Comparison of the Protective Effects of Casein Hydrolysate Containing Tyr-Pro-Val-Glu-Pro-Phe and Casein on the Behaviors and Peripheral and Brain Functions in Mice with Chronic-Stress-Induced Anxiety and Insomnia.

Chronic stress is a major inducer of anxiety and insomnia. Milk casein has been studied for its stress-relieving effects. We previously prepared a casein hydrolysate (CP) rich in the sleep-enhancing peptide YPVEPF, and this study aims to systemically investigate the different protective effects of CP and casein on dysfunction and anxiety/insomnia behavior and its underlying mechanisms in chronically stressed mice. Behavioral results showed that CP ameliorated stress-induced insomnia and anxiety more effectively than milk casein, and this difference in amelioration was highly correlated with an increase in GABA, 5-HT, GABAA, 5-HT1A receptors, and BDNF and a decrease in IL-6 and NMDA receptors in stressed mice. Furthermore, CP restored these dysfunctions in the brain and colon by activating the HPA response, modulating the ERK/CREB-BDNF-TrκB signaling pathway, and alleviating inflammation. The abundant YPVEPF (1.20 ± 0.04%) and Tyr-based/Trp-containing peptides of CP may be the key reasons for its different effects compared to casein. Thus, this work revealed the main active structures of CP and provided a novel dietary intervention strategy for the prevention and treatment of chronic-stress-induced dysfunction and anxiety/insomnia behaviors.

A comprehensive study on the regulation of Compound Zaoren Granules on cAMP/CREB signaling pathway and metabolic disorder in CUMS-PCPA induced insomnia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Zaoren Granules (CZG), an optimized herbal formulation based on the traditional Chinese medicine prescription Suanzaoren decoction, are designed specifically for insomnia treatment. However, the mechanisms underlying its efficacy in treating insomnia are not yet fully understood. AIM OF THE STUDY: The research investigated the mechanisms of CZG's improvement in insomnia by regulating cAMP/CREB signaling pathway and metabolic profiles. METHODS: The main components of CZG were characterized by liquid chromatography-mass spectrometry (LC-MS). Subsequently, these validated components were applied to network pharmacological analysis to predict signaling pathways associated with insomnia. We evaluated the effect of CZG on BV-2 cells in vitro. We also evaluated the behavioral indexes of CUMS combined with PCPA induced insomnia in rats. HE staining and Nissl staining were used to observe the pathological damage of hippocampus. ELISA was used to detect the levels of various neurotransmitters, orexins, HPA axis, and inflammatory factors in insomnia rats. Then we detected the expression of cAMP/CREB signaling pathway through ELISA, WB, and IHC. Finally, the metabolomics was further analyzed by using UHPLC-QTOF-MS/MS to investigate the changes in the hippocampus of insomnia rats and the possible metabolic pathways were also speculated. RESULTS: The results of CZG in vitro experiments showed that CZG has protective and anti-inflammatory effects on LPS induced BV-2 cells. A total of 161 chemical components were identified in CZG. After conducting network pharmacology analysis through these confirmed components, we select the cAMP/CREB signaling pathway for further investigate. The behavioral research results on insomnia rats showed that CZG significantly prolonged sleep time, mitigated brain tissue pathological damage, and exhibited liver protective properties. CZG treats insomnia by regulating the content of various neurotransmitters, reducing levels of orexin, HPA axis, and inflammatory factors. It can also treat insomnia by upregulating the expression of the cAMP/CREB signaling pathway. Hippocampus metabolomics analysis identified 69 differential metabolites associated with insomnia. The metabolic pathways related to these differential metabolites have also been predicted. CONCLUSION: These results indicate that CZG can significantly prolong sleep time. CZG is used to treat insomnia by regulating various neurotransmitters, HPA axis, inflammatory factors, cAMP/CREB signaling pathways, and metabolic disorders.

Microbiota Metabolite Profiles and Dietary Intake in Older Individuals with Insomnia of Short vs. Normal Sleep Duration.

