【作者】 施翔；

【导师】 战丽彬；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2011， 博士

【摘要】 糖尿病相关认知下降(diabetes-associated cognitive decline, DACD),也被称为糖尿病脑病,是糖尿病在脑部的一种慢性进展性并发症,临床表现为逐渐出现的认知功能下降。据统计,2030年成人糖尿病的全球患病率很可能从2010年的2.85亿增长到4.39亿,将近是2010年的2倍。中华医学会糖尿病学分会“中国糖尿病和代谢综合征研究组"2010年在《新英格兰医学杂志》发表的关于我国糖尿病患病率调查的结果显示,我国20岁以上人群中糖尿病的患病率已达9.7%,总患病人数达九千两百万以上,专家估计70岁以上年龄组患病率超过20%,形势十分严峻。目前DACD缺乏统一的诊断标准,尚无确切的患病率统计,但必将随老龄化社会的来临以及糖尿病患病率的增高而增高。认知功能损伤将给患者的生活带来不便,甚至使其丧失生活自理能力,给家庭和社会带来沉重的负担,因而DACD的防治已成为全世界亟待解决的问题。目前对DACD病机的研究涉及葡萄糖毒性、胰岛素信号障碍、钙稳态失衡、炎症、氧化应激损伤、血管病变及下丘脑-垂体-肾上腺轴异常等方面,但确切机制仍不清楚。而对本病的治疗还处于试验阶段,尚无公认的治疗措施。蛋白质是生命活动的直接执行者,细胞中各种病理改变的发生无不与蛋白质异常有关。但目前以单个蛋白质为对象的研究方式已无法满足人类对生命活动全面深入认识的需求。蛋白质组学(proteomics)作为系统生物学时代最热点的研究领域之一,是对一个基因组所表达的全部蛋白质在整体水平上的大规模研究,它通过一系列高分辨率、高通量的蛋白质分离、鉴定技术,使与疾病相关的特异蛋白分子一次性、全面的暴露出来,加快了对疾病病理改变的认识。目前关于糖尿病认知损伤的研究越来越多,但蛋白质组学方面的分析还很少。海马与学习记忆功能密切相关,也是脑组织中最易受损的部位之一。海马损伤被证明是2型糖尿病(type 2 diabetes mellitus, T2DM)患者脑部早期病变。对DACD海马蛋白质组学的研究将可能揭示糖尿病认知损伤的重要机制。糖尿病在中医学中属于“消渴”病范畴。记忆力降低等认知功能减退的表现,属于中医学“健忘”、“呆病”范畴。《圣济总录》中已有“消渴日久,健忘怔忡”等糖尿病认知损害的症状记载。因此,DACD可认为是消渴病合并健忘或呆病。消渴病机以阴虚为本,燥热为标,病变的脏腑主要在肺、胃、肾。但除此三脏之外,脾阴虚在消渴发生发展过程中的作用也不可忽视。早在《素问》中即有甘美多肥之食化热耗伤脾阴而发消渴的论述。另外郁怒化热、思虑过度皆可暗耗脾阴而致消渴。此外,脾为后天之本,“脏真濡于脾”,五脏六腑阴血津液的补充无不有赖于脾。脾阴与全身阴液在生理上息息相通,病理上相互影响。脾阴足自然能不断补充五脏之阴,使肺、胃、肾之阴亏逐渐改善而渐复；然脾阴不足,则三脏之阴无以复,导致病情加重。脾阴耗伤,脾运失健,日久气血化生不足,痰瘀阻塞脑窍,导致脑髓失养,神明失用而发呆病。从治疗角度,消渴病以阴虚为本,理当滋阴为要。而脾为太阴,乃三阴之长,故治疗阴虚当以滋养脾阴为主,脾阴充足,自能灌溉诸脏腑,而使疾病出现向愈之机。滋补脾阴方药(ZiBu PiYin Recipe, ZBPYR)源自清代医家吴澄《不居集》中的资成汤,经战丽彬教授据临床经验加减而成。全方以培补后天、滋补脾阴为原则,既能滋补脾阴而不滞腻,又兼化痰通瘀、安神益智之功效,是针对糖尿病认知损伤中的脾阴虚损、痰瘀阻窍病机治疗的良好方剂。既往研究显示ZBPYR能够通过多种途径显著改善老龄及痴呆大鼠的学习记忆能力。而ZBPYR对DACD的作用还不清楚。本研究以ZBPYR对DACD大鼠进行干预,并应用基于荧光差异凝胶电泳(DIGE)的蛋白质组学方法,对DACD大鼠的海马进行研究,希望从组织整体蛋白质水平揭示DACD可能的病变机制,并寻找ZBPYR的作用靶点,以期为DACD的防治提供有益参考。目的：1.观察ZBPYR对DACD动物模型认知功能的影响；2.寻找DACD大鼠海马蛋白质组的差异表达蛋白,以揭示疾病特异靶分子,并观察ZBPYR干预对DACD大鼠海马蛋白质组的影响,以发现ZBPYR作用靶蛋白,为DACD的防治提供线索；3.明确蛋白质组结果的可靠性,并初步分析差异表达蛋白质丰度的改变与mRNA水平变化的关系。方法：1.实验动物分为对照组、糖尿病组和治疗组。采用高脂饲养复合小剂量STZ(链脲佐菌素)腹腔注射法建立T2DM动物模型,通过监测随机血糖(RBG)、测定空腹血清胰岛素(FSI)、口服葡萄糖耐量试验(OGTT)和胰岛素耐量试验(ITT)对T2DM模型进行评估,以水迷宫、跳台实验观察动物的认知功能和ZBPYR的干预效果；2.采用DIGE方法对DACD大鼠海马蛋白质进行分离,以DIGE专用软件对凝胶扫描图进行分析,获得差异表达蛋白点；3.应用基质辅助激光解吸电离-飞行时间串联质谱(MALDI-TOF/TOF MS)和生物信息学手段对获得的感兴趣差异表达蛋白点进行鉴定,以揭示可能参与的分子机制；4.以Western blot在蛋白水平上对蛋白质组学结果进行可靠性验证,并应用实时荧光定量聚合酶链式反应(qRT-PCR)初步分析差异蛋白质丰度改变与mRNA水平变化的关系。结果：1.高脂饲料喂养4周后,糖尿病模型大鼠较对照组体重明显增加,形成肥胖。STZ注射后,糖尿病模型大鼠出现了多饮、多食、多尿等症状,RBG和FSI显著升高,葡萄糖灌胃后OGTT各点血糖值均明显高于对照组p<0.05),ITT显示血糖下降速度较对照组缓慢,以上数据提示T2DM模型形成。ZBPYR使治疗组大鼠糖尿病症状改善,饮食、饮水量减少,RBG有降低趋势,但除饮食量外均未达到统计学差异(p>0.05)。行为学测试发现糖尿病组动物的学习记忆成绩显著差于对照组(p<0.05),而治疗组大鼠学习记忆成绩与对照组水平接近,明显好于糖尿病组p<0.05)；2.经DIGE分析,糖尿病组与对照组比较共发现了13个显著差异表达蛋白点；治疗组与糖尿病组比较共发现了12个显著差异表达蛋白点；治疗组与对照组比较只发现了6个显著差异表达蛋白点；3.经MALDI-TOF/TOF MS分析和Swiss-Prot数据库检索,共有9个差异表达蛋白质被成功鉴定,分别是PDHE1α、CI-42kD、MATPSα、DRP-2、α-tubulin2、p34-ARC、G6PD、GSTM1-1和Stxbpl,其功能主要涉及能量代谢、细胞骨架动态变化和氧化应激；4.