Anmeidan Modulates Circadian Rhythm of Spontaneous Activity and Influences Clock Proteins in Sleep-deprived Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Anmeidan （AMD） on biological rhythm and related protein expression in sleep-deprived rats. MethodA total of 80 SD rats were randomized into control group （Ctrl， equivalent volume of saline）， model group （SD， equivalent volume of saline）， AMD group （9.09 g·kg-1·d-1）， and melatonin group （MT， 0.27 g·kg-1·d-1）. Insomnia was induced in rats by self-made sleep deprivation box （4 weeks）. Circadian rhythm of spontaneous activity was evaluated by spontaneous activity video analysis system. Morphology of hypothalamus was observed based on hematoxylin-eosin （HE） staining， and the histomorphology of hypothalamus neurons and the Nissl's bodies based on Nissl staining. Western blotting was employed to detect the expression of hypothalamic proteins in cAMP-response element binding protein （CREB）/clock gene period （Per） pathway， and immunohistochemistry the expression of brain and muscle ARNT-like protein 1 （Bmal1）， Clock， Per1， and cryptochrome circadian regulator 1 （Cry1）. ResultThe model group demonstrated circadian rhythm disorder， as manifested by the significant increase in activity time in 6 designated time periods compared with the control group， and the rise in the activity speed and frequency （P<0.01）. Moreover， model group showed decrease in number of neurons which were sparsely arranged with shrunken or fragmented nuclei， reduction in number and loss of Nissl's bodies with light color， and drop in the relative expression of p-CREB and Per1， and the positive rate of Bmal1， Clock， Per1， and Cry1 （P<0.01）. Compared with model group， AMD group demonstrated reduction in time， speed， and frequency of activity （P<0.01）. Moreover， the AMD group also showed alleviation of neuronal damage （P<0.01）， and increase in the number of neurons with clear nuclei and cytoplasm in some， and the number of Nissl's bodies. AMD raised the expression of p-CREB and Per1 proteins， and the positive rate of Bmal1， Clock， Per1， and Cry1 （P<0.01）. ConclusionAMD ameliorated spontaneous circadian rhythm of sleep-deprived rats by regulating CREB/Per signaling pathway and further increasing the expression of Bmal1， Clock， Per1， and Cry1.

Therapeutic Effects of Mind-tranquilizing Chinese Medicines on Sleep-disorders by Regulating Energy Metabolism： A Review / 中国实验方剂学杂志

Sleep plays an important role in energy balance. As reported， sleep disorder is an important risk factor for metabolic diseases. Controlling the relationship between energy metabolism and sleep can affect sleep homeostasis and body metabolic rate. Chinese medicine， with remarkable curative effects in the prevention and treatment of insomnia， has the characteristics of green， safety， and few side effects， and attracts extensive attention of scholars in the world. In recent years， remarkable progress has been made in the research on the mechanism of Chinese medicine in interfering with sleep. This paper reviewed the research progress of mind-tranquilizing Chinese medicines， such as compounds （pterostilbene）， Chinese medicinal drugs （Ziziphi Spinosae Semen）， and Chinese medicinal prescriptions （Jiaotaiwan， Suanzaoren tang， Tianwang Buxindan， Anmeidan， Banxia Houpotang， Qihuo decoction， Songyu Anshen prescriptions， and Shuxie Yihao prescriptions） in the treatment of sleep disorders by regulating energy metabolism. The findings revealed that Chinese medicine can intervene in the sleep deprivation model by affecting metabolism-related pathways such as material metabolism， mitochondrial function， oxidative stress and inflammatory response， appetite system， and biological clock system. In terms of frequency of use， the top drugs are Ziziphi Spinosae Semen， Poria， Schisandrae Chinensis Fructus， and Salviae Miltiorrhizae Radix et Rhizoma which affect heart and liver meridians to regulate blood circulation， ensure energy supply， and play the role of nourishing the heart and tranquilizing the mind. The present paper summarized the effects and mechanisms of Chinese medicine in the treatment of insomnia and other sleep disorders from the perspective of energy metabolism to provide references for further research and exploration of diseases in the future.

