葡萄籽原花青素B2对db/db小鼠主动脉保护机制的研究
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摘要
第一章GSPB2对db/db小鼠主动脉保护作用的蛋白质组学研究
     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。随着社会经济的发展和人民生活水平的提高,生活方式的改变和社会人口老龄化,糖尿病患病率在世界范围内呈上升趋势,已经成为继心脑血管疾病、肿瘤之后的又一严重危害人类健康的全球公共卫生问题,也成为世界各国致死、致残并造成医疗开支增高的主要原因。糖尿病不仅以持续性的高血糖为其特点,更重要的是高血糖以及合并的代谢紊乱引起的多系统损害,诸如心脏、血管、肾脏、神经、视网膜、周围神经、脑等组织器官的慢性进行性病变。
     糖尿病主动脉病变是糖尿病最常见的大血管并发症,是糖尿病患者的主要死因。糖尿病患者比非糖尿病患者发生动脉粥样硬化及其并发症的危险提高了2-4倍,糖尿病已经作为冠心病等危症越来越受到人们的重视。糖尿病主动脉病变以不断进展的动脉粥样硬化及血管重构为特征,是糖尿病心血管并发症的主要病理学基础。其发病机制尚不清楚,治疗手段也不完善。因此,在临床实践中迫切需要寻找糖尿病主动脉病变的内在分子机制与新型治疗药物,进一步完善与充实DM主动脉病变的治疗策略。
     天然药物葡萄籽原花青素(grape seed proanthocyanidin extract, GSPE)是从葡萄籽中提取的一种天然多酚类化合物,主要是以几茶素或表几茶素为单体缩合而成的聚合物,其中以低聚体(二聚、三聚、四聚体)生物活性最强。葡萄籽原花青素B2(GSPB2)是GSPE的主要成分,其生物活性在GSPE的多种成分中最强。有研究表明,GSPB2具有抗炎、抗凋亡、抗AGEs诱导的血管平滑肌细胞增殖和迁移的作用。既往研究表明,GSPE对链脲佐菌素(streptozotocin, STZ)诱导的糖尿病大鼠的主动脉具有明确的保护作用。然而,此保护作用的分子机制还不完全清楚。随着蛋白质组学技术的发展,一种基于质谱的蛋白质组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 quantitation, 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技术可对一个基因组表达的全部蛋白质或一个复杂的混合体系中的所有蛋白质进行精确定量和鉴定,寻找差异表达蛋白,并分析其蛋白功能。其已经在探讨疾病发生机制、疾病标志物的寻找和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。为了进一步探讨GSPB2对2型糖尿病主动脉损伤的保护作用及其作用靶点,本研究采用国际上公认的2型糖尿病模型小鼠——db/db小鼠为研究对象,对经GSPB2干预的db/db小鼠的主动脉进行iTRAQ蛋白质组学分析,寻找正常对照组(CC组),db/db小鼠组(DM组)与db/db小鼠GSPB2治疗组(DMT组)主动脉的差异表达蛋白,旨在揭示GSPB2对于db/db小鼠主动脉病变的保护机制,为临床糖尿病患者血管并发症的治疗寻求更为有效的药物提供候选靶点。
     研究目的
     1.研究db/db小鼠主动脉病变的特点,观察18周龄各组小鼠主动脉组织的病理学和超微结构变化；
     2.应用iTRAQ蛋白质组学技术研究db/db小鼠主动脉损伤与正常小鼠主动脉的差异表达蛋白,明确糖尿病大血管病变的病理生理机制；
     3.研究db/db小鼠经GSPB2治疗后主动脉的差异表达蛋白,筛选药物候选靶点,进一步揭示GSPB2对糖尿病主动脉的保护机制。
     研究方法
     7周龄雄性C57BLKS/J db/db小鼠16只及7周龄雄性C57BLKS/J db/m小鼠8只,适应性饲养1周。实验开始后,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：8只DM模型组(DM),每日予生理盐水灌胃；8只为GSPB2干预组(DMT), GSPB2溶于与DM组相同体积生理盐水,以30mg/kg/d灌胃,共进行10周。
     1.体重、FBG、TC、TG和AGEs的测定：实验期间每周定期测量体重(body weight, 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(fasting blood glucose, FBG)、胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)、糖基化终末产物(advanced glycation end products, AGEs)等指标。
     2.主动脉组织形态学和超微结构观察：迅速剥离主动脉,用多聚甲醛或戊二醛固定,HE染色观察主动脉病理变化,测量主动脉内中膜厚度；应用透射电镜观察主动脉组织超微结构变化。
     3.蛋白质组学(iTRAQ)分析：分别选取CC组、DM组和DMT组小鼠各4只,提取主动脉总蛋白。各组主动脉总蛋白采用FASP酶解得到相应肽段。各组取60μg肽段,按照ABI公司说明书操作与标记肽段,114标记正常对照组,115标记GSPB2干预组,117标记db/db糖尿病组。将各组已标记的肽段混合后用强阳离子交换柱(strong cation exchange, SCX)和C18柱进行分离,用串联质谱方法对在第一级质谱检测到前体离子进行碰撞诱导解离,产物离子通过第二级质谱进行分析。不同报告基团离子强度的差异就代表了它所标记的多肽的相对丰度。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Identified iTRAQ Statistic Builder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。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 (international protein index, version3.72)蛋白数据库
     4.蛋白质定位分析、功能分析及相互作用网络分析：采用GO分类工具(http://www.geneontology.org, VERSION1.8)和KOGnitor (eukaryotic orthologous groups)(http://www.ncbi.nlm.nih.gov/COG/grace/kognitor.html)分析对经鉴定的主动脉蛋白质表达谱进行定位分类与功能分析。应用Ingenuity Pathways Analysis (IPA)软件进行生物信息学分析。
     5.蛋白质免疫印迹方法(Western blot):部分主动脉组织采用、western blot方法检测鉴定出的乳凝集素(milk fat globule epidermal growth factor-8, MFG-E8)、谷胱甘肽S-转移酶(gultathione S transferases theta-1, GSTT1)和半胱氨酸甘氨酸富集蛋白1(cysteine and glycine-rich protein1, CSRP1)的蛋白表达水平以验证蛋白质组学的可靠性。
     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. GSPB2对db/db小鼠体重、FBG、TC、TG和AGEs的影响
     实验前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,一直持续至实验结束(小鼠18周龄)。DMT组小鼠与DM组相比,GSPB2能够显著改善DM小鼠的体重(p<0.01)。
     实验开始时DM组与DMT组间的FBG水平无显著性差异(p>0.05),但与CC组比较,两组FBG水平均明显升高(p<0.01)。给予GSPB2干预10周后,DMT组小鼠的FBG水平与DM组相比轻微下降,但无显著性差异(p>0.05)。