蛋白PDHE1α、DRP-2和G6PD的Western blot分析结果与蛋白质组分析结果有良好的一致性；DRP-2和G6PD的mRNA表达变化趋势与蛋白质水平变化趋势相一致,糖尿病组表达水平较其它两组为低,但都未达到统计学差异(p>0.05); PDHE1α的mRNA水平三组间非常接近,无明显差别。结论：1.在高脂饲养复合小剂量STZ腹腔注射法建立的T2DM动物模型基础上可以成功形成DACD。ZBPYR可显著减轻DACD大鼠的学习记忆损伤；2.DIGE能够成功用于DACD及ZBPYR干预后海马的比较蛋白质组学分析。DIGE发现的差异表达点很可能是与DACD发病相关的疾病特异分子或ZBPYR作用的靶蛋白；3.鉴定的9个蛋白质分子参与的细胞功能提示,DACD大鼠海马中可能存在细胞能量代谢、细胞骨架动态变化和氧化应激环节的异常而损伤认知功能,ZBPYR可作用于多靶点对病变进行修复,从而改善认知；4.Western blot分析证实了蛋白质组技术的可靠性。qRT-PCR结果提示蛋白质表达水平的改变可能不完全源于mRNA的变化,很可能也与蛋白质的翻译后修饰或降解有关。更多还原

【Abstract】 Diabetes-associated cognitive decline (DACD), also called diabetic encephalopathy, represents a complication on the diabetic brain, which manifests as a gradual decline of cognitive function. The global prevalence of diabetes among adults is expected to rise from 285 million persons in 2010 to an estimated 439 million in 2030. Chinese Diabetes and Metabolic Syndrome Study Group of Diabetes Society of Chinese Medical Association published the results of the survey on the Chinese prevalence of adult diabetes on New England Journal of Medicine in 2010. The results showed that the prevalence of diabetes has reached 9.7% and the total number of these patients has reached 92 million in the population aged over 20. And in the old age group over 70 years the prevalence has already over 20%. There is no exact prevalence report of DACD because it is still lack of uniform diagnostic criteria. But it’s sure that the DACD prevalence would rise with the advent of an aging society and the increasing of the diabetes. Cognitive impairment will cause inconvenience to the life of diabetic patients, or even self-care inability, which would bring families and the community a heavy burden. Thus the prevention and treatment of DACD have become the worldwide problems to be solved. Current studies on DACD pathogenesis involve glucose toxicity, insulin signalling dysfunction, calcium disequilibrium, inflammation, oxidative stress, vascular disorder and hypothalamus-pituitary-adrenal axis abnormalities, but the exact mechanisms remain unclear. The measures to treat DACD are still in the test stage.Proteins are the direct executors of life activities and almost participant a variety of pathological changes in cells. But currently the study of a single protein has been unable to meet the requirements of overall and in-depth understanding of life. Proteomics, as one of the hottest field in the era of systematic biology, is a kind of large-scale and overall study on all the proteins expressed by a genome. It can reveal the specific disease-related proteins with the technologies of high-resolution, high-throughput protein isolation and identification, in order to accelerate the understanding of pathology. Up to now, proteomic analysis on DACD is little regarded. Hippocampus is closely related to learning and memory function in