称为定量蛋白质组学定量蛋白质组学的重点是定性定量地解析整个细胞内所有蛋白质的动态变化,从而反映细胞功能、过程、机制等综合信息。它是功能蛋白质组学的重要内容,常被用于分析疾病与健康两种不同状态,以及药物处理前后细胞内差异表达的全体蛋白质。定量蛋白质组学技术可用于鉴定对疾病具有提示或诊断功能的蛋白质,也有助于为疾病的治疗发现具有新功能的靶标蛋白。近年来该领域的相关技术发展迅速,全新的应用和方法设计正在不断推进技术创新。最近,同位素标记相对和绝对定量(iTRAQ)技术联合质谱分析,已经在探讨疾病的发生机制、寻找疾病标志物、以及不同时段或者不同状态的多样本定量分析等蛋白质组学研究领域得到了很好的应用。
根皮苷是苹果多酚的重要组成成分,作为一种天然多酚类化合物,广泛存在于苹果属的多种植物中,在根、茎、叶与果实中均有分布。根皮苷具有多种生物活性、和药理作用,比如清除自由基、抗氧化应激、调节血糖、保护心血管以及抗肿瘤的作用。最近的体内外实验表明,根皮苷可以显著改善糖尿病心肌肥厚、糖尿病肾病、糖尿病视网膜早期周细胞损伤等并发症。本研究第一部分实验发现：根皮苷对于db/db小鼠主动脉具有明显的保护作用。但是迄今为止,根皮苷是通过什么机制及有效靶标蛋白来发挥对2型糖尿病主动脉的保护效应尚未得到解决。
本研究第二部分应用iTRAQ技术,并利用Turbo SEQUEST软件与国际蛋白质索引(international protein index, IPI)数据库等生物信息学方法,分离与鉴定正常对照组(CC组),糖尿病组(DM组)与糖尿病根皮苷干预组(DMT组)三组小鼠主动脉组织的差异表达蛋白。目的在于揭示根皮苷对于db/db小鼠主动脉的保护机制,为研发预防和治疗糖尿病大血管并发症的有效药物提供潜在靶标和新的视角。
研究目的
1.获得db/db小鼠主动脉组织的全部蛋白质的差异表达情况,进一步了解在糖尿病状态下主动脉损伤的蛋白质组学改变。
2.探讨db/db小鼠经根皮苷治疗后主动脉组织的差异表达蛋白,筛选并初步验证药物作用的关键蛋白与候选靶标,从蛋白质组学角度进一步揭示根皮苷对于糖尿病主动脉的保护机制,为开辟糖尿病大动脉病变的治疗新途径奠定实验基础。
研究方法
CC组、DM组与DMT组三组小鼠中各选取4只小鼠,分离主动脉组织。按照iTRAQ蛋白质组学技术要求的样本制备标准技术流程,如主动脉组织研磨、切碎、超声破碎、裂解等提取主动脉总蛋白,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。提取各组主动脉总蛋白后,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。各组主动脉总蛋白采用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CC组,116标记DMT组,117则标记DM组。标记结束后利用ABI4800MALDI-TOF/TOF质谱仪各组均随机选取一定数量肽段,并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。将各组已标记的肽段混合,然后用Agilent HPLC1200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,之后予以C18Cartridge (Sigma)脱盐,进行下一步质谱鉴定。本研究第二部分使用Thermo Finnigan LTQVelos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*150mmC-18反相色谱柱(RP-C18Column Technology Inc),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%)。质谱的进样方式为采用微毛细管上样,温度为200度,检测方式为正离子。一次全扫描后收集5个碎片图谱来获得多肽和多肽碎片的m/z比。原始文件用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检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。搜索使用的数抓库为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://www.geneontology.org)对经鉴定的主动脉组织蛋白质进行分类。分别从细胞组成、分子功能及生物过程对蛋白质表达谱进行定位分类与功能分析。选取两个差异表达的关键蛋白经Western blotting来验证其在糖尿病小鼠主动脉组织中的表达情况。
研究结果
1.小鼠主动脉组织总蛋白SDS-PAGE结果
结果提示各组主动脉SDS-PAGE蛋白质点迁移条带清晰,各组间平行度较好,可以进行下一步实验。
2. iTRAQ标记统计学验证
本部分以114标记的正常组小鼠主动脉蛋白样品做为对照,将117标记的糖尿病组小鼠和116标记的根皮苷治疗组小鼠主动脉蛋白样品与对照的比值进行统计分析。结果显示几种标记试剂进行的蛋白标记实验均一性好,并提示10%的蛋白有显著性差异,符合统计学的规律。
3.质谱数据解析结果
本研究使用高效液相色谱分离与电喷雾-串联质谱分析(LC-ESI-MS/MS)在线获取的MS/MS原始质谱数据,采用Turbo SEQUEST程序自动进行非冗余IPI小鼠蛋白数据库搜索,共鉴定到的唯一肽段15825条,共有3083种蛋白质,其中有定量信息的蛋白有1512种(过滤参数：蛋白质FDR≤0.01；肽段FDR≤0.01)。
本研究中,以114标记的正常组小鼠主动脉蛋白样品做为对照,将117标记的糖尿病组小鼠和116标记的根皮苷治疗组小鼠主动脉蛋白样品与对照的比值进行统计分析。比值小于0.67或大于1.5的蛋白即为根皮苷治疗(20mg/kg/day)10周后表达明显下调或者上调的主动脉组织蛋白。与正常对照小鼠组相比,DM组主动脉的差异表达蛋白557个,其中,表达增高的点463个,降低的点94个。另外,与非治疗组相比,糖尿病小鼠经根皮苷治疗后主动脉有70个差异表达蛋白的表达水平恢复,其中50个蛋白的表达降低,20个蛋白表达增高。
4.根皮苷干预后主动脉差异蛋白质的定位分析
应用UniProt蛋白数据库(http://www.uniprot.org)的GO分析以及相关文献,对糖尿病小鼠主动脉差异表达的70个蛋白分析进行定位分类。主动脉差异表达蛋白中胞浆蛋白比例最大,占23.08%。其次是核蛋白和膜蛋白,分别占15.38%和13.9%,线粒体蛋白和高尔基结构蛋白均占8.79%,内质网蛋白占7.69%,另外还有少量核糖体蛋白和溶酶体蛋白,分别占2.20%和1.10%。
5.根皮苷干预后主动脉差异蛋白质的功能分析
利用GO分析结合UniProt蛋白数据库以及相关文献,将LC-ESI-MS/MS鉴定得出的根皮苷干预后的70个主动脉差异蛋白点进行功能分析。差异蛋白中的与三大物质代谢相关的蛋白所占比例最大,达到17.28%。其次是结合蛋白,达到16.05%。参与氧化应激和ATP合成的蛋白占11.11%。介导细胞凋亡、参与细胞周期调控及增殖的蛋白占9.88%。结构蛋白和信号转导蛋白分别占8.64%和4.94%。
6. Western blotting验证差异蛋白在主动脉组织的表达改变
我们采用Western blotting的方法选择STEAP4和PDE5A两个蛋白来验证iTRAQ联合LC-ESI-MS/MS鉴定的差异蛋白的结果的可靠性。与正常对照组比较,db/db小鼠主动脉STEAP4和PDE5蛋白表达明显增加；根皮苷治疗10周后,糖尿病主动脉STEAP4和PDE5蛋白表达显著降低。因此,经Western blotting验证重点选取的差异蛋白STEAP4和PDE5A在主动脉组织上的表达与iTRAQ技术获得的蛋白质组学结果相符合,说明iTRAQ技术的实验结果真实可靠。
结论
1. iTRAQ技术能够同时对多组样本进行比较分析,可以获得更好的蛋白质组覆盖率。iTRAQ技术用于研究根皮苷对db/db小鼠主动脉保护作用的分子机制有助于发现新的药物靶标。
2.应用LC-ESI-MS/MS鉴定得到根皮苷干预db/db小鼠主动脉差异表达蛋白70个,其中下调点50个点,上调点20个,其功能涉及到细胞代谢、氧化应激、信号转导、细胞骨架、细胞周期及细胞凋亡等,提示这些过程与机制可能参与了糖尿病主动脉病变的发生与发展。
3. Western blotting证实db/db小鼠主动脉STEAP4和PDE5A蛋白表达明显增加；根皮苷治疗显著抑制这两个蛋白的表达水平,提示这些蛋白可能是根皮苷对db/db小鼠主动脉起保护作用的关键蛋白。
4.本研究提供了根皮苷对于db/db小鼠主动脉保护作用的蛋白质表达谱的重要信息,为全面揭示根皮苷作用机制提供了新的方法和新的视角,为寻找预防和治疗糖尿病大血管并发症提供了可能有意义的药物靶标,从而为临床诊治糖尿病大血管并发症奠定与充实理论基础。
Part One Effect of Phlodizin on Aorta in db/db Mice
Background