Recent evidence suggests that the gut microbiota plays a role in insomnia pathogenesis. This study compared the dietary habits and microbiota metabolites of older adults with insomnia of short vs. normal sleep duration (ISSD and INSD, respectively). Data collection included sleep assessment through actigraphy, dietary analysis using the Food Frequency Questionnaire, and metabolomic profiling of stool samples. The results show that ISSD individuals had higher body mass index and a greater prevalence of hypertension. Significant dietary differences were observed, with the normal sleep group consuming more kilocalories per day and specific aromatic amino acids (AAAs) phenylalanine and tyrosine and branch-chain amino acid (BCAA) valine per protein content than the short sleep group. Moreover, metabolomic analysis identified elevated levels of the eight microbiota metabolites, benzophenone, pyrogallol, 5-aminopental, butyl acrylate, kojic acid, deoxycholic acid (DCA), trans-anethole, and 5-carboxyvanillic acid, in the short compared to the normal sleep group. The study contributes to the understanding of the potential role of dietary and microbial factors in insomnia, particularly in the context of sleep duration, and opens avenues for targeted dietary interventions and gut microbiota modulation as potential therapeutic approaches for treating insomnia.

Herbal medicines for insomnia through regulating 5-hydroxytryptamine receptors: a systematic review.

Insomnia is a common sleep disorder without effective therapy and can affect a person's life. The mechanism of the disease is not completely understood. Hence, there is a need to understand the targets related to insomnia, in order to develop innovative therapies and new compounds. Recently, increasing interest has been focused on complementary and alternative medicines for treating or preventing insomnia. Research into their molecular components has revealed that their sedative and sleep-promoting properties rely on the interactions with various neurotransmitter systems in the brain. In this review, the role of 5-hydroxytryptamine (5-HT) in insomnia development is summarized, while a systematic analysis of studies is conducted to assess the mechanisms of herbal medicines on different 5-HT receptors subtypes, in order to provide reference for subsequent research.

No alterations in potential indirect markers of locus coeruleus-norepinephrine function in insomnia disorder.

The norepinephrine locus coeruleus system (LC NE) represents a promising treatment target in patients with insomnia disorder (ID) due to its well understood links to arousal and sleep regulation. However, consistent markers of LC NE activity are lacking. This study measured three potential indirect markers of LC NE activity - REM sleep, P3 amplitude during an auditory oddball paradigm (as a marker of phasic LC activation), and baseline pupil diameter (as a marker of tonic LC activation). The parameters were then combined in a statistical model and tested to compare LC NE activity between 20 subjects with insomnia disorder (13 female; age 44.2 ± 15.1 year) and 20 healthy, good sleeping controls (GSC; 11 female; age 45.4 ± 11.6 year). No group differences regarding the primary outcome parameters were detected. Specifically, insomnia disorder did not display the hypothesised changes in markers of LC NE function. While increased LC NE function remains an interesting speculative pathway for hyperarousal in insomnia disorder, the investigated markers do not appear closely related to each other and fail to discriminate between insomnia disorder and good sleeping controls in these samples.

Effects of electroacupuncture on the ventral tegmental area- nucleus accumbens dopamine pathway in rats with chronic sleep deprivation.

BACKGROUND: Insomnia is a well-recognized clinical sleep disorder in the adult population. It has been established that acupuncture has a clinical effects in the treatment of insomnia; however, research on the underlying neural circuits involved in these effects is limited. METHODS: The modified multiple platform method (MMPM) was used to establish a rat model of chronic sleep deprivation (CSD). Forty rats were randomly divided into a control (Con) group, (untreated) CSD group, electroacupuncture-treated CSD group (CSD + EA) and estazolam-treated CSD group (CSD + Estazolam group) with n = 10 per group. In the CSD + EA group, EA was delivered at Yintang and unilateral HT7 (left and right treated every other day) with continuous waves (2 Hz frequency) for 30 min/day over 7 consecutive days. In the CSD + Estazolam groups, estazolam was administered by oral gavage (0.1 mg/kg) for 7 consecutive days. The open field test (OFT) was used to observe behavioral changes. Immunofluorescence assays and enzyme-linked immunosorbent assay (ELISA) were used to observe the effects of EA on the ventral tegmental area (VTA)-nucleus accumbens (NAc) dopamine (DA) pathway. We also assessed the effects of EA on the expression of dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R) in the NAc, which are the downstream targets of the VTA-NAc DA pathway. RESULTS: After CSD was established by MMPM, rats exhibited increased autonomous activity and increased excitability of the VTA-NAc DA pathway, with increased VTA and NAc DA content, increased D1R expression and decreased D2R expression in the NAc. EA appeared to reduce the autonomous ability of CSD rats, leading to lower DA content in the VTA and NAc, reduced expression of D1R in the NAc and increased expression of D2R. Most importantly, EA produced effects similar to estazolam with respect to the general condition of rats with CSD and regulation of the VTA-NAc DA pathway. CONCLUSIONS: The therapeutic effect of EA in chronic insomnia may be mediated by reduced excitability of the VTA-NAc DA pathway, with lower DA content in the VTA and NAc, downregulated expression of D1R in the NAc and increased expression of D2R.

