安寐丹调控OXA/CREB/PER1信号通路改善SD模型大鼠昼夜节律紊乱  

Effects of AnMeiDan on Regulating OXA/CREB/PER1 Signaling Pathway in SD Rats Circadian Rhythm Disorder

作　　者：徐波 谢光璟 夏婧 王平 Xu Bo;Xie Guangjing;Xia Jing;Wang Ping(Institute of Geriatrics,Hubei University of Chinese medicine,Wuhan 430065,China)

机构地区：[1]湖北中医药大学老年医学研究所,武汉430065

出　　处：《世界科学技术-中医药现代化》2022年第1期298-308,共11页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会青年项目(82104712):培元固本安神法调控Orexin信号通路改善睡眠剥夺模型大鼠学习记忆的分子机制研究,负责人:徐波;湖北省教育厅科学研究计划-青年项目(Q20202003):培元固本安神法调控OXA/CREB/Per1信号通路改善睡眠剥夺模型大鼠昼夜节律紊乱的机制研究,负责人:徐波;湖北中医药大学青苗计划(2020ZZX021):基于OXA/PLCβ-1/PKC/ERK1/2信号通路研究安寐丹改善睡眠剥夺模型学习记忆的分子机制,负责人:徐波。

摘　　要：目的 观察中医经典名方安寐丹对睡眠剥夺(Sleep disruption,SD)模型大鼠食欲素(Orexin)及其介导的食欲素A(Orexin A,OXA)/cAMP反应元件结合蛋白(cAMP Response Element Binding Protein,CREB)/Period1(period circadian regulator 1,PER1)基因信号通路的作用。方法 40只雄性6月龄SD大鼠随机等分为空白组、模型组、艾司唑仑组、安寐丹组,空白组常规进食进水,其他三组采用对氯苯丙氨酸(PCPA)腹腔注射叠加多平台水环境剥夺法构建SD模型,自主活动仪监测昼夜活动节律。空白组、模型组给予等容的0.9%氯化钠灌胃,艾司唑仑组给予艾司唑仑0.09 mg·kg-1、安寐丹组给予安寐丹水煎剂9.09 mg·kg-1灌胃治疗,连续4周。4周后运用Morris水迷宫检测其学习记忆,免疫荧光检测下丘脑Orexin神经元表达,酶联免疫吸附测定法(ELISA)检测各组大鼠下丘脑组织OXA、食欲素B(Orexin B,OXB)含量,蛋白质免疫印迹(Western Blot,WB)和实时荧光定量(Quantitative Real-time PCR,RT-PCR)检测各组大鼠下丘脑组织OXA/CREB/PER1信号通路蛋白和mRNA表达。结果 与空白组比较,模型组大鼠24 h自主活动时间和活动距离均高于空白组;活动时间、活动距离显著增加(P<0.01),上平台潜伏期与游泳总路程显著延长(P<0.01),穿越站台次数和站台活动时间均减少(P<0.01);下丘脑组织OXA、OXB含量高于空白组(P<0.01);且OXA、CREB蛋白和mRNA表达均显著升高(P<0.01),PER1蛋白和mRNA表达均显著下调(P<0.01)。与模型组比较,安寐丹组大鼠上平台潜伏期缩短(P<0.01)、游泳总路程减少(P<0.05)、穿越平台数量增加(P<0.05);下丘脑组织OXA、OXB含量降低(P<0.05),OXA蛋白表达下调(P<0.05)、CREB蛋白表达下调(P<0.01)、PER1蛋白表达上调(P<0.01),OXA和CREB mRNA表达降低(P<0.05),PER1 mRNA表达升高(P<0.05)。结论 安寐丹改善SD模型大鼠昼夜节律紊乱可能与调节Orexin及其介导的OXA/CREB/PER1信号通路相关。Objective To observe the effect of AnMeiDan(AMD) on Orexin and its mediated signal pathway of Orexin A(OXA)/cAMP response element binding protein(CREB)/period 1(Per1) gene in sleep deprivation(SD) model rats.Methods 40 male 6-month-old SD rats were randomly divided into blank group, model group, estazolam group and AMD group. The blank group was fed with water, and the other three groups were injected intraperitoneally with chlorophenylalanine(PCPA) and deprived of multi-platform water environment to construct SD model. The circadian activity rhythm was monitored by autonomous activity instrument. The blank group and model group were given isovolumic 0.9% sodium chloride by gavage, the estazolam group was given estazolam 0.09 mg·kg-1, and the AMD group was given AMD Decoction 9.09 mg·kg-1by gavage for 4 weeks. Four weeks later, Morris water maze was used to detect learning and memory. Immunofluorescence was used to detect the expression of Orexin neurons in hypothalamus.Enzyme-linked immunosorbent assay(ELISA) was used to detect the contents of OXA and Orexin B(OXB) in hypothalamus. Western Blot and Quantitative real-time PCR(RT-PCR) was used to detect the Protein and mRNA expression of OXA/CREB/Per1 signal pathway in hypothalamus.Results Compared with the blank group, the 24-hour autonomous activity time and distance in the model group were higher than those in the blank group;The activity time and distance increased significantly(P<0.01), the incubation period on the platform and the total distance of swimming prolonged significantly(P<0.01), and the times of crossing the platform and the activity time on the platform decreased(P<0.01);The contents of OXA and OXB in hypothalamus were higher than those in blank group(P<0.01);The expressions of OXA, CREB protein and mRNA were significantly increased(P<0.01), and the expressions of Per1 protein and mRNA were significantly decreased(P<0.01). Compared with the model group, the latency on the platform was shortened(P<0.01), the total swimming distance was reduced(P

关 键 词：安寐丹 睡眠剥夺 昼夜节律 食欲素 

分 类 号：R285.5[医药卫生—中药学]