Type1diabetes mellitus and type2diabetes mellitus are the two main clinical types of diabetes mellitus. In the past20years, Type2diabetes has become one of the most common endocrine diseases in both developed and developing countries, due to the elevation of living standard, modern life styles and aging. Following cancers and heart diseases, diabetes mellitus has become the third disease that affects the quality of people's life in the whole world. Diabetes is caused by absolute or relative deficiencies in insulin secretion or insulin action. Chronic hyperglycemia is the key biochemical alteration in type2diabetes. The long term-lasting of high blood glucose can cause structural and functional damage of organs in diabetic patients. Moreover, individuals under type2diabetes usually develop aortic complication marked by endothelial injuries to different extents. Excessive oxidative stress has been reported to play a critical role in the development of diabetic aortic injury.
Diabetic individuals have a two-to-three fold increase risk for cardiovascular and cerebrovascular diseases such as heart attack and stroke. Diabetic vascular complication is the main pathological basis of cardiovascular and cerebrovascular diseases, and is one of the major causes of morbidity and mortality in diabetic patients. Moreover, the current teatment for diabetes including intensive insulin treatment, strict lipid control, reduction of blood hypertension and smoking cessation are not sufficient to prevent aortic deteriorative progression to arteriosclerosis. Therefore, it is important and necessary to understand the pathophysiology of type2Diabetes Mellitus and develop strategies to prevent and treat the high blood glucose and related vascular complications.
Phlorizin, a phloretin glucoside, belongs to dihydrochalcones and maily distributed in the apple trees as well as in root bark, shoots, and leaves. Phlorizin makes the major sources of flavonoid. It has been reported that phlorizin have possessed a variety of potent properties, including anti-inflammation, anti-tumor, regularing glucose concentrations, memory improvement and anti-oxidation. Phlorizin is also endothelial protective by suppressing endothelial adhesion molecule expression and platelets activation. However, the effect of phlorizin on the macrovascular complications of type2Diabetes Mellitus had not been elaborated. Here, we fed phlorizin to db/db mice to study the effects of this natural reagent on macrovascular complications of type2Diabetes Mellitus and to determine the underlying mechanism. Through this investigation perhaps new medications can be found to effectively treat macrovascular complications of type2Diabetes Mellitus.
Objectives
1. To observe the morphological changes of the aorta of db/db mice and the effects of phlorizin on the aorta of db/db mice.
2. To investigate the effects of phlorizin on the blood samples of db/db mice, including blood glucose, TC, TG, AGEs, MDA, ALT. AST, BUN and Cr, and to explore the mechanism of phlorizin's aorta preventing effects.
3. To examine the effects of phlorizin on the expression of STAT3and p-STAT3in the aorta of diabetic db/db mice, and to explore the molecular mechanism by which phlorizin prevented aortic damage by DM, further to provide new approaches for the management of macrovascular complications of Diabetes Mellitus.
Methods
Male C57BLKS/J db/m mice (n=8.6weeks old) and C57BLKS/J db/db mice (n=16,6weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu. China). The mice were kept under observation for one week prior to the start of the experiments. The db/db mice were randomly divided into2groups: untreated diabetic group (DM, n=8) and diabetic group treated by phlorizin (DMT, n=8). Phlorizin (20mg/kg Id) was given in normal saline solution intragastrically for 10weeks. C57BLKS/J db/m mice were used as control (CC, n=8). Mice of the DM group and the CC group were given the same amount of normal saline solution intragastrically every day as those of the DMT group. All mice were weighed every week during the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, and the aortas were dissected. Aortic tissues and sera were kept at-80℃for further analysis. Blood glucose, TC, TG, AGEs, MDA, ALT, AST, BUN and Cr were measured. Morphological changes of the aorta were examined by light microscopy and H-800electron microscope.
