睡眠是人体重要的生命活动，充足的睡眠有助于恢复脑力和体力。随着当代人的生活压力增大和社会节奏加快，睡眠障碍问题日益凸显。睡眠障碍包括失眠、嗜睡、阻塞性睡眠呼吸暂停综合征和昼夜节律障碍等。失眠是最常见的睡眠障碍，表现为入睡困难和睡眠难以维持。失眠会导致免疫力低下、情绪障碍和认知功能下降，增加患心血管疾病、代谢性疾病和抑郁症等疾病的风险。目前，治疗失眠的药物主要为苯二氮䓬类药物、非苯二氮䓬类药物、褪黑素及其受体激动剂和食欲素受体拮抗剂等。苯二氮䓬类药物和非苯二氮䓬类药物存在耐受性和后遗效应等不良反应，褪黑素及其受体激动剂和食欲素受体拮抗剂分别仅针对入睡困难和睡眠难以维持有效。因此，新型镇静催眠药物亟待研发。天然药物具有多成分、多靶点、多作用途径和副作用较小的优点。从天然药物中开发具有改善睡眠作用的药物或保健食品成为研究热点。《保健食品功能检验与评价方法（2023版）》中采用正常小鼠评价有助于改善睡眠的保健食品，缺乏模拟当今社会人类因长期压力导致睡眠障碍的动物模型。

参远（Shen Yuan，SY）提取物是由传统中药人参和远志（3:2）经水提醇沉工艺制得。据2020版《中国药典》记载，人参具有安神益智、主治惊悸失眠的功效；远志具有安神益智，主治失眠多梦的功效。本课题组前期研究证明，SY提取物具有显著的抗抑郁作用，但对其是否具有改善睡眠的作用，尚未进行研究。

因此，本论文旨在采用慢性束缚应激（chronic restraint stress，CRS）建立小鼠睡眠障碍模型，并阐释其发生机制。采用正常小鼠、CRS所致睡眠障碍小鼠和对氯苯丙氨酸（para-chlorophenylalanine，PCPA）所致失眠小鼠，研究SY提取物（0.5和1.0 g生药/kg）对睡眠的改善作用，并探究其作用机制。本论文的主要研究内容如下：

1. CRS致小鼠睡眠障碍模型的建立和机制研究

实验目的：建立CRS所致的小鼠睡眠障碍模型，阐述其具体机制，为改善睡眠药物和保健食品的研究提供稳定、可靠且与临床病因一致的动物模型。

实验方法：ICR雄性小鼠每天施以CRS 10 h，连续造模14、21和28天，通过空场实验和戊巴比妥钠诱导睡眠实验动态观察CRS不同造模时长对小鼠自主活动和睡眠的影响。进一步采用脑电肌电监测实验分析CRS 28天所致睡眠障碍小鼠24 h内睡眠结构的变化。通过转录组学测序技术比较正常对照组小鼠和CRS 28天小鼠下丘脑中基因表达的差异性。通过液相色谱-质谱联用（liquid chromatography-tandem mass spectrometry，LC/MS-MS）技术和酶联免疫吸附测定法（enzyme-linked immunosorbent assay，ELISA）检测神经递质和褪黑素水平的变化。采用实时荧光定量聚合酶链式反应（real-time quantitative polymerase chain reaction，qRT-PCR）和蛋白质免疫印迹（Western Blot，WB）实验对褪黑素受体的mRNA和蛋白水平，褪黑素受体下游蛋白表达，昼夜节律基因的mRNA和蛋白水平进行研究。

实验结果：CRS每天造模10 h，持续28天可导致小鼠体重下降、自主活动减少、入睡率下降、入睡潜伏期和睡眠时间减少；而CRS造模14天和21天未能对以上指标均造成显著变化。EEG/EMG实验结果显示，CRS 28天可造成小鼠觉醒占比增加，平均持续时间减少和出现次数增加；NREMS占比减少，平均持续时间减少和出现次数增加；REMS占比、平均持续时间和出现次数均增加；昼夜节律紊乱，表现出失眠和睡眠碎片化。神经递质和褪黑素水平检测结果发现，与对照组比较，CRS小鼠下丘脑中γ-氨基丁酸（γ-aminobutyric acid，GABA）、谷氨酸和褪黑素减少，乙酰胆碱、多巴胺、5-羟色胺（5-hydroxytryptamine，5-HT）、色氨酸（tryptophan，Trp）增加；皮层中GABA、5-HT和Trp减少，乙酰胆碱和组胺增加；血清中褪黑素减少。下丘脑转录组学测序结果表明，对照组和CRS组小鼠存在225个差异基因，GO和KEGG分析发现CRS致小鼠睡眠障碍可能与GABA能突触和昼夜节律通路变化有关。进一步研究发现，CRS导致小鼠下丘脑中褪黑素受体MT1和MT2的mRNA和蛋白水平减少；皮层中MT1和MT2表达减少；下丘脑中褪黑素受体下游蛋白PKCα和p-CaMKII/CaMKII减少，p-CREB/CREB增加，昼夜节律基因的mRNA和蛋白水平变化。

