Suanzaoren decoction exerts its antidepressant effect via the CaMK signaling pathway.

Calmodulin-dependent protein kinases (CaMKs) are widely regarded as "memory molecules" due to their role in controlling numerous neuronal functions in the brain, and the CaMK signaling pathway plays a crucial role in controlling synaptic plasticity. Suanzaoren decoction (SZRD) can improve depression-like behavior and thus has potential benefits in the clinical treatment of depression; however, its mechanism of action is not fully understood. In this study, we found that key proteins in the CaMK signaling pathway were regulated by the decoction used to treat depression. The purpose of this research was to ascertain if the SZRD's therapeutic efficacy in the treatment of depression is associated with the modulation of key proteins in the CaMK signaling pathway. A rat model of depression was created by exposing the animals to chronic, unexpected, mild stress. Model rats were given intragastric administration of SZRD or fluoxetine every morning once a day. Protein and mRNA relative expression levels of CaM, CaMK I, and CaMK IV in the hippocampus were measured by Western blot, quantitative polymerase chain reaction, and immunohistochemistry in the hippocampus. Our findings demonstrated that SZRD significantly improved the mood of depressed rats. This indicates that SZRD, by modulating the CaMK signaling system, may alleviate depressive symptoms and lessen work and life-related pressures.

Screening the components in multi-biological samples and the comparative pharmacokinetic study in healthy and depression model rats of Suan-Zao-Ren decoction combined with a network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Suanzaoren Decoction (SZRD) is a classic traditional Chinese prescription, which has been commonly used for treating insomnia, depression and other nerve system diseases for a long time. AIM OF THIS STUDY: The present study aimed to explore the metabolic profiles in multi-biological samples and pharmacokinetic mechanism between healthy and depression model rats combined with a network pharmacology approach after administration of SZRD. MATERIALS AND METHODS: In our study, an ultra-high performance liquid chromatography (UPLC)-Q-Exactive Orbitrap Mass Spectrometry method was firstly used to study the prototype components and metabolites of SZRD in plasma, brain, urine, and feces between healthy and depressed rats. The possible metabolic pathways were also speculated. Then a network pharmacological study was conducted on the components in the plasma of model rats. According to the above components screened by network pharmacology and the other reported representative active components, the comparative pharmacokinetic study was established for the simultaneous determination of mangiferin, spinosin, ferulic acid, liquiritin, formononetin. magnoflorine and isoliquiritin between healthy and depression model rats. Finally, molecular docking was used to validate the binding affinity between key potential targets and active components in pharmacokinetics. RESULTS: A total of 115 components were identified in healthy rats, and 101 components were identified in model rats. The prototype components and metabolites in plasma, brain, urine, and feces were also distinguished. The main metabolic pathways included phase I and phase II metabolic reactions, such as dehydrogenation, oxidation, hydroxylation, gluconaldehyde conjugation, glutathione conjugation and so on. These results provided a basis for the further study of antidepressive pharmacokinetic and pharmacological action in SZRD. Then, according to the degree value of network pharmacological study, it was predicted that 10 components and 10 core targets, which involved in the critical pathways such as neuroactive ligand-receptor interaction, cyclic adenosine monophosphate (cAMP) signaling pathway, serotonergic synapse, phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway, etc. Finally, the established pharmacokinetic method was successfully applied to compare the pharmacokinetic behavior of these 7 active components in plasma of healthy and depressed rats after oral administration of SZRD. It showed that except magnoflorine, the pharmacokinetic parameters of each component were different between healthy and depressed rats. Molecular docking analysis also indicated that the active compounds in pharmacokinetics could bind tightly to the key targets of network pharmacological study. CONCLUSION: This study may provide important information for studying the action mechanism of SZRD in treating depression.