Intervention Effect of Modified Tianwang Buxindan on Glucose and Lipid Metabolism in Mice with Chronic Sleep Deprivation Through Orexin A/OX1R / 中国实验方剂学杂志

Objective:to explore the mechanism of modified Tianwang Buxindan in improving abnormal glucose and lipid metabolism in mice with chronic sleep deprivation from the signal pathway of orexin A/ orexin receptor 1（OX1R）. Method:The 50 6-week-old male C57BL/6 mice were randomly divided into blank group ， model group ， estazolam group and Tianwang Buxindan low and high dose groups ，for ten mice of each group. Except the blank group， rats were deprived of sleep for 8 weeks by the method of multi-platform water environment. In the last 4 weeks， Tianwang Buxindan （8.5，17 g·kg-1）and estazolam solution（9.1 mg·kg-1）were given to the stomach， and the blank group and the model group were fed with pure water of the same volume. The food intake and body weight of mice were measured twice a week， on the 49th day， blood samples were collected from the tail vein for glucose tolerance test （GTT），on the 52nd day for insulin tolerance test（ITT）， was used to detect the expression of total cholesterol （TCH）， triglyceride（TG）and free fatty acid（FFA）in serum， and enzyme-linked immunosorbent assay（ELISA）was used to detect the expression of orexin A in serum and hypothalamus. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）and Western blot were used to detect the mRNA and protein expression of OX1R in hypothalamus. Result:After administration， the food intake of mice in each group was different， compared with the blank group， the body weight of model group was significantly reduced（P<0.05）， the glucose tolerance was significantly abnormal， and the TCH， TG， FFA values were significantly increased（P<0.01）. The expression of orexin A in serum and hypothalamus increased significantly（P<0.01）， and the mRNA and protein expression levels of OX1R in hypothalamus increased significantly（P<0.01）. Compared with the model group， the body weight of each group of Tianwang Buxindan was significantly increased（P<0.05）， with better glucose tolerance and insulin sensitivity， TCH， TG， FFA values were significantly reduced（P<0.05，P<0.01）， accompanied by serum and the expression of orexin A in the hypothalamus was significantly decreased（P<0.05，P<0.01）， the mRNA and protein expression levels of OX1R were significantly decreased（P<0.05，P<0.01）. Conclusion:Tianwang Buxindan can protect mice from abnormal glucose and lipid metabolism induced by chronic sleep deprivation， and its mechanism may be related to the down-regulation of orexin A/OX1R signal expression.

Mechanism of Sleep Deprivation and Cognitive Impairment and Research Progress of Traditional Chinese Medicine Intervention / 中国实验方剂学杂志

Adequate sleep is an important factor to ensure the healthy functioning of the body. A type of chronic sleep diseases characterized by insufficient sleep can be collectively referred to as sleep deprivation （SD）， which is divided into primary and secondary sources in terms of sources. As one of the most frequent types of diseases in recent years， SD has received more and more attention and attention from the whole society. SD can have a wide-ranging and far-reaching impact on cognitive behavior， such as decreased wakefulness， decreased alertness， and inattention， decreased sensory perception， decreased learning and memory capabilities， et al， involving the impact on multiple system functions of the human body， and It is closely related to the occurrence of many diseases， and may cause serious troubles to the normal life of patients and even their relatives and friends. The cognitive impairment caused by SD has been fully verified in clinical tests and various animal behavior experiments， mainly involving pathological damage such as changes in synaptic plasticity， enhanced endoplasmic reticulum stress， circadian rhythm disorders， and energy metabolism imbalance. Western medicine treatments for SD mostly have negative factors such as high side effect and strong addiction. However， Chinese medicine intervention focuses on the overall concept， has long-lasting effectiveness， significant effects， and mild side effects. It has also been widely recognized clinically for improving the complications of sleep disorders. This article reviews the current status and classification of SD research， its pathological mechanisms that lead to cognitive impairment and its molecular-level exploration directions and results. In recent 5 years， the therapeutic effect and experience of traditional Chinese medicine intervention therapy such as compound Chinese medicine， acupuncture and moxibustion as well as auxiliary therapy such as exercise and five sounds， in order to further summarize and clarify the interaction mechanism between SD and cognitive behavior， and provide a theoretical basis for the study of the pathological mechanism of SD disease and future clinical treatment.

