胰岛素降解酶介导胰岛素抵抗所致2型糖尿病轻度认知功能障碍的机制研究

发布时间：2018-04-20 15:09

本文选题：胰岛素降解酶 + 胰岛素抵抗　；参考：《东南大学》2017年博士论文

【摘要】：第一部分 IDE与胰岛素抵抗和糖尿病MCI的相关性研究背景:胰岛素降解酶(IDE)可降解胰岛素和β-淀粉样蛋白(Aβ),与2型糖尿病慢性并发症及(AD)的发生发展有密切的关系。胰岛素抵抗(IR)是2型糖尿病的始发因素之一,既往研究表明IR会增加认知功能障碍甚至AD的发生风险,也是糖尿病轻度认知障碍(MCI)向AD转化的危险因素。目的:研究IDE与胰岛素抵抗、2型糖尿病MCI的相关关系,探讨IDE对2型糖尿病MCI发生的预测价值。方法:本研究共招募239位2型糖尿病患者,根据MoCA得分将其分为MCI组和认知功能正常组。所有受试者入组后于次日早晨7点空腹抽取静脉血,测定空腹血糖、空腹C肽、糖化血红蛋白、肝肾功能、血脂全套,计算胰岛素抵抗指数(HOMA-IR)。用多维度量表评估受试者的认知功能,用聚合酶链式反应-限制性片段长度多态性法检测IDE rs4646958基因多态性,并用酶联免疫吸附法测定血清IDE浓度。结果:纳入的2型糖尿病患者中,132人符合MCI的诊断标准,107人是与之相匹配的认知功能正常组。结果表明:1、与认知功能正常组比较,2型糖尿病MCI组患者的血清IDE显著降低(P0.001),HOMA-IR显著升高(P0.001); 2、在2型糖尿病MCI患者中,血清IDE水平和MoCA得分呈显著正相关(r = 0.827;P0.001 ),HOMA-IR和MoCA得分呈显著负相关(r = -0.644;P0.001); 3、在所有2型糖尿病患者中做的相关分析表明,血清 IDE 水平和 MoCA 得分(r = 0.798;P 0.001)呈正相关,和 HOMA-IR (r =-0.586;P 0.001 )、连线试验 A (r =-0.464;P 0.001 )、连线试验 B (r = -0.399;P 0.001)、空腹血糖(r = -0.409;P0.001)、糖化血红蛋白(r = -0.237;P= 0.015)及平均血糖波动幅度(r =-0.502;P 0.001)呈负相关;4、回归分析显示,调整了年龄、性别、受教育年限、肝肾功能和血脂水平后,IDE(P=0.001)、糖化血红蛋白(P=0.024)、空腹C肽(P0.001)是2型糖尿病发生MCI的影响因素;5、IDE rs4646958基因型在2型糖尿病认知障碍组及认知功能正常组均未发现统计学差异。结论:IDE参与胰岛素抵抗相关的2型糖尿病早期认知功能障碍(特别是执行功能),可能成为早期的预测指标。IDE rs4646958基因多态性在2型糖尿病认知障碍中并未发现显著统计学结果,需要大规模样本进一步明确其在易感人群中的预测价值。第二部分 IDE参与KKAy小鼠认知功能障碍的机制及PPARy激动剂的干预效应背景:胰岛素抵抗是2型糖尿病的最显著的临床病理特征,早于显著的2型糖尿病症状发生,参与了糖尿病早期认知障碍的发生以及向AD的转化。过氧化物酶增殖物活化受体γ(PPARγ)的表达和胰岛素的敏感性呈正相关,它可以通过调节胰岛素信号通路,调控IDE的表达。PPARy激动剂可改善胰岛素抵,减少认知功能障碍和AD的发生。我们前期的研究发现2型糖尿病MCI的患者中,血清IDE水平较低,和胰岛素抵抗介导的认知功能下降显著相关,可能是2型糖尿病发生认知障碍的预测指标。在临床研究的基础上,我们进一步通过动物实验来证实IDE参与胰岛素抵抗相关的认知障碍机制和PPARy激动剂的预防和干预效应。目的:从动物水平上探讨IDE保护动物认知功能的可能机制及其与Aβ水平的相关性,以及PPARγ激动剂在胰岛素抵抗介导的2型糖尿病小鼠认知功能损伤的预防和治疗效果。方法:本研究采用高脂喂养KKAy小鼠作为2型糖尿病轻度认知功能障碍模型,罗格列酮作为PPARy激动剂对小鼠进行预防和干预。选取6周龄清洁级健康雄性KKAy小鼠和其对应的C57BL/6j (C57)小鼠(均购于中国医学科学院实验动物所),适应性喂养一周。其中随机选出三组KKAy小鼠在8周龄时分别给予低、中、高剂量罗格列酮预防,其他三组在小鼠15周龄出现认知障碍时给予罗格列酮低、中、高剂量干预,还有一组作为2型糖尿病未干预的对照组。将罗格列酮溶于0.9%生理盐水中配置成所需浓度的混悬液,低剂量组每天予1mg/ (kg·d)灌胃,中剂量组每天予3.3mg/ (kg d)灌胃,高剂量组每天予10mg/ (kg· d)灌胃,糖尿病对照组及正常组C57小鼠予等剂量生理盐水灌胃3个月。处理后进行以下检测:1、每周监测小鼠体质量、空腹血糖及空腹胰岛素水平,计算出相关的胰岛素抵抗指数;2、通过水迷宫及避暗实验观察小鼠的行为学变化;3、Western及RT-PCR法测定小鼠海马PPARγ、IDE的mRNA及蛋白水平;4、对脑组织切片进行HE染色、尼氏染色,镜下观察各组小鼠大脑组织神经元形态、尼氏体的改变;5、免疫化学法测定小鼠脑组织PPARγ、IDE、Aβ的表达。结果:1、各组小鼠体质量、空腹血糖、胰岛素和胰岛素抵抗指数水平的比较:各周龄KKAy小鼠的体质量、空腹血糖、胰岛素及胰岛素抵抗水平均高于对照组C57小鼠(P 0.05),和模型对照组相比,罗格列酮预防和干预组的体质量均无显著统计学差异(P0.05),空腹血糖、胰岛素及胰岛素抵抗水平显著下降(P0.05),高剂量效果更为显著。2、各组小鼠行为学比较:水迷宫实验结果表明,与正常对照组相比,模型对照组小鼠逃避潜伏期显著延长,穿台次数显著减少(P0.05);与模型对照组小鼠相比,罗格列酮干预和预防组小鼠逃避潜伏期显著减少,穿台次数显著延长(P0.05)。避暗实验结果表明,和正常对照组小鼠相比,模型组小鼠避暗潜伏期显著缩短,错误次数显著增加(P0.05),罗格列酮低预防和干预后,潜伏期和错误次数均有显著改善(P0.05),高剂量的效果尤其显著。3、海马组织PPARγ及IDE的mRNA及蛋白水平:模型对照组小鼠海马内PPARy及IDE的mRNA及蛋白水平显著下降(P0.05),经罗格列酮预防和干预后PPARy及IDE的mRNA及蛋白水平均显著增加(P 0.05),高剂量的效果尤其显著。4、各组小鼠神经元形态的改变:HE染色:正常对照组小鼠海马组织中,神经元细胞形态结构未见显著异常,细胞核形状呈圆形,细胞结构层次清楚且排列整齐。模型对照组小鼠海马组织的部分细胞出现形态结构的破坏,细胞核皱缩,边缘模糊,细胞结构排列松散,有变形甚至坏死。罗格列酮预防和干预后,小鼠海马组织的细胞形态结构的破坏程度有所减轻,排列松散情况好转,神经元的变性坏死程度减轻,病理损伤有所好转。尼氏染色:正常对照组小鼠海马神经元未见显著异常,胞核膜和核仁较清楚;模型对照组小鼠的海马、额叶皮质的细胞排列疏散、细胞间隙较宽,胞浆内Nissl体较少。罗格列酮预防和干预组小鼠与模型组小鼠比较,组织结构有所改善,神经元细胞排列密集、整齐,胞浆中Nissl体增加。