Genome-wide meta-analysis of insomnia prioritizes genes associated with metabolic and psychiatric pathways.

Insomnia is a heritable, highly prevalent sleep disorder for which no sufficient treatment currently exists. Previous genome-wide association studies with up to 1.3 million subjects identified over 200 associated loci. This extreme polygenicity suggested that many more loci remain to be discovered. The current study almost doubled the sample size to 593,724 cases and 1,771,286 controls, thereby increasing statistical power, and identified 554 risk loci (including 364 novel loci). To capitalize on this large number of loci, we propose a novel strategy to prioritize genes using external biological resources and functional interactions between genes across risk loci. Of all 3,898 genes naively implicated from the risk loci, we prioritize 289 and find brain-tissue expression specificity and enrichment in specific gene sets of synaptic signaling functions and neuronal differentiation. We show that this novel gene prioritization strategy yields specific hypotheses on underlying mechanisms of insomnia that would have been missed by traditional approaches.

The Molecular Relationship between Stress and Insomnia.

The bi-directional relationship between sleep and stress has been actively researched as sleep disturbances and stress have become increasingly common in society. Interestingly, the brain and underlying neural circuits important for sleep regulation may respond uniquely to stress that leads to post-traumatic stress disorder (PTSD) and stress that does not. In stress that does not lead to PTSD, the hypothalamic-pituitary-adrenal axis (HPA) pathway is activated normally that results in sympathetic nervous system activation that allows the brain and body to return to baseline functioning. However, exposure to stress that leads to PTSD, causes enhanced negative feedback of this same pathway and results in long-term physiological and psychological changes. In this review, how stress regulates glucocorticoid signaling pathways in brain glial cells called astrocytes, and then mediates stress-induced insomnia are examined. Astrocytes are critical sleep regulatory cells and their connections to sleep and stress due to disturbed glucocorticoid signaling provide a novel mechanism to explain how stress leads to insomnia. This review will examine the interactions of stress neurobiology, astrocytes, sleep, and glucocorticoid signaling pathways and will examine the how stress that leads to PTSD and stress that does not impacts sleep-regulatory processes.

Sleep deprivation leads to further impairment of hippocampal synaptic plasticity by suppressing melatonin secretion in the pineal gland of chronically unpredictable stress rats.

