【作者】 李杰；

【导师】 张松筠；

【作者基本信息】 河北医科大学 ， 内科学， 2014， 硕士

【摘要】 目的：糖尿病可导致认知功能障碍，主要表现为学习、记忆能力下降，严重者可导致痴呆。证据表明，离子型谷氨酸N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)受体的NR2B亚基通过影响海马的突触可塑性参与学习、记忆的形成，与糖尿病认知功能障碍有着密切的联系。丁苯酞是一种具有改善微循环，保护线粒体等作用的神经保护药物，已广泛应用于缺血性脑血管病，同时对糖尿病认知功能障碍也具有改善作用，但其具体机制目前尚不清楚。本实验通过预防性应用丁苯酞，观察不同剂量丁苯酞对糖尿病大鼠认知功能和海马NMDA受体亚基NR2B表达的影响，为糖尿病认知功能障碍的防治提供新思路。方法：1STZ糖尿病大鼠模型制备清洁级健康雄性Sprague Dawley（SD）大鼠，通过腹腔注射60mg/kg的链脲佐菌素（Streptozotocin，STZ）建立1型糖尿病大鼠模型，对照组注射等体积的生理盐水。72小时后测量尾静脉的血糖，血糖值≥16.7mmol/L者，表示STZ糖尿病大鼠模型形成。2分组适应性喂养一周后，55只SD大鼠随机分为正常对照组（NC组，n=10）和糖尿病组（DM组，n=45）。糖尿病组又随机分为3组：糖尿病对照组（DC组，n=15），丁苯酞低剂量组（DL组，60mg.kg-1.d-1， n=15）和丁苯酞高剂量组（DH组，120mg.kg-1.d-1， n=15）。成模后第一天开始干预，丁苯酞原液用玉米油溶解后灌胃，NC组和DC组以等体积的玉米油灌胃，每日1次。各组均干预12周。3一般观察每周测量大鼠血糖及体重。观察大鼠精神状态、进食水量及尿量等一般情况。4行为学测试应用丁苯酞干预12周末行Morris水迷宫测试，包括定位航行和空间探索两部分。记录各组大鼠逃避潜伏期及穿越平台次数。分析各组大鼠的学习、记忆能力。5形态学及mRNA、蛋白观察指标水迷宫测试后处死大鼠，取大脑海马组织。应用HE染色法观察海马组织形态，实时定量聚合酶链反应（Real-Time polymerase chain reaction，Real-Time PCR）、蛋白质免疫印迹（Western-Blot）和免疫组织化学染色法检测大鼠海马组织中NMDA受体亚基NR2B mRNA和蛋白水平的表达。6统计学方法采用SPSS13.0软件进行统计分析，应用完全随机设计的单因素方差分析；水迷宫实验结果用重复测量方差分析统计，使用重复测量数据多重比较配对的t检验法(Bonferroni法)，可进行每个分组每个时间点上作用的两两比较；使用多元方差分析的方法，可进行每个时间点上每个分组之间作用的两两比较。因数据符合正态分布（P＞0.1），故应用均数±标准差表示结果，应用p＜0.05表示差异有统计学意义。结果：1一般情况观察正常对照组大鼠精神状态良好，反应灵敏，毛色纯白有光泽，进食水及尿量正常；糖尿病组大鼠精神状态差，精神萎靡，易激惹，毛色稀疏枯黄无光泽，有多食、多饮、多尿症状。比较成模前及成模后12周末的血糖和体重情况：成模前：NC组和DM组大鼠血糖（mmol/L）均正常，差异无统计学意义（7.19±0.54VS7.18±0.46，P>0.05）；体重（g）变化也无统计学意义（130.46±4.98VS132.08±4.36，P>0.05）成模后12周末：与同期NC组相比，DM各组大鼠血糖均保持明显升高（30.13±2.08，29.35±2.57，28.18±2.19VS7.44±0.47，P<0.01），体重明显降低（237.77±14.87,242.73±14.75,245.00±13.07VS340.14±6.65，P<0.01），均有统计学意义；但糖尿病各亚组之间血糖及体重比较，差异无统计学意义(P>0.05)。2行为学观察（Morris水迷宫测试结果）2.1基础游泳速度水迷宫测试前，为排除大鼠个体游泳速度差异，各组中随机选取5只大鼠，让其自由游泳60S，测量其基础游泳速度，结果显示：各组大鼠之间基础游泳速度差异无统计学意义（30.97±1.96，30.73±2.04，29.72±2.95和30.00±2.36，P>0.05）。2.2定位航行第1天各组大鼠逃避潜伏期无明显区别（P>0.05）；第2-5天DC组逃避潜伏期较NC组明显延长（P<0.05），DL和DH组较DC组逃避潜伏期明显缩短，但仍高于NC组（P<0.05），而DH组从第3天到第5天，逃避潜伏期较DL组明显缩短，差异有统计学意义(p<0.05)，组别和天数之间不存在交互作用（P>0.05）。各组间第5天与第1天逃避潜伏期的变化：DC组逃避潜伏期无明显变化（P＞0.05）NC、DL和DH组，逃避潜伏期明显缩短（P＜0.05）。各组间逃避潜伏期的顺序为：NC组<DH组<DL组<DC组。2.3空间探索与NC组相比，DC组穿越平台次数明显减少（3.00±0.64VS15.68±1.57，P<0.05）；与DC组相比，DL和DH组（9.01±1.1和11.68±1.57）穿越平台次数明显增加，但仍少于NC组（P<0.05）；与DL组相比，DH组穿越平台次数明显增加，差异有统计学意义(P<0.05)；各组间穿越平台次数的顺序为：NC组>DH组>DL组>DC组。3HE染色NC组大鼠海马椎体神经元细胞排列整齐，层次清楚，形态完整，核大而圆。DC组神经元细胞排列紊乱，层次不清，细胞可见空泡变性，出现细胞膜断裂，细胞质减少，细胞核固缩。丁苯酞治疗组上述改变较DC组明显减轻，且DH组优于DL组。4海马组织中NR2B mRNA及蛋白的表达4.1海马组织NR2B mRNA的表达与NC组相比，DC组，DL组和DH组海马NR2B mRNA的表达均显著降低（0.14±0.01，0.23±0.01，0.29±0.01VS0.35±0.02，P<0.05）；与DC组相比，DL和DH组海马NR2B mRNA的表达均显著增加（P<0.05）；与DL组相比，DH组海马NR2B mRNA的表达显著增加（P<0.05）4.2海马组织NR2B蛋白表达与NC组相比，DC组，DL组和DH组海马NR2B蛋白的表达均显著降低（0.37±0.03，0.49±0.04，0.60±0.04VS0.68±0.05，P<0.05）；与DC组相比，DL和DH组海马NR2B蛋白的表达均显著增加（P<0.05）；P与DL组相比，DH组海马NR2B蛋白的表达亦显著增加（P<0.05）5免疫组织化学染色与NC组相比，DC组大鼠海马组织中的NR2B阳性细胞表达显著减少（平均光密度值：0.16±0.01VS0.47±0.10，P<0.05）；与DC组相比，DL和DH组海马组织中的NR2B阳性细胞表达均显著增加（平均光密度值：0.27±0.11，0.37±0.14VS0.47±0.10，P<0.05），但仍低于NC组（P<0.05）。与DL组相比，DH组海马组织中的NR2B阳性细胞表达显著增加，有统计学意义（P<0.05）。结论：1STZ糖尿病大鼠12周末学习记忆能力明显下降，出现认知功能障碍。2糖尿病大鼠出现认知功能障碍时，海马组织中NR2B的表达下降。3预防性应用丁苯酞，可改善糖尿病认知功能障碍，提高海马组织中NR2B的表达，并且丁苯酞高剂量组（120mg/kg）的疗效优于丁苯酞低剂量组（60mg/kg）。推测丁苯酞可能通过上调海马NR2B的表达改善糖尿病认知功能。更多还原

