【作者】 许玲；

【导师】 高海青；

【作者基本信息】 山东大学 ， 内科学， 2007， 博士

【摘要】 目的：到2010年，全球糖尿病（Diabetes Mellitus，DM）患者总数将增加到2.2亿。在25-74岁的人口中，中国DM患者每年增加754，000人，21世纪将仅位于印度之后成为DM第二大国。在糖尿病中枢神经系统并发症中，对糖尿病引起的昏迷、糖尿病脑血管病变研究较多，而对糖尿病引起的认知功能障碍即糖尿病脑病认识不足。糖尿病脑病是慢性进展性疾病，是一种中枢神经系统退行性变。糖尿病可通过非酶蛋白糖基化、氧化应激、多元醇旁路和蛋白激酶C激活多种途径对中枢神经系统产生影响，具体机制不明，更缺乏特异性的治疗。近年来的研究表明，晚期糖基化终末产物（advanced glycation end-products，AGEs）、晚期糖基化终末产物受体（receptor for advanced glycation end-products，RAGE）和转录核因子-kappa B（nuclear factor-kappa B，NF-κB）在糖尿病并发症的发生、发展中具有重要意义，但对其在糖尿病中枢神经病变，特别是在糖尿病脑病发病机制中的作用研究较少。葡萄籽原花青素（grape seed proanthocyanidin extracts，GSPE）具有抗氧化、抑制糖尿病非酶性糖基化、减少氧化应激反应和细胞修复等作用，有望成为治疗糖尿病并发症的新药。本研究利用链脲佐菌素（streptozotocin，STZ）所致糖尿病大鼠模型，试图从高血糖诱发的AGEs／RAGE／NF-κB信号转导途径入手，探讨糖尿病脑病-主要是糖尿病大鼠海马退行性变的发病机制，以及GSPE改善糖尿病海马神经元细胞、胶质星行细胞退行性变的可能机制，为预防、治疗糖尿病脑病寻找新的方向。材料与方法：健康雄性Wistar大鼠60只，体重180-220g。实验动物饲以标准颗粒饲料，分笼喂养，饮食自由，自然昼夜光线照明，实验室内通风良好，温度保持在18-25℃，相对湿度为40-70％。实验前动物测定随机血糖、称重。按空腹体重编号，随机选出10只作为正常对照组（C1组），10只为正常对照加GSPE治疗组（C2组，予以GSPE 250mg／kg/日灌胃）。余40只大鼠适应性喂养7天后，禁食12h。以0.1mol／L、PH4.2的柠檬酸盐缓冲液溶解STZ，配成新鲜的2％的STZ溶液，经滤菌器除菌。实验组按55mg／Kg一次性尾静脉注射，正常对照组注射同等剂量的柠檬酸盐缓冲液。注射后每天后取鼠尾微量血测定血糖（采用强生公司One-Touch Ultra血糖仪），尿糖试纸检测定性尿糖。尿糖在+++以上，血糖＞16.7mmol／L者定为糖尿病大鼠。成模后每周测定一次定性尿糖，每周测定一次血糖和体重。成模糖尿病大鼠30只，平衡一周后，随机分为2组：15只作为糖尿病组（DM1组），15只为正常对照并GSPE治疗组（DM2组，予以GSPE 250mg／kg／日灌胃）。每周检测空腹血糖、体重，标准饲养24周后取血测定血清AGEs和糖化血红蛋白，大鼠断头取脑并分离海马，利用HE染色、神经生长因子和胶质原纤维酸性蛋白（glial fibrillary acid protein，GFAP）免疫组织化学染色观察海马形态，逆转录一聚合酶链式反应检测AGEs／RAGE和NF-κB mRNA表达，免疫组织化学和Western Blot方法研究海马神经元AGEs／RAGE和NF-κB蛋白表达。统计学分析：所有数据均以均数±标准差（x±SD）表示，用SPSS13.0软件进行分析。样本间均数的比较采用t检验或方差分析。P＜0.05表示有统计学意义。结果：1．一般观察糖尿病大鼠污秽无泽，生长缓慢，血糖波动在较高水平（＞20mmol／L），其中多只因严重感染而死亡，未纳入最后统计。GSPE治疗组糖尿病大鼠上述表现减轻，血糖降低，但仍高于正常对照组大鼠。正常对照组大鼠无糖尿病症状，生长及营养状况良好。2．血糖、糖化血红蛋白及体重的比较实验前4组大鼠的血糖、体重均无统计学差异（p＞0.05）。实验终止时，糖尿病大鼠体重明显低于正常对照组大鼠同期体重（t=16.791，P＜0.001），而血糖和HbA_1C则显著高于对照组（P＜0.05）；DM2组大鼠与DM1相比，体重有增长但无显著差异（p＞0.05），血糖控制好转但无显著差异（p＞0.05）。糖尿病组FPG、HbA_1C和体重与正常组比较均有显著性差异，说明糖尿病模型动物是可靠的。C1和C2组肝、肾功均正常，无差异（p＞0.05），说明GSPE干预剂量适当，未产生毒副作用。3．海马神经元形态学观察糖尿病大鼠海马神经元发生退形性改变：神经元细胞层不完整，其中许多细胞胞体缩小，胞浆呈淡红色，胞核固缩深染，排列亦疏散不规则。正常对照组海马神经元较大，形态完整，排列规则且密集，层次清晰。糖尿病组海马CA区神经元较对照组减少（t=3.672，P=0.002），GSPE干预后DM2组DM1组相比神经元数目增加（t=-2.702，p=0.015）。这种改变提示：糖尿病大鼠脑内存在海马及皮层神经元退行性改变，造成神经元数目减少，GSPE可以改善、修复被损伤的神经元，对海马有保护作用。4．海马星形细胞增生性改变实验结束时，免疫组织化学结果显示：与对照组相比，糖尿病组海马星形细胞GFAP染色灰度值减少（t=3.065，p=0.007），GFAP阳性细胞个数增多（t=-3.524，p=0.005），表明GFAP蛋白表达增加。GSPE治疗后DM2和DM1组相比海马星形细胞GFAP染色灰度值增加（t=-2.360，p=0.030），GFAP阳性细胞个数减少（t=2.981，p=0.013），表明GFAP蛋白表达减少。糖尿病大鼠海马CA区GFAP表达增加，提示糖尿病组存在脑损伤的激活和胶质星形细胞增生，GSPE对胶质星形细胞增生有改善作用。5．GSPE对STZ致糖尿病大鼠RAGE／AGEs和NF-κB表达的影响5．1海马RAGE mRNA表达与对照组相比，糖尿病组大鼠海马内RAGE mRNA表达增加（t=-3.795，P=0.002），经GSPE治疗后，DM2组和DM1组相比海马RAGEmRNA表达降低，差别均有统计学意义（t=2.588，P=0.019）。5．2海马NF-κB mRNA表达与对照组相比，糖尿病组大鼠海马内NF-κBmRNA表达增加（t=-3.972，P=0.002），经GSPE治疗后，DM2组和DM1组相比大鼠海马NF-κB mRNA表达降低，差别均有统计学意义（t=3.480，P=0.003）。5．3海马RAGE蛋白表达免疫组化染色显示，实验结束时，糖尿病组大鼠海马CA区神经元细胞上RAGE阳性染色颗粒丰富，而DM2组较DM1组RAGE阳性染色颗粒明显减少。糖尿病组较对照组海马RAGE灰度值减少（t=2.482，p=0.027），阳性细胞数增加（t=-5.218，p＜0.001），表明蛋白表达增加；GSPE治疗后DM2和DM1组相比海马RAGE灰度值增加（t=-2.437，p=0.026），阳性细胞数减少（t=3.609，p=0.003），表明RAGE蛋白表达减少。5．4海马NF-κB蛋白表达免疫组化染色显示，糖尿病组大鼠海马CA区神经元细胞NF-κB阳性染色颗粒丰富，而DM2组较DM1组RAGE阳性染色颗粒明显减少。