There has been ample research showing that insomnia is a potential trigger of depression as well as a symptom of depression. These two factors contribute to behavioural problems and are closely related to the plasticity of hippocampal synapses. Although depression and insomnia impair hippocampal synaptic plasticity, the mechanism by which this happens remains a mystery. This study aimed to investigate the pathogenesis of insomnia comorbidity in depression and the regulatory effect of venlafaxine combined with melatonin on hippocampal synaptic plasticity in chronic unpredictable mild stress (CUMS) with sleep deprivation (SD) rats. Thus, rats were subjected to 14 days of chronic mild unpredictable stress, gradually acclimated to sleep deprivation on days 12-14. Followed by 21 consecutive days of sleep deprivation, 18 h per day, with daily gavage of venlafaxine (13.5 mg/kg) + melatonin (72 mg/kg) on days 15-36. Venlafaxine + melatonin treatment improves depression-like behaviour, pentobarbital sodium experimental sleep latency, and sleep duration in CUMS +SD rats. In addition to improving depressive-like behaviors, sleep deprivation also upregulates the expression of caspase-specific cysteine protein 3 (Caspase 3) in the pineal glial cells of chronic mild rats, as well as in hippocampal microglia. Expression of ionic calcium-binding adaptor 1 (iba-1), downregulates the secretion of several synaptic plasticity-related proteins, notably cAMP response element binding protein (CREB), glial cell line-derived neurotrophic factor (GDNF), and the synaptic scaffolding protein Spinophiline (Spinophiline). Hematoxylin-eosin staining showed that the structure of the pineal gland and hippocampus was damaged, and Golgi staining showed that the dendrites and spines in the DG area of the hippocampus were destroyed, vaguely aggregated or even disappeared, and the connection network could not be established. Western blot analysis further revealed a positive correlation between low melatonin levels and reduced Spinophiline protein. Interestingly, venlafaxine + melatonin reversed these events by promoting hippocampal synaptic plasticity by regulating melatonin secretion from the pineal gland. Therefore, it exerted an antidepressant effect in sleep deprivation combined with CUMS model rats. Overall, the results of this study suggest that the pathophysiology of depressive insomnia comorbidity is mediated by impaired pineal melatonin secretion and impaired hippocampal synaptic plasticity. In addition, these responses are associated with melatonin secretion from the pineal gland.

Variant-to-gene-mapping analyses reveal a role for pancreatic islet cells in conferring genetic susceptibility to sleep-related traits.

We investigated the potential role of sleep-trait associated genetic loci in conferring a degree of their effect via pancreatic α- and ß-cells, given that both sleep disturbances and metabolic disorders, including type 2 diabetes and obesity, involve polygenic contributions and complex interactions. We determined genetic commonalities between sleep and metabolic disorders, conducting linkage disequilibrium genetic correlation analyses with publicly available GWAS summary statistics. Then we investigated possible enrichment of sleep-trait associated SNPs in promoter-interacting open chromatin regions within α- and ß-cells, intersecting public GWAS reports with our own ATAC-seq and high-resolution promoter-focused Capture C data generated from both sorted human α-cells and an established human beta-cell line (EndoC-ßH1). Finally, we identified putative effector genes physically interacting with sleep-trait associated variants in α- and EndoC-ßH1cells running variant-to-gene mapping and establish pathways in which these genes are significantly involved. We observed that insomnia, short and long sleep-but not morningness-were significantly correlated with type 2 diabetes, obesity and other metabolic traits. Both the EndoC-ßH1 and α-cells were enriched for insomnia loci (p = .01; p = .0076), short sleep loci (p = .017; p = .022) and morningness loci (p = 2.2 × 10-7; p = .0016), while the α-cells were also enriched for long sleep loci (p = .034). Utilizing our promoter contact data, we identified 63 putative effector genes in EndoC-ßH1 and 76 putative effector genes in α-cells, with these genes showing significant enrichment for organonitrogen and organophosphate biosynthesis, phosphatidylinositol and phosphorylation, intracellular transport and signaling, stress responses and cell differentiation. Our data suggest that a subset of sleep-related loci confer their effects via cells in pancreatic islets.

Utilizing network pharmacology and experimental validation to explore the potential molecular mechanisms of BanXia-YiYiRen in treating insomnia.

BanXia-YiYiRen (Pinellia Ternata and Coix Seed, BX-YYR) has been clinically proven to be an effective Chinese medicine compatible with the treatment of insomnia. However, the underlying mechanism of BX-YYR against insomnia remains unclear. This study aimed to explore the pharmacological mechanisms of BX-YYR in treating insomnia based on network pharmacology and experimental validation. The drug-disease targets were obtained using publicly available databases. The analysis revealed 21 active compounds and 101 potential targets of BX-YYR from the pharmacological database of Chinese medicine system and analysis platform (TCMSP) and 1020 related targets of insomnia from the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. Furthermore, 38 common targets of BX-YYR against insomnia were identified, and these common targets were used to construct a protein-protein interaction (PPI) network. The visual PPI network was constructed by Cytoscape software. The top three genes from PPI according to degree value are FOS, AKT1, and CASP3. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were applied to reveal the potential targets and signaling pathways involved in BX-YYR against insomnia, especially the serotonergic pathway. In addition, molecular docking revealed that baicalein, beta-sitosterol, and stigmasterol displayed strong binding to AKT1, FOS, PRKCA, and VEGFA. Experimental study found that BX-YYR against insomnia might play a role in improving sleep by modulating the serotonergic pathway. In summary, our findings revealed the underlying mechanism of BX-YYR against insomnia and provided an objective basis for further experimental study and clinical application.