Results
1. General characteristics
Mice of the CC group showed good condition and grew up sound with smooth furs. The db/db mice of the DM group featured polydipsia, polyphagia and polyuris with shaggy furs and a rapid growth. Phlorizin treated db/db mice also displayed abnormal, but much better than the db/db mice of the DM group.
2. Effects of phlorizin on body weights
Baseline body weights were similar in mice of the DM group and the DMT group, and higher than the mice of the CC group. At as early as week2of the study, the body weight in db/db mice group was significantly than that of the control mice(P<0.01). This trend did not change until the end of the experiment. However, the body weight was significantly inhibited at2th week,4th week,6th week,8th week, and11th week after phlorizin administration in the DMT group compared to the DM group (PO.01).
3. Effects of phlorizin on FBG、TC、TG. AGE、MDA、ALT、AST、BUN and Cr
Baseline FBG, AGEs and MDA were similar between mice of the DM group and the DMT group (P>0.05), and higher than the mice of the CC group (P<0.05). In the10th week, the FBG, AGEs and MDA of diabetic mice were higher than those of control mice (P<0.01). Phlorizin significantly reduced FBG, AGEs and MDA in the diabetic mice (P<0.05). Serum TC. TG. AGEs、MDA. ALT、AST、BUN and Cr were similar among the three groups.
4. Histological and electron micrographical findings
The microscopic images of the aortic sections of the control group appeared normal, with organized intimal surfaces and normal ultrastructure. Vascular wall of the DM group thickened with impaired intima and a narrowed lumen. The ultrastructure shows that in untreated diabetic mice, the vascular smooth muscle cells migrated into the elastic laminar, and an increase of heterochromatin could be observed. With phlorizin treatment, the micro-and ultra-structure changes were less severe.
5. Effects of phlorizin on the expression of STAT3and p-STAT3
Western blotting showed that the expression of p-STAT3in the aortic section of the DM group was much higher than that of the CC group. With phlorizin treatment, the expression of p-STAT3was much decreased.
Conclusion
Phlorizin can decrease the body weight of the db/db mice. Phlorizin also decreased the serum glucose, TC, TG, AGEs, and MDA of the db/db mice, and had no effects on ALT.. AST、BUN and Cr. In addition, phlorizin ameliorated the aortic damage of Diabetes Mellitus. The protective effects of phlorizin on diabetic aortas can be attributed to its antiglycation and antioxidant effect. Phlorizin may be a safe natural reagent for diabetes mellitus treatment. Perhaps new medications can be found to effectively treat macrovascular complications of type2Diabetes Mellitus through intensive study on phlorizin.
Part Two iTRAQ-based proteomic study of the effects of phlorizin on Aortic Arteriosclerosis in db/db mice
Background
With the achievement of human genome sequencing project, life science has turned to the post-genomic era. Proteome is consisting of the total proteins present in a cell or organism at a given time. It includs both proteins translated directly from genetic material and the modified proteins arising from extensive posttranslational processing. Thus, proteome is regarded as the most critical and promising part in the post-genomic studies.
Quantitative proteomics is the major part of functional proteomics, qualitatively and quantitively identifying the whole proteins of the cells. Quantitative proteomics focusing on the dynamic structural and functional alterations of the proteins in a given cell. Theremore, quantitative proteomics profiling is applied to identify the differentially expressed proteins under disease or after drug intervention. Quantitative proteomics is being developed rapidly, with new technological innovations and protocol design applied. The rapidly emerging field of quantitative proteomics provides a powerful technique-isobaric tag for relative and absolute quantitation (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the identification and characterization of protein profiles. This technique can lessen variation, enhance throughput and enable quantitative analysis.
Phlorizin as a member of the chalcone class of organic compounds is mainly distributed in the plants of the genus Malus. Phlorizin is proved to possess multiple pharmacological activities, such as estrogenic and anti-estrogenic activities, anti-oxidative, regulation of blood sugar level, memory improvement and cardioprotective activities. Phlorizin has been shown to inhibit intestinal glucose uptake and renal glucose reabsorption by inhibiting the sodium D-glucose cotransporter. In the first part of our experiment, Phlorizin has demonstrated protective effects on aortic tissue in the db/db mice. But the molecular targets and the underlying mechanism of phlorizin are unclear.
In the second part, we identified differentially expressed proteins in aortas of the control db/m mice, diabetic db/db mice and phlorizin treated db/db mice applying 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 targeting proteins of phlorizin and the underlying mechanism by which phlorizin improved diabetic aorta injury, to develop potential for treating diabetes induced retinal lesions.
Objective
1. The purpose of the study was to obtain the differentially expressioned proteins in the aorta of diabetic mice and to explore the the pathogenetic mechanism of macrovascular complications caused by diabetes.
2. The objective of this study was to identify the effective multifunctional targets and therapeutic strategies against diabetic macrovascular complications after phlorizin treatment.
Methods
The aortic tissue was dissected among the control, diabetic db/db mice, phlorizin treated db/db mice. The processes of proteomic analysis were described as follows: The separated aortic tissue sample were prepared according to the iTRAQ procedure, including tissue disruption, sample clean-up, and pretein solubilization. The protein concentrations were estimated by Bradford arrary. Then the aortic tissues were kept at-80℃for further analysis. About20ug peptides of each group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were digested and iTRAQ labeled, 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 the labeled samples were finally mixed together, separated by Strong Cation Exchange (SCX) chromatography into10fractions and finally desalted by an offline fraction collector and C18cartridges (Sigma). After being labeled, 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, which was based on the observation of the peak for reporter ions encoded in the low mass-to-charge ratio portion of the MS/MS spectrum. In the second part, mass spectrometric analysis was performed by 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 by data-dependent acquisition conditions. The acquired MS/MS spectra were automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version3.72) by Turbo SEQUEST program in the BioWorksTM3.1software suite. 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 according to the molecular function, biologicai process, and cellular component by AmiGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi Version1.8). Finally, some candidated differentially expressed proteins in the aorta were confirmed by Western blottingting analysis.
Results
1. SDS-PAGE image
Proteins brand loaded on the gels separared by SDS-PAGE were observed clearly, available for the following experiments.
2. iTRAQ labeling statistical validation
Our results indicated that only10percent of the peptides between two groups were significantly different.