     实验结束时测量血清TC、TG和AGEs。DM组的TC、TG和AGEs与CC组相比明显升高(p<0.01)。GSPB2干预10周后,TC、TG和AGEs水平明显下降(p<0.01)。
     3. GSPB2对主动脉组织形态学的影响
     HE染色：光镜下CC组主动脉内膜光滑,内皮细胞形态规则、排列整齐,中膜主要由同心排列的弹性纤维组成,无增厚；DM组主动脉壁层次不清,内皮细胞损伤、突起、形态不规则,中膜明显增厚,平滑肌细胞增生。DMT组内皮细胞损伤减轻,中膜增厚明显减轻,平滑肌细胞和弹性纤维排列较规则。
     4. GSPB2对主动脉超微结构的影响
     电镜下CC组主动脉内皮细胞形态正常,平滑肌细胞核染色质分布正常,可见线粒体、内质网、高尔基复合体等细胞器,形态正常。DM组内皮细胞损伤,可见插入的平滑肌细胞；弹力膜断裂,管壁胶原纤维增多；平滑肌细胞核固缩,异染色质增多；线粒体固缩,棒状线粒体出现；内质网肿胀。DMT组内皮下结构基本正常,平滑肌细胞插入和细胞器异常明显减轻。
     5. iTRAQ质谱鉴定结果
     本研究经质谱鉴定蛋白1530个。CC组和DM组小鼠主动脉差异表达蛋白557个(±1.5倍),其中糖尿病小鼠主动脉组织表达上调的点463个,下调的点94个,经GSPB2治疗后回调的蛋白139个6. GSPB2干预后差异蛋白的定位分析
     对质谱鉴定得出的139个差异蛋白点(DM组和DMT组之间)经过蛋白质组学工具分析,差异表达蛋白定位主要包括胞浆蛋白(40%),胞核蛋白(18.8%),胞膜蛋白(12.1%),内质网蛋白(7.9%),线粒体蛋白(7.3%),分泌蛋白(6.7%),中心体蛋白(6.0%),核糖体蛋白(3.0%),高尔基体蛋白(2.4%)和溶酶体蛋白(1.2%)。
     7. GSPB2干预后差异蛋白的功能分析
     对质谱鉴定得出的139个差异蛋白点(DM组和DMT组之间)进行功能分析。差异表达蛋白功能主要包括信号转导蛋白(17%),细胞骨架蛋白(12%),翻译后修饰、蛋白转换和分子伴侣蛋白(9%),翻译、核糖体结构蛋白(7%),细胞内运输、分泌蛋白(6%),细胞周期、细胞分化蛋白(4%),核苷酸的转运和代谢蛋白(4%),脂质转运和代谢蛋白(4%)。还有一部分蛋白分布于13个其他功能类别。
     8.差异表达蛋白的IPA分析
     应用IPA软件将差异表达蛋白进行PATHWAY分析,分别绘制包括细胞凋亡、氧化应激和脂质代谢等通路,这些通路有利于更好的理解糖尿病血管重塑的分子机制及GSPB2的干预靶点。
     9.筛选部分差异蛋白在主动脉组织的表达改变
     为验证蛋白质组学鉴定出的部分差异蛋白在主动脉组织的表达,应用Western blot方法检测其中差异蛋白如MFG-E8、GSTT1和CSRP1在各组小鼠主动脉组织中的表达。结果显示,与正常对照组小鼠比较,MFG-E8和CSRP1在DM小鼠中表达明显增高(p<0.01),应用GSPB2治疗后,表达增高的蛋白有所下调(p<0.01)。另外,GSTT1在DM小鼠中表达中与正常对照组比较表达后明显降低(p<0.01),经过GSPB2干预后该蛋白表达回调(p<0.01)。该结果与iTRAQ鉴定结果是一致的。这些蛋白为我们将来的深入研究提供了思路。
     结论
     1.与正常对照组比较,db/db小鼠体重显著增加,GSPB2干预可以显著改善db/db小鼠的肥胖趋势。
     2.与正常对照组比较,db/db小鼠的FBG、TC、TG和AGES水平显著升高,经GSPB2治疗后能够显著降低其TC、TG与AGES水平。
     3.光镜和电镜显示：db/db小鼠主动脉内皮损伤,平滑肌增殖,血管重塑,各种细胞器异常,GSPB2能够显著改善db/db小鼠主动脉损伤。
     4.作为定量蛋白质组学的新技术iTRAQ具有高灵敏性、高通量的特点,能够对多组样本同时进行比较分析。因此,iTRAQ可用于研究GSPB2对db/db小鼠主动脉保护作用的分子机制,寻找新的药物靶点,获得可靠的各组差异表达蛋白。
     5.质谱鉴定CC组和DM组小鼠主动脉差异表达蛋白557个,其中DM组主动脉组织表达上调的点463个,下调的点94个,经GSPB2治疗后回调的蛋白139个。以胞浆蛋白为主,其功能涉及细胞凋亡、氧化应激和脂质代谢,提示这些过程参与了T2DM主动脉病变的发生发展。
     6. Western blot验证结果显示,与CC组比较,MFG-E8和CSRP1在DM组小鼠主动脉中表达明显增高,应用GSPB2治疗后,表达增高的蛋白有所下调。GSTT1在DM组小鼠中表达中与正常对照组比较表达后明显降低,经过GSPB2干预后该蛋白表达回调。这为深入研究GSPB2对主动脉的保护作用提供了关键的候选靶点。
     第二章GSPB2对db/db小鼠主动脉的保护机制及对MFG-E8的干预研究
     研究背景
     随着人类基因组计划的完成,生命科学研究已进入了后基因组时代。蛋白质是生理功能的执行者,是生命现象的直接体现者,对蛋白质结构和功能的研究将直接阐明生命在生理或病理条件下的变化机制。传统的对单个蛋白质进行研究的方式已无法满足后基因组时代的要求。不同生理和病理状态下蛋白质的表现是多样的、动态的,因此要对生命的复杂活动有全面和深入的认识,必然要在整体、动态、网络的水平上对蛋白质进行研究。蛋白质组学的研究是其中的关键内容之一。但蛋白质组学对成百上千的蛋白质的识别只是这一过程的初始阶段,对大量的差异蛋白进行数据分析和筛选,进行功能验证以及研究其相互作用和信号通路,从而确定潜在的靶点才是其核心内容。
     葡萄籽原花青素B2(GSPB2)是迄今为止发现的最强效的自由基清除剂之一。抗氧化及清除自由基的能力与其分子结构中含有较多的酚羟基,并与其特定分子立体化学结构密切相关。本课题组前期研究发现,GSPB2能够抑制AGEs诱导的活性氧自由基(reactive oxygen species, ROS)的产生和血管平滑肌细胞的增殖；GSPE对1型糖尿病大鼠主动脉具有明确的保护作用。但是对2型糖尿病主动脉的保护机制尚未深入研究,其分子靶点也尚未明确。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,无法实现对于药物作用的靶点的全面分析。而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。本研究第一部分采用iTRAQ蛋白质组学技术,鉴定出CC组与DM组的差异蛋白557个,经GSPB2治疗后,此差异蛋白回调139个。我们根据已有知识、数据库注释和文献资料进行筛选,发现其中MFG-E8蛋白表达DM组升高,GSPB2治疗后表达降低,在DM/CC中的比值为2.41,DMT/DM的比值为0.59。
     MFG-E8是最早在乳脂球表面发现的一种亲脂性糖蛋白,在很多组织细胞中均有表达。有研究表明,MFG-E8在炎症损伤及动脉斑块中有表达；MFG-E8随年龄增长而显著增高；在对乳腺癌及膀胱癌等肿瘤的研究中,体内MFG-E8的表达升高。但在糖尿病及其靶器官病变中,MFG-E8的作用尚未明确。因此,本研究拟在蛋白质组学鉴定的基础上,进一步研究MFG-E8对糖尿病主动脉损伤的作用,在2型糖尿病模型db/db小鼠体内采用慢病毒介导的RNA干扰技术降低体内MFG-E8蛋白的表达,以及体内注射重组MFG-E8蛋白增加小鼠MFG-E8蛋白表达,观察MFG-E8对T2DM主动脉的作用,研究其内在机制,从而为临床早期监测和干预,以及T2DM主动脉并发症的治疗提供新的药物靶点。
     研究目的
     1.深入研究GSPB2对db/db小鼠主动脉的保护机制,进一步明确糖尿病大血管病变的病理生理机制；
     2.构建MFG-E8慢病毒干扰载体,以及体内注射重组蛋白过表达MFG-E8,研究MFG-E8降低或升高对主动脉病理学和超微结构变化的作用,并深入研究MFG-E8参与糖尿病主动脉损伤的作用机制。
     研究方法
     1.主动脉内皮细胞原代培养：8周龄雄性C57BLKS/J db/m小鼠剥离主动脉进行主动脉内皮细胞原代培养。将主动脉纵行剪开,平展在平皿内,用手术刀将其切成2mm×2mm小动脉片,然后贴入无菌的细胞培养瓶中,动脉内膜面与培养瓶玻璃面接触,加入5ml含20%胎牛血清的内皮细胞培养基(ECM),放于37℃5%二氧化碳培养箱中培养。7-9天细胞形成细胞单层,用0.25%的胰酶消化传代。使用3.6代细胞用于研究。
