【作者】 宝轶；

【导师】 刘志民；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 糖尿病(diabetes mellitus, DM)是一种常见的代谢性疾病,常伴发严重的微血管并发症,如肾衰竭、视网膜病变、周围神经病变等。近些年研究表明高血糖可以累及中枢神经系统,称为“糖尿病脑病”,其通常表现为认知功能的障碍和神经电生理的异常,同时伴有神经生化、神经结构和脑的退行性改变。目前,研究发现控制欠佳的糖尿病可以增加血管性痴呆和阿尔茨海默病的发病风险,导致学习和记忆障碍。但由于诊断DE仍缺乏明确的标准,因此很难被确诊,而“糖尿病性认知障碍”更容易被接受,对其病理生理机制的深入研究,已成为糖尿病并发症领域的研究热点之一。Smad蛋白家族是TGF-β超家族细胞内信号转导过程中极其重要的介导分子,主要介导TGF-β1的生物学信息。Smad蛋白家族分为3个不同的亚族：受体调节型Smad蛋白(R-Smads,包括Smad2和Smad3)、共调节型Smad蛋白(Co-Smads,包括Smad4)和抑制性蛋白(I-Smads,包括Smad6和Smad7)。Smad2/3磷酸化与Smad4形成异聚复合体,将信号从胞浆转移到细胞核中,与其它转录协同因子和抑制因子共同调节下游靶基因的转录。TGF-β1/Smad通路参与胚胎发育、间质纤维化、肿瘤发生发展和炎症修复反应等诸多生物学过程,该通路的异常激活与诸多糖尿病相关并发症的病因密切相关,且此通路在β-淀粉样前体蛋白(β-amyloid precursor protein, APP)和β-淀粉样蛋白(β-amyloid, Aβ)的转录调控中具有重要作用。因此,探讨TGF-β1/Smad2/3通路调控星形胶质细胞的生物学行为并促进糖尿病认知功能障碍的发生发展,有助于阐明其病理生理机理,并为临床上寻找新的治疗靶标提供坚实的理论依据。本研究拟通过观察糖尿病大鼠的认知功能状态及Smad蛋白在糖尿病大鼠海马组织星形胶质细胞中的确切表达情况,并通过AGEs干预原代培养大鼠星形胶质细胞,进一步研究TGF-β1/Smad2/3通路在糖尿病性认知功能障碍发生发展过程中的功能角色,探讨其可能的病理生理机制。实验共分为三个部分：第一部分,观察糖尿病大鼠认知功能状态以及海马组织的超微结构；第二部分,观察pSmad2、pSmad3在糖尿病认知功能障碍大鼠海马组织星形胶质细胞中的表达水平以及GFAP、TGF-β1等的表达水平；第三部分,通过AGEs干预原代培养大鼠星形胶质细胞,不同时间点、不同浓度观察pSmad2、pSmad3、TGF-β1及目标蛋白APP、Aβ的表达水平,应用通路抑制剂干预,观察pSmad2和APP蛋白的表达水平。第一部分糖尿病大鼠的认知功能状态及组织病理结构的观察目的：研究糖尿病大鼠发病早期的认知功能状态和氧化应激相关酶类的活性水平,观察海马组织的病理变化。方法：通过腹腔注射STZ(60mg·kg-1)制作糖尿病大鼠模型,72h后测定尾静脉末梢血糖≥16.7mmol·L-1为诱导糖尿病模型成功。将造模成功的大鼠随机分成4周DM和8周DM,正常对照组随机分成4周CON和8周CON,各组大鼠10只。于糖尿病发病8周时,利用Morris水迷宫实验检测其学习和记忆能力。实验连续5天,每天训练1次,每次将大鼠面向池壁从不同的四个方向作为入水点,观察并记录大鼠寻找并爬上平台所需的时间(逃避潜伏期)及距离。第5天实验结束后撤除平台,进行探索实验检测大鼠对原平台的记忆,测量其在60s内跨过原平台所在象限的时间占总时间的比例(Platform%)。Morris水迷宫实验结束后,腹主动脉采血,断头取脑,迅速留取双侧海马组织,测定大鼠血浆及海马组织中SOD、GSH-Px活性和MDA含量水平,并应用HE染色光镜和透射电镜观察海马组织结构的病理变化。结果：在Morris水迷宫实验中,糖尿病大鼠在发病8周时出现认知功能障碍,逃避潜伏期(P<0.01)、游出距离显著大于CON(P<0.01),探索实验中Platform%显著低于CON(P<0.01)。DM大鼠血浆SOD(108.64±25.20 vs.209.10±28.06,P<0.01)、GSH-Px(280.69±33.64 vs.395.29±52.56,P<0.01)活性明显下降,与CON相比差异显著。而MDA水平较CON显著升高(7.21±1.33 vs.3.22±0.78,P<0.01)。海马组织SOD(10.85±2.11 Vs.20.05±4.17,P<0.01)、GSH-Px(19.85±3.96 vs.29.00±4.26,P<0.01)活性明显下降,MDA水平显著升高(5.08±0.78 vs.2.7±1.04,P<0.01),与CON相比差异显著。透射电镜观察下,CON大鼠海马组织神经元及胶质细胞形态正常,核内常染色质丰富,线粒体嵴呈平行排列,突触间隙正常。而DM组胞质内空泡形成明显,部分出现裸核,线粒体肿胀,嵴大部分消失,基质呈空泡状。突触间隙增宽,电子密度降低。H-E染色光镜下观察两组大鼠海马组织未发现异常病理改变。结论：长期高血糖可导致学习和记忆能力下降,出现认知功能障碍的表现,同时伴有氧化应激相关酶类活性水平的变化和超微结构的病理改变。第二部分糖尿病认知功能障碍大鼠海马组织Smad2/3蛋白的表达及调节目的：研究转化生长因子胞内蛋白Smad2/3及其活性形式pSmad2/3在糖尿病认知功能障碍大鼠海马组织中的蛋白表达水平,并通过观察其与星形胶质细胞的标志蛋白GFAP的共表达情况,研究其细胞定位水平。方法：应用免疫印迹、免疫组织化学等方法检测：Smad2、Smad3及pSmad2、pSmad3在糖尿病认知功能障碍大鼠发病4周及8周时的蛋白表达水平,8周时应用免疫荧光染色技术检测pSmad2、pSmad3在大鼠海马组织中的细胞定位水平及其与GFAP蛋白的定位关系。同时应用蛋白印迹实验检测’TGFβ1、GFAP蛋白表达情况。结果：蛋白印迹实验证实pSmad2在糖尿病认知功能障碍大鼠发病4周和8周时,在海马组织的表达水平显著高于正常大鼠(4w：0.10±0.23 vs 0.0±0.0；P<0.05；8w：2.04±0.23 vs 0.0±0.0；P<0.05)。