基于Cyt C调节细胞凋亡途径探讨安寐丹改善老年睡眠剥夺模型致认知损伤的作用机制  

Mechanism of Anmeidan in Ameliorating Cognitive Impairment in Geriatric Sleep Deprivation Model Based on Cyt C Signaling Pathway on Apoptosis

作　　者：张军路 孙康 吴艺璇 王平[2,3] 谢光璟 ZHANG Junlu;SUN Kang;WU Yixuan;WANG Ping;XIE Guangjing(School of Basic Medical Sciences,Hubei University of Chinese Medicine,Wuhan 430065,China;Ministry of Education Engineering Research Centre of Chinese Medicine Protection Technology and New Product Development for Elderly Brain Health,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Shizhen Laboratory,Wuhan 430065,China)

机构地区：[1]湖北中医药大学基础医学院,武汉430065 [2]湖北中医药大学老年脑健康中医药防护技术与新产品研发教育部工程研究中心,武汉430065 [3]湖北时珍实验室,武汉430065

出　　处：《中国实验方剂学杂志》2024年第19期1-9,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82305354)。

摘　　要：目的:探讨安寐丹(AMD)对老年睡眠剥夺模型的认知功能及细胞色素C(Cyt C)信号通路蛋白表达和细胞凋亡影响。方法:60只老年C57小鼠随机分为空白组、模型组、安寐丹高、中、低剂量组(26.26、13.13、6.565 g·kg^(-1)·d^(-1))和褪黑素组(1.3 mg·kg^(-1)·d^(-1)),每组10只。通过自制睡眠剥夺箱进行连续性睡眠剥夺4周。以Morris水迷宫检测小鼠的认知功能水平,苏木素-伊红(HE)染色和尼氏染色观察海马CA1区的锥体细胞形态学改变。透射电镜观察海马神经元线粒体形态结构;蛋白免疫印迹法(Western blot)检测海马中Cyt C、胱天蛋白酶-3(Caspase-3)、胱天蛋白酶-9(Caspase-9)、脑源性神经营养因子(BDNF)、线粒体转录因子A(TFAM)、电压依赖性阴离子通道1(VDAC1)蛋白表达。免疫组化检测Cyt C、Caspase-3、Caspase-9的蛋白表达水平。免疫荧光检测海马B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的蛋白表达。结果:与空白组比较,模型组上平台潜伏期延长(P<0.01),穿越平台次数,目标象限活动时间和路程显著减少(P<0.01),线粒体结构损坏,嵴消失或断裂,肿胀变形;Cyt C、Caspase-3、Caspase-9、Bax、VDAC1蛋白表达显著增加(P<0.01),BDNF、TFAM、Bcl-2蛋白表达显著减少(P<0.01)。与模型组比较,安寐丹高、中、低剂量组改善老年睡眠剥夺模型小鼠空间探索和定位航行水平(P<0.05,P<0.01);缓解线粒体损伤,增加尼氏小体数量;Cyt C、Caspase-3、Caspase-9、Bax、VDAC1蛋白表达明显减少(P<0.05,P<0.01),BDNF、TFAM、Bcl-2蛋白表达明显增加(P<0.05,P<0.01)。结论:安寐丹能够改善老年睡眠剥夺模型小鼠认知功能,其作用可能与降低Cyt C信号通路介导的细胞凋亡途径有关。Objective:To investigate the effects of Anmeidan(AMD)on cognitive function,cytochrome C(Cyt C)signaling pathway protein expression,and apoptosis in a geriatric sleep deprivation model.Method:Sixty aged C57 mice were randomly divided into a blank group,a model group,AMD high,medium,and low dose(26.26,13.13,6.565 g·kg^(-1)·d^(-1))groups,and a melatonin group(1.3 mg·kg^(-1)·d^(-1)),with 10 mice in each group.Continuous sleep deprivation was performed for 4 weeks using a homemade sleep deprivation box.Cognitive function was assessed using the Morris water maze,and morphological changes in pyramidal cells in the CA1 area of the hippocampus were observed by hematoxylin-eosin(HE)staining and Nissl staining.Transmission electron microscopy was used to observe the mitochondrial morphology and structure of hippocampal neurons.Western blot was used to detect Cyt C,cysteine-aspartate protease-3(Caspase-3),cysteine-aspartate protease-9(Caspase-9),brain-derived neurotrophic factor(BDNF),mitochondrial transcription factor A(TFAM),and voltage-dependent anion channel 1(VDAC1)protein expression.Immunohistochemistry was used to detect protein expression levels of Cyt C,Caspase-3,and Caspase-9,and immunofluorescence was used to detect the protein expression of B-cell lymphoma 2(Bcl-2)and Bcl-2-associated X protein(Bax).Result:Compared with the blank group,the model group showed prolonged platform latency(P<0.01),reduced number of platform crossings,and reduced time and distance in the target quadrant(P<0.01).The mitochondrial structure was damaged,with disappearance or breakage of cristae,and increased swelling and deformation.The protein expression levels of Cyt C,Caspase-3,Caspase-9,Bax,and VDAC1 were significantly increased(P<0.01),while BDNF,TFAM,and Bcl-2 protein expression levels were decreased(P<0.01).Compared with the model group,the AMD high,medium,and low dose groups improved spatial exploration and navigation abilities in geriatric sleep-deprived mice(P<0.05,P<0.01),alleviated mitochondrial damage,and increased the nu

关 键 词：安寐丹 老年睡眠剥夺 细胞凋亡 线粒体 细胞色素C(CytC)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33