     2.慢病毒干扰载体的构建及细胞内转染：构建针对小鼠MFG-E8基因4个靶点的慢病毒干扰载体,并构建含GFP的载体作为对照载体。分别在MOI(multiplicity of infection)50、75、100的情况下进行转染主动脉内皮细胞,并于12h、24h、48h和72h在荧光显微镜下观察转染效率。确定最佳MOI值为100。转染3天后,收集细胞使用western blot检测MFG-E8蛋白表达下调水平,筛选出干扰效率最高的靶点。大规模包装此靶点病毒,进行db/db小鼠体内实验。
     3. db/db小鼠体内实验研究MFG-E8缺失与过表达对主动脉的影响：7周龄雄性C57BLKS/J db/db小鼠32只及7周龄雄性C57BLKS/J db/m小鼠8只,适应性饲养1周。实验开始后,C57BLKS/J db/m小鼠8只作为正常对照组(CC),C57BLKS/J db/db小鼠随机分为四组：8只DM模型组(DM)；8只为GFP干预组(GFP),于小鼠14周给予GFP尾静脉注射一次；8只为MFG-E8慢病毒干扰载体=F预组(M-RNAi),f小鼠14周给予LV-MFG-E8尾静脉注射一次；8只为重组MFG-E8蛋白干预组(rmMFG-E8),从小鼠14周开始,每周两次尾静脉注射MFG-E8重组蛋白(20μg/kg),共4周。
     4. MFG-E8干预对主动脉组织形态学及超微结构的影响：实验周期结束,所有小鼠禁食12h后处死,迅速剥离主动脉,多聚甲醛或戊二醛固定,HE染色观察主动脉病理变化,测量主动脉内中膜厚度；应用透射电镜观察主动脉组织超微结构变化。
     5. MFG-E8干预对主动脉MFG-E8和p-ERK蛋白表达的影响：部分主动脉组织分离后立即投入液氮,然后.80℃保存。Western blot检测主动脉MFG-E8和p-ERK蛋白表达情况。
     6. MFG-E8干预对血清MCP-1表达的影响：采血并分离血清,检测各组血清MCP.1表达情况。
     研究结果
     1.MFG-E8对主动脉组织形态学的影响
     HE染色：光镜下CC组主动脉内膜光滑,内皮细胞形态规则、排列整齐,中膜主要由同心排列的弹性纤维组成,无增厚；DM组和GFP组主动脉壁层次不清,内皮细胞损伤、突起、形态不规则,中膜明显增厚,平滑肌细胞增生；M-RNAi组内皮细胞损伤减轻,中膜增厚明显减轻,平滑肌细胞和弹性纤维排列较规则；rmMFG-E8组较DM组损伤更为明显,内皮细胞肿胀,血管壁增厚,炎症细胞如中性粒细胞和淋巴细胞浸润,并有局部坏死。
     2. MFG-E8对主动脉超微结构的影响
     电镜下CC组主动脉内皮细胞形态正常,平滑肌细胞核染色质分布正常,可见线粒体、内质网、高尔基复合体等细胞器,形态正常；DM组和GFP组内皮细胞损伤,可见插入的平滑肌细胞；弹力膜断裂,管壁胶原纤维增多；平滑肌细胞核固缩,异染色质增多；线粒体固缩,棒状线粒体出现；内质网肿胀；M-RNAi组内皮下结构基本正常,平滑肌细胞插入和细胞器异常明显减轻；rmMFG-E8组较DM组病变加重,并可见更多的异染色质和异常细胞器,平滑肌细胞迁移入内弹力膜。
     3. MFG-E8干预对主动脉MFG-E8蛋白表达的影响
     Western blot分析表明,与正常组小鼠相比,DM组和GFP组主动脉组织MFG-E8的蛋白表达水平明显升高(p<0.01)；M-RNAi干扰组MFG-E8表达较GFP组明显降低(p<0.01)；而重组蛋白rmMFG-E8组的蛋白表达较DM组明显升高(p<0.01)
     4. GSPB2和MFG-E8干预对主动脉p-ERK蛋白表达的影响
     Western blot分析表明,与正常组小鼠相比,DM组和GFP组主动脉组织p-ERK的蛋白表达水平明显升高(p<0.05或p<0.01)；GSPB2治疗显著抑制p-ERK的蛋白表达水平(p<0.01)；M-RNAi干扰组p-ERK表达较GFP组明显降低(p<0.01)；而重组蛋白rmMFG-E8组的p-ERK蛋白表达较DM组明显升高(p<0.01)
     5. GSPB2和MFG-E8干预对血清MCP-1表达的影响
     ELISA法检测血清MCP-1表达情况显示,与正常组小鼠相比,DM组和GFP组MCP-1的表达水平明显升高(p<0.01)；GSPB2治疗显著抑制MCP-1的表达(p<0.01)；M-RNAi干扰组MCP-1表达较GFP组明显降低(p<0.01)；而重组蛋白rmMFG-E8组的MCP-1表达较DM组明显升高(p<0.01)
     结论
     1.构建了可靠的慢病毒MFG-E8干扰载体,并在细胞及动物水平验证了其转染效率
     2. GSPB2和慢病毒MFG-E8干扰载体能够显著抑制体内MFG-E8蛋白表达,从而减轻2型糖尿病主动脉的损伤；而体内注射MFG-E8重组蛋白加重主动脉损伤程度。
     3. GSPB2和慢病毒MFG-E8干扰载体能够显著降低主动脉组织p-ERK蛋白表达；而体内注射MFG-E8重组蛋白增加主动脉p-ERK蛋白表达。
     4. GSPB2和慢病毒MFG-E8干扰载体能够显著降低血清MCP-1表达；而体内注射MFG-E8重组蛋白增加MCP-1表达。
     研究背景
     药物成瘾是一种顽固且极易复发的精神疾病,具有长期记忆的特点。随着对成瘾行为生物机制研究的深入,药物成瘾已经越来越受到人们的重视。在成瘾药物的作用下,神经系统发生长程适应性变化,并发生行为、细胞分子及基因表达与调控的改变。研究表明成瘾药物诱导行为敏化的形成涉及新的基因转录、新的蛋白合成以及组蛋白乙酰化修饰的改变。
     代谢型谷氨酸受体(metabotropic glutamate receptors, mGluRs)是通过G蛋白偶联,调节细胞内第二信使的产生而导致代谢改变的谷氨酸受体。根据氨基酸序列的同源性及其药理学特征和信号转导机制的不同,可将其分为三组：mGluRsⅠ组、mGluRs Ⅱ组和mGluRs Ⅲ组。mGluRs Ⅱ组在糖尿病神经病变中起保护作用,能够防止糖尿病外周神经系统的细胞损伤；mGluRs Ⅱ组被谷氨酸盐激活后,还能导致胰腺酶类分泌增加,这对糖尿病的治疗提供了一种新的途径。mGluRs Ⅰ组包括mGluRl和mGluR5。mGluRs拮抗剂能够减轻药物成瘾所致的行为学改变,包括可卡因成瘾。然而,其治疗成瘾的作用机制尚未完全明了。已有研究表明激活mGluR5能够增加突触蛋白合成,并且已经在脆性X综合征的发病中得到证实,但mGluRs Ⅰ组介导的蛋白合成是否与药物成瘾有关还未得到证实。
     mGluRs Ⅰ组与细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK)和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路有关。激活这两个通路能够促进翻译起始和蛋白合成。记忆的形成和加强涉及蛋白的重新合成,药物成瘾的记忆和行为也与新蛋白的合成有关。但学习记忆与药物成瘾导致蛋白合成的信号机制尚未阐明。其中一个可能的机制是学习记忆与药物成瘾导致谷氨酸盐的释放,从而激活Ⅰ组mGluRs增加蛋白合成。
     中脑腹侧被盖区(ventral tegmental area, VTA)及其多巴胺能神经元的投射在成瘾行为和奖赏回路(reward circuit)中起关键作用。mGluR1和mGluR5在中脑多巴胺神经元中均有表达,而mGluR1的表达更多。我们提出假设,VTA区的mGluR1介导的蛋白合成与可卡因条件性位置偏爱(conditioned place preference, CPP)有关。为了验证此假说,本研究采用电生理、生物化学及行为学手段对SD大鼠进行可卡因成瘾研究,揭示药物成瘾的潜在机制,为治疗药物成瘾提供新的思路。
     研究目的
     本实验旨在研究mGluR1在可卡因成瘾及其行为学改变中的作用机制,为药物成瘾的治疗提供新的途径。
     研究方法
     1.18-30天SD大鼠处死后制备VTA区脑片,使用电生理设备进行全细胞电压钳记录。记录抑制性突触后电流(inhibitory postsynaptic currents, IPSCs)。