SuanZaoRen decoction alleviates neuronal loss, synaptic damage and ferroptosis of AD via activating DJ-1/Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: SuanZaoRen Decoction (SZRD), a famous herbal prescription, and has been widely proven to have positive therapeutic effects on insomnia, depression and Alzheimer's disease (AD). However, the anti-AD molecular mechanism of SZRD remains to be further investigated. AIM OF THE STUDY: To elucidate the molecular mechanism of SZRD's improvement in AD's neuronal loss, synaptic damage and ferroptosis by regulating DJ-1/Nrf2 signaling pathway. MATERIALS AND METHODS: LC-MS/MS was used to detect the active ingredients from SZRD. APP/PS1 mice was treated with SZRD and a ferroptosis inhibitor (Liproxstatin-1), respectively. Upon the completion of behavioral tests, Nissl staining, FJB staining, Golgi staining, immunofluorescence, immunohistochemistry, and transmission electron microscopy were preformed to evaluate the effects of SZRD on neuronal loss, synaptic damage, Aß deposition. Iron staining, transmission electron microscopy, and iron assay kit was performed to estimate the effects of SZRD on ferroptosis. SOD kit, MDA kit, GSH kit, and GSH/GSSG kit were utilized to measure the oxidative stress levels in the hippocampus. The protein expression of TfR1, FTH1, FTL, FPN1, DJ-1, Nrf2, GPX4, SLC7A11, and ACSL4 were detected by Western blot. RESULTS: A total of 16 active ingredients were identified from SZRD extract. SZRD SZRD significantly alleviated learning and memory impairment in APP/PS1 mice. SZRD improved the hippocampal neuronal loss and degenerated neurons in APP/PS1 mice via inhibiting the Aß deposit. SZRD mitigated the hippocampal synaptic damage in APP/PS1 mice. SZRD inhibited iron accumulation, and alleviated the oxidative stress level in the hippocampus of APP/PS1 mice. Meanwhile, SZRD could up-regulate the protein expression level of FPN1, DJ-1, Nrf2, GPX4 and SLC7A11 in the hippocampus, and inhibit TfR1, FTH1, FTL, and ACSL4 protein expression. CONCLUSION: SZRD alleviated neuronal loss, synaptic damage and ferroptosis in AD via activating DJ-1/Nrf2 signaling pathway.

[Effect of Suanzaoren Decoction on expression of ionotropic glutamate receptors and synaptic plasticity in hippocampus of anxiety rats].

This study investigated the effect of Suanzaoren Decoction on the expression of N-methyl-D-aspartate receptors(NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors(AMPAR) in the hippocampus and synaptic plasticity in rats with conditioned fear-induced anxiety. The effect of Suanzaoren Decoction on rat behaviors were evaluated through open field experiment, elevated plus maze experiment, and light/dark box experiment. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of glutamate(Glu) and γ-aminobutyric acid(GABA) in the rat hippocampus. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to assess the gene and protein expression of ionotropic glutamate receptors in the hippocampal region. Transmission electron microscopy was utilized to observe the changes in the ultrastructure of synaptic neurons in the hippocampal region. Long-term potentiation(LTP) detection technique was employed to record the changes in population spike(PS) amplitude in the hippocampal region of mice in each group. The behavioral results showed that compared with the model group, the Suanzaoren Decoction group effectively increased the number of entries into open arms, time spent in open arms, percentage of time spent in open arms out of total movement time, number of entries into open arms out of total entries into both arms(P<0.01), and significantly increased the time spent in the light box and the number of shuttle crossings(P<0.01). There was an increasing trend in the number of grid crossings, entries into the center grid, and time spent in the center grid, indicating a significant anxiolytic effect. ELISA results showed that compared with the model group, the Suanzaoren Decoction group exhibited significantly reduced levels of Glu, Glu/GABA ratio(P<0.01), and significantly increased levels of GABA(P<0.01) in the rat hippocampus. Furthermore, Suanzaoren Decoction significantly decreased the gene and protein expression of NMDAR(GluN2B and GluN2A) and AMPAR(GluA1 and GluA2) compared with the model group. Transmission electron microscopy results demonstrated improvements in synapses, neuronal cells, and organelles in the hippocampal region of the Suanzaoren Decoction group compared with the model group. LTP detection results showed a significant increase in the PS amplitude changes in the hippocampal region of Suanzaoren Decoction group from 5 to 35 min compared with the model group(P<0.05, P<0.01). In conclusion, Suanzaoren Decoction exhibits significant anxiolytic effects, which may be attributed to the reduction in NMDAR and AMPAR expression levels and the improvement of synaptic plasticity.

Suanzaoren decoction improves depressive-like behaviors by regulating the microbiota-gut-brain axis via inhibiting TLR4/NFκB/NLRP3 inflammation signal pathway.

Depression is a common but serious sickness which causes a considerable burden on individuals and society. Recently, it has been well established that the occurrence of depression was related to the microbiota-gut-brain axis. The toll-like receptor 4 (TLR4)/ nuclear factor kappa-B kinase (NFκB)/ NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway is closely associated with the regulation of microbiota-gut-brain axis. Suanzaoren Decoction (SZRD), which recorded in Jin Gui Yao Lve in Han dynasty, has been used for treating insomnia and depression for a long time. However, the action mechanism of the depression regulation through the TLR4/NFκB/NLRP3 pathway by SZRD was still unclear. In this study, SZRD was firstly performed on a chronic unpredictable mild stress (CUMS) mice model. The results of behavioral tests showed that SZRD treatment could ameliorate the depressive-like behaviors of CUMS mice effectively. According to our previous researches about the components of SZRD in vitro and in vivo, the identification of serum metabolites in depression model rats was further analyzed qualitatively using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry. 27 prototypes and 44 metabolites were identified. The main types of metabolic reactions are glucuronization, sulfation, and so on. Then, using immunohistochemistry and western blotting to monitor the difference in activation of TLR4/NFκB/NLRP3 signaling pathway in mice brain and colon. The results showed that SZRD treatment could reduce expression levels of related factors. Additionally, the SZRD treatment could also inhibit the histopathological damage in the path morphology of the hippocampus and colon. The results of 16SrRNA demonstrated that SZRD could reduce the dysbiosis of the intestinal flora of depressive mice. The above results provided important information for studying the action mechanism of SZRD in treating depression by regulating microbiota-gut-brain axis via inhibiting TLR4/NFκB/NLRP3 pathway.

