糖化β-淀粉样蛋白对阿尔茨海默病样大鼠认知功能的影响

Effect of glycated β-amyloid protein on cognitive function of Alzheimer’s disease-like rats

目的探讨糖化β-淀粉样蛋白(Aβ-AGE)对阿尔茨海默病样大鼠认知功能的影响。方法将β-淀粉样蛋白(Aβ)与甲基乙二醛于37℃下温育1个月合成Aβ-AGE。将40只SpragueDawley大鼠随机分为Aβ组、Aβ+小鼠单克隆高级糖化终产物受体抗体(Anti-RAGE)组、Aβ-AGE组、Aβ-AGE+Anti-RAGE组,每组10只。行立体定向左侧侧脑室注射制备阿尔茨海默病样模型,其中Aβ组大鼠给予Aβ5μg,Aβ+Anti-RAGE组大鼠给予Aβ5μg和RAGE抗体Anti-RAGE50μg,Aβ-AGE组大鼠给予Aβ-AGE5μg,Aβ-AGE+Anti-RAGE组大鼠给予Aβ-AGE5μg和Anti-RAGE50μg。注射后第2~7天进行Morris水迷宫实验,记录大鼠寻找隐匿平台的潜伏期;注射后第9天,记录各组大鼠穿越平台次数和目标象限停留时间;Westernblot法检测各组大鼠海马组织中高级糖化终产物受体(RAGE)蛋白的表达,免疫组织化学染色法检测各组大鼠大脑皮层中RAGE的表达。结果在注射后第2天,各组大鼠潜伏期比较差异无统计学意义(F=3.767,P>0.05)。注射后第3~7天,各组大鼠潜伏期比较差异均有统计学意义(F=9.167、25.050、56.980、62.380、122.200,P<0.05);Aβ-AGE组大鼠潜伏期显著长于Aβ组(P<0.05),Aβ+Anti-RAGE组大鼠潜伏期显著短于Aβ组(P<0.05),Aβ-AGE+Anti-RAGE组大鼠潜伏期显著短于Aβ-AGE组(P<0.05)。注射后第9天,各组大鼠穿越平台次数和目标象限停留时间比较差异有统计学意义(F=12.930、13.560,P<0.05);Aβ-AGE组大鼠穿越平台次数和目标象限停留时间显著短于Aβ组(P<0.05),Aβ+Anti-RAGE组大鼠穿越平台次数和目标象限停留时间显著长于Aβ组(P<0.05),Aβ-AGE+Anti-RAGE组大鼠穿越平台次数和目标象限停留时间显著长于Aβ-AGE组(P<0.05)。注射后第9天,各组大鼠海马组织中RAGE蛋白相对表达量比较差异有统计学意义(F=8.626,P<0.05);Aβ-AGE组大鼠海马组织中RAGE蛋白相对表达量显著高于Aβ组(P<0.05),Aβ+Anti-RAGE组大鼠海马组织中RAGE蛋白相对表达量与Aβ组比较差异无统计学意义(P>0.05),Aβ-AGE+Anti-RAGE组大鼠海马组织中RAGE蛋白相对表达量显著低于Aβ-AGE组(P<0.05)。免疫组织化学染色结果显示,各组大鼠大脑皮层中RAGE阳性细胞数比较差异有统计学意义(F=76.370、P<0.01);Aβ-AGE组大鼠大脑皮层中RAGE阳性细胞数显著高于Aβ组(P<0.01),Aβ+Anti-RAGE组大鼠皮层中RAGE阳性细胞数显著低于Aβ组(P<0.01);Aβ-AGE+Anti-RAGE组大鼠皮层中RAGE阳性细胞数显著低于Aβ-AGE组(P<0.01)。结论Aβ-AGE可能通过激活RAGE信号转导通路介导加重AD样大鼠的认知功能障碍,Aβ-AGE和RAGE可能成为治疗AD的新靶点。