     2.采用10-11周SD大鼠(300-350g)进行双侧VTA区手术插管。术后恢复一周。然后进行条件性位置偏爱(CPP)实验。使用三箱体CPP装置进行训练。
     (1)第一天预测试(Pre-test)20min,记录大鼠分别在黑色和白色箱子停留时间,时间相差180s以上从本实验中剔除。
     (2)第2-9天为训练阶段(Conditioning)。大鼠分为六组：空白对照+可卡因训练组；空白对照+生理盐水训练组；颅内注射mGluR1拮抗剂JNJ16259685(0.1ng,每侧1μl)+可卡因训练组；颅内注射mGluR1拮抗剂JNJ16259685(0.1ng,每侧1μl)+生理盐水训练组；颅内注射蛋白合成抑制剂环己酰亚胺(cycloheximide,100ng,每侧1μl)+可卡因训练组；颅内注射蛋白合成抑制剂环己酰亚胺(cycloheximide,100ng,每侧1μl)+生理盐水训练组。使用2μl微量注射器双侧同时给药,给药时间不少于2min,给药完毕后,额外保留注射器在颅内2main以保证药物充分扩散。
     可卡因训练：大鼠在第2、4、6、8天颅内注射30min后,腹腔注射可卡因(15mg/kg),然后放入其中一个箱子进行训练20min,第3、5、7、9天接受颅内注射30min后,腹腔注射生理盐水(1ml／kg),然后放入与可卡因训练时相反的箱子中进行训练20min；
     生理盐水训练：大鼠在第2、4、6、8天颅内注射30min后,腹腔注射生理盐水(1ml/kg),然后放入其中一个箱子进行训练20min,第3、5、7、9天接受颅内注射30min后,腹腔注射生理盐水(1ml/kg),然后放入与2、4、6、8天训练相反的箱子中进行训练20min。
     (3)第10天测试(Test)20min。所有大鼠在三个箱子中自由活动,记录在每个箱子中的停留时间。Preference Score (s)计算=可卡因训练箱中停留时间-生理盐水训练箱中停留时间。
     3.制备18-30天大鼠脑片,给予mGluR1激动剂DHPG (100μM)10min。随后脑片分为空白对照和JNJ6259685(100nM)组。Western blot测定ERK和mTOR磷酸化水平。
     4.CPP实验结束后1小时,大鼠切VTA脑片进行western blot测p-ERK、 p-mTOR、p-p70S6K、p-S6、p-eIF4E和eEFIA的表达。
     研究结果
     1.在脑片VTA区记录抑制性突触后电流(IPSCs)
     mGluRs Ⅰ组兴奋剂DHPG (100μM,10min)能够诱导IPSCs的长时程抑制(long-term depression, LTD)(n=7, p<0.01vs baseline)。 mGluR1选择性拮抗剂LY367385(100μM)能够阻断DHPG诱导的IPSCs抑制(n=7,p<0.05vscontrol);另外一种mGluR1选择性拮抗剂JNJ16259685(100nM)也能够阻断DHPG诱导的IPSCs抑制(n=6, p<0.05vs control)。而mGluR5选择性拮抗剂MTEP (10μM)并不能影响DHPG诱导的IPSCs抑制(n=8, p>0.05vs control)。
     2. DHPG诱导的I-LTD需要蛋白合成
     在脑片VTA区记录抑制性突触后电流(IPSCs)。蛋白合成抑制剂anisomycin (30μM, n=8, p<0.05vs control)和cycloheximide (80μM, n=6, p<0.05vs control)均能阻断DHPG诱导的I-LTD,但在给药初期对IPSCs抑制无明显影响(p>0.05vs control)。
     3. DHPG诱导的I-LTD需要ERK和mTOR信号通路的激活
     MEK抑制剂U0126(20μM)部分阻断DHPG诱导的I-LTD,但无统计学意义(n=8, p>0.05vs control);其无活性异构体U0124(20μM)对DHPG诱导的I-LTD无影响(n=7, p>0.05vs control)。mTOR抑制剂雷帕霉素(rapamycin,100Nm)对DHPG诱导的LTD无影响(n=7, p>0.05vs control)。然而,同时给予rapamycin (100nM)和U0126(20μM)能够阻断DHPG诱导的LTD (n=8, p<0.05vs control)。
     4. DHPG通过mGluRl激活ERK和mTOR磷酸化
     VTA脑片western blot结果显示DHPG (100μM,10min)升高p-ERK和p-mTOR水平,而此升高作用能够被mGluRl拮抗剂JNJ16259685(100nM)阻断(p<0.001)。
     5.CPP结果
     颅内注射JNJ16259685或cycloheximide能够明显减轻可卡因训练大鼠的CPP(n=7-10,p<0.001),但对生理盐水训练大鼠的CPP无影响(n=7-10,p>0.05)。颅内注射JNJ6259685或cycloheximide对大鼠自主活动量(locomotor activity)无明显影响(n=7-10,p>0.05)。
     6.对可卡因CPP实验后1小时制备的VTA标本进行western blot测定
     与盐水训练的大鼠相比,可卡因训练的大鼠VTA中p-ERK1/2和p-mTOR水平明显升高(p<0.001),而JNJ组能够降低p-ERK1/2和p-mTOR水平(p<0.001)；与盐水训练的大鼠相比,可卡因训练的大鼠VTA中p-p70S6K、p-S6和p-eIF4E水平明显升高(p<0.001),而JNJ组能够阻断其升高作用(p<0.001或p<0.01)；与盐水训练的大鼠相比,可卡因训练的大鼠VTA中eEF1A水平明显升高(p<0.001),而JNJ组和cycloheximide (Cyc)组能够阻断其升高作用(p<0.001)。
     结论
     研究结果表明,mGluR1抑制剂JNJ16259685能够明显减轻可卡因成瘾导致的CPP改变。对VTA脑片电生理及大鼠CPP实验后的VTA标本进行研究后表明,可卡因成瘾与蛋白重新合成增加有关。ERK1／2及mTOR信号通路的激活在可卡因成瘾的形成中起了关键作用。本研究可为进一步探索药物成瘾的机制提供新的思路,为治疗药物成瘾开拓新的视野。
Chapter One Proteomic Analysis of Protective Effects of GSPB2on Aorta in db/db Mice
     Background
     Diabetes mellitus (DM) is a disease of metabolic disorder characterized by hyperglycemia and insulin resistance accompany with insufficiency of secretion or action of endogenous insulin. With the development of economy and the improvement of living standard, and the aging population increased, the prevalence of DM has become a global public health problem, resulting in morbidity and mortality and increased medical expenses. The damage of DM is not only for its persistent hyperglycemia, but also the multi-system damage caused by hyperglycemia and metabolic disorders, such as chronic lesions of heart, blood vessels, kidneys, nerves, retina, peripheral nerve, brain and other tissues.