【Abstract】 Objective: Diabetes mellitus is a known cause of cognitive dysfunctionthat involves a decline of learning and memory, eventually ends in dementia.Studies have shown that NR2B subunit of the N-methyl-D-aspartate receptor(NMDAR), which is ionic glutamate receptor, has been proved to be animportant factor for cognitive function.L-3-n-Butylphthalide (NBP) is a nerve protective drug, which canimprove microcirculation, protect mitochondria and has been widely used inclinical treatment of ischemic cerebrovascular diseases, meanwhile it canimprove cognitive dysfunction induced by diabetes mellitus, but itspathogenesis is not well-established. The aim of this study was to investigatethe preventive effects of NBP on cognitive dysfunction and NR2B expressionin hippocampus of STZ-induced diabetic rats, which may provide aninteresting direction for diabetic cognitive dysfunction in the future.Method:1STZ-induced diabetic rat modelSingle intraperitoneal injection (IP) of streptozotocin (60mg/kg) wasused for induction of diabetes in male Sprague Dawley (SD) rats. The controlgroup received the equal volume of citrate buffer solution by intrapertonealinjection. The induction of diabetes was confirmed after72h by monitoringthe blood glucose of tail-vein sampling. The rats with blood glucose above16.7mmol/L were enrolled in the T1DM group.2GroupsAfter adaptive feed about one week, fifty-five SD rats were randomlydivided into the normal control group (NC, n=10) and the diabetes mellitus group (DM, n=45); DM group again were randomly divided into3groups: i)diabetic control group (DC, n=15), ii) diabetic+low-dose NBP group (DL,administered NBP60mg/(kg· d), gavage for12weeks, n=15), iii) diabetic+high-dose NBP group (DH, administered NBP120mg/(kg· d), gavage for12weeks, n=15). NBP was dissolved with corn oil. The rats of NC and DC groupwere given the same volume corn oil for12weeks.3General observationsThe body weight and the blood glucose of the rats were monitored onceevery week. General situations of rats were observed during the research, suchas mental state, food intake, water intake, urine volume, etc.4Behavioral testsAll rats accepted the Morris water maze test at the12week (w) point,including place navigation tests and space probe tests. The escape latency andthe number of crossing platform were recorded in order to analyzing learningand memory ability of different group rats.5Morphology and observation indicatorsThe rats were sacrificed after Morris water maze test and thehippocampus tissues were removed for biochemical assays. Morphology ofthe hippocampus tissue was observed by hematoxylin-eosin staining (HE), theexpressions of NR2B mRNA and protein were assessed by Real-Time PCR、Western-blot and immunohistochemical staining(IHC).6Statistical analysisAll analyses