Insomnia and depressive behavior of MyD88-deficient mice: Relationships with altered microglial functions.

Myeloid differentiation primary response gene 88 (MyD88) is essential for microglial activation. Despite the significant role of microglia in regulating sleep homeostasis, the contribution of MyD88 to sleep is yet to be determined. To address this, we performed electroencephalographic and electromyographic recordings on MyD88-KO mice and wild-type mice to investigate their sleep/wake cycles. In the daytime, MyD88-KO mice exhibited prolonged wakefulness and shorter non-rapid eye movement sleep duration. Tail suspension and sucrose preference tests revealed that MyD88-KO mice displayed a depressive-like phenotype. We determined monoamines in the prefrontal cortex (PFC) using high-performance liquid chromatography and observed a decreased content of serotonin in the PFC of MyD88-KO mice. Flow cytometry revealed that CD11b, CD45, and F4/80 expressions were elevated at Zeitgeber time (ZT) 1 compared to at ZT13 only in wild-type mice. Furthermore, MFG-E8 and C1qB-tagged synapses were enhanced at ZT1 in the PFC of wild-type mice but not in MyD88-KO mice. Primary cultured microglia from MyD88-KO mice revealed decreased phagocytic ability. These findings indicate that genetic deletion of MyD88 induces insomnia and depressive behavior, at least in part, by affecting microglial homeostasis functions and lowering the serotonergic neuronal output.

Metabolic outcomes in adults with type 2 diabetes and sleep disorders.

PURPOSE: Insomnia is frequently co-morbid with obstructive sleep apnea (OSA); the effect of insomnia or co-morbid insomnia and OSA (OSA + I) on associated metabolic outcomes in adults with type 2 diabetes (T2D) remains unclear. This study in adults with T2D compared metabolic outcomes among persons with OSA, insomnia, or OSA + I. METHODS: This study analyzed baseline data from the Diabetes Sleep Treatment Trial of persons recruited for symptoms of OSA or poor sleep quality. Home sleep studies determined OSA presence and severity. Insomnia was evaluated using the Insomnia Severity Index. Height and weight to calculate body mass index (BMI) and blood for laboratory values were obtained. Multivariate general linear models were used to examine the impact of the type of sleep disorder and sociodemographic, lifestyle, and sleep risk factors on metabolic outcomes. RESULTS: Participants (N = 253) were middle-aged (56.3 ± 10.5 years), white (60.5%), obese (mean BMI of 35.3 ± 7.1 kg/m2), and male (51.4%) with poor glucose control (mean HbA1c of 8.0 ± 1.8%). Most participants had OSA + I (42.7%) or insomnia only (41.0%). HbA1c and BMI differed among the sleep disorder groups. In addition, in the adjusted models, having insomnia only, compared to OSA only, was associated on average with higher HbA1c levels (b = 1.08 ± 0.40, p < 0.007) and lower BMI (b = - 7.03 ± 1.43, p < 0.001). CONCLUSIONS: Findings suggest that insomnia frequently co-exists with OSA, is independently associated with metabolic outcomes in adults with T2D, and should be considered in investigations of the effects of OSA in persons with T2D. TRIAL REGISTRATION: Diabetes-Obstructive Sleep Apnea Treatment Trial (NCT01901055), https: Clinicaltrials.gov/ct2/show/NCT01901055; Registration date: July 17, 2013.

Pharmacokinetic and pharmacodynamic assessment of histamine H3 receptor occupancy by enerisant: a human PET study with a novel H3 binding ligand, [11C]TASP457.