pSmad3的蛋白表达水平升高,与正常大鼠海马组织相比差别显著(4w：0.10±0.23 vs 0.0±0.0；P<0.05；8w：2.04±0.23 vs 0.0±0.0；P<0.05)。在8周时免疫组织化学染色显示DM大鼠海马组织星形胶质细胞GFAP表达水平较正常组显著性升高(P<0.01),而pSmad2及pSmad3表达水平较正常组无明显差异。免疫荧光染色提示pSmad2和pSmad3表达于海马组织星形胶质细胞胞核,在海马CA1、CA3、DG区域pSmad2+的星形胶质细胞所占的比值显著性升高,与正常大鼠相比差异显著(CA1：31.45±3.06%vs.9.77±1.87%,P<0.01；CA3：34.98±4.33%vs.11.06±3.97%,P<0.01；DG：36.87±5.17%vs.15.01±3.10%,P<0.01)。DM大鼠海马CA1、CA3、DG区域pSmad3+的星形胶质细胞所占的比值较正常大鼠升高(CA1：23.33±4.53%vs.7.35±1.14%,P<0.01；CA3：18.32±6.34%vs.11.36±4.53%,P<0.05；DG：20.64±4.45%vs.14.39±4.10%,P<0.05)。同时,蛋白印迹实验显示DM大鼠TGFβ1、GFAP表达水平升高,与正常组相比差异显著(P<0.01)。结论：胞内转化生长因子Smad2/3的活性形式pSmad2/3在糖尿病大鼠海马组织星形胶质细胞中的表达升高,提示该通路的激活可能是糖尿病认知功能障碍的病理生理机制之一。第三部分AGEs对大鼠原代培养星形胶质细胞的Smad信号途径及其目标蛋白APP的调节目的：研究AGEs对原代培养的大鼠海马星形胶质细胞的Smad信号通路相关蛋白的表达水平和细胞亚定位水平,及其调节的靶基因APP的蛋白表达水平。方法：应用AGEs干预原代培养的大鼠星形胶质细胞,MTT法测定干预后24、48、72小时原代星形胶质细胞增殖率,分别应用AGEs在0、50、100ug·ml-1的不同浓度干预原代培养星形胶质细胞,应用免疫荧光染色检测pSmad2和pSmad3在原代星形胶质细胞中的细胞亚定位水平及其与GFAP的定位关系。采用无血清饥饿法同步细胞于G0期,蛋白印迹实验测定AGEs干预后1、24、48小时pSmad2、Smad2、pSmad3、Smad3、TGFβ1、APP、Aβ的蛋白表达水平变化。应用RAGE抗体及TβRI抑制剂在AGEs前30min干预胶质细胞,观察pSmad2和APP的蛋白表达水平。结果：MTT法检测显示AGEs可以促进星形胶质细胞的增殖活性,细胞免疫荧光染色显示pSmad2和pSmad3的表达在AGEs干预后1h即出现升高,并持续至24h时达到高峰,48h出现回落。蛋白印迹实验显示pSmad2和pSmad3在AGEs干预星形胶质细胞后1小时开始出现升高,在24h蛋白表达达到峰值,而在48h时表达有所下降。在0、50(P<0.05)、100(P<0.01) ug·ml-1不同浓度AGEs刺激星形胶质细胞24小时后,结果显示pSmad2蛋白表达显著性增高,这种作用是浓度依赖性的。AGEs可以诱导TGFβ1在星形胶质细胞中的表达,在24h和48h显著性增高,与阴性对照相比差异显著(P<0.01)。100ug·ml-1浓度的AGEs干预星形胶质细胞24小时后, APP和Aβ的蛋白表达水平显著性增加,均较干预前相比差异性显著(P<0.01)。24h时TβRI抑制剂及RAGE抗体可以抑制pSmad2及APP的蛋白表达,提示AGEs激活Smad信号通路是通过RAGE介导的TGFβ依赖性的途径而实现的。结论：通过AGEs可以活化星形胶质细胞；激活星形胶质细胞Smad信号通路,进而促进靶基因APP的蛋白表达,而这个过程是TGFβ1介导实现的。由此可见,抑制该通路的激活以及AGEs的作用可能作为未来糖尿病认知障碍的选择之一。更多还原

【Abstract】 Diabetes mellitus represents a common metabolic disorder, which may lead to severe microvascular complications, such as kidney failure, retinopathy, and peripheral nerve system. Recently, accumulating evidence suggests that diabetes may also affect the central nervous system and lead to memory and learning impairment accompanied by the alterations of structure, function and metabolism of the brain, referred to as diabetic encephalopathy. Recently, ample evidence suggests that uncontrolled diabetes may increase the risk of vascular dementia and Alzheimer’s disease, leading to memory and learning impairment in diabetic patients. But the diagnosis diabetic encephalopathy lacks clear criteria and is therefore difficult to ascertain.The Smad family comprises receptor-regulated Smads (R-Smads, including Smad2 and Smad3), common partner Smads (CO-Smads, including Smad4) and inhibitory Smads (I-Smads, including Smad6 and Smad7). They modulate the activity of transforming growth factor beta ligands. The phosphorylation of Smads (to form pSmads) triggers