5、免疫化学法测定小鼠脑组织PPARγ、IDE、Aβ的表达:正常组小鼠海马组织细胞排列紧密,胞体小,Aβ蛋白着色较浅,表达较弱。模型对照组小鼠海马组织细胞排列疏松,胞体较大,Aβ着色深,表达呈强阳性,PpAaγ及IDE的IOD值显著减少(P0.05)。罗格列酮预防和干预组小鼠PPARy及IDE的IOD值显著增加,Aβ蛋白显著减少(P0.05)。结论:IDE参与胰岛素抵抗所致的糖尿病早期认知障碍,对认知功能起到保护作用。PPARγ激动剂可能是通过升高IDE和降低Aβ水平来预防和改善KKAy小鼠的认知功能障碍的。第三部分 IDE介导的PPARy激动剂对PC12细胞APP和Aβ的影响背景:前期实验证实PPARγ激动剂可能是通过升高IDE和降低Aβ水平来预防和改善KKAy小鼠的认知功能障碍的。但在细胞水平,PPARy激动剂对神经细胞的保护作用机制尚未明确。目的:研究IDE介导的PPARγ激动剂对PC12细胞淀粉样前体蛋白(APP)和Aβ的影响,探讨PPARy激动剂神经保护作用可能的潜在机制。方法:选取未分化的PC12细胞株,以神经生长因子诱导为神经元细胞。预试验采用1Oμmol/L的罗格列酮、20μmol/L的氯喹、12.5μmol/L的T0070907分别处理后,处理组和对照组相比具有显著的统计学差异,后续的实验就用该浓度作为理想浓度来处理PC12细胞。在细胞稳定生长24小时后随机分为六组:正常对照组、罗格列酮干预组、T0070907处理组、T0070907预处理+罗格列酮组、氯喹处理组、氯喹预处理+罗格列酮组,每个组设6个平行复孔。干预处理后收集各组相应时间点的细胞,用RT-PCR以及Western blotting分别检测PPARγ、IDE、APP等指标mRNA和蛋白的表达水平;用ELISA法检测细胞培养液上清液Aβ水平。结果:结果显示:1、和正常对照组相比,罗格列酮干预组的PPARy和IDE的mRNA和蛋白含量显著增加(P 0.05),APP的mRNA和蛋白含量显著减少(P0.05); 2、单独用T0070907处理组和用T0070907与罗格列酮联合处理组相比,PPARγ、IDE和APP的mRNA和蛋白含量未见显著统计学差异(P0.05); 3、和氯喹处理组相比,用氯喹与罗格列酮联合处理组PPARy的mRNA和蛋白含量显著升高,IDE和APP的mRNA和蛋白含量未见显著统计学差异(P 0.05); 4、和正常对照组相比,罗格列酮干预组的PPARy和IDE的培养液上清液的Aβ含量显著减少(P0.05),其他各组培养液上清液的Aβ含量未见显著统计学差异(P0.05)。结论:PPARy激动剂可诱导DDE的表达,从而促进APP和Aβ的降解。但是,PPARy激动剂对IDE诱导的神经细胞损伤的保护作用,均可被PPARy抑制剂T0070907及IDE抑制剂氯喹所拮抗,表明PPARγ激动剂的神经细胞损伤的保护机制是通过PPARy激活IDE途径参与调节的。
[Abstract]:The first part of the study on the correlation between IDE and insulin resistance and diabetes MCI: insulin degrading enzyme (IDE) degrading insulin and beta amyloid (A beta) is closely related to the development of chronic complications of type 2 diabetes and (AD). Insulin resistance (IR) is one of the initiation factors of type 2 diabetes. Previous studies have shown that IR will increase. The risk of cognitive dysfunction or even AD is also a risk factor for mild cognitive impairment of diabetes (MCI) to AD. Objective: To study the correlation between IDE and insulin resistance and MCI in type 2 diabetes, and to explore the predictive value of IDE for the occurrence of MCI in type 2 diabetes. Methods: a total of 239 patients with type 2 diabetes were recruited and the score of MoCA was based on the score of MoCA. It was divided into the MCI group and the normal cognitive function group. All the subjects took the venous blood at 7 a. m. on the next morning and measured the fasting blood glucose, fasting C peptide, glycosylated hemoglobin, liver and kidney function, blood lipid whole set, and calculated the insulin resistance index (HOMA-IR). The cognitive function of the subjects was evaluated by the multidimensional scale and the polymerase chain reaction restriction was used. The polymorphism of IDE rs4646958 gene was detected by sex fragment length polymorphism and serum IDE concentration was measured by ELISA. Results: among the patients with type 2 diabetes, 132 were in accordance with the diagnostic criteria of MCI and 107 were matched with the normal cognitive function group. The results showed that: 1, compared with the normal cognitive function