PURPOSE: Histamine H3 receptor antagonists and inverse agonists have been extensively developed to treat sleep-wake, neurocognitive, and allied disorders. However, potential adverse effects, including insomnia, hampered the clinical use of these drugs, possibly due to their persistent interaction with the target molecules. The purpose of the present study was to estimate the pharmacokinetics and pharmacodynamics of enerisant, a novel antagonist and inverse agonist for histamine H3 receptors. METHODS: To measure the histamine H3 receptor occupancy by enerisant, positron emission tomography studies using [11C]TASP457, a specific radioligand for histamine H3 receptors, were performed in 12 healthy men at baseline and at 2 h after oral administration of enerisant hydrochloride. For three of these subjects, two additional scans were performed at 6 and 26 h after the administration. Relationships between the receptor occupancy by enerisant and its dose and plasma concentrations were then analyzed. RESULTS: Administration of enerisant hydrochloride decreased the radioligand binding in a dose-dependent manner. The estimated receptor occupancy values at 2 h varied as a function of its dose or plasma concentration. The time course of the occupancy showed persistently high levels (> 85%) in the two subjects with higher doses (25 and 12.5 mg). The occupancy was also initially high at 2 h and 6 h with the lower dose of 5 mg, but it decreased to 69.7% at 26 h. CONCLUSION: The target engagement of enerisant was demonstrated in the brains of living human subjects. The occupancy of histamine H3 receptors by enerisant at 2 h can be predicted by applying the plasma concentration of enerisant to Hill's plot. The preliminary time-course investigation showed persistently high brain occupancy with high doses of enerisant despite the decreasing plasma concentration of the drug. Five milligrams or less dose would be appropriate for the treatment for narcolepsy with initially high occupancy allowing for effective treatment of narcolepsy, and then the occupancy level would be expected to decrease to a level to avoid this drug's unwanted side effect of insomnia at night, although further research is warranted to confirm the statement since the expected decrease is based on the finding in one subject. TRIAL REGISTRATION: This study was retrospectively registered with ClinicalTrials.gov (NCT04631276) on November 17, 2020.

GWAS Meta-Analysis Reveals Shared Genes and Biological Pathways between Major Depressive Disorder and Insomnia.

Major depressive disorder (MDD) is one of the most prevalent and disabling mental disorders worldwide. Among the symptoms of MDD, sleep disturbance such as insomnia is prominent, and the first reason patients may seek professional help. However, the underlying pathophysiology of this comorbidity is still elusive. Recently, genome-wide association studies (GWAS) have begun to unveil the genetic background of several psychiatric disorders, including MDD and insomnia. Identifying the shared genomic risk loci between comorbid psychiatric disorders could be a valuable strategy to understanding their comorbidity. This study seeks to identify the shared genes and biological pathways between MDD and insomnia based on their shared genetic variants. First, we performed a meta-analysis based on the GWAS summary statistics of MDD and insomnia obtained from Psychiatric Genomics Consortium and UK Biobank, respectively. Next, we associated shared genetic variants to genes using two gene mapping strategies: (a) positional mapping based on genomic proximity and (b) expression quantitative trait loci (eQTL) mapping based on gene expression linkage across multiple tissues. As a result, a total of 719 shared genes were identified. Over half (51%) of them are protein-coding genes. Functional enrichment analysis shows that the most enriched biological pathways are related to epigenetic modification, sensory perception, and immunologic signatures. We also identified druggable targets using a network approach. Together, these results may provide insights into understanding the genetic predisposition and underlying biological pathways of comorbid MDD and insomnia symptoms.

Exploration of the anti-insomnia mechanism of Ganoderma by central-peripheral multi-level interaction network analysis.