3. Mass spectrometry identify the differently expressed proteins
Protein profiling was analyzed by iTRAQ approach followed by LC-MS/MS identification and IPI database searching. A total of3083proteins were identified, of which1512proteins meet the standard. Among which557proteins were differentially expressed in aorta of db/db mice compared with control db/m mice, of which463proteins were up-regulated and94proteins were down-regulated. After phlorizin treatment,50proteins were down-regulated while20proteins up-regulated in aorta of diabetic db/db mice.
4. Subcellular localization analysis phlorizin associated retina proteins in db/db mice
We performed the localization analysis of the identified proteins in aorta of db/db mice by AmiGO (Version1.8).23.08%of the total proteins were in cytoplasm,15.38%in nucleus and13.9%in plasma membrane.9.68%of the proteins were in mitochondrion,7.69%in endoplasmic reticulum.
5. Subcellular bioinformatic functional analysis phlorizin associated retina proteins in db/db mice
Most of the back-regulated proteins were involved in material metabolism, oxidative stress, apoptosis, cell growth and signaling transduction. Among the functional assignment of the proteins,17.28%of the proteins were in metabolic processes, and16.05%were binding proteins.11.11%of the back-regulated proteins participate in oaxidative stress and ATP synthesis;8.64%mediate the apoptosis, cell cycle and growth.
6. Confirmation of selected candidate proteins of iTRAQ by Western blottingting analysis
To confirme the differentially expressed protein, PDE5A and STEAP4were validated by Western blottingting analysis. PDE5A and STEAP4were significantly increased in aorta of diabetic db/db mice compared to the control group. After phlorizin treatment, PDE5A and STEAP4were regulated near to normal.
Conclusion
1. As an advanced technology of quantitative proteomics, iTRAQ can analyze four samples synchronously with higher accuracy and sensitivity. In the second part, with the help of iTRAQ, we explored the mechanism underlying the protection of phlorizin against diabetic aortic injury.
2. A total of70differentially expressed proteins were identified in aorta of db/db mice after phlorizin treatment by LS-ESI-MS/MS methods. Of these,50proteins were down-regulated while20were up-regulated after phlorizin administration. Most of these back-regulated proteins were located in cytoplasm and plasma membrane, related to diverse functions, including metabolic process, signal transduction, apoptosis and oxidative stress. Our results suggested that these processes may be linked to the pathogenesis of diabetic macrovascular complications.
3. PDE5A and STEAP4were increased in aorta of db/db mice compared to the control group. After phlorizin treatment, PDE5A and STEAP4were regulated near to normal.
4. Our second part provided new insight into the mechanism and new promising treatment for diabetic macrovascular complications.
研究背景
糖尿病以慢性血糖水平增高为特征,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缺陷而导致的代谢性疾病。糖尿病临床分型主要包括1型和2型糖尿病。其中,2型糖尿病患病率最高,发病机制最为复杂。过去的20年,随着人们生活水平的提高、生活方式的改变以及人口的老龄化,2型糖尿病的发病率在全球范围内逐年增加。目前我国己成为糖尿病第一大国。糖尿病威胁人们健康与生命,严重影响人类的生活质量,已经成为世界性公共卫生问题。
长期的高血糖可使一些组织或器官发生结构改变和功能障碍,导致各种并发症的产生。糖尿病血管病变是糖尿病的主要并发症之一,是糖尿病致死和致残的首要原因。其中大血管病变表现为主动脉、冠状动脉、脑基底动脉等大、中型血管的动脉粥样硬化,这些血管的损害是糖尿病心脑血管疾病的病理基础。糖尿病患者的动脉粥样硬化相对非糖尿病患者患病率高,发病年龄早,病情进展快。几乎超过半数的2型糖尿病患者最终会并发糖尿病的大血管病变。但是迄今为止,针对糖尿病大血管病变的防治手段仍然集中于代谢危险因素的控制(如严格控制血糖、血脂、血压),戒烟,血管紧张素转换酶抑制剂的应用等。然而上述治疗方法并不能完全阻断动脉粥样硬化的发展。因此,深刻理解2型糖尿病及其血管并发症的病理生理,开发研制新型药物,在积极控制血糖的同时,关注并加强对其血管并发症的干预和治疗有着极为重要的意义。