Exploring the mechanism of Suanzaoren decoction in treatment of insomnia based on network pharmacology and molecular docking.

Objective: To explore the functional mechanisms of Suanzaoren decoction (SZRD) for treating insomnia using network pharmacology and molecular docking. Methods: The active ingredients and corresponding targets of SZRD were obtained from the Traditional Chinese Medicine Systems Pharmacology database, and then, the names of the target proteins were standardized using the UniProt database. The insomnia-related targets were obtained from the GeneCards, DisGeNET, and DrugBank databases. Next, a Venn diagram comprising the drug and disease targets was created, and the intersecting targets were used to draw the active ingredient-target network diagram using Cytoscape software. Next, the STRING database was used to build a protein-protein interaction network, followed by cluster analysis using the MCODE plug-in. The Database for Annotation, Visualization, Integrated Discovery (i.e., DAVID), and the Metascape database were used for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. AutoDock Vina and Pymol software were used for molecular docking. Results: SZRD contained 138 active ingredients, corresponding to 239 targets. We also identified 2,062 insomnia-related targets, among which, 95 drug and disease targets intersected. The GO analysis identified 490, 62, and 114 genes related to biological processes, cellular components, and molecular functions, respectively. Lipid and atherosclerosis, chemical carcinogen-receptor activation, and neuroactive ligand-receptor interaction were the most common pathways in the KEGG analysis. Molecular docking demonstrated that the primary active components of SZRD for insomnia had good binding capabilities with the core proteins in PPI network. Conclusion: Insomnia treatment with SZRD involves multiple targets and signaling pathways, which may improve insomnia by reducing inflammation, regulating neurotransmitters.

Screening the effective components of Suanzaoren decoction on the treatment of chronic restraint stress induced anxiety-like mice by integrated chinmedomics and network pharmacology.

BACKGROUND: Suanzaoren decoction (SZRD) is a classical traditional Chinese prescription. It is widely used to treat mental disorders, including insomnia, anxiety, and depression, in China and other Asian countries. However, the effective components and mechanisms underlying SZRD remained unclear. PURPOSE: We aimed to develop a new strategy to discover the effects and potential mechanisms of SZRD against anxiety and to further reveal the effective components of SZRD in treating anxiety. STUDY DESIGN AND METHODS: First, the chronic restraint stress (CRS)-induced mouse model of anxiety was orally administered SZRD, and behavioral indicators and biochemical parameters were applied to assess efficacy. A chinmedomics strategy based on UHPLC-Q-TOF-MS technology and network pharmacology were then used to screen and explore potentially effective components and therapeutic mechanisms. Finally, molecular docking was applied to further confirm the effective components of SZRD, and a multivariate network for anxiolytic effects was constructed. RESULTS: SZRD exerted anxiolytic effects by increasing the percentage of entries into open arms and the time spent in open arms; improving hippocampal 5-HT, GABA, and NE levels; and increasing serum corticosterone (CORT) and corticotropin-releasing hormone (CRH) levels caused by CRS challenge. Beside, SZRD exerted a sedative effect by decreasing sleep time and prolonging sleep latency with no muscle relaxation effect in CRS mice. A total of 110 components were identified in SZRD, 20 of which were absorbed in the blood. Twenty-one serum biomarkers involved in arachidonic acid, tryptophan, sphingolipid, and linoleic acid metabolism were identified after SZRD intervention. Finally, a multivariate network including prescription-effective components-targets-pathway of SZRD treating anxiety, including 11 effective components, 4 targets and 2 pathway was constructed. CONCLUSION: The current study demonstrated that integrating chinmedomics and network pharmacology was a powerful approach to investigating the effective components and therapeutic mechanisms of SZRD and provided a solid basis for the quality marker (Q-marker) of SZRD.