BACKGROUND: Ganoderma (Lingzhi in Chinese) has shown good clinical outcomes in the treatment of insomnia, restlessness, and palpitation. However, the mechanism by which Ganoderma ameliorates insomnia is unclear. We explored the mechanism of the anti-insomnia effect of Ganoderma using systems pharmacology from the perspective of central-peripheral multi-level interaction network analysis. METHODS: The active components and central active components of Ganoderma were obtained from the TCMIP and TCMSP databases, then screened to determine their pharmacokinetic properties. The potential target genes of these components were identified using the Swiss Target Prediction and TCMSP databases. The results were matched with the insomnia target genes obtained from the GeneCards, OMIM, DisGeNET, and TCMIP databases. Overlapping targets were subjected to multi-level interaction network analysis and enrichment analysis using the STRING, Metascape, and BioGPS databases. The networks analysed were protein-protein interaction (PPI), drug-component-target gene, component-target gene-organ, and target gene-extended disease; we also performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: In total, 34 sedative-hypnotic components (including 5 central active components) were identified, corresponding to 51 target genes. Multi-level interaction network analysis and enrichment analysis demonstrated that Ganoderma exerted an anti-insomnia effect via multiple central-peripheral mechanisms simultaneously, mainly by regulating cell apoptosis/survival and cytokine expression through core target genes such as TNF, CASP3, JUN, and HSP90αA1; it also affected immune regulation and apoptosis. Therefore, Ganoderma has potential as an adjuvant therapy for insomnia-related complications. CONCLUSION: Ganoderma exerts an anti-insomnia effect via complex central-peripheral multi-level interaction networks.

Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis.

AIM: Modified Suanzaoren Decoction (MSZRD) is obtained by improving Suanzaoren Decoction (SZRT), a traditional Chinese herbal prescription that has been used to treat insomnia for more than thousands of years. Our previous study showed that MSZRD can improve the gastrointestinal discomfort related insomnia by regulating Orexin-A. This study is the first study to evaluate the effects and possible mechanisms of MSZRD in mice with insomnia caused by p-chlorophenylalanine (PCPA) combined with multifactor random stimulation. METHODS: After 14 days of multifactor stimulation to ICR mice, a PCPA suspension (30 mg/mL) was injected intraperitoneally for two consecutive days to establish an insomnia model. Three different doses of MSZRD (3.6, 7.2, and 14.4 g/kg/day) were given to ICR mice for 24 days. The food intake and back temperature were measured, and behavioral tests and pentobarbital sodium-induced sleep tests were conducted. The levels of Orexin-A, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and adrenocortical hormones (CORT) in the serum and 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in hypothalamus were measured using enzyme-linked immunosorbent assay (ELISA) kits. The levels of γ-aminobutyric acid (GABA) and glutamic acid (Glu) were measured by high-performance liquid chromatography (HPLC). The expression of 5HT1A receptor (5-HTRIA) and orexin receptor 2 antibody (OX2R) was measured by Western blot (WB) and immunohistochemical staining (ICH). Hematoxylin and eosin (H&E) staining and Nissl staining were used to assess the histological changes in hypothalamus tissue. RESULTS: Of note, MSZRD can shorten the sleep latency of insomnia mice (P < 0.05, 0.01), prolonged the sleep duration of mice (P < 0.05, 0.01), and improve the circadian rhythm disorder relative to placebo-treated animals. Furthermore, MSZRD effectively increased the content of 5-HT and 5-HTR1A protein in the hypothalamus of insomnia mice (P < 0.05, 0.01), while downregulated the content of DA and NE (P < 0.05, 0.01). Importantly, serum GABA concentration was increased by treatment with MSZRD (P < 0.05), as reflected by a decreased Glu/GABA ratio (P < 0.05). Moreover, MSZRD decreased the levels of CORT, ACTH, and CRH related hormones in HPA axis (P < 0.05, 0.01). At the same time, MSZRD significantly downregulated the serum Orexin-A content in insomnia mice (P < 0.05), as well as hypothalamic OX2R expression (P < 0.05). In addition, MSZRD also improved the histopathological changes in hypothalamus in insomnia mice. CONCLUSION: MSZRD has sleep-improvement effect in mice model of insomnia. The mechanism may be that regulating the expression of Orexin-A affects the homeostasis of HPA axis and the release of related neurotransmitters in mice with insomnia.

Molecular Regulation of Betulinic Acid on α3ß4 Nicotinic Acetylcholine Receptors.