根皮苷是一种天然酚类物质,可由苹果树的根、皮、茎、嫩叶及果实中提取获得。研究资料表明,苹果树体内酚类物质总含量中,根皮苷占到95%。根皮苷具有多种潜在的功效,包括降血糖,抗氧化,抗衰老,抗肿瘤等。根皮苷抑制内皮粘附分子和血小板激活,对血管内皮有保护作用。然而,根皮苷对糖尿病的大血管是否有保护作用,目前国内外尚未见报道。本实验以db/db小鼠作为2型糖尿病动物模型,给予根皮苷干预,观察根皮苷对2型糖尿病大血管并发症的影响,讨论可能的作用机制,以探索根皮苷治疗糖尿病的可能性,为临床治疗糖尿病大血管并发症提供新的思路和途径。
研究目的
1.观察db/db小鼠主动脉组织病理学变化和超微结构变化,以及根皮苷对db/db小鼠主动脉损害的影响。
2.观察db/db小鼠根皮苷干预对血糖、血脂、甘油三酯、胆固醇、糖基化终末产物、丙二醛、谷丙转氨酶、谷草转氨酶、肌酐、尿素氮等指标的影响,探讨根皮苷对db/db小鼠主动脉保护作用的可能的机制及其安全性。
3.研究根皮苷对于STAT3蛋白与P-STAT3蛋白在主动脉组织表达的影响,初步探讨根皮苷对db/db小鼠主动脉保护作用的分子机制,为开辟糖尿病大血管病变治疗的新途径完善理论基础。
研究方法
7周龄雄州C57BLKS/J db/m小鼠8只以及7周龄雄性C57BLKS/J db/db小鼠16只,均饲喂颗粒饲料,饮水不限,观察1周进入实验。16只db/db小鼠随机分为两组：一组8只为糖尿病根皮苷干预组(DMT),每日给予根皮苷(20mg/kg/d)生理盐水溶液灌胃;另一组8只为糖尿病组(DM),给予相同体积的生理盐水灌胃；db/m小鼠8只作为正常对照组(CC),给予相同体积的生理盐水灌胃10周。实验期间每周定期测量体重(BW)并记录。于10周末,所有小鼠空腹过夜,10%水合氯醛麻醉后(350mg/kg),腹主动脉取血处死。留取血清检测空腹血糖(FBG),甘油三酯(TG)、胆固醇(TC)、糖基化终末产物(AGEs)、(?)丙二醛(MDA)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、尿素氮(BUN)、肌酐(Cr)等生化指标。留取的主动脉用4%多聚甲醛固定后,石蜡包埋、切片,HE染色,进行光学显微镜及电子显微镜检查。部分主动脉组织分离后速存于液氮,速冻后-80℃保存,用作蛋白质组学实验。
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减轻。
2.体重变化
实验开始时,DM组和DMT组基础体重无显著性差异(P>0.05),均明显高于CC组(P<0.01)。自实验第2周开始,DM组小鼠体重逐渐增加,该趋势一直持续至第10周实验结束。然而,DMT组小鼠与DM组相比,在实验的第
2、4、6、8、10周体重均明显降低(P<0.01),表明根皮苷能够显著改善DM小鼠的体重。
3. FBG、TC、TG、AGEs、MDA、ALT、AST、BUN、Cr含量检测结果
实验开始时DM组与DMT组间的FBG、AGEs、MDA水平无显著性差异(P>0.05),但与CC组比较,两组FBG、AGES和MDA水平均明显升高(P<0.05)。10周末,DM组小鼠FBG、AGEs和MDA均明显高于CC组(P<0.01),DMT组小鼠FBG、AGEs和MDA显著低于DM组(P<0.05)。表明根皮苷显著降低了FBG、TC、TG、AGEs和MDA水平。ALT、AST、BUN、Cr在各组间无差异。
4.主动脉组织学观察
正常对照组小鼠主动脉内膜光滑,无异常表现。糖尿病组小鼠主动脉管壁增厚,血管内膜受损,血管平滑肌细胞增殖、移行。根皮苷干预组小鼠比糖尿病小鼠主动脉病变程度减轻。表明根皮苷对糖尿病主动脉有保护作用。
5.根皮苷对db/db小鼠主动脉STAT3蛋白与p-STAT3蛋白表达的影响
蛋白质免疫印迹(Western blotting)染色显示,10周末,与CC组小鼠主动脉组织p-STAT3蛋白表达相比较,DM组主动脉组织p-STAT3蛋白表达明显增多。根皮苷治疗后,p-STAT3蛋白表达则显著减少。表明根皮苷对STAT3传导通路有抑制作用。
结论
根皮苷能够有效减轻db/db小鼠体重,减轻主动脉损害。能够降低小鼠的FBG、改善TC、TG、AGEs、MDA等生化指标,对肝、肾功能无影响。根皮苷对db/db小鼠主动脉的保护作用与其降糖调脂、降低AGEs水平和抗氧化有关。根皮苷抑制db/db小鼠主动脉STAT3的磷酸化,提示根皮苷可以通过抑制STAT3信号转导通路发挥其血管保护作用。
根皮苷为纯天然物质,安全,副作用小,能够减轻2型糖尿病的血管损害,具有降糖调脂、抗氧化等多种功效。可望作为一种新型的功能成分,为2型糖尿病并发症的防治提供新的思路和手段。
第二部分应用TRAQ技术探讨根皮苷对db/db小鼠主动脉的保护机制
研究背景
随着人类基因组工作框架的完成,生命科学的研究进入了后基因组时代。科学家逐渐意识到基因只是遗传信息的载体,而蛋白质才是生命活动的最终执行者,是生命现象的直接体现者,介导几乎所有的生命活动。因此,除了了解蛋白质的数量、结构和性质以外,全面动态的了解蛋白质的生物功能及不同蛋白质相互关系的变化,才能直接阐明生理或病理条件下生命活动的变化机制,从而揭示生命活动的规律和生命现象的本质。因此生命科学的工作重心从获取生命的全部遗传信息而渐渐转移到对生命功能的整体探索上来。对蛋白质组学研究也逐渐成为后基因组研究中的核心内容。
精确鉴定及定量整个细胞基因组所表达的全部蛋白质称为定量蛋白质组学定量蛋白质组学的重点是定性定量地解析整个细胞内所有蛋白质的动态变化,从而反映细胞功能、过程、机制等综合信息。它是功能蛋白质组学的重要内容,常被用于分析疾病与健康两种不同状态,以及药物处理前后细胞内差异表达的全体蛋白质。定量蛋白质组学技术可用于鉴定对疾病具有提示或诊断功能的蛋白质,也有助于为疾病的治疗发现具有新功能的靶标蛋白。近年来该领域的相关技术发展迅速,全新的应用和方法设计正在不断推进技术创新。最近,同位素标记相对和绝对定量(iTRAQ)技术联合质谱分析,已经在探讨疾病的发生机制、寻找疾病标志物、以及不同时段或者不同状态的多样本定量分析等蛋白质组学研究领域得到了很好的应用。
根皮苷是苹果多酚的重要组成成分,作为一种天然多酚类化合物,广泛存在于苹果属的多种植物中,在根、茎、叶与果实中均有分布。根皮苷具有多种生物活性、和药理作用,比如清除自由基、抗氧化应激、调节血糖、保护心血管以及抗肿瘤的作用。最近的体内外实验表明,根皮苷可以显著改善糖尿病心肌肥厚、糖尿病肾病、糖尿病视网膜早期周细胞损伤等并发症。本研究第一部分实验发现：根皮苷对于db/db小鼠主动脉具有明显的保护作用。但是迄今为止,根皮苷是通过什么机制及有效靶标蛋白来发挥对2型糖尿病主动脉的保护效应尚未得到解决。
本研究第二部分应用iTRAQ技术,并利用Turbo SEQUEST软件与国际蛋白质索引(international protein index, IPI)数据库等生物信息学方法,分离与鉴定正常对照组(CC组),糖尿病组(DM组)与糖尿病根皮苷干预组(DMT组)三组小鼠主动脉组织的差异表达蛋白。目的在于揭示根皮苷对于db/db小鼠主动脉的保护机制,为研发预防和治疗糖尿病大血管并发症的有效药物提供潜在靶标和新的视角。
研究目的
1.获得db/db小鼠主动脉组织的全部蛋白质的差异表达情况,进一步了解在糖尿病状态下主动脉损伤的蛋白质组学改变。
2.探讨db/db小鼠经根皮苷治疗后主动脉组织的差异表达蛋白,筛选并初步验证药物作用的关键蛋白与候选靶标,从蛋白质组学角度进一步揭示根皮苷对于糖尿病主动脉的保护机制,为开辟糖尿病大动脉病变的治疗新途径奠定实验基础。
研究方法