结论：CRS每天10 h，持续28天，通过影响脑内神经递质水平和Trp/5-HT/褪黑素/昼夜节律通路导致小鼠睡眠障碍。

2. SY提取物改善睡眠的药效作用研究

实验目的：探究SY提取物是否具有改善睡眠的作用。

实验方法：采用正常小鼠、CRS所致睡眠障碍小鼠和PCPA所致失眠小鼠，通过空场实验和戊巴比妥钠诱导睡眠实验研究SY提取物对小鼠自主活动和睡眠的影响。

实验结果：SY提取物（0.5和1.0 g生药/kg）单次给药对正常小鼠的睡眠无明显作用，而给药14天和28天则可显著缩短正常小鼠的入睡潜伏期和延长睡眠时间；给药28天可显著延长CRS小鼠的睡眠时间；给药7天和14天显著缩短PCPA小鼠的入睡潜伏期。空场实验发现，SY提取物（0.5和1.0 g生药/kg）对正常小鼠和CRS小鼠的自主活动无明显影响。

结论：SY提取物具有改善睡眠的作用。

3. SY提取物改善睡眠的机制研究

实验目的：探究SY提取物改善睡眠的作用机制，为SY提取物开发成为改善睡眠的保健食品或药物提供理论基础。

实验方法：采用LC/MS-MS技术和WB实验探究SY提取物对正常小鼠皮层中神经递质水平和色氨酸羟化酶（tryptophan hydroxylase，TPH）表达的影响。采用LC/MS-MS、qRT-PCR、WB、ELISA和戊巴比妥钠诱导睡眠实验对SY提取物改善CRS所致小鼠睡眠障碍的机制进行研究。

实验结果：SY提取物（0.5和1.0 g生药/kg）灌胃给药14天可以增加正常小鼠皮层中Trp的含量和TPH2的表达，从而增加5-HT水平；增加GABA含量，降低谷氨酸和去甲肾上腺素水平。对于CRS所致睡眠障碍小鼠，SY提取物（0.5和1.0 g生药/kg）通过降低血清皮质酮浓度，增加雌激素受体β的mRNA和蛋白水平，促进TPH2的转录和翻译；增加Trp水平，进而增加5-HT水平；通过上调褪黑素合成酶AANAT的表达，增加下丘脑中褪黑素水平；进一步增加MT2的mRNA和蛋白水平，促进昼夜节律基因Clock和Cry1的表达，从而改善CRS小鼠的睡眠。当MT2受体拮抗剂4-P-PDOT和SY提取物共同作用时，SY提取物（0.5和1.0 g生药/kg）增加CRS小鼠睡眠时间的作用减弱。

结论：SY提取物（0.5和1.0 g生药/kg）通过改变神经递质水平，调节皮质酮/雌激素受体β/TPH2、Trp/5-HT/褪黑素和昼夜节律相关通路发挥改善睡眠的作用。

综上所述，本论文建立了CRS所致的小鼠睡眠障碍模型，并发现其发生机制与神经递质水平改变和褪黑素及昼夜节律通路变化相关。在正常小鼠、CRS所致睡眠障碍小鼠和PCPA所致失眠小鼠中，首次发现SY提取物具有改善睡眠的作用，其作用机制与调节神经递质水平、皮质酮/雌激素受体β/TPH2通路、Trp/5-HT/褪黑素及昼夜节律通路有关。本研究为改善睡眠保健食品和药物的研发提供稳定、可靠的动物模型和机制上的理论支持，为SY提取物开发成为改善睡眠的保健食品或药物奠定了坚实的基础。