Betulinic acid (BA) is a major constituent of Zizyphus seeds that have been long used as therapeutic agents for sleep-related issues in Asia. BA is a pentacyclic triterpenoid. It also possesses various anti-cancer and anti-inflammatory effects. Current commercially available sleep aids typically use GABAergic regulation, for which many studies are being actively conducted. However, few studies have focused on acetylcholine receptors that regulate wakefulness. In this study, we utilized BA as an antagonist of α3ß4 nicotinic acetylcholine receptors (α3ß4 nAChRs) known to regulate rapid-eye-movement (REM) sleep and wakefulness. Effects of BA on α3ß4 nAChRs were concentration-dependent, reversible, voltage-independent, and non-competitive. Site-directed mutagenesis and molecular-docking studies confirmed the binding of BA at the molecular level and showed that the α3 subunit L257 and the ß4 subunit I263 residues affected BA binding. These data demonstrate that BA can bind to a binding site different from the site for the receptor's ligand, acetylcholine (ACh). This suggests that BA may be an effective antagonist that is unaffected by large amounts of ACh released during wakefulness and REM sleep. Based on the above experimental results, BA is likely to be a therapeutically useful sleep aid and sedative.

Acupuncture stimulation at HT7 as a non-pharmacological therapy for sleep disorder caused by caffeine administration in rats.

OBJECTIVES: Insomnia is one of the most common sleep disorders and is difficult to completely treat because of the undesirable side effects of hypnotics. The present study was designed to investigate the hypnotic effect of acupuncture stimulation at HT7 on caffeine-induced sleep disorders and locomotor activity in rats. We also evaluated neuronal activity changes in the arousal region of the basal forebrain. METHODS: Rats received intraperitoneal injections of caffeine, and then electroencephalogram power spectrum analysis and locomotor activity measurements were performed. Stimulation at HT7 was performed using a mechanical acupuncture instrument (MAI) before caffeine injection, and its effects on caffeine-induced changes in sleep architecture, locomotor activity and c-Fos expression were examined. RESULTS: Caffeine injection (7.5 mg/kg) produced a significant decrease in slow-wave sleep and an increase in wake time compared with saline injection. Caffeine injection also increased locomotor activity and c-Fos expression in the medial septum-vertical limb of the diagonal band of Broca (MS-VDB), one of the arousal regions of the basal forebrain. Stimulation at HT7 with the MAI alleviated the caffeine-induced sleep disturbance and the increase in locomotor activity. In addition, MAI treatment at HT7, compared with treatment at a location not corresponding to any traditional acupuncture point, reduced the caffeine-induced increase in c-Fos expression. CONCLUSION: These results indicate that the hypnotic effect of HT7 acupuncture stimulation on caffeine-induced insomnia was associated with suppression of neuronal activity in the basal forebrain.

Causes and Consequences of Chronic Sleep Deficiency and the Role of Orexin.

Sleep is one of the pillars of health. Experimental models of acute sleep loss, of chronic partial sleep deprivation, and of sleep fragmentation in healthy sleepers are helpful models of sleep deficiency produced by insufficient sleep duration, sleep timing, and sleep disorders. Sleep deficiency is associated with changes in markers associated with risk for disease. These include metabolic, inflammatory, and autonomic markers of risk. In addition, sleep disruption and sleep deficits lead to mood instability, lack of positive outlook, and impaired neurobehavioral functioning. On a population level, insufficient sleep is associated with increased risk for hypertension and diabetes. Sleep disturbance is very common, and about half the population will report that they have experienced insomnia at some time in their lives. Approximately 10% of the population describe daytime impairment due to sleep disturbance at night, consistent with a diagnosis of insomnia disorder. The hypothalamic neuropeptides, orexin-A and orexin-B, act through G-protein-coupled receptors (orexin-1 and orexin-2 receptors). Dual and selective orexin-2 receptor antagonists have shown efficacy in inducing sleep in men and women with insomnia disorder by accelerating sleep onset and improving sleep efficiency and total sleep time. Further study comparing these medications, in short- and longer-term use models, is recommended. Greater understanding of comparative effects on mood, neurobehavioral, and physiological systems will help determine the extent of clinical utility of dual versus selective orexin receptor antagonists.