     Aorta complications are the major cause of mortality in patients with DM. The risk of diabetic patients to atherosclerosis and its complications is2to4times than that of nondiabetic patients. DM has been regarded as an equivalent to coronary disease. The hallmarks of macrovascular damage are accelerated atherosclerosis and remodeling of large arteries characterized by thickening and stiffening of the arterial wall. Thus far, the underlying mechanism remains poorly understood, and there is no cure available for DM and its cardiovascular complications. Therefore, elucidation of the molecular mechanism of aorta damage associated with DM and identification of the target protein critically involved in this process could lead to a more specific strategy in fighting against the vascular complications of DM.
     Procyanidins are a complex family of polyphenol polymers widely existing in natural products. Grape seed proanthocyanidin extracts (GSPE) derived from grape seeds. Dimeric procyanidin B2is one of the main components of GSPE, composed of two molecules of the flavan-3-ol (-)-epicatechin linked by a4b→8bonds. Studies including ours have shown that procyanidin B2has properties including anti-inflammation, anti-oxidant, anti-AGEs-induced proliferation and migration of aortic smooth muscle cells. Previous data showed that GSPE has protective effect on aorta of STZ induced diabetic rats. However, the molecular mechanism remains unrevealed. With the development of proteomics, a new quantitative approach based on the mass spectrum using isobaric tag for relative and absolute quantitation (iTRAQ) has become the main method with its unique superiority. iTRAQ enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. It has been widely used in finding the pathogenesis, disease markers, the differentially expressed proteins at different state, and so on. In an effort to understand the protective effect and the potential target protein of GSPB2on aorta in type2diabetes (T2DM), we resorted to iTRAQ technique of the aorta using db/db diabetic mice. db/db mice are well-established animal model to study T2DM complications. The purpose of this experiment was to explore the protective mechanisms of GSPB2on aorta in db/db mice by searching the differential protein among CC group, DM group and DMT group, thus provide candidate targets for clinical treatment of diabetic vascular complications.
     Objective
     1. To study the characteristic of aorta in db/db mice, observe the morphology changes by light and electron microscope.
     2. To explore the differentially expressed proteins of aorta in db/db mice by iTRAQ, further defined the physiopathologic mechanisms of diabetic macrovascular.
     3. To establish the aortic differentially expressed proteins of db/db mice after treatment of GSPB2, evaluate the protective effects of GSPB2on diabetic aortic damage.
     Methods
     Male C57BLKS/J db/db mice (n=16,7weeks old) and db/m mice (n=8,7weeks 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. C57BLKS/J db/m mice were selected as control group (CC, n=8). The db/db mice were divided into2groups:an untreated diabetic group (DM, n=8) administrated with normal saline solution by intragastric administration and GSPB2-treated group with a dosage of30mg/kg/d (DMT, n=8) for10weeks.
     1. The measurement of body weight, FBG, TC, TG and AGEs: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, fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured.
     2. Aortic morphology and ultratructure:The aortas were dissected from the controls, db/db mice and db/db mice treated with GSPB2, then fixed in4%paraformaldehyde and embedded in paraffin for HE staining and intima-media thickness measurement, or fixed in glutaraldehyde for ultrastructure observation using electron microscope.
     3. Proteomic analysis (iTRAQ):About50mg Aortic tissue from each of four mice per group was pooled and homogenized in the presence of liquid nitrogen. In filter-aided sample preparation (FASP) detergents are removed by ultrafiltration, and after protein digestion, peptides are separated from undigested material. About60μg peptides of each group were labeled with iTRAQ reagents (114for the peptides of CC group,115for the peptides of DMT group, and117for the peptides of DM group respectively) following the manufacturer's instructions (Applied Biosystems). Strong Cation Exchange (SCX) chromatography was performed to separate the labeled samples into10fractions. Eluted peptides were collected and desalted by an offline fraction collector and C18cartridges. The precursor ion was through collision-induced dissociation by tandem mass spectrometry, product ion was analyzed by secondary MS. The different ionic strength of reporter group represents the difference in the relative abundance of its labeled polypeptides. 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. Use114as the reference (CC group).
     4. Subcellular localization analysis, functional analysis and ingenuity pathway analysis:The localization analysis of the identified proteins in aortas was performed by using AmiGO (Version1.8). The functional analysis was performed by KOGnitor (http://www.ncbi.nlm.nih.gov/COG/grace/kognitor.html). The global protein changes data in the aorta of db/db mice treatment with GSPB2were analyzed through the use of Ingenuity Pathways Analysis (Ingenuity Systems, CA). Ingenuity software uses the data to navigate the Ingenuity pathways database for interactions between these focus proteins and all the other protein stored in the database to generate biological networks.
     5. Western blot:Some differentially expressed proteins were validated using western blot analysis.
     Results
     1. General Data
     In the course of experiment, the mice of CC group showed good condition with smooth furs. No diabetic symptoms were shown. The mice of DM group showed polydipsia, polyphagia and hyperdiuresis with filthy furs and obesity. GSPB2treated db/db mice also displayed abnormal, but better than the db/db mice.
     2. Effects of GSPB2on Body Weight, FBG, TC, TG and AGEs
     There was no statistical significance of body weight between DM group and DMT group prior to the experiment (p>0.05), but were significantly higher than CC group (p<0.01). At week2of the study, the body weight of DM group was significantly increased. This trend did not change until the end of the experiment (18weeks old). However, the increase of body weight was significantly inhibited by GSPB2administration in the DMT group compared to the DM group (p<0.01).
     There was no statistical significance of FBG between DM group and DMT group at the beginning of experiment (p>0.05), but were significantly higher than CC group (p<0.01). GSPB2decreased the level of FBG in DMT group, but there was no significant difference (p>0.05).
     The serum TC, TG and AGEs were measured at the end of experiment. The TC. TG and AGEs of DM group were significantly higher than CC group (p<0.01). GSPB2significantly reduced the serum TC, TG and AGEs of db/db mice (p<0.01).