实验结束时，糖尿病组较对照组海马NF-κB灰度值减少（t=2.747，p=0.019），阳性细胞数增加（t=-3.720，p=0.002），表明蛋白表达增加；GSPE治疗后DM2和DM1组相比海马NF-κB灰度值增加（t=-2.859，p=0.017），阳性细胞数减少（t=4.302，p=0.001），表明蛋白表达减少。5．5 Pearson相关分析表明，糖尿病大鼠海马CA区NF-κB、RAGE表达和血清AGEs水平呈正相关。结论：1、糖尿病组大鼠海马与非糖尿病组相比神经元细胞数目减少，发生明显退行性改变，经GSPE干预后神经元形态改善，数目增加。2、糖尿病组大鼠血清AGEs水平增高，海马CA区RAGE mRNA表达与正常对照组相比明显增加，经GSPE治疗后其表达降低。3、糖尿病组大鼠海马CA区NF-κB mRNA表达与正常对照组相比明显增加，经GSPE治疗后其表达降低。4、糖尿病组大鼠海马CA区RAGE蛋白表达与正常对照组相比明显增加，经GSPE治疗后其表达降低。5、糖尿病组大鼠海马CA区NF-κB蛋白表达与正常对照组相比明显增加，经GSPE治疗后其表达降低。6、糖尿病大鼠海马CA区RAGE、NF-κB表达与血清AGEs水平呈正相关。AGEs／RAGE和NF-κB在糖尿病脑病的发生中起了重要的作用。GSPE通过阻断AGEs／RAGE的结合效应，使海马CA区NF-κB表达减少，使神经元数目增多，星行细胞增生减少，退行性变程度降低，对糖尿病大鼠脑病有治疗、改善作用。以上研究结果提示慢性高血糖能引起大鼠海马AGEs／RAGE和NF-κB表达增加，糖尿病大鼠海马CA区RAGEmRNA、NF-κB蛋白表达含量与血清AGEs呈正相关，长期高血糖通过激活AGEs／RAGE和NF-κB通路使神经元细胞结构和功能产生退行性改变。GSPE治疗后，海马CA区RAGE和NF-κB表达均降低，神经元退行性变得到改善，GSPE通过阻断AGEs／RAGE／NF-κB信号转导通路对糖尿病大鼠脑病起保护作用，可望成为治疗糖尿病脑病的新药。更多还原

【Abstract】 Aims/hypothesis:The association between long-term hyperglycemia and chronic complications of diabetes mellitus has been reported in both type 1 and type 2 diabetes. Mechanisms involved including excess sorbitol-aldose reductase pathway flux, hyperactivity of protein kinase C (PKC) isoforms, increased oxidative stress and microangiopathic changes leading to ischemia. Advanced glycation end-products (AGEs) are unstable, reactive and toxic compounds that alter the extracellular matrix and exacerbate oxidative stress which has been known as the major factors that contribute to the pathogenesis of diabetic complications. AGEs interactions with the receptor for AGEs (RAGE) modify proinflammatory cytokine expression, increase free radical production via quenching of nitric oxide contributing to defective vasodilatation. induce irreversible crosslinks in extracellular matrix structural proteins and transform intracellular signaling pathways in part through nuclear factor-kappa B (NF-κB). Whereas kidney, retina, blood vessels and peripheral nerves are the primary targets of long-term diabetes, brain damage was previously considered secondary to vascular disease. With the increased understanding of diabetes, it has been disclosed that humans with long-term, particularly poorly controlled diabetes, however, develop cognitive dysfunction and an increased risk for dementia and cerebral atrophy, which is now an accepted concept as encephalopathy that still needs a defination. To prevent the development of this disease and to improve advanced brain injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts (GSPE), an antioxidant derived from grape seeds have been reported to possess a variety of potent properties may have therapeutic potential in the prevention and treatment of complications in patients with diabetes. In this study, we examined whether GSPE could attenuate the degeneration changes in the diabetic brain by modulating the AGEs/RAGE and NF-κB pathway.Methods:Male Wistar rats weighting 180-220 g were purchased from the Animal Centre of Shandong University. Animals were kept in individual cages on a 12-h light-dark cycle with an ambient temperature of 22±1℃, with free access to food and water. 