【作者】 马志伟；

【导师】 周新文；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2013， 硕士

【摘要】 研究背景阿尔茨海默病（Alzheimer’s disease, AD）是导致老年人群痴呆最重要的原因，其特征性的病理改变为聚集于神经细胞外的老年斑（Senile plaque，SP）和细胞内的神经原纤维缠结（Neurofibrillary tangle, NFT）以及进行性的学习记忆障碍。糖尿病患者AD发病危险性比普通人群明显升高，且糖尿病可通过多种途径引起神经元病理和认知功能损伤。随着老化和糖代谢失调，多元醇通路和醛糖还原酶（Aldose reductase, AR）明显激活，但AR是否参与AD病理过程以及抑制AR活性能否改善AD相关的神经病理改变目前仍不清楚。目的探讨醛糖还原酶抑制剂是否能改善链脲佐菌素（Streptozotocin, stz）引起的AD样病理改变及其可能的机制。材料与方法为探讨醛糖还原酶抑制剂对stz引起的AD样病变的影响极其作用机制，我们选用3月龄Sprague Dawley（SD）大鼠（体重约为300±30g）进行双侧脑室注射stz（Intracerebroventricular-streptozotocin, icv-stz,3mg/kg）建立AD样动物模型。造模成功后根据分组用不同的试剂对实验动物进行连续3周的灌胃处理。实验动物分为四组，分别为：（1）空白对照组，侧脑室注射人工脑脊液，生理盐水灌胃；（2）stz组，侧脑室注射stz，生理盐水灌胃；（3）stz+zop组，侧脑室注射stz，醛糖还原酶抑制剂zopolrestat灌胃（50mg/kg·day）；（4）stz+sob组，侧脑室注射stz，醛糖还原酶抑制剂sorbinil灌胃（25mg/kg·day）；每组动物数量均为8-10只。模型建立一月后用免疫印迹法检测动物海马tau蛋白磷酸化程度和tau蛋白磷酸化相关蛋白激酶表达水平，用试剂盒检测相应脑区组织细胞氧化应激相关指标超氧化物歧化酶（Superoxide Dismutase, SOD）活力和丙二醛（MaleicDialdehyde, MDA）含量，并用Morris水迷宫实验评估动物的空间学习记忆能力。结果1.醛糖还原酶抑制剂改善stz引起的空间记忆能力障碍。水迷宫实验表明，侧脑室注射stz不影响大鼠空间学习能力，但严重损害大鼠空间记忆能力；醛糖还原酶抑制剂显著改侧脑室注射stz引起的空间记忆障碍。2.醛糖还原酶抑制剂降低stz诱导的tau蛋白过度磷酸化。免疫印迹结果表明，侧脑室注射stz引起大鼠海马tau蛋白多个位点磷酸化程度加强，醛糖还原酶抑制剂降低icv-stz大鼠海马tau蛋白在这些位点的磷酸化水平。3.醛糖还原酶抑制剂通过丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）途径改善stz引起的AD样病变。免疫印迹结果显示，侧脑室注射stz引起大鼠海马MAPK途径的C-Jun基末端激酶（C-Jun N-terminal kinase, JNK）和激活细胞外调节蛋白激酶（extracellular regulated protein kinase, ERK1/2）磷酸化水平升高，提示其活性增强；两种醛糖还原酶抑制剂则不同程度地减弱上述激酶的磷酸化程度。此外，stz组大鼠海马糖原合酶激酶-3（glycogen synthase kinase-3, GSK-3）在serine9位点磷酸化程度加强，提示其活性减弱，说明侧脑室注射stz可能不通过GSK-3影响tau蛋白磷酸化。4.氧化应激不参与stz引起的大鼠AD样病理改变。模型建立后一个月，与对照组相比，stz组大鼠脑部SOD活力和MDA含量均无显著差异，说明侧脑室注射stz此时并不引起大鼠脑部氧化应激，stz诱导的AD样病变可能不是由氧化应激介导的。此外，醛糖还原酶抑制剂对大鼠脑部SOD活力和MDA含量亦无影响。结论1.醛糖还原酶抑制剂对stz诱导的AD样病变有改善作用。2.醛糖还原酶可能通过MAPK通路改善stz诱导的AD样病变。更多还原