UPLC-Q-Orbitrap-MS based metabolomics and analysis of the effect of Suanzaoren Decoction on serum of chronic unpredictable mild stress depression rats / 药学学报

A UPLC-Q-Orbitrap-MS based metabolomic approach combined with biochemical assay and histopathological inspection were employed to study the intervention effects of Suanzaoren Decoction (SZRD) on chronic unpredictable mild stress (CUMS) depression rats, and to clarify the metabolic regulation pathway of SZRD. The rats were randomly divided into normal control group, CUMS model group, positive drug venlafaxine group, SZRD high (24 g·kg-1) and low (12 g·kg-1) dose groups, respectively. The CUMS model was replicated by subjecting to a variety of stimulus, such as thermal stimulation, ice water swimming, ultrasonic stimulation, tail clamping, day and night reversal, plantar electric shock and so on for rats. After oral administration of drugs for 28 days, the behavioral indexes of rats in each group were observed and the hippocampus and serum samples of rats were collected for biochemical assay and histopathological inspection. Compared with the CUMS model group, low dose and high dose SZRD groups can significantly reduce the immobility time of forced swimming (P < 0.001, P < 0.001), increase the sucrose preference rate (P < 0.01, P < 0.05), the number of crossings (P < 0.05, P < 0.01) and the number of uprights (P < 0.05, P < 0.01) in the open field test, suggesting that SZRD can significantly improve the depression-like behavior of CUMS model rats. In addition, SZRD could significantly reduce the levels of serum IL-6, IL-1β and TNF-α of CUMS model rats. A total of 21 differential metabolites in serum were identified by comparison with the data from the literature and databases. In addition, low-dose SZRD and high-dose SZRD improved the 8 and 11 perturbed potential serum biomarkers that were induced by CUMS, respectively, which related to alanine, aspartic acid and glutamic acid, tryptophan and arachidonic acid metabolism. This study provides a scientific basis for expanding the clinical indications of SZRD. This experiment was approved by the Animal Ethics Committee of Shanxi University (Approval No. SXULL2020028).

SuanZaoRen decoction amliorates mitochondrial dysfunction of APP/PS1 mice via activating AMPK/SIRT1/PGC-1α signaling pathway / 中国药理学通报

Aim To explore the effect of Suanzaoren decoction(SZRD) on mitochondrial dysfunction in AD model of APP/PS1 mice via AMPK/SIRT1/PGC-1α signaling pathway and to reveal the possible mechanism. Methods Thirty APP/PS1 mice were randomly divided into app /PS1 group, low-dose SZRD group(L-SZRD) and high-dose SZRD group(H-SZRD). Ten C57BL/6JNju mice were set as control group(WT). Morris water maze test was used to detect the learning and memory ability of mice. Thioflavin T staining was used to observe senile plaques hippocampus. Immunohistochemistry was performed to detect the expression level of Aβ in hippocampus. Transmission electron microscope was used to observe the mitochondrial morph hology in hippocampus. Kits were employed to detect the contents of ATP and ROS in hippocampus; Western blot was employed to detect the expression levels of AMPK, p-AMPKThrK172, SIRT1, PGC-1α, NRF1, NRF2 and TFAM in hippocampus. Results Compared to the APP/PS1 group, L-SZRD and H-SZRD induced mouse cognitive impairment, reduced the deposition of senile plaques, inhibited the expression of Aβ, improved the damage of mitochondrial structure, increased the content of ATP in the hippocampus, reduced the expression level of ROS in hippocampus and increased the expression of p-AMPK-ThrK172, SIRT1, PGC-1α, NRF1, NRF2, TFAM Conclusions SZRD could improve the cognitive impairment, senile plaque deposition and mitochondrial dysfunction of AD mice, and its mechanism may be involved in the up-regulation of the expression of AMPK/SIRT1/PGC-1α signaling pathway.Reduced the Deposition of Senile Plaques, Inhibited the Expression of Aβ, Improved The Damage of Mitochondric Structure, Increased the Content of At in TH. E hippocampus, Reduced the Expression level of Ros in Hippocampus and Increased The Expression of P-Ampk-Thrk172, SIRT1, SIRT1 PGC-1α, NRF1, NRF2, TFAM. Conclusions SZRD could improve the cognitive impairment, senile plaque deposition and mitochondrial dysfunction of AD mice, and its mechanism may be involved in the up-regulation of the expression of AMPK/SIRT1/PGC-1α signaling pathway.Reduced the Deposition of Senile Plaques, Inhibited the Expression of Aβ, Improved The Damage of Mitochondric Structure, Increased the Content of At in TH. E hippocampus, Reduced the Expression level of Ros in Hippocampus and Increased The Expression of P-Ampk-Thrk172, SIRT1, SIRT1 PGC-1α, NRF1, NRF2, TFAM. Conclusions SZRD could improve the cognitive impairment, senile plaque deposition and mitochondrial dysfunction of AD mice, and its mechanism may be involved in the up-regulation of the expression of AMPK/SIRT1/PGC-1α signaling pathway.Senile plaque deposition and mitochondrial dysfunction of AD mice, and its mechanism may be involved in the up-regulation of the expression of AMPK/SIRT1/PGC-1α signaling pathway.Senile plaque deposition and mitochondrial dysfunction of AD mice, and its mechanism may be involved in the up-regulation of the expression of AMPK/SIRT1/PGC-1α signaling pathway.