40 randomly selected rats were divided into 2 groups: Control group 1(C1) and control group treated with GSPE (C2, administrated with GSPE with a dosage of 250mg/kg).Streptozotocin ( STZ) induced diabetic rats received a single dose of STZ (55 mg/kg, injected into tail veins) freshly dissolved in 0.1 M sodium citrate buffer (pH 4.5 ) after a 12 hours’ overnight fasting. Control animals received a single tail vein injection of 0.1M citrate buffer only. Glucose concentration from fasting animals was measured daily, in a blood sample obtained from the tail by pinprick, with a glucose oxidase-impregnated test strip and a reflectance meter (One Touch II, Lifescan, USA). Hippocampus of the brains were immunohistochemically stained for RAGE and for the detection of mRNA of RAGE and NF-κB by reverse transcriptase coupled to polymerase chain reaction (RT-PCR). For morphological observations, hippocampus of the brains were stained by immunohistochemistry for neuron growth factor (NGF) and glial fibrillary acidic protein (GFAP).Results:1、At the end of this study, the weight of diabetic rats was lower than that of the controls . Mouse glycated hemoglobin, fasting plasma glucose and AGEs were increased in diabetic mice relative to controls. After treated with GSPE, the serum AGEs decreased in DM2 group than that of DM1, while there were no differences in their weights and FPG (21.35±3.59 vs 19.04±3.24mmol/L, t=1.494, P=0.153).2、It was found that the number of neurons of hippocampus in STZ induced rats decreased (t=3.672,P=0.002) and had degenerative changes: the layers of neurons were not intact, the nuclei of neurons were shrinked and in irregular orders, the cytoplasma of neurons turned into pink-colored. After treated with GSPE, the above changes were improved, and the number of neurons in DM2 group increased comparing to DM1 group (t=-2.702,p=0.015) .3、By GFAP immunochemistry, it was found that the grey value of astrocytes in CA region of hippocampus in STZ induced rats decreased comparing to the non-diabetic controls (t=3.065, p=0.007) , while the number of GFAP positive cells increased (t=-3.524, p=0.005); After treated with GSPE, the grey value of astrocytes in CA region of hippocampus in STZ induced rats increased comapring to DM1 group(t=-2.360, p=0.030) , while the number of GFAP positive cells decreased (t=2.981, p=0.013). Recent evidence suggests that the upregulation of GFAP, an astrocyte-specific intermediate filament component, is a biological marker of neurotoxicity after cerebral injury. It is suggested that there is proliferation of astrocytes in the hippocampus of diabetic rats, and GSPE can decrease the proliferation of astrocytes.4、By RT-PCR, it was found that the expression of RAGE mRNA in CA region of hippocampus of STZ induced rats increased comparing to the non-diabetic controls(t=-3.795,P=0.002); After treated with GSPE, the expression of RAGE mRNA in CA region of hippocampus decreased comapring to DM1 group(t=2.588,P=0.019).5、By RT-PCR, it was found that the expression