group, the MCI group of type 2 diabetes mellitus was developed. Serum IDE was significantly decreased (P0.001) and HOMA-IR significantly increased (P0.001); 2, in type 2 diabetic MCI patients, serum IDE levels were positively correlated with MoCA scores (r = 0.827; P0.001), and HOMA-IR and MoCA scores were negatively correlated (r =;); 3, in all patients with type 2 diabetes, serum levels and levels MoCA score (r = 0.798; P 0.001) was positive correlation, and HOMA-IR (R =-0.586; P 0.001), connection test A (R =-0.464; P 0.001), connection test B (0.001), fasting blood glucose, glycated hemoglobin (0.015) and average blood glucose fluctuations (0.001) negative correlation; 4, regression analysis showed After adjusting age, sex, years of education, liver and kidney function and blood lipid levels, IDE (P=0.001), glycosylated hemoglobin (P=0.024), and fasting C peptide (P0.001) were the factors affecting MCI in type 2 diabetes; 5, IDE rs4646958 genotypes were not found in the cognitive impairment group of type 2 diabetes and the normal cognitive function group. Conclusion: IDE participates in the pancreas. The early cognitive impairment (especially executive function) of type 2 diabetes associated with Isle resistance may be an early predictor of the.IDE rs4646958 gene polymorphism in the cognitive impairment of type 2 diabetes, which requires a large sample to further clarify its predictive value in the susceptible population. Second part IDE The mechanism of participation in cognitive dysfunction in KKAy mice and the intervention effect of PPARy agonists: insulin resistance is the most significant clinicopathological feature of type 2 diabetes, earlier than the onset of type 2 diabetes, the occurrence of early cognitive impairment in diabetes and the conversion to AD. The peroxidase proliferator activated receptor gamma (PPAR gamma) ) expression is positively related to insulin sensitivity, and it can regulate the insulin signaling pathway by regulating the expression of IDE.PPARy agonists to improve insulin resistance and reduce the occurrence of cognitive dysfunction and AD. Our previous study found that in patients with type 2 diabetes MCI, the level of IDE in blood albumin was lower and the cognitive work mediated by insulin resistance was found in patients with type 2 diabetes. Reduced significant correlation, may be a predictor of cognitive impairment in type 2 diabetes. On the basis of clinical studies, we further confirmed the mechanism of cognitive impairment associated with insulin resistance and the prevention and intervention effects of PPARy agonists by IDE. Objective: To explore the cognitive function of IDE protection from animal levels. The possible mechanisms and their correlation with A beta levels, as well as the preventive and therapeutic effects of PPAR gamma agonists on cognitive impairment in type 2 diabetic mice induced by insulin resistance. Methods: This study used high fat