生慧颗粒通过抑制PI3K/Akt/mTOR信号通路改善阿尔茨海默病小鼠学习记忆能力

Shenghui Granules Improve Learning Memory in Alzheimer's Disease Mice by Inhibiting PI3K/Akt/mTOR Signaling Pathway

目的基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路探讨生慧颗粒对阿尔茨海默病(AD)小鼠学习记忆能力的影响。方法将30只6月龄雄性APP/PS1双转基因小鼠随机分为模型组、生慧颗粒组(7.02 g/kg)、多奈哌齐组(0.65 mg/kg),每组10只;C57BL/6J小鼠10只,设为对照组,对照组和模型组均给予等体积生理盐水,每日1次,连续给药4周。采用Morris水迷宫检测学习记忆能力,HE染色观察海马CA1区病理形态变化,免疫组化检测海马CA1区Aβ1-42和P-Tau416的表达水平,Western Blot检测海马区PI3K/Akt/mTOR通路蛋白及其磷酸化蛋白和Beclin 1、p62、LC3蛋白表达水平。结果与对照组比较,模型组小鼠平台潜伏期、游泳总路程增加(P<0.01),穿越平台次数和目标象限停留时间减少(P<0.01);海马CA1区细胞数量减少、排列疏松,细胞层次减少;Aβ1-42和P-Tau416蛋白表达增多(P<0.01);PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR、p62蛋白表达水平升高(P<0.01),Beclin 1蛋白表达水平及LC3Ⅱ/LC3Ⅰ比值降低(P<0.01)。与模型组比较,生慧颗粒组和多奈哌齐组小鼠平台潜伏期、游泳总路程均减少(P<0.01,P<0.05),穿越平台次数和目标象限停留时间均增加(P<0.01,P<0.05);海马CA1区细胞数目均增多,结构相对完整;p-Tau416、PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR、p62蛋白表达水平均降低(P<0.01,P<0.05),Beclin 1蛋白表达水平均上调(P<0.01);生慧颗粒组Aβ1-42蛋白表达水平降低(P<0.01)、LC3Ⅱ/LC3Ⅰ比值上调(P<0.01)。结论生慧颗粒能抑制PI3K/Akt/mTOR信号通路,诱导细胞自噬,促进Aβ、磷酸化Tau蛋白清除,改善AD小鼠学习记忆能力。

Objective To investigate the effect of Shenghui Granules on the learning memory ability of Alzheimer's disease(AD)mice based on the Phosphatidylinositol 3-kinase(PI3K)/Protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway.Methods Thirty six-month-old male APP/PS1 double transgenic mice were randomly divided into the model group,the Shenghui Granules group(7.02 g/kg)and the Donepezil group(0.65 mg/kg),with 10 mice in each group,and 10 C57BL/6J mice were selected as the control group.the drugs were administered once daily for 4 weeks,and both of the control and model groups were given equal volume of saline.Morris water maze was used to test the learning memory ability,HE staining was used to observe the pathological morphological changes in hippocampal CA1 area,Immunohistochemistry was used to detect the expression level of Aβ1-42 and P-Tau416 in hippocampal CA1 area,and Western Blot was used to detect the expression level of PI3K/Akt/mTOR pathway proteins and their phosphorylated proteins and Beclin 1,p62 and LC3 proteins in hippocampal area.Results Compared with the control group,mice in the model group showed an increase in platform latency and total swimming distance(P<0.01)and a decrease in the number of platform crossings and dwell time in the target quadrant(P<0.01);a decrease in the number of cells in the hippocampal CA1 region,a sparse arrangement and a decrease in cellular hierarchy;an increase in the expression of Aβ1-42 and P-Tau416 proteins(P<0.01);an increase in PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR and p62 protein expression levels(P<0.01);and a decrease in Beclin 1 protein expression levels and LC3Ⅱ/LC3Ⅰratio(P<0.01).Compared with the model group,the platform latency and total swimming distance of mice in the Shenghui Granules group and Donepezil group were reduced(P<0.01,P<0.05),and the number of platform crossings and dwell time in the target quadrant were increased(P<0.01,P<0.05);the number of cells in the CA1 area of the hippocampus was increased,with relatively intact structure;the levels of p-Tau416,PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR and p62 protein expression were all decreased(P<0.01,P<0.05),and the levels of Beclin 1 protein expression were all upregulated(P<0.01);Aβ1-42 protein expression level was decreased and LC3Ⅱ/LC3Ⅰratio was up-regulated in the Shenghui Granules group(P<0.01).Conclusion Shenghui Granules inhibits the PI3K/Akt/mTOR signaling pathway,induces cellular autophagy,promotes the clearance of Aβ,phosphorylated Tau proteins,and improves learning and memory in AD mice.