the recruitment of the cofactor Smad4, and the entire Smad complex is translocated to the nucleus where it binds to DNA response elements and, together with transcription factors and various cofactors, promotes transcription of a rangeof target genes. This pathway is associated with the transcriptional activity of APP. Therefore, investigation of functional role of Smad signaling pathway in astrocyte of diabetic associated cognitive decline may offer a better understanding of pathogenesis in cognitive impairment and important targets for treatment strategies.The aim of this study is to clarify the exact role for Smad signaling pathway in the hippocampus of cognitive impairment diabetic rat and effect of advanced glycation end products on astrocytes in primary culture. This study is consisted of three main parts:the first part is to investigate the effect of streptozotocin-induced diabetes on cognitive function and hippocampal ultrastructure in the rat, the second is to see whether Smad signallingwas activated in the hippocampus of streptozotocin-induced diabetic cognitive impairment rats and investigate the expression of pSmad2、pSmad3、GFAP and TGFβ1 in hippocampus of diabetic cognitive impairment rat. Finally, the effect of AGEs and TGFβ1 on astrocytes in primary culture and observe the expression of pSmad2、pSmad3、APP、Aβ, further investigation underlying molecules mechanism of diabetic astrocyts. Part I Effect of Streptozotocin-induced diabetes on cognitive function and hippocampal ultrastructure in the ratsObjective:To evaluate the effect of streptozotocin-induced diabetes on cognitive function and hippocampal ultrastructure in the rat.Methods:Diabetes was induced by a single intraperitoneal injection of streptozotocin at a dose of 60mg·kg-1 body weight dissolved in citrate buffer. The streptozotocin-injected rats with blood glucose levels≥16.7 mmol·L-1 3 days after injection were included in the study and were randomly divided into two groups:4w DM (n=10) and 8w DM (n=10). Control rats were divided into two groups:4w CON (n=10) and 8w CON (n=10). After 8 weeks of experiment, rats were tested in Morris water maze. The animals were trained to locate the platform on 5 consecutive days, and both latency times and distances to reach the platform were recorded. On the 5th day, the rat was given a maximum of 60 s to locate the hidden platform and the time spent in the quadrant of the pool containing the hidden platform was recorded. SOD、GSH-Px and MDA were measured in plasma and hippocampal tissues, and the cellular ultrastructure of the hippocampus were observed through light microscope and transmission electronic microscope.Results:In