Effect of Suanzaoren Decoction on expression of ionotropic glutamate receptors and synaptic plasticity in hippocampus of anxiety rats / 中国中药杂志

This study investigated the effect of Suanzaoren Decoction on the expression of N-methyl-D-aspartate receptors(NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors(AMPAR) in the hippocampus and synaptic plasticity in rats with conditioned fear-induced anxiety. The effect of Suanzaoren Decoction on rat behaviors were evaluated through open field experiment, elevated plus maze experiment, and light/dark box experiment. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of glutamate(Glu) and γ-aminobutyric acid(GABA) in the rat hippocampus. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to assess the gene and protein expression of ionotropic glutamate receptors in the hippocampal region. Transmission electron microscopy was utilized to observe the changes in the ultrastructure of synaptic neurons in the hippocampal region. Long-term potentiation(LTP) detection technique was employed to record the changes in population spike(PS) amplitude in the hippocampal region of mice in each group. The behavioral results showed that compared with the model group, the Suanzaoren Decoction group effectively increased the number of entries into open arms, time spent in open arms, percentage of time spent in open arms out of total movement time, number of entries into open arms out of total entries into both arms(P<0.01), and significantly increased the time spent in the light box and the number of shuttle crossings(P<0.01). There was an increasing trend in the number of grid crossings, entries into the center grid, and time spent in the center grid, indicating a significant anxiolytic effect. ELISA results showed that compared with the model group, the Suanzaoren Decoction group exhibited significantly reduced levels of Glu, Glu/GABA ratio(P<0.01), and significantly increased levels of GABA(P<0.01) in the rat hippocampus. Furthermore, Suanzaoren Decoction significantly decreased the gene and protein expression of NMDAR(GluN2B and GluN2A) and AMPAR(GluA1 and GluA2) compared with the model group. Transmission electron microscopy results demonstrated improvements in synapses, neuronal cells, and organelles in the hippocampal region of the Suanzaoren Decoction group compared with the model group. LTP detection results showed a significant increase in the PS amplitude changes in the hippocampal region of Suanzaoren Decoction group from 5 to 35 min compared with the model group(P<0.05, P<0.01). In conclusion, Suanzaoren Decoction exhibits significant anxiolytic effects, which may be attributed to the reduction in NMDAR and AMPAR expression levels and the improvement of synaptic plasticity.

Network pharmacology strategy to investigate the pharmacological effects of Suanzaoren Decoction on insomnia

Abstract Suanzaoren Decoction (SZRD) is an ancient prescription used in the treatment of insomnia. This study aimed to investigate the components and targets of SZRD in treating insomnia. First, the compounds of five herbs in SZRD were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the putative targets for treating insomnia were obtained from DrugBank to construct the herb-compound-target- disease network. A protein-protein interaction (PPI) network was constructed in the STRING database, and then Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to predict the mechanism of action of intersection target. Finally, 30 mice were divided into five groups: control, model, and quercetin groups (100, 50, 25 mg/kg). The sleep latency and duration of pentobarbital-induced sleeping were measured. The production of interleukin-6 (IL-6) and γ-aminobutyric acid (γ-GABA) was detected by using an enzyme-linked immunosorbent assay kit (ELISA), and Gamma-aminobutyric acid type a receptor subunit alpha1 (GABRA1) was tested by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). A total of 152 active ingredients, including 80 putative targets of SZRD, were obtained. The main active compounds included quercetin and kaempferol, and the key targets involved IL-6 and nitric oxide synthase 3 (NOS3). The results of pathway enrichment analysis indicated that the putative targets of SZRD mainly participated in Neuroactive ligand-receptor interaction. The experiment of P-chlorophenylalanine (PCPA)-induced insomnia model showed that quercetin obviously shortened the sleep latency and prolonged the sleep duration of the insomnia model. The production of IL-6, γ-GABA, and GABRA1 mRNA was significantly increased in mice treated with quercetin. This study predicted the active ingredients and potential targets of SZRD on insomnia on the basis of a systematic network pharmacology approach and illustrated that SZRD might exert hypnotic effects via regulating IL-6, γ-GABA, and GABRA1

Study on the active components and mechanism of Suanzaoren decoction in improving cognitive impairment caused by sleep deprivation.

