解郁安神法调节失眠模型大鼠睡眠时相及中缝核5-HT信号通路的机制研究

Based on the analysis on the pathogeny 、pathogenesis 、constitution 、syndrome and treatment of traditional Chinese medicine（TCM），it is currently accepted that liver depression occurs predominantly in insomnia and jieyu anshen method is considered as the fundamental therapy for insomnia. Previous evidence suggested that Song-Yu-An-Shen-Fang，the herbal compound of prescription for jieyu anshen method，had a reliable curative effect on insomnia and the underlying mechanism may be relevant to regulate the level of 5-hydroxytryptamine（5-HT）. On the basis of early researches，we propose that the rat model of insomnia is induced by electrical stimulation. The treatment start，consisting of daily oral gavage with Song-Yu-An-Shen-Fang or vehicle(tap water) only for a subsequent period of 14 days. The electroencephalogram（EEG） and electromyogram（EMG）signals are recorded before and after treatment to verify the effect of the jieyu anshen method on states of sleep-waking cycle in rats.Rats are decapitated using a guillotine.Brains are immediately removed and dorsal raphe nucleus（DRN）tissues are separated. High performanee liquid dehromatography-eleetroehemical determination（HPLC-ECD）is used to detect 5-HT/5-HIAA coneeniration of DRN tissues and calculate the 5-HT inversion ratio . Serotonintransporter（SERT）and 5-HT1A receptor （5-HT1AR） mRNA expression is investigated by real-time RT-PCR.The level of 5-HT1AR protein expression is also measured by western blot method. In the other rats，coronal DRN slices are prepared.We use whole cell patch-clamp recordings of 5-HT-induced G-proteincoupled inwardly rectifying potassium (GIRK) currents as a measure of 5-HT1AR functionality，so as to explore the mechanism of 5-HT signaling pathway in DRN upon insomnia rats treated by jieyu anshen method. The exploration may provide reliable experimental basis for TCM collaborative solution the clinical dilemma of insomnia, and are of certain value in guiding rational clinical therapy.

失眠多肝郁，解郁安神法是防治失眠的根本大法。前期研究证实以解郁安神法为载体的中药组方松郁安神方治疗失眠疗效确切，其机制与调节5-HT含量有关。本课题拟在前期研究基础上，采用电刺激法复制失眠大鼠模型，运用松郁安神方进行干预，通过对大鼠EEG和EMG生理信号特征的分析，确定解郁安神法对失眠模型大鼠睡眠时相的影响。大鼠断头取脑分离中缝核，随机选取部分中缝核，采用HPLC-ECD测定5-HT及5-HIAA含量，计算5-HT转化率，运用RealTime RT-PCR法检测SERT、5-HT1ARmRNA表达，采用Western-blot方法检测5-HT1AR蛋白质的表达；另一部分制备中缝核脑片，应用膜片钳全细胞记录技术观察5-HT1AR介导的抑制性GIRK电流的变化，探讨解郁安神法调控失眠模型大鼠中缝核5-HT信号通路的作用机制，为中医药协同解决失眠这一临床难题提供可靠的实验依据，指导临床合理治疗。

失眠多肝郁，解郁安神法是防治失眠的根本大法。前期研究证实以解郁安神法为载体的中药组方松郁安神方治疗失眠疗效确切，其机制与调节5-HT含量有关。本课题采用电刺激法复制失眠大鼠模型，运用松郁安神方进行干预，通过对大鼠EEG和EMG生理信号特征的分析，明确解郁安神法对失眠模型大鼠睡眠时相的影响。结果发现：松郁安神方可缩短失眠大鼠睡眠潜伏期（SOL），延长总睡眠时间（TST）。在睡眠时相上主要表现为延长SWS和REMS。与经典的苯二氮卓类药物艾司唑仑（1mg）、新型的非苯二氮卓类药物唑吡坦（10mg）和具有镇静作用的抗抑郁药物曲唑酮（100mg）相比，松郁安神方在改善REMS以及恢复失眠大鼠正常的睡眠结构方面具有明显优势。选择性5-HT1AR拮抗剂p-MPPI(1mg/kg)单用对失眠模型大鼠的睡眠-觉醒周期影响不大，但与松郁安神方合用后可以显著改善睡眠；选择性5-HT1AR激动剂8-OH-DPAT(0.1mg/kg)能够显著减少失眠模型大鼠的睡眠时间，而松郁安神方可拮抗这种睡眠抑制作用。这一结果提示松郁安神方调控睡眠-觉醒周期可能与5-HT1AR有关。课题研究进一步分离大鼠中缝核，采用HPLC-ECD测定5-HT以及代谢产物5-HIAA含量，计算5-HT转化率（5-HIAA/5-HT），运用RT-qPCR法检测SERT、5-HT1AR mRNA表达，采用Western-blot方法检测5-HT1AR蛋白质的表达，应用膜片钳全细胞记录技术观察原代培养的大鼠中缝核5-HT能神经元上5-HT1AR介导的抑制性GIRK电流的变化，探讨解郁安神法介导中缝核5-HT信号通路调控睡眠-觉醒周期的作用机制。结果显示：松郁安神方降低中缝核5-HT及其代谢产物5-HIAA水平，提高5-HT1AR mRNA及蛋白质的表达水平，增强原代培养的大鼠中缝核5-HT能神经元上5-HT1AR介导的抑制性GIRK电流从而降低5-HT能神经元兴奋性，而对5-HT代谢水平（5-HIAA/5-HT）和SERT mRNA表达未产生影响。该项研究为中医药协同解决失眠这一临床难题提供了可靠的实验依据，有助于指导临床合理治疗。