【Abstract】 BackgroundAlzheimer’s disease（AD）, the most common type of dementia among old people,is characterized by extracellular-aggregated senile plaque （SP）, intracellularneurofibrillary tangle （NFT） and progressive cognitive disorder. Numerous studieshave shown that patients with diabetes exhibit increased risk of developing AD thannondiabetic individuals, and hyperglycemia may do harm to cognitive function bymultiple ways, one of which is to increase oxidative stress in the brain. However, it isstill unclear whether aldose reductase （AR）, which is activated by hyperglycemia inthe polyol pathway and may play a role in diabetic stress, is involved in ADdevelopment and inhibiting its activity could ameliorateAD-like neuropathology.ObjectiveTo explore the effects of AR inhibitors on streptozotocin-induced AD-likeneuropathology and its underlying mechanism.Materials and methodsTo establish AD-like animal model, a certain number of3-month-old SpragueDawley（SD）rats （300±30g） were treated with streptozotocin （stz） via bilateralintracerebroventricular（icv）injection（3mg/kg）. Recovering from surgery, animalswere administered with AR inhibitors or normal saline （NS） by gavage for threeweeks, once a day. Animals were divided into four groups:（1）control group, whichwere injected with artificial cerebral spinal fluid and gavaged with NS;（2）stz group,which were injected with stz and gavaged with NS;（3） stz+zop group, which wereinjected with stz and gavaged withAR inhibitor zopolresta（tzop,50mg/kg·day）;（4）stz+sob group, which were injected with stz and gavaged with AR inhibitor sorbinil（sob,25mg/kg·day）. Each group consisted of8-10rats. One month after the icvinjection, the phosphorylation status of tau protein and related kinases were detectedvia western blot, oxidative stress markers via assay kits and cognitive function viabehavioral tests.Results1. AR inhibitors ameliorated stz-induced spatial memory dysfunction.While stz-treated rats exhibited severely impaired spatial memory, those alsogavaged with AR inhibitors performed as excellent as normal rats without icv-stzin Morris water maze tests. However, rats in stz group performed quite normalcompared with control group in the learning task, indicating unchanged learningability.2. AR inhibitors reduced stz-induced hyperphosphrylation of tau protein. Compared with control group, tau protein in the rat hippocampus of stz groupwas hyperphosphorylated at threonine205and serine404. Treatment with eitherzopolrestat or sorbinil reduced the phosphorylation of tau protein at the residuesabove.3. AR inhibitors ameliorated stz-induced AD-like neuropathology viamitogen-activated protein kinase （MAPK） signal pathway.The phosphorylation levels of C-Jun N-terminal kinase（JNK）and extracellularregulated protein kinase （Erk1/2）, the major kinases in MAPK signal pathway,were elevated as a result of icv-stz, and this hyperphosphorylation was attenuatedfollowing AR inhibitor gavage. Notably, the phosphorylation level of glycogensynthase kinase-3（GSK-3） at serine9was increased, suggesting a reduction inthe activity of this kinase, which implied GSK-3was not responsible for thehyperphosphorylation of tau protein in icv-stz model.4. Oxidative stress was not involved in stz-induced AD-like pathology.Compared with control group, neither SOD activity nor MDA content in thehippocampus of icv-stz rats displayed significant difference in statistic analysis.Meanwhile, neither of the two AR inhibitors exerted any effect on the abovemarkers for oxidative stress.Conclusion1. AR inhibitors may be used to treat diabetes-related cognitive dysfunction.2. The meliorative effect of AR inhibitors on AD-like neuropathology may be basedon the functions of insulin and MAPK signal pathway.更多还原