     3. Effects of GSPB2on Aortic Morphology
     HE staining:Under light microscopy, aortic remodeling and proliferation of vascular smooth cells (VSMC) and endothelial injury were observed in the aorta of db/db mice. Moreover, GSPB2suppressed the endothelial injury, aortic remodeling and proliferation of VSMC and led to light microscopic findings similar to those of the control mice.
     4. Effects of GSPB2on Aortic Ultrastructure
     Under electron microscopy, the impaired endothelial cells and the rod-like mitochondria in endothelial cells were seen in aortic tissue in the diabetic mice. Moreover, the rupture of vascular elastic membrane and the increased collagen fibers were observed in the diabetic aorta. Many smooth muscle cells showed pyknotic nucleus, increased heterochromatin, mitochondrial condensation, swelling of the endoplasmic reticulum, and many autophagosome appeared, whereas normal ultrastructure was observed in the aortic tissue of the control db/m mice. GSPB2tended to improve the preservation of the fine structure of aortic tissue.
     5. Mass Spectrometry Identify the Differentially Expressed Proteins
     We identified1530proteins in this study.557proteins were shown to have significantly different abundance between control group and DM group (±1.5-fold). Of these557proteins, the levels of139proteins were normalized by GSPB2treatment.
     6. Subcellular Localization Analysis of Differentially Abundant GSPB2Associated Arterial Proteins
     The localization analysis of the identified proteins was performed using AmiGO (Version1.8). Among these proteins, some are located in one or more subcompartments of the cell.40%were in cytoplasm,18.8%in nucleus,12.1%in plasma membrane,7.9%in endoplasmic reticulum,7.3%in mitochondrion,6.7%in extracellular,6%in centrosome,3%in ribosome,2.4%in Golgi,1.2%in lysosome.
     7. Bioinformatic Functional Analysis of Differentially Abundant GSPB2Associated Arterial Proteins
     Among the functional assignment of the proteins,17%were involved in signal transduction mechanisms,12%in cytoskeleton,9%in posttranslational modification, protein turnover, chaperones,7%in translation, ribosomal structure and biogenesis,6%in intracellular trafficking, secretion, and vesicular transport,4%in cell cycle control, cell division, chromosome partitioning,4%in nucleotide transport and metabolism,4%in lipid transport and metabolism, and the remaining proteins were identified as dispersed across13remaining categories.
     8. Ingenuity Pathway Analysis of Differentially Abundant GSPB2Associated Arterial Proteins
     Ingenuity Pathway Analysis (IPA) showed that the primary pathway involved was cell death, lipid metabolism and oxidative stress. The pathways would facilitate the understanding of diabetes biomarkers for further study.
     9. Validation of iTRAQ Data on Other Selected Proteins
     To validate the proteomic analysis of aorta using iTRAQ we performed, milk fat globule epidermal growth factor-8(MFG-E8), cysteine and glycine-rich protein1(CSRP1) and gultathione S transferases theta-1(GSTT1) were validated using western blotting analysis. MFG-E8and CSRP1were found to be inhibited whereas GSTT1was enhanced in the DMT group compared to the DM group (p<0.01). The significance of those proteins would be interesting topic for our future studies.
     Conclusion
     1. GSPB2intervention could significantly improve the obesity trend of db/db diabetic mice.
     2. After treated with GSPB2, the TC, TG and AGEs decreased in DMT group than those of DM group.
     3. GSPB2suppressed the aortic remodeling and endothelial injury, and led to light and electron microscopic findings similar to those of the control mice.
     4. As a new technique of quantitative proteomics, iTRAQ enables comparative analysis for many groups. We obtained the reliable differentially expressed proteins from CC group, DM group and DMT group.
     5.557proteins were shown to have significantly different abundance between control group and DM group. Of these557proteins, the levels of139proteins were normalized by GSPB2treatment. Most of the proteins were in cytoplasm, the pathway involved were cell death, lipid metabolism and oxidative stress. The pathways would facilitate the understanding of aortic damage in diabetes.
     6. Western blotting showed MFG-E8and CSRP1were found to be inhibited whereas GSTT1was enhanced in the DMT group compared to the DM group. The significance of those proteins would be critical candidate targets for future studies of GSPB2on diabetic aorta.
     Chapter Two Protective Mechanism of GSPB2on Aorta and the Intervention Study of MFG-E8in db/db Mice
     Background
     With the completion of human genome project, life science research has entered the post genomic era. Protein is the executor of physiological functions. Research on the structure and function of proteins will directly clarify the physiological or pathological mechanism. Traditional method on individual protein has been unable to meet the requirement of post genomic era. The proteins are multiple and dynamic at different conditions. Therefore we must explore changes of global and dynamic proteins for comprehensive understanding of complicated conditions. Proteomics is one of the critical researches in post genomic era.
     Grape seed procyanidin B2is one of the most powerful drugs of free radical scavenger. The ability related to its phenolic hydroxyl of molecular structure. Our previous studies found that GSPB2could inhibit the generation of AGEs-induced reactive oxygen species (ROS) and proliferation of vascular smooth muscle cell, GSPE possess definite protective effect on aorta of type1diabetic rats. However, the protective mechanism of GSPB2and its molecular target on type2diabetes remain poorly understood. The traditional drug research pays more attention on drug phenotypic observation or the activity of single molecular or enzyme, unable to realize the comprehensive analysis of drug targets. But the combination of quantitative proteomics and pharmaceutical research provide a means for potential development of new pharmacological molecular. In part1of this study,557proteins were shown to have significantly different abundance between control group and DM group. Of these557proteins, the levels of139proteins were normalized by GSPB2treatment. According to our prior knowledge, database and literature material, we found that MFG-E8was increased by2.4-fold in abundance in db/db mice, which was normalized by GSPB2treatment, protein expression ratio:DMT/DM0.59.
     Justified to its name, milk fat globule-epidermal growth factor-8(MFG-E8) was initially identified as an indispensable component of the milk fat globule. It is expressed in many histocyte. Studies have shown that MFG-E8was expressed in inflammation and artery plaque, the level of MFG-E8was significantly increased with ages, and elevated in breast cancer and bladder cancer. Its effect on diabetes still remains unknown. Therefore, this part of our study will further explore the effect of MFG-E8on diabetic aortic damage based on the identification of iTRAQ. We evaluated the function of MFG-E8on aorta of T2DM by inhibiting of MFG-E8by RNA interference and exogenous recombinant MFG-E8administration in db/db mice. Approaches targeting MFG-E8could potentially lead to new modality in the prevention and treatment of vascular complications in DM patients.
     Objective
     1. Further establish the protective mechanism of GSPB2on aorta of db/db mice, define the pathophysiological mechanism of macro vascular disease in T2DM.
     2. Construction of lentiviral vector with short hairpin RNA for MFG-E8, and overexpression MFG-E8with recombinant, observe the aortic morphologic changes and further explore the mechanism for MFG-E8involved in the diabetic aortic damage.
     Methods
     1. Primary culture of aortic endothelial cell:The aorta of male C57BLKS/J db/m mice (8weeks old) was isolated. Longitudinal dividing the aortic wall and cut into small pieces, stick the lining surface in the cell culture flasks. Then put it in the incubator with endothelial cell medium (ECM, ScienCell) with an atmosphere of5%CO2/95%air. After7-9days, cell fused into a single layer, dealt with0.25%trypsin digestion and passage. Cells from passages3to6were used in this study.
