目的 基于cAMP/PKA/CREB 通路探讨柴胡皂苷A 对失眠大鼠的改善作用及机制。方法 将75 只SD 大鼠随机分为空白组、模型组、柴胡皂苷A 低剂量组（0.625 mg·kg-1）、柴胡皂苷A 高剂量组（2.500 mg·kg-1）、艾 司唑仑组（0.1 mg·kg-1），每组15 只。采用腹腔注射苯丙氨酸（PCPA，0.1 mg·kg-1）复制失眠大鼠模型。观察大 鼠一般情况及昼夜节律；采用戊巴比妥钠翻正实验测定大鼠睡眠潜伏期及睡眠持续时间；观测大鼠睡眠时相， 记录慢波睡眠第1 期（SWS1）、慢波睡眠第2 期（SWS2）、快速眼球运动睡眠期（REMS）时长以及总睡眠时长 （TST）；qRT-PCR 法测定下丘脑节律基因Clock、Bmal1 mRNA 及钟控基因Rev-erbα、Rorα mRNA 的表达水 平；免疫荧光法测定海马组织NeuN 表达水平；ELISA 法测定脑组织中的cAMP 水平；Western Blot 法测定脑 组织中Clock、Bmal1、Rev-erbα、Rorα 及cAMP/PKA/CREB 通路相关蛋白表达水平。结果 与空白组比较，模 型组大鼠昼伏夜出的节律紊乱，极度兴奋，易激惹，睡眠减少；睡眠潜伏期明显延长（P＜0.05），睡眠持续时 间及SWS1、SWS2、REMS、TST 均明显缩短（P＜0.05）；神经元排列紊乱，NeuN 阳性神经元IOD 值明显降低 （P＜0.05）； 脑组织Clock、Bmal1、Rev-erbα、Rorα mRNA 及蛋白表达水平明显降低（P＜0.05）； 脑组织 cAMP、p-PKA/PKA、p-CREB/CREB 蛋白表达水平明显降低（P＜0.05）。与模型组比较，给药组大鼠的攻击性 明显减弱，昼伏夜出有节律性，活动减少，睡眠增多；睡眠潜伏期明显缩短（P＜0.05），睡眠持续时间及 SWS1、SWS2、REMS、TST 均明显延长（P＜0.05）；神经元排列紊乱情况有所恢复，NeuN 阳性神经元IOD 值 明显升高（P＜0.05）；脑组织Clock、Bmal1、Rev-erbα、Rorα mRNA 及蛋白表达水平明显升高（P＜0.05）；脑 组织cAMP、p-PKA/PKA、p-CREB/CREB 蛋白表达水平明显升高（P＜0.05）。结论 柴胡皂苷A 可能通过激活 cAMP/PKA/CREB 通路改善失眠大鼠的昼夜节律。

Abstract: Objective To explore the improving effect and mechanism of saikosaponin A on insomnia rats based on cAMP/PKA/CREB pathway. Methods Seventy-five SD rats were randomly divided into blank group， model group， low-dose saikosaponin A group （0.625 mg·kg-1），high-dose saikosaponin A group （2.500 mg·kg-1） and Estazolam group （0.1 mg·kg-1）， with 15 rats in each group. Insomnia rat model was established by intraperitoneal injection of Phenylalanine （PCPA， 0.1 mg·kg-1） . The general condition and circadian rhythm of rats were observed； the sleep latency and sleep duration of rats were measured by pentobarbital sodium righting experiment. The sleep phase of rats was observed，and the duration of slow wave sleep phase 1 （SWS1），slow wave sleep phase 2 （SWS2），rapid eye movement sleep phase （REMS） and total sleep time （TST） were recorded. The mRNA expression levels of hypothalamic circadian genes Clock，Bmal1 and clock-controlled genes Rev-erbα and Rorα were determined by qRTPCR. The expression level of NeuN in hippocampus was determined by immunofluorescence. The level of cAMP in brain tissue was determined by ELISA. The expression levels of Clock， Bmal1， Rev-erbα， Rorα and cAMP/PKA/CREB pathway-related proteins in brain tissue were determined by Western Blot. Results Compared with the blank group，the rats in the model group had disordered circadian rhythms，extreme excitement，irritability，and reduced sleep ；the sleep latency was significantly prolonged （P＜0.05），and the sleep duration and SWS1，SWS2，REMS and TST were significantly shortened （P＜0.05） . The arrangement of neurons was disordered，and the IOD value of NeuN positive neurons was significantly decreased （P＜0.05） . The mRNA and protein expression levels of Clock， Bmal1， Reverbα and Rorα in brain tissue were significantly decreased （P＜0.05） . The expression levels of cAMP，p-PKA/PKA and p-CREB/CREB in brain tissue were significantly decreased （P＜0.05） . Compared with the model group， the aggressiveness of the rats in the administration group was significantly weakened，the circadian rhythm was rhythmic， the activity was reduced，and the sleep was increased. The sleep latency was significantly shortened （P＜0.05），and the sleep duration and SWS1， SWS2， REMS and TST were significantly prolonged （P＜0.05） . The disorder of neuronal arrangement was restored， and the IOD value of NeuN positive neurons was significantly increased （P＜ 0.05） . The mRNA and protein expression levels of Clock， Bmal1， Rev-erbα and Rorα in brain tissue were significantly increased （P＜0.05） . The protein expression levels of cAMP，p-PKA/PKA and p-CREB/CREB in brain tissue were significantly increased （P＜0.05） . Conclusion Saikosaponin A may improve the circadian rhythm of insomnia rats by activating cAMP/PKA/CREB pathway.

R285.5

2021年河南省卫生健康委国家中医临床研究基地科研专项（2021JDZX2111）。