of NF-κB mRNA in CA region of hippocampus of STZ induced rats increased comparing to the non-diabetic controls(t=-3.972,P=0.001); After treated with GSPE, the expression of NF-κB mRNA in CA region of hippocampus in STZ induced rats decreased comapring to DM1 group(t=3.480,P=0.003).6、By immunochemistry, it was found that the grey value of RAGE in CA region of hippocampus of STZ induced rats decreased comparing to the non-diabetic controls (t=2.482,P=0.027) while the number of RAGE positive cells increased (t=-5.218, p<0.001); After treated with GSPE, the grey value of RAGE in CA region of hippocampus in STZ induced rats increased comapring to DM1 group(t=-2.437, P=0.026), while the number of RAGE positive cells decreased (t=3.609, p=0.003).7、By immunochemistry, it was found that the grey value of NF-κB in CA region of hippocampus of STZ induced rats decreased comparing to the non-diabetic controls (t=2.747,P=0.019), while the number of NF-κB positive cells increased (t=-3.720, p=0.002); After treated with GSPE, the grey value of NF-κB in CA region of hippocampus in STZ induced rats increased comapring to DM1 group(t=-2.859, P=0.017), while the number of NF-κB positive cells decreased (t=4.302, p=0.001).8、By Pearson’s relationship test, it was found that the expression of RAGE and NF-κB was realted to serum AGEs, respectively.Conclusions:1、Long term chronic hyperglycemia can cause degenerative changes in CA region of hippocampus in STZ induced diabetic rats, including the decrease of neuron numbers and the proliferation of glial astrocytes.2、At both mRNA and protein levels, long term chronic hyperglycemia can cause the overexpression of RAGE and NF-κB in CA region of hippocampus in STZ induced diabetic rats. The serum AGEs increased and was related to the the expression of RAGE and NF-κB, which suggusted that the signal transdcution pathway of AGEs/RAGE/NF-κB plays an important role in the pathogenesis of diabetic encepholopathy.3、Comparing with the controls, in STZ induced diabetic rats , after treated with GSPE, the number of neurons increased, the serum AGEs decreased, the degeneration in CA regions of hippocampus were improved , while the expression of RAGE and NF-κB also decreased in both mRNA and protien levels , which suggests that GSPE can improve and protect diabetic encepholopathy while modulating the AGEs/RAGE/NF-κB pathway. In summary, long term chronic hyperglycemia can cause the over-expression of AGEs/RAGE and NF-κB in CA region of hippocampus in STZ induced diabetic rats, hyperglycemia induced activation of AGEs/RAGE/NF-κB pathway takes an important role in the pathogenesis of the degenerative changes of diabetic hippocampus. GSPE improves some changes including neuron degeneration and the proliferation of astrocytes compared with non-treated diabetic rats, via decreasing the expression of AGEs/RAGE and NF-κB in the CA region of hippocampus in diabetic rats at a daily oral dosage of 250mg/kg and act as an antagonist to RAGE, which suggests that GSPE be a useful remedy in the treatment of diabetic encephalopathy.更多还原