feeding KKAy mice as a mild cognitive impairment model of type 2 diabetes, and rosiglitazone was used as a PPARy agonist to mice. Prevention and intervention. Select 6 week old clean healthy male KKAy mice and their corresponding C57BL/6j (C57) mice (all purchased in the laboratory animal Institute of Chinese Academy of Medical Sciences) for one week. Among them, three groups of KKAy mice were randomly selected to be given low, medium and high dose rosiglitazone at 8 weeks of age, and the other three groups were identified at 15 weeks of age. A low, medium and high dose intervention was given to rosiglitazone, and a group of untreated type 2 diabetes was given as a control group. Rosiglitazone was dissolved in 0.9% physiological saline to form a desired concentration of suspension. The low dose group was given 1mg/ (kg. D) every day. The medium dose group was given 3.3mg/ (kg d) every day, and the high dose group was given 10mg/ (kg. D) every day. Gavage, the diabetic control group and the normal group C57 mice were given the same dose of normal saline for 3 months. After treatment, the following tests were performed: 1, the body mass, fasting blood glucose and fasting insulin level were monitored weekly, and the related insulin resistance index was calculated; 2, the behavior changes of mice were observed by water maze and dark experiment; 3, Western and RT-PC were observed. The R method was used to determine the PPAR gamma, mRNA and protein levels of IDE in the hippocampus of mice. 4, the brain tissue sections were stained with HE, Nissl staining, and the changes of neuron morphology and Nissl body were observed under the microscope. 5, the expression of PPAR gamma, IDE, and A beta in the brain tissue of mice was measured by immunochemistry. Results: 1, the mass of mice in each group, fasting blood glucose, insulin and islets of insulin and islet. Comparison of the level of vegetal resistance index: body mass, fasting blood glucose, insulin and insulin resistance at all weeks of age of KKAy mice were higher than that of C57 mice in the control group (P 0.05). Compared with the model control group, there was no significant difference in body mass between the prophylaxis and intervention groups (P0.05), fasting blood glucose, insulin and insulin resistance levels were significant. Decrease (P0.05), high dose effect is more significant.2, mice Behavior Comparison in each group: water maze test results show that compared with the normal control group, the escape latency of the model control group was significantly prolonged, and the number of wear stage decreased significantly (P0.05), compared with the model control group, the incubation period of rosiglitazone intervention and prevention group was significantly reduced. The test results showed that the dark latency of the mice in the model group was significantly shortened and the number of errors increased significantly (P0.05) compared with the normal