CC组、DM组与DMT组三组小鼠中各选取4只小鼠,分离主动脉组织。按照iTRAQ蛋白质组学技术要求的样本制备标准技术流程,如主动脉组织研磨、切碎、超声破碎、裂解等提取主动脉总蛋白,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。提取各组主动脉总蛋白后,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。各组主动脉总蛋白采用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CC组,116标记DMT组,117则标记DM组。标记结束后利用ABI4800MALDI-TOF/TOF质谱仪各组均随机选取一定数量肽段,并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。将各组已标记的肽段混合,然后用Agilent HPLC1200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,之后予以C18Cartridge (Sigma)脱盐,进行下一步质谱鉴定。本研究第二部分使用Thermo Finnigan LTQVelos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*150mmC-18反相色谱柱(RP-C18Column Technology Inc),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%)。质谱的进样方式为采用微毛细管上样,温度为200度,检测方式为正离子。一次全扫描后收集5个碎片图谱来获得多肽和多肽碎片的m/z比。原始文件用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检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。搜索使用的数抓库为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://www.geneontology.org)对经鉴定的主动脉组织蛋白质进行分类。分别从细胞组成、分子功能及生物过程对蛋白质表达谱进行定位分类与功能分析。选取两个差异表达的关键蛋白经Western blotting来验证其在糖尿病小鼠主动脉组织中的表达情况。
研究结果
1.小鼠主动脉组织总蛋白SDS-PAGE结果
结果提示各组主动脉SDS-PAGE蛋白质点迁移条带清晰,各组间平行度较好,可以进行下一步实验。
2. iTRAQ标记统计学验证
本部分以114标记的正常组小鼠主动脉蛋白样品做为对照,将117标记的糖尿病组小鼠和116标记的根皮苷治疗组小鼠主动脉蛋白样品与对照的比值进行统计分析。结果显示几种标记试剂进行的蛋白标记实验均一性好,并提示10%的蛋白有显著性差异,符合统计学的规律。
3.质谱数据解析结果
本研究使用高效液相色谱分离与电喷雾-串联质谱分析(LC-ESI-MS/MS)在线获取的MS/MS原始质谱数据,采用Turbo SEQUEST程序自动进行非冗余IPI小鼠蛋白数据库搜索,共鉴定到的唯一肽段15825条,共有3083种蛋白质,其中有定量信息的蛋白有1512种(过滤参数：蛋白质FDR≤0.01；肽段FDR≤0.01)。
本研究中,以114标记的正常组小鼠主动脉蛋白样品做为对照,将117标记的糖尿病组小鼠和116标记的根皮苷治疗组小鼠主动脉蛋白样品与对照的比值进行统计分析。比值小于0.67或大于1.5的蛋白即为根皮苷治疗(20mg/kg/day)10周后表达明显下调或者上调的主动脉组织蛋白。与正常对照小鼠组相比,DM组主动脉的差异表达蛋白557个,其中,表达增高的点463个,降低的点94个。另外,与非治疗组相比,糖尿病小鼠经根皮苷治疗后主动脉有70个差异表达蛋白的表达水平恢复,其中50个蛋白的表达降低,20个蛋白表达增高。
4.根皮苷干预后主动脉差异蛋白质的定位分析
应用UniProt蛋白数据库(http://www.uniprot.org)的GO分析以及相关文献,对糖尿病小鼠主动脉差异表达的70个蛋白分析进行定位分类。主动脉差异表达蛋白中胞浆蛋白比例最大,占23.08%。其次是核蛋白和膜蛋白,分别占15.38%和13.9%,线粒体蛋白和高尔基结构蛋白均占8.79%,内质网蛋白占7.69%,另外还有少量核糖体蛋白和溶酶体蛋白,分别占2.20%和1.10%。
5.根皮苷干预后主动脉差异蛋白质的功能分析
利用GO分析结合UniProt蛋白数据库以及相关文献,将LC-ESI-MS/MS鉴定得出的根皮苷干预后的70个主动脉差异蛋白点进行功能分析。差异蛋白中的与三大物质代谢相关的蛋白所占比例最大,达到17.28%。其次是结合蛋白,达到16.05%。参与氧化应激和ATP合成的蛋白占11.11%。介导细胞凋亡、参与细胞周期调控及增殖的蛋白占9.88%。结构蛋白和信号转导蛋白分别占8.64%和4.94%。
6. Western blotting验证差异蛋白在主动脉组织的表达改变
我们采用Western blotting的方法选择STEAP4和PDE5A两个蛋白来验证iTRAQ联合LC-ESI-MS/MS鉴定的差异蛋白的结果的可靠性。与正常对照组比较,db/db小鼠主动脉STEAP4和PDE5蛋白表达明显增加；根皮苷治疗10周后,糖尿病主动脉STEAP4和PDE5蛋白表达显著降低。因此,经Western blotting验证重点选取的差异蛋白STEAP4和PDE5A在主动脉组织上的表达与iTRAQ技术获得的蛋白质组学结果相符合,说明iTRAQ技术的实验结果真实可靠。
结论
1. iTRAQ技术能够同时对多组样本进行比较分析,可以获得更好的蛋白质组覆盖率。iTRAQ技术用于研究根皮苷对db/db小鼠主动脉保护作用的分子机制有助于发现新的药物靶标。
2.应用LC-ESI-MS/MS鉴定得到根皮苷干预db/db小鼠主动脉差异表达蛋白70个,其中下调点50个点,上调点20个,其功能涉及到细胞代谢、氧化应激、信号转导、细胞骨架、细胞周期及细胞凋亡等,提示这些过程与机制可能参与了糖尿病主动脉病变的发生与发展。
3. Western blotting证实db/db小鼠主动脉STEAP4和PDE5A蛋白表达明显增加；根皮苷治疗显著抑制这两个蛋白的表达水平,提示这些蛋白可能是根皮苷对db/db小鼠主动脉起保护作用的关键蛋白。
4.本研究提供了根皮苷对于db/db小鼠主动脉保护作用的蛋白质表达谱的重要信息,为全面揭示根皮苷作用机制提供了新的方法和新的视角,为寻找预防和治疗糖尿病大血管并发症提供了可能有意义的药物靶标,从而为临床诊治糖尿病大血管并发症奠定与充实理论基础。
Part One Effect of Phlodizin on Aorta in db/db Mice
Background
Type1diabetes mellitus and type2diabetes mellitus are the two main clinical types of diabetes mellitus. In the past20years, Type2diabetes has become one of the most common endocrine diseases in both developed and developing countries, due to the elevation of living standard, modern life styles and aging. Following cancers and heart diseases, diabetes mellitus has become the third disease that affects the quality of people's life in the whole world. Diabetes is caused by absolute or relative deficiencies in insulin secretion or insulin action. Chronic hyperglycemia is the key biochemical alteration in type2diabetes. The long term-lasting of high blood glucose can cause structural and functional damage of organs in diabetic patients. Moreover, individuals under type2diabetes usually develop aortic complication marked by endothelial injuries to different extents. Excessive oxidative stress has been reported to play a critical role in the development of diabetic aortic injury.