the brain. Hippocampal damage has been proved to be an early change in type 2 diabetic patients. Proteomic research on DACD hippocampus may reveal some important mechanisms of cognitive impairment.For thousands of years, traditional Chinese medicine (TCM) has played an indispensable role in the fight against diseases in China. TCM aims to modulate human body to homeostasis using prescriptions with unique effects. Its biggest advantage is improving the function without obvious side effects. The ZiBu PiYin Recipe (ZBPYR) is derived from Zicheng Decoction, a TCM formula recorded in the book of Bujuji, written by Wu Cheng in the Qing dynasty and used for clinical treatment of amnesia. Previous studies have shown that ZBPYR treatment improved learning and memory abilities in aged and dementia rats through multiple metabolisms. However, the effect of ZBPYR on DACD is unclear. Here, using fluorescence-based difference gel electrophoresis (DIGE)-based proteomic method, we investigated the protein alterations in the hippocampus in DACD and in ZBPYR treated rats and hope to reveal the possible pathological mechanisms in DACD and ZBPYR interventional targets to provide some useful information for the prevention and treatment of DACD.Objectives:1. To observe the effect of ZBPYR on the cognitive function of DACD rats.2. To find the changed proteins in DACD hippocampal proteome in order to reveal the disease-specific molecular targets, and to observe the effect of ZBPYR intervention on the DACD proteome and find targets of ZBPYR. 3. To confirm the reliability of proteomic analysis, and preliminarily analyze the relationship between changed protein and its mRNA.Methods:1. Animals were divided into control group, diabetes group and ZBPYR-treated group (DM/ZBPYR). The method of high fat diet combined with low dose of streptozotocin (STZ) intraperitoneal injection was used to establish T2DM animal models. And the model was evaluated by random blood glucose (RBG), fasting serum insulin (FSI), oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). The cognitive function and the effect of ZBPYR were evaluated using Morris water maze and step down test.2. DIGE was used to isolate DACD hippocampal proteins, and Decyder 6.5 software analyzed the gel images to find the changed protein spots.3. Matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF MS) and bioinformatic tools were used to identify the identities of changed protein spots of interest. 