control group, and the latency and error times of rosiglitazone were significantly improved (P0.05), and the effect of high dose was particularly significant (.3, PPA), PPA in the hippocampus, and PPA in the hippocampus. The level of mRNA and protein of R gamma and IDE: the mRNA and protein levels of PPARy and IDE in the hippocampus of the model control group decreased significantly (P0.05). The mRNA and protein levels of PPARy and IDE increased significantly after the prophylaxis and intervention of rosiglitazone (P 0.05). In the hippocampus of the mice, the morphological structure of the neurons was not remarkable, the nucleus shape was round, the structure of the cell was clear and orderly. The part of the hippocampus tissue in the model control group was damaged by the morphological structure, the nucleus crinkled, the edge was blurred, the cell structure was loosely arranged, and the rosiglitazone was deformed and even necrotic. Prevention and prognosis, the damage degree of cell morphology and structure in the hippocampus of mice was reduced, loosely arranged, the degree of degeneration and necrosis of neurons reduced and pathological damage improved. Nissl staining: no significant abnormalities were found in the hippocampus neurons in normal control group, and the cell nuclear membrane and nucleolus were clearer; the hippocampus of model control mice In the frontal cortex, the cells in the frontal cortex were arranged to evacuate, the cell space was wide and the Nissl body in the cytoplasm was less. The tissue structure of the mice in the rosiglitazone prevention and intervention group was better than the model mice, the cells were arranged densely and neatly, the Nissl body in the cytoplasm increased.5. The expression of PPAR gamma, IDE and A beta in the brain tissue of the mice was measured by immunochemistry: the normal group was small. The cells in the hippocampus of the rat were arranged closely, the cell body was small, the expression of A beta protein was shallow, and the expression was weak. The cells in the hippocampus of the model control group were loosely arranged, the cell body was large, the expression of A beta was strong positive, the IOD value of PpAa gamma and IDE decreased significantly (P0.05). The IOD value of PPARy and IDE in the mice of rosiglitazone prevention and intervention group was significantly increased, A beta protein showed significant. Reduction (P0.05). Conclusion: IDE participates in the early cognitive impairment induced by insulin resistance. The protective effect of.PPAR gamma agonist on cognitive function may be to prevent and improve the cognitive impairment of KKAy mice by increasing IDE and reducing A beta. The effect of PPARy agonists on PC12 cell APP and A beta in the third part of IDE Background: earlier experiments confirmed that PPAR gamma agonists could prevent and improve the cognitive impairment of KKAy mice by raising IDE and reducing A beta