Diabetic individuals have a two-to-three fold increase risk for cardiovascular and cerebrovascular diseases such as heart attack and stroke. Diabetic vascular complication is the main pathological basis of cardiovascular and cerebrovascular diseases, and is one of the major causes of morbidity and mortality in diabetic patients. Moreover, the current teatment for diabetes including intensive insulin treatment, strict lipid control, reduction of blood hypertension and smoking cessation are not sufficient to prevent aortic deteriorative progression to arteriosclerosis. Therefore, it is important and necessary to understand the pathophysiology of type2Diabetes Mellitus and develop strategies to prevent and treat the high blood glucose and related vascular complications.
Phlorizin, a phloretin glucoside, belongs to dihydrochalcones and maily distributed in the apple trees as well as in root bark, shoots, and leaves. Phlorizin makes the major sources of flavonoid. It has been reported that phlorizin have possessed a variety of potent properties, including anti-inflammation, anti-tumor, regularing glucose concentrations, memory improvement and anti-oxidation. Phlorizin is also endothelial protective by suppressing endothelial adhesion molecule expression and platelets activation. However, the effect of phlorizin on the macrovascular complications of type2Diabetes Mellitus had not been elaborated. Here, we fed phlorizin to db/db mice to study the effects of this natural reagent on macrovascular complications of type2Diabetes Mellitus and to determine the underlying mechanism. Through this investigation perhaps new medications can be found to effectively treat macrovascular complications of type2Diabetes Mellitus.
Objectives
1. To observe the morphological changes of the aorta of db/db mice and the effects of phlorizin on the aorta of db/db mice.
2. To investigate the effects of phlorizin on the blood samples of db/db mice, including blood glucose, TC, TG, AGEs, MDA, ALT. AST, BUN and Cr, and to explore the mechanism of phlorizin's aorta preventing effects.
3. To examine the effects of phlorizin on the expression of STAT3and p-STAT3in the aorta of diabetic db/db mice, and to explore the molecular mechanism by which phlorizin prevented aortic damage by DM, further to provide new approaches for the management of macrovascular complications of Diabetes Mellitus.
Methods
Male C57BLKS/J db/m mice (n=8.6weeks old) and C57BLKS/J db/db mice (n=16,6weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu. China). The mice were kept under observation for one week prior to the start of the experiments. The db/db mice were randomly divided into2groups: untreated diabetic group (DM, n=8) and diabetic group treated by phlorizin (DMT, n=8). Phlorizin (20mg/kg Id) was given in normal saline solution intragastrically for 10weeks. C57BLKS/J db/m mice were used as control (CC, n=8). Mice of the DM group and the CC group were given the same amount of normal saline solution intragastrically every day as those of the DMT group. All mice were weighed every week during the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, and the aortas were dissected. Aortic tissues and sera were kept at-80℃for further analysis. Blood glucose, TC, TG, AGEs, MDA, ALT, AST, BUN and Cr were measured. Morphological changes of the aorta were examined by light microscopy and H-800electron microscope.
Results
1. General characteristics
Mice of the CC group showed good condition and grew up sound with smooth furs. The db/db mice of the DM group featured polydipsia, polyphagia and polyuris with shaggy furs and a rapid growth. Phlorizin treated db/db mice also displayed abnormal, but much better than the db/db mice of the DM group.
2. Effects of phlorizin on body weights
Baseline body weights were similar in mice of the DM group and the DMT group, and higher than the mice of the CC group. At as early as week2of the study, the body weight in db/db mice group was significantly than that of the control mice(P<0.01). This trend did not change until the end of the experiment. However, the body weight was significantly inhibited at2th week,4th week,6th week,8th week, and11th week after phlorizin administration in the DMT group compared to the DM group (PO.01).
3. Effects of phlorizin on FBG、TC、TG. AGE、MDA、ALT、AST、BUN and Cr
Baseline FBG, AGEs and MDA were similar between mice of the DM group and the DMT group (P>0.05), and higher than the mice of the CC group (P<0.05). In the10th week, the FBG, AGEs and MDA of diabetic mice were higher than those of control mice (P<0.01). Phlorizin significantly reduced FBG, AGEs and MDA in the diabetic mice (P<0.05). Serum TC. TG. AGEs、MDA. ALT、AST、BUN and Cr were similar among the three groups.
4. Histological and electron micrographical findings
The microscopic images of the aortic sections of the control group appeared normal, with organized intimal surfaces and normal ultrastructure. Vascular wall of the DM group thickened with impaired intima and a narrowed lumen. The ultrastructure shows that in untreated diabetic mice, the vascular smooth muscle cells migrated into the elastic laminar, and an increase of heterochromatin could be observed. With phlorizin treatment, the micro-and ultra-structure changes were less severe.
5. Effects of phlorizin on the expression of STAT3and p-STAT3
Western blotting showed that the expression of p-STAT3in the aortic section of the DM group was much higher than that of the CC group. With phlorizin treatment, the expression of p-STAT3was much decreased.
Conclusion
Phlorizin can decrease the body weight of the db/db mice. Phlorizin also decreased the serum glucose, TC, TG, AGEs, and MDA of the db/db mice, and had no effects on ALT.. AST、BUN and Cr. In addition, phlorizin ameliorated the aortic damage of Diabetes Mellitus. The protective effects of phlorizin on diabetic aortas can be attributed to its antiglycation and antioxidant effect. Phlorizin may be a safe natural reagent for diabetes mellitus treatment. Perhaps new medications can be found to effectively treat macrovascular complications of type2Diabetes Mellitus through intensive study on phlorizin.