     2. Construction of MFG-E8RNAi and endothelial cell transfection:Lentiviral vectors with short hairpin RNAs (shRNAs) for MFG-E8were designed and chemically synthesized. The lentiviral vector with green fluorescence protein (GFP) was used as the RNAi control. Aortic endothelial cell were transfected with lentiviral vectors at a multiplicity of infection (MOI=50,75,100) according to the manufacturer's instructions. Fluorescence microscope was used to measure transfection efficiency at time points of12,24,48and72hours after transfection. The optimal MOI was determined for100. The most effective target site of RNA interference was confirmed by using western blot for the expression levels of MFG-E8after3days.
     3. Treatment of MFG-E8RNAi and recombinant MFG-E8in db/db mice:Male C57BLKS/J db/db and db/m mice (n=40,7weeks old) were used in this study. C57BLKS/J db/m mice were selected as control group (CC, n=8). The db/db mice were divided into4groups:an untreated diabetic group (DM, n=8), LV-GFP treated db/db group (GFP, n=8), MFG-E8RNAi treated db/db mice group (M-RNAi, n=8) and recombinant MFG-E8treated db/db mice group (rmMFG-E8, n=8). MFG-E8RNAi (M-RNAi group) or LV-GFP (GFP group) was diluted to a total volume of300μl containing4×107TU was injected into the tail vein of fourteen-week-old male db/db mice. Recombinant mouse MFG-E8was diluted in PBS and300ul of the solution (20μg/kg) was injected through the tail vein of fourteen-week-old male db/db mice twice a week for4weeks (rmMFG-E8group). Each group of mice was observed from week13to week18without any administration of hypoglycemic therapy.
     4. Aortic morphology and ultrastructure:At the end of the intervention, all mice were fasted overnight and then sacrificed. the aortas were dissected, then fixed in4%paraformaldehyde and embedded in paraffin for HE staining and intima-media thickness measurement, or fixed in glutaraldehyde for ultrastructure observation using electron microscope.
     5. MFG-E8and p-ERK expression in aorta using western blot:Some aortic tissues were kept at-80℃for MFG-E8and p-ERK expression analysis.
     6. ELISA of MCP-1in db/db mice serum:Sera were separated for ELISA of MCP-1.
     Results
     1. Effects of MFG-E8RNAi and recombinant MFG-E8on Aortic Morphology
     Under light microscopy, aortic remodeling and proliferation of vascular smooth cells (VSMC) and endothelial injury were observed in the aorta of DM and GFP group. Moreover, MFG-E8RNAi suppressed the endothelial injury, aortic remodeling and proliferation of VSMC and led to light microscopic findings similar to those of the control mice. In aorta of recombinant MFG-E8protein-treated group, the deteriorative changes were observed. The blood vessel was thicker, characterized by more inflammatory cells infiltrated such as neutrophil and lymphocyte and focal necrosis. Endothelial cell swelling and perivascular inflammation were also observed.
     2. Effects of MFG-E8RNAi and recombinant MFG-E8on Aortic Ultrastructure
     Under electron microscopy, the impaired endothelial cells and the rod-like mitochondria in endothelial cells were seen in aortic tissue in the diabetic or GFP treated mice. Moreover, the rupture of vascular elastic membrane and the increased collagen fibers were observed in the diabetic aorta. Many smooth muscle cells showed pyknotic nucleus, increased heterochromatin, mitochondrial condensation, swelling of the endoplasmic reticulum, and many autophagosome appeared, whereas normal ultrastructure was observed in the aortic tissue of the control db/m mice. MFG-E8RNAi tended to improve the preservation of the fine structure of aortic tissue, but in the rmMFG-E8group, the deteriorative changes and the migration of smooth muscle cells in the vascular elastic membrane were observed.
     3. Effects of MFG-E8RNAi and Recombinant MFG-E8on the Expression of MFG-E8
     Western blotting analysis showed protein expression of MFG-E8in DM or GFP group was significantly higher than that of control db/m group (p<0.01); after treatment with MFG-E8RNAi, the expression of MFG-E8decreased compared to GFP treated db/db mice (p<0.01), which confirmed the transfection efficiency in db/db mice in vivo. However, the rmMFG-E8group displayed increased expression of MFG-E8compared to that of both control db/m group and DM group (p<0.01).
     4. Effects of GSPB2, MFG-E8RNAi and Recombinant MFG-E8on ERK1/2phosphorylation levels
     To understand the mechanism of atherosclerosis in T2DM, we further examined the ERK1/2phosphorylation (p-ERK1/2) in aorta of db/db mice by Western blotting. The expression of p-ERK1/2in db/db mice were higher than that of control db/m mice (p <0.05); after treatment with GSPB2, the expression of p-ERK1/2was decreased compared to that of untreated db/db mice. MFG-E8RNAi decreased in the phosphorylated ERK1/2(p<0.01). Treatment with recombinant MFG-E8increased ERK1/2phosphorylation (p<0.01).
     5. Effects of GSPB2. MFG-E8RNAi and Recombinant MFG-E8on the Levels of MCP-1
     The expression of MCP-1in serum was measured by ELISA. The expression of MCP-1in serum was significantly increased in DM group compared with that of control group (p<0.01). GSPB2and MFG-E8RNAi decreased the level of MCP-1(p <0.01). Recombinant MFG-E8significantly increased the expression of MCP-1(p<0.01).
     Conclusion
     1. Construct a reliable lentiviral vectors for MFG-E8interference, and verify the transfection efficiency in cell and animal.
     2. GSPB2and MFG-E8RNAi significantly inhibit the protein expression of MFG-E8, thus reduce aortic injury of T2DM, and recombinant MFG-E8aggravate the aortic damage.
     3. GSPB2and MFG-E8RNAi significantly decrease the protein expression of p-ERK, and recombinant MFG-E8increase the p-ERK.
     4. GSPB2and MFG-E8RNAi significantly decrease the expression of MCP-1in serum, and recombinant MFG-E8increase the MCP-1.
     Background
     Drug addiction is a kind of stubborn and easily recurrence of mental illness which has the characteristics of long-term memory. With the development of the biological mechanism of addictive behaviors research, drug addiction has been got more and more attention. Under the effects of addictive drugs, the long-range adaptive changes in nervous system, and behavioral, cell and molecular and gene expression changes. Studies have shown that addictive drugs inducing the formation of behavioral sensitization involving new gene transcription, protein synthesis and modification of histone acetylation.
     Metabotropic glutamate receptors (mGluRs) are coupled to G-protein, regulate intracellular second messenger and lead to metabolic changes. According to the amino acid sequence homology, its pharmacological characteristics and the signal transduction mechanism, mGluRs can be divided into three groups:mGluRs Ⅰ group, mGluRs Ⅱ group and mGluRs Ⅲ group. mGluRs Ⅱ group could prevent the cell damage of diabetic peripheral nervous system. mGluRs Ⅱ group can induce increased secretion of pancreatic enzymes after glutamate activation, which provides a new thought for the treatment of diabetes. mGluRs Ⅰ group includes mGluRl and mGluR5. mGluRs antagonist can alleviate behavioural changes caused by drug addiction, including cocaine addiction. However, its mechanism of treating addiction has not yet been fully understood. The activation of group Ⅰ mGluRs. mGluR5in particular, increases protein synthesis at synapses, and mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome. However, fewer studies have investigated the role of mGluR1in cocaine addiction.
     mGluRs Ⅰ group was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation and protein synthesis. The formation and consolidation of memories require de novo protein synthesis. The development of drug-associated memories and addictive behavior also requires new protein synthesis. However, the upstream signaling mechanisms by which learning and drug exposure lead to protein synthesis remain poorly understood. One possibility is that learning and cue-drug pairing cause the release of glutamate, which activates group Ⅰ mGluRs to increase protein synthesis.