Sleep is a vital life activity for the human body. Adequate sleep helps restore mental and physical energy. With the increasing stress of modern life and the accelerated pace of society, sleep disorders are becoming more pronounced. These disorders include insomnia, narcolepsy, sleep apnea syndrome, circadian rhythm disorders, etc. Insomnia, the most prevalent type of sleep disorder, is characterized by difficulty in sleep initiation and maintenance. It can induce immune hypofunction, mood disorders, and cognitive decline, increasing the risk of cardiovascular diseases, metabolic disorders, depression, etc. Currently, medications for treating insomnia primarily include benzodiazepines, non-benzodiazepines, melatonin and its receptor agonists, and orexin receptor antagonists. However, benzodiazepines and non-benzodiazepines have adverse reactions such as tolerance and hangover effects the next day. Melatonin and its receptor agonists and orexin receptor antagonists are only effective for difficulty falling asleep or maintaining sleep, respectively. Therefore, new sedative-hypnotic drugs are urgently needed to be developed. Herbal medicines have the advantages of multiple ingredients, multiple targets, multiple action pathways, and fewer side effects. Herbal sleep aids have gained popularity worldwide as an alternative to prescription drugs for the treatment of insomnia.

The method described in the “Functional Evaluation Methods of Health Food (2023 version)” utilizes normal mice to evaluate dietary supplements that ameliorate sleep. However, there is a lack of animal models that accurately simulate sleep disorders caused by chronic stress in humans. Shen Yuan (SY) extract, composed of a 3:2 ratio of traditional Chinese herbs Ginseng Radix et Rhizoma and Polygalae Radix, has drawn attention. According to the “Chinese Pharmacopoeia (2020 edition)”, Ginseng has the effects of calming the mind and treating insomnia, while Polygalae is used to treat insomnia, calm the mind, and intensify intelligence. In our previous experiments, SY extract has demonstrated antidepressant effects, but whether it can ameliorate sleep has not yet been studied.

Therefore, the purpose of this study is to establish a mouse sleep disorder model induced by chronic restraint stress (CRS) and elucidate its mechanism. The effects and mechanisms of SY extract (0.5 and 1.0 g/kg) on sleep were studied using normal mice, mice with sleep disorders induced by CRS, and mice with insomnia resulting from para-chlorophenylalanine (PCPA). The main research content and results of this paper are as follows:

1. Establishment of a mouse sleep disorder model induced by CRS and its relative mechanisms

Objective: This study aims to establish a mouse sleep disorder model caused by CRS and elucidate its specific mechanisms, thereby providing a stable, reliable, and clinically etiological animal model for research on sleep-improving drugs and dietary supplements.

Methods: ICR male mice were subjected to 10 h of restraint each day for 14, 21, or 28 days.  The locomotor activity and sleep of CRS-treated mice were tested through open field test (OFT) and pentobarbital-induced sleep test (PST). The effect of CRS treatment on the sleep/wake architecture of the mice was investigated by consecutive electroencephalogram (EEG) and electromyography (EMG) recordings for 24 h (started at ZT 0 on day 29). Hypothalamic RNA sequencing was performed to explore changes in gene transcription between the control group and the CRS 28d group. The neurotransmitter and melatonin levels were measured using liquid chromatography coupled with tandem mass spectrometry. spectrometry (LC/MS-MS) analysis and enzyme-linked immunosorbent assay (ELISA). The mRNA levels of melatonin receptors and circadian rhythm genes were detected using real-time quantitative polymerase chain reaction (qRT-PCR) experiments. The expressions of melatonin receptors, melatonin receptor downstream proteins, and circadian rhythm genes were detected using Western Blot (WB) experiments.

Results: The results showed that CRS treatment for 28 days (10 h/day) induced weight loss, decreased locomotor activity, reduced falling asleep rate, and shortened sleep latency and sleep duration in mice. However, CRS for 14 days and 21 days failed to cause significant changes in these indicators. In the EEG/EMG experiment, CRS for 28 days caused increased percentage and episode number of wakefulness, decreased episode duration of wake, increased episode number of NREMS, decreased percentage and episode duration of NREMS, increased percentage, episode number and episode duration of REMS, and circadian rhythm disorders. The CRS mice exhibited insomnia and sleep fragmentation. The test results of neurotransmitter and melatonin levels found that compared with the mice in the control group, CRS caused the increase of acetylcholine, dopamine, 5-hydroxytryptamine (5-HT), tryptophan (Trp) levels and the decrease of γ-aminobutyric acid (GABA), glutamic acid, melatonin levels in the hypothalamus. In the cortex of CRS mice, acetylcholine and histamine levels were increased, while the GABA, 5-HT, and Trp levels were decreased. The melatonin level was also reduced in the serum of CRS mice. The transcriptome sequencing study of the hypothalamus identified 225 differentially expressed genes. GO and KEGG analyses suggested that CRS-induced sleep disorders in mice may be related to GABAergic synapses and circadian rhythm signaling pathways. Further studies found that the mRNA and protein levels of melatonin receptors MT1 and MT2 in the hypothalamus and their expressions in the cortex were reduced in CRS mice. The melatonin receptor downstream proteins, PKCα and p-CaMKII/CaMKII expressions were downregulated, while p-CREB/CREB was upregulated in the hypothalamus of CRS mice. CRS thus caused changes in the mRNA and protein levels of circadian rhythm genes, inducing sleep disorders in mice.

