大补元煎联合运动通过调控PI3K/Akt/GSK-3β信号通路改善AD大鼠学习记忆能力

Da Buyuanjian Combined with Exercise Improves Learning Memory of AD Rats by Modulating PI3K/Akt/GSK-3βSignaling Pathway

目的:研究大补元煎灌胃联合4周的跑台运动对阿尔茨海默病(AD)大鼠磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/糖原合成酶激酶-3β(GSK-3β)信号通路及相关细胞凋亡蛋白的调节作用,探讨其改善AD大鼠认知功能的作用机制。方法:将50只雄性SD大鼠随机均分为假手术组(Sham组)、运动组(EX组)、模型组(AD组)、大补元煎组(TCM组)、运动+大补元煎组(EX+TCM组),每组10只。采用海马区注射β淀粉样蛋白25-35(Aβ25-35)寡聚体溶液建立AD大鼠模型,EX组进行6 d/周的跑台训练。TCM组予大补元煎水煎液灌胃(5.36 g·kg^(-1)),EX+TCM组在跑台训练同时进行大补元煎水煎液灌胃,Sham组、AD组及EX组给予等容纯水灌胃。Morris水迷宫评价大鼠的学习记忆,苏木素-伊红(HE)染色观察大鼠海马神经元的形态变化,透射电镜观察海马神经元超微结构的改变,蛋白免疫印迹法(Western blot)检测大鼠海马中蛋白激酶B(Akt)、磷酸化(p)-Akt、GSK-3β、p-GSK-3β、β-连环蛋白(β-catenin)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、剪切的胱天蛋白酶-3(cleaved Caspase-3)蛋白表达水平。结果:与Sham组比较,AD组大鼠逃避潜伏期和游泳总路程显著增加,目标象限停留时间显著减少(P<0.01),海马锥体细胞排列疏松紊乱,密度降低;海马超微结构出现髓鞘样改变,线粒体肿胀模糊;Bcl-2、p-Akt、p-GSK-3β的水平显著降低、β-catenin表达明显下降(P<0.05,P<0.01)、cleaved Caspase-3表达明显上调(P<0.05)。与AD组比较,EX组、TCM组、EX+TCM组大鼠逃避潜伏期和游泳路程明显减少,目标象限停留时间明显增加(P<0.05,P<0.01);锥体细胞排列规律,细胞数量增加,透射电镜下可见髓鞘样改变减轻,线粒体肿胀改善;Bcl-2、p-Akt、p-GSK-3β表达升高,cleaved Caspase-3表达水平明显降低(P<0.05,P<0.01),且EX+TCM组β-catenin水平升高而EX组、TCM组与AD组比较差异无统计学意义。与EX+TCM组比较,EX组与TCM组逃避潜伏期和游泳路程增加(P<0.05,P<0.01),目标象限停留时间显著减少(P<0.01);锥体细胞排列疏松,电镜下突触完整性下降;Bcl-2、p-Akt、p-GSK-3β、β-catenin的水平明显降低(P<0.05,P<0.01),cleaved Caspase-3表达明显升高(P<0.05)。结论:有氧运动结合大补元煎可改善AD大鼠认知功能障碍,其机制可能与升高PI3K/Akt/GSK-3β信号通路中的关键蛋白表达有关,且联合使用优于运动或药物单独使用。

Objective:To investigate the effects of 4 weeks of aerobic exercise combined with traditional Chinese medicine(TCM)on the regulation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/glycogen synthase kinase-3β(GSK-3β)signaling pathway and related apoptotic proteins in AD rats,and to explore its mechanism of action in improving cognitive function of AD rats.Method:Fifty male SD rats were randomly divided into Sham group,exercise(EX)group,Alzheimer’s disease(AD)group(model),TCM group(Dabuyuan Jian),and EX+TCM group,10 in each group.Aβ25-35 oligomer solution was injected into the hippocampal region to establish the AD rat model,and the EX group underwent 6 days/week of running table training.The TCM group was given Dabuyuan Jian(5.36 g·kg^(-1))by gavage,and the EX+TCM group received running table training combined with intragastric administration of Dabuyuan Jian.An equal volume of purified water was given to the Sham,AD,and EX groups by gavage.The rats’learning memory was evaluated by Morris water maze.The morphological changes and ultrastructural changes of hippocampal neurons were observed by hematoxylin-eosin(HE)staining and transmission electron microscopy,respectively.Western blot was performed to detect the protein expression levels of Akt,p-Akt,GSK-3β,p-GSK-3β,β-catenin,Bcl-2,Bax,and cleaved Caspase-3 in the hippocampus of rats.Result:Compared with the Sham group,the AD group showed an increase in escape latency and total swimming distance,and a decrease in the residence time in the target quadrant(P<0.01),with loose and disorganized arrangement of hippocampal pyramidal cells,a decrease in density,myelin-like changes in hippocampal ultrastructure,and a blurring of mitochondrial swelling.In addition,the expression of Bcl-2,p-Akt,p-GSK-3βandβ-catenin was down-regulated(P<0.05,P<0.01)while the expression of cleaved Caspase-3 was up-regulated(P<0.05).Compared with the conditions in the AD group,the escape latency and swimming distance were reduced in the EX,TCM,and EX+TCM groups,while the residence time in the target quadrant was increased(P<0.05,P<0.01),with regularly arranged pyramidal cells,elevated number of cells,and reduced myelin-like changes and improved mitochondrial swelling as seen by transmission electron microscopy.Moreover,the expression of Bcl-2,p-Akt and p-GSK-3βwas up-regulated,while the expression of cleaved Caspase-3 was down-regulated(P<0.05,P<0.01).The EX+TCM group had increased level ofβ-catenin while the EX and TCM groups were not statistically significant compared with the AD group.Compared with the EX+TCM group,the EX and TCM groups had increased escape latency and swimming distance and decreased residence time in the target quadrant(P<0.05,P<0.01),with loosely arranged pyramidal cells and reduced synaptic integrity under transmission electron microscopy(P<0.01).Furthermore,the levels of Bcl-2,p-Akt,p-GSK-3βandβ-catenin were lowered(P<0.05,P<0.01)while the level of cleaved Caspase-3 was elevated(P<0.05).Conclusion:Aerobic exercise combined with Dabuyuan Jian could improve the cognitive dysfunction of AD rats,and the mechanism might be related to the elevated expression of key proteins in the PI3K/Akt/GSK-3βsignaling pathway.The combined use was better than the use of exercise or drugs alone.