Morris water maze test, in diabetic rats the latency (P<0.001) and distance (P<0.01) swum reach to the platform increased significantly compared to controls, and the percentage of time spent in the quadrant of the pool with platform decreased notably (P<0.01). The levels of GSH-Px and SOD decreased significantly (P<0.001) and the levels of MDA increased significantly in diabetic groups compared to controls (P<0.001). Conspicuous intracytoplasmic vacuole formation was present in neurons and astrocytes of diabetic rats’hippocampus. Obvious chromosome aberrations, dilatate, widened synaptic clefts as well as disappeared mitochondriales crista were also observed in the diabetic rats’ hippocampus by TEM. However, H-E staining is not obvious pathological change by light microscope.Conclusion:In summary, we demonstrate that moderate impairment of learning and memory occurred in diabetic rats. At the same time, oxidative stress induced by hyperglycemia and ultrastructure of diabetic rats’hippocampus are abnormal. PartⅡExpression changes of phosphorylated Smad2 and Smad3 in the hippocampus of streptozotocin-induced diabetic cognitive impairment ratsObjective:Activation of the Smad signalling pathway has been implicated in the pathological process of diabetic associated complications.The current study was designed to see whether Smad signallingwas activated in the hippocampus of streptozotocin-induced diabetic cognitive impairment rats.Methods:We determined the expression level of phosphorylated Smad2 and Smad3 in the hippocampus by western blot analysis and double immunofluorescene labeling assay. At same time, we determined the expression level of TGFβ1 in the hippocampus by western blot analysis, and pSmad2、pSmad3、GFAP staining by immunohistochemistry.Results:Compared with the normal controls, immunoblot analysis showed that pSmad2 averagely increased by 0.10-fold at week 4 and 2.04-fold at week 8 post STZ induction in the hippocampal homogenates (P<0.01 at week 8), pSmad3 increased by 0.66-fold at week 4 and 1.91-fold at week 8 of diabetic duration (P<0.01). pSmad2 and pSmad3 immunoreactivity were predominantly detected in the nuclei of astrocyte in hippocampus The percentage of pSmad2+astrocyteswas signifi cantly increased in CA1 region (31.45±3.06%vs.9.77±1.87%, P<0.01), CA3 region (34.98±4.33%vs.11.06±3.97%, P< 0.01) and DG region (36.87±5.17%vs.15.01±3.10%, P<0.01) in the hippocampus of diabetic rats as compared to controls at week 8. Similarly, double immunofl uorescence labeling assay revealed a signifi cant increase in the percentage of pSmad3 immunoreactive astrocytes in CA1 region (23.33±4.53%vs.7.35±1.14%, P<0.01), CA3 region (18.32±6.34%vs.11.36±4.53%, P<0.05) and DG region (20.64±4.45% vs.14.39±4.10%, P<0.05) in the hippocampus of diabetic rats as compared with controls at week 8. Expression levels of TGFβ1 and GFAP increased in hippocampus of diabetic rats, whereas expression levels of pSmad2 and pSmad3 are not changed by IHC.Conclusion:These data indicate that Smad signalling is enhanced in hippocampal astrocytes of diabetic rats, and may thereby represent a clue to explore its exact role in the development of diabetic associated cognitive decline. PartⅢEffect of AGEs on smad signaling pathway and APP in primary cultured astrocytesObjective:To investigate the effects of AGEs on smad signaling pathway and APP in primary cultured astrocytes.Methods:Primary cultured astrocytes were treated by advanced glycation end products at different concentration of 0、50、100ug·ml-1. Cell proliferative activity was assessed by MTT assay. Immunofluorescene labelling assay was employed to investigate the celluar location of pSmad2 and pSmad3 protein in primary cultured astrocytes. A neutralizing anti-RAGE antibody and TβR I inhibitor were added into cells 30 min before AGEs stimulations. The expression levels of pSmad2、Smad2、pSmad3、Smad3、APP、Aβprotein were measured by western blot.Results:Proliferation of astrocytes was significantly increased compared with that of control at 24h (0.078±0.019 vs.0.117±0.028, P<0.05),48h (0.109±0.044 vs.0.284±0.043, P<0.01),72h (0.178±0.034 vs.0.512±0.103, P<0.01) post AGEs stimulation. By immunofluorescene, pSmad2 and pSmad3 were predominantly detected in the nuclei of astrocytes, and expression of pSmad2 and pSmad3 increased at 1h (P<0.01),24h (P<0.01), 48h (P<0.01), peaking at 24h. Western blotting showed that AGEs induced Smad2 and Smad3 phosphorylation by astrocytes in a dose-dependent manner, being at 1h, peaking at 24h and 48h after AGEs stimulation. AGEs were able to induce TGFβ1 expression by astrocytes, being significant at 24h (P<0.01) and 48h (P<0.01). At the same time, AGEs induced marked APP and Ap protein expression at 24h (P<0.01). Finally, western blot analysis showed that the neutralizing anti-RAGE antibody and TβR I inhibitor completely inhibited AGE-induced Smad2 phosphorylation and APP at 24h. This indicates that AGEs signal through RAGE to activate smad signaling via TGFβ-dependent pathways.Conclusion:Overall, our results indicate that AGEs can induce astrocytes proliferation and act by activating smad signaling to mediate diabeteic cognitive dysfunction via TGFβ-dependent pathways. Therefore, inhibit smad pathway and AGEs may represent a potentially treatment strategy against diabetic cognitive dysfunction.更多还原