Conclusion: Mice are subjected to CRS for 28 days, 10 hours a day, which can cause sleep disorders by affecting neurotransmitter levels and the Trp/5-HT/melatonin/circadian rhythm pathways.

2. Study on the pharmacodynamic effects of SY extract on ameliorating sleep

Objective: This study aims to explore whether SY extract can ameliorate sleep.

Methods: The effects of SY extract on locomotor activity and sleep were studied using normal mice, mice with sleep disorders induced by CRS, and mice with insomnia induced by PCPA through OFT and PST.

Results: The results showed that a single administration of SY extract (0.5 and 1.0 g/kg) had no obvious effect on the sleep of normal mice. Administration of SY extract (0.5 and 1.0 g/kg) for 14 and 28 days could significantly shorten the sleep latency and prolong the sleep duration of normal mice. For CRS mice, SY administration for 28 days significantly prolonged their sleep duration. In addition, Administration of SY extract (0.5 and 1.0 g/kg) for 7 and 14 days significantly shortened the sleep latency of PCPA mice. However, SY extract (0.5 and 1.0 g/kg) had no significant effect on the locomotor activity of normal mice and CRS mice in OFT.

Conclusion: SY extract has the effect of ameliorating sleep.

3. Study on the mechanism of SY extract in ameliorating sleep

Objective: This study aims to explore the mechanisms of SY extract in ameliorating sleep and provide a theoretical basis for the development of SY extract into dietary supplements or drugs.

Methods: The effects of SY extract on neurotransmitter levels and tryptophan hydroxylase (TPH) in the cortex of normal mice were analyzed by LC/MS-MS and WB experiments. The mechanisms of SY extract ameliorating sleep disorders in mice induced by CRS were studied using LC/MS-MS, qRT-PCR, WB, ELISA, and PST.

Results: The results showed that SY extract (0.5 and 1.0 g/kg) was intragastrically administered to normal mice for 14 days could increase Trp level and TPH2 expression, thereby increasing 5-HT levels. It could also increase GABA level and reduce glutamic acid and norepinephrine levels in the cortex of normal mice. For CRS mice, the results showed that SY extract (0.5 and 1.0 g/kg) promoted the transcription and translation of TPH2 by reducing serum corticosterone concentration and increasing the mRNA and protein levels of estrogen receptor β. Because SY extract (1.0 g/kg) could also increase Trp level, it in turn increased the 5-HT level in the cortex of CRS mice. In addition, SY extract (0.5 and 1.0 g/kg) could increase melatonin level in the hypothalamus by upregulating the expression of melatonin synthase arylalkylamine N-acetyltransferase. SY extract (0.5 and 1.0 g/kg) also directly regulated melatonin receptor 2, increasing its mRNA and protein levels, thereby promoting the expressions of circadian rhythm genes Clock and Cry1. When the MT2 receptor antagonist 4-P-PDOT and SY extract acted together, the effect of SY extract (0.5 and 1.0 g/kg) on increasing the sleep duration of CRS mice was weakened. Therefore, SY extract (0.5 and 1.0 g/kg) ameliorates sleep by regulating neurotransmitter levels, corticosterone/estrogen receptor β/TPH2, Trp/5-HT/melatonin, and circadian rhythm pathways.

Conclusion: SY extract (0.5 and 1.0 g/kg) ameliorates sleep by regulating neurotransmitter levels, corticosterone/estrogen receptor beta/TPH2, Trp/5-HT/melatonin, and circadian rhythm pathways.

In summary, this study established a mouse model of sleep disorder induced by CRS and elucidated its related mechanism, which is related to neurotransmitters and melatonin levels and circadian rhythm pathways. The ameliorating sleep effects of SY extract were first found in normal mice, mice with sleep disorders induced by CRS, and mice with insomnia induced by PCPA. Its mechanisms were related to the regulation of neurotransmitter levels, corticosterone/estrogen receptor β/TPH2, Trp/5-HT/melatonin, and circadian rhythm pathways. This study provides a stable and reliable animal model and mechanistic theoretical support for the development of sleep-ameliorating dietary supplements and medicines, while also providing a solid foundation for the development of SY extract as a sleep-ameliorating dietary supplement or medicine.