Part Two iTRAQ-based proteomic study of the effects of phlorizin on Aortic Arteriosclerosis in db/db mice
Background
With the achievement of human genome sequencing project, life science has turned to the post-genomic era. Proteome is consisting of the total proteins present in a cell or organism at a given time. It includs both proteins translated directly from genetic material and the modified proteins arising from extensive posttranslational processing. Thus, proteome is regarded as the most critical and promising part in the post-genomic studies.
Quantitative proteomics is the major part of functional proteomics, qualitatively and quantitively identifying the whole proteins of the cells. Quantitative proteomics focusing on the dynamic structural and functional alterations of the proteins in a given cell. Theremore, quantitative proteomics profiling is applied to identify the differentially expressed proteins under disease or after drug intervention. Quantitative proteomics is being developed rapidly, with new technological innovations and protocol design applied. The rapidly emerging field of quantitative proteomics provides a powerful technique-isobaric tag for relative and absolute quantitation (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the identification and characterization of protein profiles. This technique can lessen variation, enhance throughput and enable quantitative analysis.
Phlorizin as a member of the chalcone class of organic compounds is mainly distributed in the plants of the genus Malus. Phlorizin is proved to possess multiple pharmacological activities, such as estrogenic and anti-estrogenic activities, anti-oxidative, regulation of blood sugar level, memory improvement and cardioprotective activities. Phlorizin has been shown to inhibit intestinal glucose uptake and renal glucose reabsorption by inhibiting the sodium D-glucose cotransporter. In the first part of our experiment, Phlorizin has demonstrated protective effects on aortic tissue in the db/db mice. But the molecular targets and the underlying mechanism of phlorizin are unclear.
In the second part, we identified differentially expressed proteins in aortas of the control db/m mice, diabetic db/db mice and phlorizin treated db/db mice applying 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 targeting proteins of phlorizin and the underlying mechanism by which phlorizin improved diabetic aorta injury, to develop potential for treating diabetes induced retinal lesions.
Objective
1. The purpose of the study was to obtain the differentially expressioned proteins in the aorta of diabetic mice and to explore the the pathogenetic mechanism of macrovascular complications caused by diabetes.
2. The objective of this study was to identify the effective multifunctional targets and therapeutic strategies against diabetic macrovascular complications after phlorizin treatment.
Methods
The aortic tissue was dissected among the control, diabetic db/db mice, phlorizin treated db/db mice. The processes of proteomic analysis were described as follows: The separated aortic tissue sample were prepared according to the iTRAQ procedure, including tissue disruption, sample clean-up, and pretein solubilization. The protein concentrations were estimated by Bradford arrary. Then the aortic tissues were kept at-80℃for further analysis. About20ug peptides of each group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were digested and iTRAQ labeled, 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 the labeled samples were finally mixed together, separated by Strong Cation Exchange (SCX) chromatography into10fractions and finally desalted by an offline fraction collector and C18cartridges (Sigma). After being labeled, 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, which was based on the observation of the peak for reporter ions encoded in the low mass-to-charge ratio portion of the MS/MS spectrum. In the second part, mass spectrometric analysis was performed by 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 by data-dependent acquisition conditions. The acquired MS/MS spectra were automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version3.72) by Turbo SEQUEST program in the BioWorksTM3.1software suite. 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 according to the molecular function, biologicai process, and cellular component by AmiGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi Version1.8). Finally, some candidated differentially expressed proteins in the aorta were confirmed by Western blottingting analysis.
Results
1. SDS-PAGE image
Proteins brand loaded on the gels separared by SDS-PAGE were observed clearly, available for the following experiments.
2. iTRAQ labeling statistical validation
Our results indicated that only10percent of the peptides between two groups were significantly different.
3. Mass spectrometry identify the differently expressed proteins
Protein profiling was analyzed by iTRAQ approach followed by LC-MS/MS identification and IPI database searching. A total of3083proteins were identified, of which1512proteins meet the standard. Among which557proteins were differentially expressed in aorta of db/db mice compared with control db/m mice, of which463proteins were up-regulated and94proteins were down-regulated. After phlorizin treatment,50proteins were down-regulated while20proteins up-regulated in aorta of diabetic db/db mice.
4. Subcellular localization analysis phlorizin associated retina proteins in db/db mice
We performed the localization analysis of the identified proteins in aorta of db/db mice by AmiGO (Version1.8).23.08%of the total proteins were in cytoplasm,15.38%in nucleus and13.9%in plasma membrane.9.68%of the proteins were in mitochondrion,7.69%in endoplasmic reticulum.
5. Subcellular bioinformatic functional analysis phlorizin associated retina proteins in db/db mice
Most of the back-regulated proteins were involved in material metabolism, oxidative stress, apoptosis, cell growth and signaling transduction. Among the functional assignment of the proteins,17.28%of the proteins were in metabolic processes, and16.05%were binding proteins.11.11%of the back-regulated proteins participate in oaxidative stress and ATP synthesis;8.64%mediate the apoptosis, cell cycle and growth.
6. Confirmation of selected candidate proteins of iTRAQ by Western blottingting analysis
To confirme the differentially expressed protein, PDE5A and STEAP4were validated by Western blottingting analysis. PDE5A and STEAP4were significantly increased in aorta of diabetic db/db mice compared to the control group. After phlorizin treatment, PDE5A and STEAP4were regulated near to normal.
Conclusion
1. As an advanced technology of quantitative proteomics, iTRAQ can analyze four samples synchronously with higher accuracy and sensitivity. In the second part, with the help of iTRAQ, we explored the mechanism underlying the protection of phlorizin against diabetic aortic injury.
2. A total of70differentially expressed proteins were identified in aorta of db/db mice after phlorizin treatment by LS-ESI-MS/MS methods. Of these,50proteins were down-regulated while20were up-regulated after phlorizin administration. Most of these back-regulated proteins were located in cytoplasm and plasma membrane, related to diverse functions, including metabolic process, signal transduction, apoptosis and oxidative stress. Our results suggested that these processes may be linked to the pathogenesis of diabetic macrovascular complications.
3. PDE5A and STEAP4were increased in aorta of db/db mice compared to the control group. After phlorizin treatment, PDE5A and STEAP4were regulated near to normal.
4. Our second part provided new insight into the mechanism and new promising treatment for diabetic macrovascular complications.
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