were performed using SPSS for Windows, version13.0,which includes One-way analysis of variance (ANOVA). In the results ofMorris water maze, we use repeated-measure analysis of variance. Themethod of Bonferroni should be used to do pairwise comparisons of therepeatedly measured data in different measurement time of each treated group.With multivariate ANOVA, data in different treated group of eachmeasurement time could be compared pairwise. As the data passed thenormality test (P>0.10), the results were expressed as the means±standarddeviation. P<0.05was considered to indicate a statistically significant difference.Results:1General situationThe rats of NC group: good mental state, agility, white and glossy fur,drinking, eating and urine volume were normal; the rats of DM group: poormental state, depression, irritability, yellow, sparse and lusterless fur,polyphagia, polydipsia, polyuria.Compare blood glucose and body weight of different groups before beingmade into model and at the end of12w:Before being made into model: there were no significant differences inthe blood glucose (mmol/L) between the NC group and the DM group(7.19±0.54VS7.18±0.46, P>0.05); the body Weight (g) change also had nostatistical significance(130.46±4.98VS132.08±4.36，P>0.05).At the end of12w:Compared to NC group, the blood glucose of DM group increasedsignificantly (30.13±2.08,29.35±2.57,28.18±2.19VS7.44±0.47, P<0.01) andthe body weight decreased significantly (237.77±14.87,242.73±14.75,245.00±13.07VS340.14±6.65, P<0.01); while among subgroups of DMgroup, the blood glucose and body weight had no statistical significance(P>0.05).2Behavioral observations (Morris water maze)2.1The basic swimming speedIn order to exclude individual swimming speed difference, we randomlyselected from five rats of each group to swim60s freely to test the basicswimming speed. The result showed that the swimming speed of rats amongfour groups was not significantly different (30.97±1.96,30.73±2.04,29.72±2.95and30.00±2.36, P>0.05).2.2place navigation testsOn the first day, there were no significant differences in the escapelatencies between DM group and NC group (P>0.05);From the second day to the fifty day, the escape latencies of DL group and DH group were significantly shorter than DC group, but were still longerthan NC group (P<0.05). Meanwhile, the escape latency of DH group wassignificantly shorter than DL from the third to fifth day (P<0.05). There wasno interaction between the days and the groups (P>0.05).The escape latency variations from the fifth day to the first day: DCgroup, no significant changes in the escape latency (P>0.05); NC group, DLgroup and DH group were significantly shorter in the escape latency (P<0.05).The order of the escape latency each group: NC group<NH group<DLgroup<DC group.2.3space probe testsCompare with NC group, the numbers of rats crossing the platform weremarkedly reduced in DC group (3.00±0.64VS15.68±1.57, P<0.05).Compare with DC group, the