摘要
目的:探讨六味地黄丸通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/叉头框转录因子O3a(FoxO3a)通路调控自噬对快速老化小鼠8型(SAMP8)小鼠记忆功能的影响。方法:6月龄SPF级抗快速老化亚系小鼠(SAMR1)雄性小鼠6只设为正常组,6月龄SPF级SAMP8雄性小鼠24只随机均分为模型组、多奈哌齐组(0.747 mg·kg^(-1))、六味地黄丸高、低剂量组(2.700、1.350 g·kg^(-1)),灌胃给药2个月。Morris水迷宫法检测各组小鼠学习和记忆能力;尼氏染色观察小鼠皮层、海马神经元;免疫组化(IHC)检测皮层、海马微管相关蛋白1轻链3B(LC3B)阳性表达;蛋白免疫印迹法(Western blot)检测各组小鼠皮层自噬关键分子酵母ATG6同系物(Beclin1)、B细胞淋巴瘤-2(Bcl-2)、自噬相关基因5(ATG5)、胱天蛋白酶-3(Caspase-3)、Caspase-9、Akt、p-Akt、FoxO3a、p-FoxO3a的蛋白表达水平。结果:与正常组小鼠比较,模型组小鼠逃避潜伏期明显延长(P<0.05,P<0.01),穿越平台次数、目标象限停留时间显著减少(P<0.01);皮层、海马神经细胞数量明显减少、体积缩小,分布疏散,LC3B阳性表达显著增多(P<0.01);皮层Beclin1、ATG5表达显著升高(P<0.01),Bcl-2表达水平降低(P<0.01),Caspase-3、Caspase-9表达水平显著升高(P<0.01),p-Akt/Akt、p-FoxO3a/FoxO3a表达水平显著降低(P<0.01)。与模型组比较,多奈哌齐组及六味地黄丸高、低剂量组小,3 d逃避潜伏期明显缩短(P<0.05,P<0.01);穿越平台次数显著增加(P<0.01);目标象限停留时间显著增加(P<0.01);多奈哌齐组皮层海马神经元神经细胞数量增多,六味地黄丸高、低剂量组小鼠神经元数目及尼氏体明显增加且分布致密,细胞损伤程度较低;多奈哌齐组及六味地黄丸高、低剂量组皮层、海马LC3B阳性表达显著降低(P<0.01);六味地黄丸高、低剂量组Beclin1表达显著降低(P<0.01),多奈哌齐组和六味地黄丸低剂量组ATG5表达显著降低(P<0.01),多奈哌齐组及六味地黄丸高、低剂量组皮层Bcl-2表达水平显著升高(P<0.01),Caspase-3表达水平显著降低(P<0.01),Caspase-9表达明显降低(P<0.05,P<0.01),p-Akt/Akt、p-FoxO3a/FoxO3a表达水平显著升高(P<0.01)。结论:六味地黄丸有效改善了快速老化SAMP8 AD模型小鼠的学习记忆能力,保护神经元,其机制可能与调控PI3K/Akt/FoxO3a信号通路,下调自噬相关因子ATG5、Beclin1和LC3B表达和减少凋亡有关。
Objective:To investigate the effect of Liuwei Dihuangwan on memory function of senescence-accelerated mouse prone 8(SAMP8)mice by regulating autophagy through the phosphatidylinositol3-kinase(PI3K)/protein kinase B(Akt)/forkhead box O3a(FoxO3a)pathway.Method:Six male senescenceaccelerated mouse resistant 1(SAMR1)mice of SPF grade aging 6 months were assigned to a normal group,and 24 male SAMP8 mice of SPF grade aging 6 months were randomly divided into a model group,a donepezil group(0.747 mg·kg^(-1)),and high-and low-dose Liuwei Dihuangwan groups(2.700 and 1.350 g·kg^(-1)),with6 mice in each group.The mice were treated with drugs by gavage for 2 months.Morris water maze was used to detect the learning and memory abilities of mice in each group.Nissl staining was used to observe the neurons in the cortex and hippocampus.The positive expression of microtubule-associated protein 1 light chain 3B(LC3B)in the cortex and hippocampus was detected by immunohistochemistry(IHC).Western blot was used to detect the protein expression of the mammalian ortholog of yeast ATG6(Beclin-1),B cell lymphoma-2(Bcl-2),autophagy-related gene 5(ATG5),cysteinyl aspartate-specific protease 3(Caspase-3),Caspase-9,Akt,p-Akt,FoxO3a,and p-FoxO3a.Result:Compared with the normal group,the model group showed prolonged escape latency(P<0.05,P<0.01),reduced number of platform crossings and the residence time in the target quadrant(P<0.01),decreased neurons with reduced volume and dispersed distribution in the cortex and hippocampus,increased positive expression of LC3B(P<0.01),elevated expression of Beclin-1 and ATG5 in the cortex(P<0.01),declined Bcl-2 expression(P<0.01),up-regulated Caspase-3 and Caspase-9 expression(P<0.01),and decreased expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a(P<0.01).Compared with the model group,the donepezil group and the Liuwei Dihuangwan groups showed shortened 3 d escape latency(P<0.05,P<0.01),increased number of platform crossings(P<0.01),and prolonged residence time in the target quadrant(P<0.01).In the donepezil group,the number of neurons in the cortex and hippocampus was increased.In the Liuwei Dihuangwan groups,the number of neurons and Nissl bodies increased with denser distribution and lower degree of cell damage.The positive expression of LC3B in the cortex and hippocampus was decreased in the donepezil group and Liuwei Dihuangwan groups(P<0.01).The expression of Beclin-1 was decreased in the Liuwei Dihuangwan groups(P<0.01).The expression of ATG5 was decreased in the donepezil group and the low-dose Liuwei Dihuangwan group(P<0.01).The donepezil group and the Liuwei Dihuangwan groups showed the increased expression level of Bcl-2 in the cortex(P<0.01),decreased expression level of Caspase-3(P<0.01),reduced expression level of Caspase-9(P<0.05,P<0.01),and elevated expression levels of p-Akt/Akt and p-FoxO3a/FoxO3a(P<0.01).Conclusion:Liuwei Dihuangwan can effectively improve the learning and memory abilities of the SAMP8 mice and protect neurons.Its mechanism may be related to the regulation of the PI3K/Akt/FoxO3a signaling pathway,down-regulation of the expression of ATG5,Beclin-1,and LC3B,and the inhibition of apoptosis.
作者
宋军营
丁蕊
李俊霖
蔡菊
袁永
贾亚泉
张振强
SONG Junying;DING Rui;LI Junlin;CAI Ju;YUAN Yong;JIA Yaquan;ZHANG Zhenqiang(Academy of Chinese Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450046,China;School of Medicine,Henan University of Chinese Medicine,Zhengzhou 450046,China;Henan Engineering Research Center for Prevention and Treatment of Neurodegenerative Diseases,Zhengzhou 450046,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第17期1-8,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(U1504829)
中原科技创新领军人才项目(204200510022)
河南省高校科技创新团队支持计划项目(21IRTSTHN026)
河南省中医药科学研究专项重点课题(2018ZY1009)
河南省科技攻关项目(212102311084)。