4. The reliability of proteomics were confirmed by Western blot at the protein level, and quantitive real-time polymerase chain reaction (qRT-PCR) was used to analyze the relationship between the changes in protein abundance and its mRNA level.Results:1. After 4 weeks of high fat diet, diabetic model rats gained more weight than the control and became fat. After STZ injection, diabetic model rats appeared the symptoms of diabetes, such as drinking, eating and urining more. RBG and FSI of diabetic model rats were significantly elevated, and their blood glucose levels in OGTT were significantly higher than the control (p<0.05), but those in ITT decresed slower than the control. These data suggested the formation of T2DM model. ZBPYR treatment improved the rats’status in DM/ZBPYR group and their diet, water intakes were reduced, and the RBG had a trend of reduction. But except for food intake, the data didn’t reach statistical significance (p>0.05). Behavioral test showed that performance of diabeties group in Morris water maze were significantly worse than the control (p<0.05), while the performance of DM/ZBPYR rats was similar to the control, and significantly better than the diabetes group (p<0.05).2. The DIGE analysis found 13 significantly changed protein spots between the diabetes and control group,12 ones between DM/ZBPYR and diabetes group and 6 ones between DM/ZBPYR and control group.3.9 protein spots identities were successfully identified by MALDI-TOF/TOF MS. They are PDHE1α, CI-42kD, MATPSα, DRP-2,α-tubulin 2, and p34-ARC, G6PD, GSTM1-1 and Stxbpl. These proteins’functional categories include energy metabolism, cytoskeleton dynamics, and oxidative stress.4. Western blot analysis of interested proteins PDHE1α, DRP-2 and G6PD showed good consistency with proteomic results. The mRNA expressions of DRP-2 and G6PD in the diabetes group were lower than the other two groups, which had consistent trends with protein levels, but did not reach statistical significance (p>0.05). The mRNA level of PDHE1αwas very close among the three groups.Conclusions:1. The method of high fat diet combined with low dose of streptozotocin (STZ) intraperitoneal injection established good T2DM animal model and this model could successfully formed DACD. ZBPYR could significantly improve the learning and memory abilities in DACD rats. 2. DIGE could be used for comparative proteomics analysis of hippocampus in DACD and ZBPYR-treated rats. The differentially produced protein spots found by DIGE may be the specific moleculars involved in pathogenesis of DACD and the targets of ZBPYR.3. The nine proteins identified here indicated that DACD rat hippocampus may exist disorders in energy metabolism, cytoskeleton dynamics and oxidative stress, which caused the cognitive impairment. ZBPYR can act on multiple targets to reduce the lesions, thereby improving cognition. 4. Western blot analysis confirmed the reliability of proteomic results. The results of qRT-PCR suggested that changes in protein levels may not be entirely due to the mRNA change. It may be related to protein post-translational modification or degradation, too.更多还原