numbers of rats crossing the platform weresignificantly increased in NBP-treated groups (9.01±1.1and11.68±1.57);however compare with NC, the numbers were still markedly reduced(P<0.05).Compare with DL group, the numbers of rats crossing the platform weresignificantly increased in DH group, with statistical difference (P<0.05).The order of the numbers of rats crossing the platform: NC group>NHgroup>DL group>DC group.3HE stainingIn NC group, the hippocampal pyramidal cells were arranged in rows andclear in layers, and its structure was integrated, cell nucleus was large andround. On the contrary, the hippocampal pyramidal cells of DC group becamedisorganized in rows and vaguer in layers, with membrane rupture, decreasingof endochylema, and karyopycnosis, even vacuolar degeneration. The changeof HE staining in NBP-treated groups were significantly reduced compare toDC group and DH group was superior to the DL group.4The expression of NR2B mRNA and protein in hippocampus4.1The expression of NR2B mRNA in hippocampus Compare with NC group, the expressions of NR2B mRNA in DC, DLand DH group were significantly declined (0.14±0.01,0.23±0.01,0.29±0.01VS0.35±0.02, P<0.05).Compare with DC group, the expressions of NR2B mRNA inNBP-treated groups were significantly increased (P<0.05).Compare with DL group, the expression of NR2B mRNA in DH groupwas significantly increased (P<0.05).4.2The expression of NR2B protein in hippocampusCompared with NC group, the expression of NR2B protein wassignificantly decreased in the hippocampus in DC, DL and DH groups(0.37±0.03,0.49±0.04,0.60±0.04VS0.68±0.05, P<0.05).Compared with NC group, the expressions of NR2B protein inNBP-treated groups were significantly increased (P<0.05).Compared with DL group, the expression of NR2B protein in DH groupwas also significantly increased (P<0.05).5Immunohistochemical stainingCompared with NC group, the expression of NR2B positive cells weresignificantly decreased in the hippocampus in DC group (the average opticaldensity value:0.16±0.01VS0.47±0.10, P<0.05).Compared with NC group, the expression of NR2B positive cells inNBP-treated groups were significantly increased (the average optical densityvalue:0.27±0.11,0.37±0.14VS0.47±0.10, P<0.05), but was stillsignificantly decreased compare to NC group (P<0.05).Compared with DL group, the expression of NR2B positive cells in DHgroup was also significantly increased, with Statistical significance (P<0.05).Conclusions:1STZ-induced diabetic rat caused learning and memory deficits at theend of12week.2The expressions of NR2B mRNA and protein in the hippocampus werealso decreased in STZ-induced diabetic rats at the end of12week, inaccordance with the cognitive dysfunction. 3NBP could improve cognitive impairment and increase the expressionof NR2B, which speculated that NBP might prevent the rats from gettingcognitive impairment by up-regulating NR2B expression, and the preventiveeffect of120mg/kg is better than that of60mg/kg.更多还原