安寐丹通过线粒体介导的海马神经细胞凋亡改善睡眠剥夺模型大鼠的学习记忆水平  

Effect of Anmeidan in Improving Learning and Memory Levels of Sleep Deprived Rats Through Mitochondrial Mediated Hippocampal Neuronal Apoptosis

作　　者：谢光璟 徐波 黄攀攀 王平 XIE Guang-jing;XU Bo;HUANG Pan-pan;WANG Ping(School of Basic Medicine,Hubei University of Traditional Chinese Medicine,Wuhan 430065,China)

机构地区：[1]湖北中医药大学基础医学院,武汉430065

出　　处：《中国实验方剂学杂志》2020年第24期38-44,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家科技部重点研发计划“中医药现代化研究”项目(2018YFC1705600)。

摘　　要：目的:探讨安寐丹通过线粒体介导海马神经细胞凋亡对睡眠剥夺大鼠学习记忆的影响。方法:48只SD大鼠随机分为正常组、模型组、安寐丹低、中、高剂量(4.86,9.72,19.44 g·kg^-1·d^-1)组和艾司唑仑组(0.1 mg·kg^-1·d^-1),每组8只。通过自制睡眠剥夺箱进行持续性睡眠剥夺14 d。以Morris水迷宫检测大鼠的学习记忆水平,酶联免疫吸附测定(ELISA)检测海马组织B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)的含量。透射电镜观察海马区神经元线粒体形态结构,免疫荧光和实时荧光定量聚合酶链式反应(Real-time PCR)分别检测海马区细胞色素C(Cyt-C),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白及Bcl-2,Bax mRNA表达。结果:与正常组比较,模型组上平台潜伏期、游泳总路程延长,初次到达平台时间延长,跨越平台次数减少,目标象限时间显著降低(P<0.01),Bcl-2蛋白及mRNA水平下降,Bax蛋白及mRNA水平显著升高(P<0.01),线粒体结构破坏,嵴断裂,肿胀变形;Cyt-C,Caspase-3蛋白及mRNA表达显著升高(P<0.01);与模型组比较,安寐丹低、中、高剂量组明显改善SD大鼠空间探索和定位航行水平(P<0.05,P<0.01),Bcl-2蛋白及mRNA水平升高,Bax蛋白及mRNA水平明显降低(P<0.05,P<0.01);线粒体损伤改善,水肿减轻;Cyt-C,Caspase-3蛋白及mRNA表达显著下降(P<0.01)。结论:安寐丹能够改善睡眠剥夺大鼠学习记忆水平,其作用与线粒体介导的海马神经细胞凋亡,降低Cyt-C,Caspase-3等蛋白表达相关。Objective:To explore the effect of Anmeidan(AMD)on the learning and memory levels of sleep deprived rats through mitochondrial mediated hippocampal neuronal apoptosis pathway. Method:Fortyeight SD rats were randomly divided into blank group,model group,low,medium,high-dose AMD groups(4.86,9.72,19.44 g·kg^-1·d^-1)and estazolam group(0.1 mg·kg^-1·d^-1). Insomnia model was prepared by selfmade sleep deprivation box for 14 days. Morris water maze was used to detect learning and memory levels,enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions of cytochrome C(Cyt-C),cysteine aspartic acid protease-3(Caspase-3)in hippocampus. Transmission electron microscopy(TEM)was used to observe the morphological structure of mitochondria in hippocampus. Protein and mRNA expressions of Cyt-C, Caspase-3, Bcl-2, Bax were detected by immunofluorescence(IF)and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)respectively. Result: In the model group, the incubation period of the platform and the total distance of swimming and the time of first arriving platform were prolonged,the number of platform crossing and the time of target quadrant movement were reduced,protein and mRNA expressions of Bcl-2 dropped, protein and mRNA expressions of Bax increased(P<0.01), and mitochondrial structure was abnormal with crista fracture,swelling and deformation. And protein and mRNA expressions of Cyt-C,Caspase-3 increased significantly(P<0.01). Low,medium and high-dose AMD groups could improve levels of space exploration and navigation of SD rats(P<0.01),increase protein and mRNA expressions of Bcl-2,decrease protein and mRNA expressions of Bax,improve the damage of mitochondria,and decrease the protein and mRNA expressions of Cyt-C,Caspase-3(P<0.01). Conclusion: AMD can improve the learning and memory levels of SD rats, the effect is related to the mitochondrial mediated hippocampal neuronal apoptosis pathway and decrease of Cyt-C and Caspase-3 expressions.

关 键 词：安寐丹 睡眠剥夺 细胞凋亡 学习记忆 细胞色素C(Cyt-C) 半胱氨酸天冬氨酸蛋白酶-3(Caspase-3) 

分 类 号：R2-0[医药卫生—中医学] R22