高血糖通过AMPK/SIRT1调控CDK5通路介导阿尔兹海默症样分子病理改变

Hyperglycemia promotes Alzheimer's disease-like pathological changes by upregulating the CDK5 expression through AMPK/SIRT1 action

目的阐明高血糖诱导对阿尔兹海默症(Alzheimer’s Disease,AD)样病理改变的潜在分子机制。方法选取18周龄的Sprague Dawley(SD)大鼠30只,将其随机分为对照组(Control)、模型组(STZ+HFD)以及模型+AMPK激动剂AICAR(5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside)组(STZ+HFD+AICAR),每组均为10只大鼠。模型组在高脂饮食(High-Fat Diet,HFD)喂养的基础上,经腹腔注射的方式给予大鼠50 mg/kg链脲佐菌素(Streptozotocin,STZ)建立2型糖尿病(TypeⅡDiabetes Mellitus,T2MD)/高血糖模型,STZ+HFD+AICAR组另给予大鼠100 mg/kg的AICAR,Control组给予常规饲料喂养并给予等量的柠檬酸钠缓冲液腹腔注射作为对照。8周内连续测量大鼠空腹血糖,8周后对各组大鼠进行糖耐量检测。通过Morris水迷宫实验分析各组大鼠认知功能的变化。采用Western Blot方法检测三个实验组大鼠脑组织AMPK/SIRT1通路分子、CDK5和H3acK9的表达变化。脑组织免疫组织化学染色检测AD特征性分子病理蛋白MAPT/Tau的磷酸化活性。结果与Control组相比,8周高脂喂养联合STZ注射3天后,大鼠空腹血糖于第2周开始升高直至第8周(P<0.05),且8周后糖耐量显著受损(P<0.05),血清胰岛素水平显著升高(P<0.01)。STZ+HFD大鼠的水迷宫定位航行试验逃避潜伏时间明显延长(P<0.05),而水迷宫空间探索时间显著降低(P<0.05)。Western Blot结果显示,与Control组相比,STZ+HFD大鼠脑组织磷酸化的AMPK蛋白以及SIRT1蛋白的表达水平显著降低,而H3acK9和CDK5蛋白表达水平显著升高。脑组织免疫组织化学结果显示,Tau蛋白磷酸化水平显著升高。AICAR可部分改善脑组织分子病理改变,如:增加磷酸化AMPK蛋白以及SIRT1蛋白的表达,减少H3acK9和CDK5蛋白表达,进而降低Tau蛋白磷酸化水平。结论AMPK/SIRT1通路失活促进去乙酰化蛋白H3acK9及CDK5的表达,进而引起Tau蛋白活性增加,该机制是高血糖引起脑组织AD样分子病理改变的潜在机制之一。

Objective This study aims to elucidate the underlying mechanism of hyperglycemia induced Alzheimer's disease(AD)-like pathological changes.Methods Thirty Sprague Dawley rats(aged 18 weeks)were randomly divided into one control groups and two experimental groups(n=10 for each group),including control group,STZ+HFD group,and STZ+HFD+AICAR group.Control rats(n=10)was fed with the standard diet and experimental rats(n=20)was fed with high fat diet(HFD)for 8 weeks.8 weeks after feeding,we estimated the reliability of the experimental model by measuring blood glucose levels(each week)of rats,serum insulin concentrations and oral glucose tolerance test(OGTT)test.Then experimental rats were injected with 50 mg/kg/day Streptozotocin(STZ)intraperitoneally for three days,whereas the control rats were given the same dose of citric acid buffer for comparison.Morris water maze tests was performed to observe the functional changes of brain regions related to spatial learning and memory in each group.Western blot was performed to detect the abundances of AMPK,p-AMPK,SIRT1,H3acK9 and CDK5 in rats.Immunohistochemistry staining was performed to evaluate the levels of MAPT/Tau which can reflect the AD-like pathological changes.Furthermore,AMPK agonist(AICAR)was used to assess whether AMPK/SIRT1 pathway,which participate in the AD-like pathological changes,is the upstream molecules of H3acK9 and CDK5.Results We found treatment with HFD and STZ significantly increased blood glucose and serum insulin levels,and impaired glucose tolerance in rats.Hyperglycemia led to a decrease in the activity of AMPK and expression of SIRT1.The expression levels of underlying molecules of AMPK including H3acK9 and CDK5 significantly increased.The levels of phosphorylated Tau significantly increased in brain tissues.Additonally,AICAR administration partial reversed hyperglycemia-induced AD-like molecular pathologic changes through activating AMPK/SIRT1,decreasing expression of H3acK9 and CDK5,and inhibiting Tau protein hyperphosphorylation in rat brain tissues.Conclusion Hyperglycemia induces inactivation of AMPK/SIRT1 pathway which promotes excessive deacetylation of H3K9 and over expression of CDK5,these changes in turn lead to Tau protein hyperphosphorylation and AD-like molecular pathologic changes.