Objective To investigate the effect of glycated amyloid β(Aβ-AGE) on cognitive function of Alzheimer′s disease-like rats. Methods Aβ-AGE was synthesized by incubating with methylglyoxal under 37 ℃ for 1 month.Forty Sprague Dawley(SD) rats were randomly divided into Aβ group,Aβ+ mouse monoclonal advanced glycation end product receptor antibody (Anti-RAGE) group,Aβ-AGE group,Aβ-AGE+Anti-RAGE group,with 10 rats in each group.The Aβ( 5 μg ) were stereotaxically injected into the lateral ventricle of rats in Aβ group,the Aβ(5 μg) and Anti-RAGE(50 μg) were stereotaxically injected into the lateral ventricle of rats in Aβ+Anti-RAGE group,the Aβ-AGE(5 μg) were stereotaxically injected into the lateral ventricle of rats in Aβ-AGE group,the Aβ-AGE(5 μg) and Anti-RAGE(50 μg) were stereotaxically injected into the lateral ventricle of rats in Aβ-AGE+ Anti-RAGE group.Morris water maze test was performed on the 2-7 days after administration to detect the latency of rats for searching the hidden platform.The number of crossing platform and the time stayed in the target quadrant of rats were detected on the ninth day after administration.The expression of receptor for advanced glycation endproducts (RAGE) protein in hippocampus of rats were examined by Western blot.The expression of RAGE in the cortex of rats in each group was detected by immunohistochemistry. Results There was no significant difference in the latency period of rats among the groups on the second day after administration ( F =3.767, P >0.05).There was statistic difference in the latency period of rats in each group on the 3-7 day after administration ( F =9.167,25.050,56.980,62.380,122.200;P <0.05);the latency period of rats in the Aβ-AGE group was significantly longer than that in the Aβ group ( P <0.05);the latency period of rats in the Aβ+Anti-RAGE group was significantly shorter than that in the Aβ group ( P <0.05);the latency period of rats in the Aβ-AGE+Anti-RAGE group was significantly shorter than that in the Aβ-AGE group ( P <0.05).There was significant difference in the number of crossing the platform and the time stayed in the target quadrant of rats among the groups on the ninth day after administration ( F =12.930,13.560;P <0.05).The number of crossing the platform and the time stayed in the target quadrant of rats in Aβ-AGE group were significantly lower than those in the Aβ group ( P <0.05);in the Aβ+Anti-RAGE group the number of crossing the platform was more and the time stayed in the target quadrant of rats was loger than those in the Aβ group ( P <0.05);in the Aβ-AGE+Anti-RAGE group the number of crossing the platform was more and the time stayed in the target quadrant of rats was longer than those in the Aβ-AGE group ( P <0.05).There was statistic difference in the relative expression of RAGE protein in hippocampus of rats among the groups on the ninth day after administration ( F =8.626, P <0.05).The relative expression of RAGE protein in hippocampus of rats in the Aβ-AGE group was significantly higher than that in the Aβ group ( P <0.05);there was no statistic difference in the relative expression of RAGE protein in hippocampus of rats between the Aβ+Anti-RAGE group and Aβ group ( P >0.05);the relative expression of RAGE protein in hippocampus of rats in the Aβ-AGE+Anti-RAGE group was significantly lower than that in the Aβ-AGE group ( P <0.05).Immunohistochemical staining results showed that there was statistic difference in the number of RAGE positive cells in the cerebral cortex of rats among the groups ( F =76.370, P <0.01).The number of RAGE positive cells in the cerebral cortex of rats in the Aβ-AGE group was significantly more than that in the Aβ group ( P <0.01);the number of RAGE positive cells in the cortex of rats in the Aβ+Anti-RAGE group was significantly less than that in the Aβ group ( P <0.01);the number of RAGE positive cells in the cortex of rats in the Aβ-AGE+Anti-RAGE group was significantly less than that in the Aβ-AGE group( P <0.01). Conclusion Aβ-AGE may aggravate cognitive dysfunction of AD-like rats through activating RAGE pathway.The Aβ-AGE and RAGE may be new therapeutic targets for AD.