ETHNOPHARMACOLOGICAL RELEVANCE: Suanzaoren Decoction (SZRD) is a traditional and classic prescription for the treatment of insomnia, with a history of more than 1,000 years. It replenishes blood components, calms the nerves, reduces fever and irritability. It is commonly used in the clinical treatment of chronic fatigue syndrome, cardiac neurosis, and menopausal syndromes. Modern pharmacological studies have shown that it improves cognitive impairment; however, its mechanism of action remains unclear. AIM OF THE STUDY: This study preliminarily investigated the potential bioactive components and mechanism of SZRD in improving cognitive impairment by exploring network pharmacology, molecular docking, and conducting in vivo experiments. MATERIALS AND METHODS: The components of various Chinese herbs in SZRD and their disease-related targets were identified through network pharmacology and literature. Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of intersection targets were performed using the relevant database. Next, the "Components-Targets-Pathways" (C-T-P) and "Protein-Protein interaction" networks were constructed using the enrichment analysis results to further identify potential pathways, bioactive components, and hub genes. At the same time, molecular docking was used to further distinguish the key bioactive components and genes of SZRD responsible for improving cognitive impairment. Finally, the potential mechanism of action was further analysed and verified using in vivo experiments. RESULTS: A total of 117 potential active components and 138 intersection targets were identified by network pharmacology screening. The key bioactive components, including calycosin, 5-Prenylbutein, licochalcone G, glypallichalcone, and ZINC189892, were identified by analysing the networks and molecular docking results. Hub genes included ACHE, CYP19A1, EGFR, ESR1, and ESR2. The oestrogen signalling pathway was the most important in the enrichment analysis. In vivo experiments further proved that SZRD could improve cognitive impairment by affecting the oestrogen signalling pathway and the expression of ACHE and CYP19A1. CONCLUSIONS: Network pharmacology and in vivo experiments demonstrate that SZRD improves cognitive impairment caused by sleep disturbance through estrogen receptor pathway, which provides a basis for its clinical application.

[Metabonomic study of biochemical changes in serum of PCPA-induced insomnia rats after treatment with Suanzaoren Decoction].

Suanzaoren Decoction(SZRD) is a classical formula for the clinical treatment of insomnia. This study analyzed the effect of SZRD on endogenous metabolites in insomnia rats based on metabonomics and thereby explored the anti-insomnia mechanism of SZRD. To be specific, DL-4-chlorophenylalanine(PCPA) was used to induce insomnia in rats. Then pathological changes of the liver and brain were observed and biochemical indexes such as 5-hydroxytryptamine(5-HT), dopamine(DA), glutamate(Glu), γ-aminobutyric acid(GABA), and norepinephrine(NE) in the hippocampus and prostaglandin D2(PGD2), tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), and IL-6 in the serum of rats were detected. On this basis, the effect of SZRD on PCPA-induced insomnia rats was preliminarily assessed. The metabolic profile of rat serum samples was further analyzed by ultra-performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS). Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were combined with t-test and variable importance in projection(VIP) to identify differential metabolites, and MetaboAnalyst 5.0 was employed for pathway analysis. The results showed that SZRD could improve the pathological changes of brain and liver tissues, increase the levels of neurotransmitters 5-HT, DA, and GABA in hippocampus and the level of PGD2 in hypothalamic-pituitary-adrenal axis(HPA axis), and reduce the levels of IL-1ß and TNF-α in serum of insomnia rats. Metabonomics analysis yielded 12 significantly changed potential metabolites: 5-aminovaleric acid, N-acetylvaline, L-proline, L-glutamate, L-valine, DL-norvaline, D(-)-arginine, pyroglutamic acid, 1-methylguanine, L-isoleucine, 7-ethoxy-4-methylcoumarin, and phthalic acid mono-2-ethylhexyl ester(MEHP), which were related with multiple biochemical processes including metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of arginine and proline, arginine biosynthesis, glutathione metabolism. These metabolic changes indicated that SZRD can improve the metabolism in insomnia rats by regulating amino acid metabolism.

[Effect of Suanzaoren Decoction on molecular levels of bile acids in serum, liver, and ileum of insomnia mice].

To explore the mechanism of Suanzaoren Decoction in the treatment of insomnia from endogenous bile acid regulation, the present study investigated the hepatoprotective effect of Suanzaoren Decoction and the molecular changes of bile acids in the serum, liver, and ileum of insomnia model mice and Suanzaoren Decoction treated mice. The insomnia model in mice was established by the sleep deprivation method. After Suanzaoren Decoction(48.96 mg·kg~(-1)·d~(-1)) intervention by gavage for 7 days, the related indicators, such as water consumption, food intake, body weight, aspartate aminotransferase(AST), alanine transaminase(ALT), and total bile acid(TBA) were detected, and the pathological changes of the liver and ileum were observed. The molecular levels and distribution of 23 bile acids in the serum, liver, and ileum were analyzed by UPLC-MS/MS combined with principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA). The results showed that Suanzaoren Decoction could improve the decreased water consumption and food intake, weight loss, and increased AST and ALT in the model group, and effectively reverse the injury and inflammation in the liver and ileum. The bile acids in the liver of the insomnia model mice were in the stage of decompensation, and the bile acids in the serum, liver, and ileum of the mice decreased or increased. Suanzaoren Decoction could regulate the anomaly of some bile acids back to normal. Seven bile acids including glycoursodeoxycholic acid(GUDCA), glycodesoxycholic acid(GDCA), tauro-α-MCA(T-α-MCA), α-MCA, taurodeoxycholate(TDCA), T-ß-MCA, and LCA were screened out as the main discriminant components by PLS-DA. It is concluded that Suanzaoren Decoction possesses the hepatoprotective effect and bile acids could serve as the biochemical indicators to evaluate the drug efficacy in the treatment of abnormal liver functions caused by insomnia. The mechanism of Suanzao-ren Decoction in soothing the liver, resolving depression, tranquilizing the mind, and improving sleep may be related to the molecular regulation of bile acid signals.