Effect of Anmeidan in Improving Learning and Memory Levels of Sleep Deprived Rats Through Mitochondrial Mediated Hippocampal Neuronal Apoptosis / 中国实验方剂学杂志

Objective:To explore the effect of Anmeidan （AMD） on the learning and memory levels of sleep deprived rats through mitochondrial mediated hippocampal neuronal apoptosis pathway. Method:Forty-eight SD rats were randomly divided into blank group， model group， low， medium， high-dose AMD groups （4.86， 9.72， 19.44 g·kg-1·d-1） and estazolam group （0.1 mg·kg-1·d-1）. Insomnia model was prepared by self-made sleep deprivation box for 14 days. Morris water maze was used to detect learning and memory levels， enzyme-linked immunosorbent assay （ELISA） was used to detect the expressions of cytochrome C （Cyt-C）， cysteine aspartic acid protease-3 （Caspase-3） in hippocampus. Transmission electron microscopy （TEM） was used to observe the morphological structure of mitochondria in hippocampus. Protein and mRNA expressions of Cyt-C， Caspase-3， Bcl-2， Bax were detected by immunofluorescence （IF） and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） respectively. Result:In the model group， the incubation period of the platform and the total distance of swimming and the time of first arriving platform were prolonged， the number of platform crossing and the time of target quadrant movement were reduced， protein and mRNA expressions of Bcl-2 dropped， protein and mRNA expressions of Bax increased （P<0.01）， and mitochondrial structure was abnormal with crista fracture， swelling and deformation. And protein and mRNA expressions of Cyt-C， Caspase-3 increased significantly （P<0.01）. Low， medium and high-dose AMD groups could improve levels of space exploration and navigation of SD rats （P<0.01）， increase protein and mRNA expressions of Bcl-2， decrease protein and mRNA expressions of Bax， improve the damage of mitochondria， and decrease the protein and mRNA expressions of Cyt-C， Caspase-3 （P<0.01）. Conclusion:AMD can improve the learning and memory levels of SD rats， the effect is related to the mitochondrial mediated hippocampal neuronal apoptosis pathway and decrease of Cyt-C and Caspase-3 expressions.

Effect of Modified Tianwang Buxindan on Learning and Memory Function and Expression of Inflammatory Factors in Sleep Deprived Rats / 中国实验方剂学杂志

Objective:By studying the effects of Tianwang Buxindan on sleep quality， cognitive function， inflammatory factors and immune-related gene expression in sleep deprivation model rats， explore the effect of Tianwang Buxindan on the learning and memory process under sleep deprivation and its anti-inflammatory effects possible mechanism. Method:The 40 male SPF rats were used to simulate the sleep deprivation model by multi-platform water environment method， and were randomly divided into model group， Tianwang Buxindan group （20 g·kg-1） and estazolam group （0.1 mg·kg-1）， set up a normal group， 10 in each group. A total of 4 weeks of sleep deprivation modeling was performed， and drug intervention was performed 2 weeks later. The model group and the blank group were given equal volumes of pure water. Electroencephalogram （EEG） evaluation of modeling and analysis of sleep structure and quality of rats， Morris water maze positioning navigation and space exploration experiment analysis of learning and memory ability of rats， application of enzyme-linked immunosorbent assay （ELISA） was used to detect the serum inflammatory factor interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， monocyte chemokine-1 （MCP-1） expression， Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA levels of EB virus inducible gene 3 （EBI3）， extracellular signal-regulated kinase 5 （ERK5）， and p21 activated protein kinase 4 （PAK4）. Result:The sleep deprivation model was successfully built. Compared with blank group， the total sleep time， total duration of slow wave sleep and the duration of the first and second phases of slow wave sleep in the model group were significantly shortened （P<0.01）. The incubation period on the upper platform， the total swimming distance and the time to reach the original platform for the first time increased significantly， while the number of times to cross the platform and the target quadrant significantly decreased （P<0.05，P<0.01）. The expression levels of IL-1β，TNF-α and MCP-1 increased significantly （P<0.01）， the mRNA expression levels of EBI3， ERK5 and PAK4 in the hypothalamus of the model group decreased significantly （P<0.01）. Compared with the model group， the sleep quality of the rats in the Tianwang Buxindan group was significantly improved. The total sleep time， the total duration of slow wave sleep and the duration of the first phase of slow wave sleep were significantly increased （P<0.01）. The incubation period on the platform， the total swimming distance and the time to reach the original platform for the first time are shortened， the number of times to cross the platform and the target quadrant time are extended （P<0.05，P<0.01）， IL-1β， TNF-α， MCP-1 expression levels were significantly reduced （P<0.05，P<0.01）， mRNA expression levels of EBI3， ERK5 and PAK4 in rat hypothalamus were significantly increased （P<0.05，P<0.01）. Conclusion:Tianwang Buxindan can improve the sleep quality and learning and memory ability of sleep deprivation model rats， which may be related to the increase of the expression level of related inflammatory factors and its anti-inflammatory effect.