     The ventral tegmental area (VTA) of the midbrain and its dopaminergic projection play a critical role in reward processing and addictive behavior. Both mGluR1and mGluR5are expressed in midbrain dopamine neurons, although mGluR1is expressed at much higher density. We hypothesized that mGluRl-dependent protein synthesis in the ventral tegmental area (VTA) is required for cocaine-induced conditioned place preference (CPP). In the present study, we tested this hypothesis using a combination of electrophysiological, biochemical and behavioral approaches. These results provide evidence that mGluR1-dependent protein synthesis is critically involved in behavioral effects of cocaine.
     Objective
     To explore the underlying mechanism of mGluR1on cocaine addiction and behavioral changes, and provide new avenues for treating drug addiction,
     Methods
     1. Male Sprague-Dawley rats were used for slice electrophysiology (P18-30of age,50-1OOg). Whole-cell voltage-clamp recording were made from VTA dopamine neurons in midbrain slices. Inhibitory postsynaptic currents (IPSCs) were evoked by stimulating inhibitory synaptic afferents.
     2. Male Sprague-Dawley rats (10-11weeks,300-350g) were anesthetized and placed in a stereotaxic device. Guide cannulae were bilaterally implanted2.8mm above the VTA using aseptic techniques. After the surgery, rats were allowed to recover for about1week. CPP experiments were performed using three-chamber CPP apparatus. The CPP protocol consisted of the following sessions:
     (1) Pre-test (day1):animals were allowed to explore both chambers for20min and time spent in each side was recorded. Rats showing unconditioned side preference (≥180s disparity) were excluded (n=4).
     (2) Conditioning (day2-9):Rats received bilateral intra-VTA infusions of vehicle, JNJ16259685or cycloheximide via the pre-implanted cannulae. Injector cannulae (33-gauge) were inserted into the guide cannulae. The intra-VTA infusions were made via C313C connectors to2μl-Hamilton micro-syringes. Vehicle (1μl per side), JNJ16259685(0.1ng,1μl per side) or cycloheximide (100ng,1μl per side) was manually injected at a rate of1μl over2min, and the injectors were kept in place for an additional2min to ensure adequate drug diffusion from the injector tip. Thirty minutes after the microinjections, rats received cocaine or saline conditioning.
     Cocaine conditioning. Rats received saline injection (0.9%NaCl,1ml/kg, i.p.) on days2,4,6,8and were immediately confined to one chamber for20min. On days3,5,7and9, rats received cocaine injection (15mg/kg, i.p.) and were immediately confined to the opposite chamber for20min.
     Saline conditioning. Rats received daily saline injection and were immediately confined to one chamber for20min on days2,4,6, and8and were confined to the opposite chamber for20min on days3,5,7, and9.
     (3) CPP test (day10):all of the animals were allowed to explore freely for20min among the three chambers and time spent on each side is recorded.
     3. VTA slices from male Sprague-Dawley rats (P18-30of age) were used for western blot. Slices were treated with vehicle or various drugs (see Results section). Western blot were performed using the antibody of phospho-ERK and phospho-mTOR.
     4. Rats were anesthetized with isoflurane and rapidly decapitated-1hour after the CPP tests. The brains were immediately removed and placed in oxygenated ACSF at4℃. Midbrain slices were prepared, and the VTA was dissected out and homogenized in lysis buffer. Western blot was performed using the antibody of phospho-ERK, phospho-mTOR, phospho-p70S6K, phospho-S6, phospho-eIF4E and eEF1A.
     Results
     1. DHPG-induced depression of IPSCs in VTA dopamine neurons is mediated mainly by mGluRl. Bath application of group Ⅰ mGluR agonist DHPG (100μM,10min) induced long-term depression (LTD) of evoked IPSCs (n=7, p<0.01vs. baseline). The mGluR1-selective antagonist LY367385(100μM) blocked DHPG-induced depression of IPSCs (n=7, p<0.05vs. control). Another mGluRl-selective antagonist, JNJ16259685(100nM) blocked DHPG-induced depression of IPSCs (n=6, p<0.05vs. control). The mGluR5-selective antagonist MTEP (10μM) did not affect DHPG-induced depression of IPSCs (n=8, p>0.05vs. control).
     2. Protein synthesis is required for DHPG-induced I-LTD in the VTA.
     The protein synthesis inhibitor anisomycin (30μM; n=8, p<0.05vs. control) or cycloheximide (80μM; n=6,p<0.05vs. control) blocked DHPG-induced I-LTD but did not significantly affect the early component of DHPG-induced depression of IPSCs(p>0.05vs. control).
     3. DHPG-induced-I-LTD requires the activation of ERK1/2and mTOR signaling pathways.
     The MEK inhibitor U0126(20μM) exerted only a modest effect on DHPG-induced I-LTD that did not reach statistical significance (n=8, p>0.05vs. control). U0124(20μM), an inactive analog of U0126, had no effect on DHPG-I-LTD (n=7, p>0.05vs. control). The mTOR inhibitor rapamycin (Rapa,100nM) also had no significant effect on DHPG-induced I-LTD (n=7, p>0.05vs. control), whereas co-application of rapamycin (100nM) and U0126(20μM) blocked DHPG-induced I-LTD (n=8,p <0.05vs. control).
     4. DHPG induced ERK and mTOR phosphorylation and activation in the VTA via mGluR1. DHPG (100μM,10min) increased p-ERK1/2levels in VTA homogenates compared, this effect was blocked by the JNJ16259685(JNJ,100nM, p<0.001). DHPG also increased p-mTOR levels in VTA homogenates compared with control, this effect was blocked by JNJ16259685(100nM,p<0.001).
     5. Intra-VTA infusions of the mGluRl antagonist JNJ16259685or protein synthesis inhibitor cycloheximide during the conditioning phase attenuated the acquisition of CPP to cocaine. Intra-VTA infusions of JNJ16259685or cycloheximide significantly attenuated CPP in cocaine-conditioned rats (n=7-10, p<0.001) but did not affect CPP scores in saline-conditioned rats (n=7-10, p>0.05). Intra-VTA infusions of JNJ16259685or cycloheximide did not significantly affect locomotor activity in cocaine-or saline-conditioned rats (p>0.05, n=7-10).
     6. Western blot was performed on VTA samples collected-1hour after the CPP tests. Cocaine CPP activates ERK and mTOR signaling pathways, p-ERK1/2and p-mTOR levels in the VTA were significantly increased in cocaine-conditioned rats compared with those in saline-conditioned rats, and the increase was blocked by intra-VTA infusions of JNJ16259685(p<0.001). p-p70S6K, p-S6and p-eIF4E levels in the VTA were significantly increased in cocaine-conditioned rats compared with those in saline-conditioned rats, and these increases were blocked by intra-VTA infusions of JNJ16259685during the conditioning phase(p<0.01,p<0.001). eEF1A was significantly increased in cocaine-conditioned rats compared with that in saline-conditioned rats (p<0.001), and this increase was blocked by intra-VTA infusions of JNJ16259685or cycloheximide (Cyc) during the conditioning phase (p<0.001).
     Conclusion
     Intra-VTA microinjections of mGluRl antagonist JNJ16259685significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP). These results suggest that mGluR1antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine. mGluR1was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. This study might provide new avenues for treating drug addiction.
引文
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