levels. But at the cellular level, the mechanism of the protective action of PPARy agonists on the nerve cells is not clear. Objective: To study the IDE mediated PPAR gamma agonist on the amyloid precursor protein (APP) of PC12 cells (APP) and to the IDE mediated PPAR gamma agonist. The effects of A beta on the potential mechanism of the neuroprotective effect of PPARy agonists. Methods: an undifferentiated PC12 cell line was selected to be induced by nerve growth factor as neuron cell. The pretreatment was treated with 1O mu mol/L rosiglitazone, chloroquine 20 mu mol/L, and T0070907 of 12.5 mu mol/L respectively. The treatment group was significantly more than the control group. PC12 cells were treated with the concentration as ideal concentration. After 24 hours of stable growth, the cells were randomly divided into six groups: the normal control group, the rosiglitazone intervention group, the T0070907 treatment group, the T0070907 preconditioning + rosiglitazone group, the chloroquine treatment group, the chloroquine preconditioning + rosiglitazone group, each group of 6 parallel compounds. RT-PCR and Western blotting were used to detect the expression level of mRNA and protein of PPAR gamma, IDE, APP and so on, and the level of A beta in the supernatant of cell culture liquid was detected with ELISA method. Results: 1, the PPARy and IDE mRNA and eggs of the rosiglitazone intervention group were compared with the normal control group. The white content increased significantly (P 0.05), and the content of mRNA and protein in APP decreased significantly (P0.05). 2, there was no significant difference in the mRNA and protein content of PPAR gamma, IDE and APP (P0.05) compared with the T0070907 treatment group and the combination of T0070907 and rosiglitazone combined treatment group. 3, chloroquine and rosiglitazone were treated with chloroquine and rosiglitazone in combination with the chloroquine treatment group. The content of mRNA and protein in Y increased significantly. The mRNA and protein content of IDE and APP had no significant statistical difference (P 0.05). 4, the content of A beta in the supernatant of the culture liquid of rosiglitazone intervention group decreased significantly compared with the normal control group (P0.05), and no significant statistical difference was found in the A beta content of the supernatant of other groups (P0.05). On the other hand, PPARy agonists can induce the expression of DDE and promote the degradation of APP and A beta. However, the protective effect of PPARy agonists on IDE induced neuronal damage can be antagonized by the PPARy inhibitor T0070907 and chloroquine, a IDE inhibitor, indicating that the protective mechanism of the PPAR gamma agonist's nerve cell damage is to participate in the modulation by PPARy activation IDE pathway. The festival.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2

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