Bioinformatics Analysis on Mechanism of Modified Tianwang Buxindan Intervention on Energy Metabolism in Sleep Deprived Rats / 中国实验方剂学杂志

Objective:To detect the changes of functional expression profile of energy metabolism related differential genes in sleep deprived rats before and after intervention by Tianwang Buxindan by microarray sequencing technology， so as to provide possible ideas and theoretical basis for the prevention and treatment of sleep deprivation. Method:The rats were randomLy divided into two groups： the Tianwang Buxindan group and the model group. The Tianwang Buxindan group was given the decoction of Tianwang Buxindan at the dose of 20 g·kg-1， and the model group was given the pure water of equal volume for 14 days. Taking liver， heart and hypothalamus as samples， high-throughput sequencing was used to obtain differential genes. Gene Ontology（Go）classification and kyoto Encyclopedia of Genes and Genomes （KEGG）pathway enrichment analysis were used to construct a co expression network with lncrna. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）was used to detect the mRNA expression levels of neuropeptide Y（NPY）， bispecific phosphatase 1/mitogen-activated protein kinase phosphatase-1（DUSP1/MKP-1）and alpha-L-iduronidase（IDUA）， three key genes with significant differences in energy metabolism. Result:The 321 differentially expressed genes were obtained， 231 of which were up-regulated and 90 down regulated， which mainly promoted the process of lipid metabolism， glucose metabolism and protein metabolism， participated in the synthesis and expression of fibrinogen， vitamin B6 and mesencephalic astrocyte-derived neurotrophic factor（MANF）， and involved mitogen activated protein kinases（MAPK）， p53 gene（p53）， cyclic adenosine monophosphate（cAMP） and other signal pathways. Compared with the model group， the expression of IDUA significantly increased in the Tianwang Buxindan group （P<0.05）， but decreased significantly in NPY and DUSP1（P<0.01）. Conclusion:Tianwang Buxindan can interfere with the energy metabolism mechanism of sleep deprived rats in many ways. By down regulating the mRNA expression level of NPY and DUSP1 genes， it may activate the p38 MAPK signal pathway and affect the lipid metabolism.

Mechanism of Modified Tianwang Buxindan on Trx System Oxidative Damage in PCPA Insomnia Model Rats / 中国实验方剂学杂志

Objective:To explore the mechanism of modified Tianwang Buxindan on oxidative damage of Trx system in parachlorophenylalanine (PCPA) insomnia model rats. Method:Sixty male SD rats were randomly divided into blank group and model group. Insomnia model was prepared through intraperitoneal injection with PCPA (150 mg·kg-1). The discontinuous injection lasted for 7 d. After successful modeling, the rats were divided into model group (the same volume of normal saline), low, medium, high-dose Tianwang Buxindan groups (8.8, 17.6, 35.2 g·kg-1·d-1) and estazolam group (0.1mg·kg-1·d-1), with 10 in each group. Autonomous activity video was used to detect circadian activity rhythm. Transmission electron microscopy (TEM) was used to observe supra chiasmatic nucleus (SCN) morphology and Organelle integrity. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in serum. Expressions of Trx2, TrxR2 in SCN cells were detected by immunofluorescence (IF) and Western blot. Result:Compared with blank group, the activity rhythm of model group was irregular, the activity time increased (PPPPPPPPConclusion:The effect of modified Tianwang Buxindan on insomnia model rats is related to the regulation of Trx2 and TrxR2 protein expressions in Trx system.