[Mechanism of Suanzaoren Decoction in improving insomnia rats by integrating metabolomics and intestinal flora analysis].

To explore the mechanism of Suanzaoren Decoction(SZRD) in improving the insomnia rat model induced by DL-4-chlorophenylalanine(PCPA). The insomnia model was established by single intraperitoneal injection with PCPA(400 mg·kg~(-1)), UPLC-Q-TOF-MS/MS was used to analyze the profile of metabolites in rat hippocampus samples, combined with multivariate statistical analysis and screening of differential metabolites, and related metabolic pathways were constructed with MetaboAnalyst 5.0. The high-throughput sequencing of V3-V4 regions of 16 S rRNA gene was used to predict the structure and relative abundance of intestinal flora by LEfSe, OPLS-DA and PICRUSt2. A total of 22 differential hippocampus metabolites were identified by metabolomics analysis, including amino acids, fatty acids, nucleosides, organic acids, vitamins, and others. Pathway analysis showed that alanine, aspartate and glutamic metabolism, D-glutamine and D-glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis were the main pathways. 16 S rRNA gene sequencing showed that Ruminococcus and Eubacterium were the differences between SZRD group and model group. Ruminococcus might be the sign of SZRD improving PCPA insomnia on analysis of PICRUSt2 and LEfSe. Furthermore Spearman correlation analysis showed that the differential metabolites 2-oxo-4-methylthiobutyric acid and palmitic acid intervened by SZRD were significantly positively correlated with the differential flora. In conclusion, SZRD indirectly improves insomnia by affecting metabolic pathways such as amino acids metabolic pathways and regulating the structure of flora. The results of this study provide a new mechanism and new idea for elucidating the mechanism of classic famous prescription SZRD in improving insomnia from the perspective of intestinal flora.

Metabonomic study of biochemical changes in serum of PCPA-induced insomnia rats after treatment with Suanzaoren Decoction / 中国中药杂志

Suanzaoren Decoction(SZRD) is a classical formula for the clinical treatment of insomnia. This study analyzed the effect of SZRD on endogenous metabolites in insomnia rats based on metabonomics and thereby explored the anti-insomnia mechanism of SZRD. To be specific, DL-4-chlorophenylalanine(PCPA) was used to induce insomnia in rats. Then pathological changes of the liver and brain were observed and biochemical indexes such as 5-hydroxytryptamine(5-HT), dopamine(DA), glutamate(Glu), γ-aminobutyric acid(GABA), and norepinephrine(NE) in the hippocampus and prostaglandin D2(PGD2), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and IL-6 in the serum of rats were detected. On this basis, the effect of SZRD on PCPA-induced insomnia rats was preliminarily assessed. The metabolic profile of rat serum samples was further analyzed by ultra-performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS). Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were combined with t-test and variable importance in projection(VIP) to identify differential metabolites, and MetaboAnalyst 5.0 was employed for pathway analysis. The results showed that SZRD could improve the pathological changes of brain and liver tissues, increase the levels of neurotransmitters 5-HT, DA, and GABA in hippocampus and the level of PGD2 in hypothalamic-pituitary-adrenal axis(HPA axis), and reduce the levels of IL-1β and TNF-α in serum of insomnia rats. Metabonomics analysis yielded 12 significantly changed potential metabolites: 5-aminovaleric acid, N-acetylvaline, L-proline, L-glutamate, L-valine, DL-norvaline, D(-)-arginine, pyroglutamic acid, 1-methylguanine, L-isoleucine, 7-ethoxy-4-methylcoumarin, and phthalic acid mono-2-ethylhexyl ester(MEHP), which were related with multiple biochemical processes including metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of arginine and proline, arginine biosynthesis, glutathione metabolism. These metabolic changes indicated that SZRD can improve the metabolism in insomnia rats by regulating amino acid metabolism.

Effect of Suanzaoren Decoction on molecular levels of bile acids in serum, liver, and ileum of insomnia mice / 中国中药杂志

To explore the mechanism of Suanzaoren Decoction in the treatment of insomnia from endogenous bile acid regulation, the present study investigated the hepatoprotective effect of Suanzaoren Decoction and the molecular changes of bile acids in the serum, liver, and ileum of insomnia model mice and Suanzaoren Decoction treated mice. The insomnia model in mice was established by the sleep deprivation method. After Suanzaoren Decoction(48.96 mg·kg~(-1)·d~(-1)) intervention by gavage for 7 days, the related indicators, such as water consumption, food intake, body weight, aspartate aminotransferase(AST), alanine transaminase(ALT), and total bile acid(TBA) were detected, and the pathological changes of the liver and ileum were observed. The molecular levels and distribution of 23 bile acids in the serum, liver, and ileum were analyzed by UPLC-MS/MS combined with principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA). The results showed that Suanzaoren Decoction could improve the decreased water consumption and food intake, weight loss, and increased AST and ALT in the model group, and effectively reverse the injury and inflammation in the liver and ileum. The bile acids in the liver of the insomnia model mice were in the stage of decompensation, and the bile acids in the serum, liver, and ileum of the mice decreased or increased. Suanzaoren Decoction could regulate the anomaly of some bile acids back to normal. Seven bile acids including glycoursodeoxycholic acid(GUDCA), glycodesoxycholic acid(GDCA), tauro-α-MCA(T-α-MCA), α-MCA, taurodeoxycholate(TDCA), T-β-MCA, and LCA were screened out as the main discriminant components by PLS-DA. It is concluded that Suanzaoren Decoction possesses the hepatoprotective effect and bile acids could serve as the biochemical indicators to evaluate the drug efficacy in the treatment of abnormal liver functions caused by insomnia. The mechanism of Suanzao-ren Decoction in soothing the liver, resolving depression, tranquilizing the mind, and improving sleep may be related to the molecular regulation of bile acid signals.

Efficacy and safety of electroacupuncture combined with Suanzaoren decoction for insomnia following stroke: study protocol for a randomized controlled trial.

BACKGROUND: Insomnia is a common but frequently overlooked sleep disorder after stroke, and there are limited effective therapies for insomnia following stroke. Traditional Chinese medicine (TCM), including acupuncture and the Chinese herbal medication (CHM) Suanzaoren decoction (SZRD), has been reported as an alternative option for insomnia relief after stroke in China for thousands of years. Here, this study aims to investigate the efficacy and safety of electroacupuncture (EA) in combination with SZRD in the treatment of insomnia following stroke. METHODS: A total of 240 patients with post-stroke insomnia will be included and randomized into four groups: the EA group, SZRD group, EA & SZRD group, and sham group. The same acupoints (GV20, GV24, HT7, and SP6) will be used in the EA group, EA & SZRD group, and sham group, and these patients will receive the EA treatment or sham manipulation every other day for 4 consecutive weeks. SZRD treatments will be given to participants in the SZRD group and EA & SZRD group twice a day for 4 consecutive weeks. The primary outcome measures include Pittsburgh Sleep Quality Index scores and polysomnography. Secondary outcome measures include the Insomnia Severity Index, the National Institutes of Health Stroke Scale, the Hospital Anxiety and Depression Scale, brain magnetic resonance imaging, functional magnetic resonance imaging, and nocturnal melatonin concentrations. The primary and secondary outcomes will be assessed at baseline (before treatment), during the 2nd and 4th weeks of the intervention, and at the 8th and 12th weeks of follow-up. Safety assessments will be evaluated at baseline and during the 4th week of the intervention. DISCUSSION: This study will contribute to assessing whether the combination of these two therapies is more beneficial for post-stroke insomnia than their independent use, and the results of this clinical trial will improve our understanding of the possible mechanisms underlying the effects of combination therapies. TRIAL REGISTRATION: Chinese Clinical Trials Register ChiCTR2000031413 . Registered on March 30, 2020.

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.

[Review of chemical constituents, pharmacological effects and clinical applications of Suanzaoren Decoction and prediction and analysis of its Q-markers].

Suanzaoren Decoction is a classic prescription for nourishing the heart and liver, nourishing blood and tranquilizing the mind. It has the functions of sedation and hypnosis, anti-anxiety, anti-depression, anti-convulsion and so on. Modern clinic is mostly used to treat different types of insomnia, depression, neurasthenia, tension headache and vertigo. In this paper, the chemical consti-tuents, pharmacological effects and clinical application of Suanzaoren Decoction are reviewed. Based on this, the quality marker(Q-marker) of Suanzaoren Decoction was predicted and analyzed according to the "five principles" of Q-marker of traditional Chinese medicine--transmission and traceability, specificity, effectiveness, measurability and compatibility environment of compound prescriptions. The results indicated that jujuboside, spinosin, ferulic acid, senkyunolide Ⅰ, sarsasapogenin, mangiferin, liquiritoside and glycyrrhizic acid were predicted and analyzed, and those can be used as Q-markers of Suanzaoren Decoction. Subsequently, the above components can be selected as indicators to control and evaluate the quality of Suanzaoren Decoction and its preparations, and establish a quality traceability system.