Combining network pharmacology and experimental verification to reveal the mechanism of Chaigui granules in the treatment of depression through PI3K/Akt/mTOR signaling pathways.

INTRODUCTION: Chaigui granules are a novel manufactured traditional Chinese antidepressant medicine, which is originated from the ancient classical prescription of Xiaoyaosan. It ameliorated depression-like behavior and concomitant symptoms in animal models. But its antidepressant mechanism is still unclear. Therefore, network pharmacology and molecular biology were used to explore underlying antidepressant mechanism in this study. METHODS: Firstly, network pharmacology was used to screen main active ingredients and potential targets in the treatment of depression with Chaigui granules, and to perform pathway enrichment analysis. Secondly, chronic and unpredictable mild stress-induced depression model rats were used, and behavioral tests were used to evaluate the antidepressant effect of Chaigui granules. Finally, the core targets and key pathways predicted by network pharmacology were validated by qRT-PCR and Western blot to determine the relevant gene and protein expression levels in rat hippocampus. RESULTS: The results of network pharmacology indicated that the PI3K/Akt signaling pathway may play a key role in antidepressant of Chaigui granules. The results of animal experiments showed that Chaigui granules significantly modulated behavioral indicators. Subsequently, the upregulation of relative mRNA levels of mTOR, Akt and PI3K and downregulation of GSK-3ß and FoxO3a were observed in rat hippocampus by molecular biology diagnosis. In addition, the decreased expression of Akt and mTOR in CUMS rats hippocampus was significantly reversed, and the expression levels of GSK-3ß and FoxO3a were upregulated. CONCLUSIONS: Based on the results of network pharmacology and animal experiment validation, Chaigui granules may reverse CUMS-induced depression-like behavior in rats through PI3K/Akt/mTOR signaling pathway.

Stable Isotope-Resolved Metabolomics Studies on Corticosteroid-Induced PC12 Cells: A Strategy for Evaluating Glucose Catabolism in an in Vitro Model of Depression.

Depression is a common psychopathological state or mood disorder syndrome. The serious risks to human life and the inadequacy of the existing antidepressant drugs have driven us to understand the pathogenesis of depression from a new perspective. Our research group has found disturbances in glucose catabolism in both depression and nephrotic syndrome. What are the specific metabolic pathways and specificities of glucose catabolism disorders caused by depression? To address the above scientific questions, we creatively combined traditional metabolomics technology with stable isotope-resolved metabolomics to research the glucose catabolism of the corticosterone-induced PC12 cell damage model and the adriamycin-induced glomerular podocyte damage model. The results showed an increased flux of pyruvate metabolism in depression. The increased flux of pyruvate metabolism led to an activation of gluconeogenesis in depression. The disturbed upstream metabolism of succinate caused the tricarboxylic acid cycle (TCA cycle) to be blocked in depression. In addition, there were metabolic disturbances in the purine metabolism and pentose phosphate pathways in depression. Compared with nephrotic syndrome, pyruvate metabolism, the TCA cycle, and gluconeogenesis metabolism in depression were specific. The metabolic pathways researched above are likely to be important targets for the efficacy of antidepressants.

Stable Isotope-Resolved Metabolomics Reveals the Abnormal Brain Glucose Catabolism in Depression Based on Chronic Unpredictable Mild Stress Rats.

The severe harm of depression to human life has attracted great attention to neurologists, but its pathogenesis is extremely complicated and has not yet been fully elaborated. Here, we provided a new strategy for revealing the specific pathways of abnormal brain glucose catabolism in depression, based on the supply of energy substrates and the evaluation of the mitochondrial structure and function. By using stable isotope-resolved metabolomics, we discovered that the tricarboxylic acid cycle (TCA cycle) is blocked and gluconeogenesis is abnormally activated in chronic unpredictable mild stress (CUMS) rats. In addition, our results showed an interesting phenomenon that the brain attempted to activate all possible metabolic enzymes in energy-producing pathways, but CUMS rats still exhibited a low TCA cycle activity due to impaired mitochondria. Depression caused the mitochondrial structure and function to be impaired and then led to abnormal brain glucose catabolism. The combination of the stable isotope-resolved metabolomics and mitochondrial structure and function analysis can accurately clarify the mechanism of depression. The mitochondrial pyruvate carrier and acetyl-CoA may be the key targets for depression treatment. The strategy provides a unique insight for exploring the mechanism of depression, the discovery of new targets, and the development of ideal novel antidepressants. Data are available via ProteomeXchange with identifier PXD025548.

Comprehensive Analysis Strategy of Nervous-Endocrine-Immune-Related Metabolites to Evaluate Arachidonic Acid as a Novel Diagnostic Biomarker in Depression.

Depression is one of the most complex multifactorial diseases affected by genetic and environmental factors. The molecular mechanism underlying depression remains largely unclear. To address this issue, a novel nervous-endocrine-immune (NEI) network module was used to find the metabolites and evaluate the diagnostic ability of patients with depression. During this process, metabolites were acquired from a professional depression metabolism database. Over-representation analysis was performed using IMPaLA. Then, the metabolite-metabolite interaction (MMI) network of the NEI system was used to select key metabolites. Finally, the receiver operating characteristic curve analysis was evaluated for the diagnostic ability of arachidonic acid. The results show that the numbers of the nervous system, endocrine system, and immune system pathways are 10, 19, and 12 and the numbers of metabolites are 38, 52, and 13, respectively. The selected shared metabolite-enriched pathways can be 97.56% of the NEI-related pathways. Arachidonic acid was extracted from the NEI system network by using an optimization formula and validated by in vivo experiments. It was indicated that the proposed model was good at screening arachidonic acid for the diagnosis of depression. This method provides reliable evidences and references for the diagnosis and mechanism research of other related diseases.

Metabolic profiling of RB-2 and RB-4, two analogs of polyacetylene from Bupleurum.

RB-2 and RB-4 are two structural analogs of polyacetylene from Radix Bupleuri that show antidepressant effects. However, no metabolic data are available to elucidate their systemic homeostasis. Mass spectrometry combined with liver microsomes and recombinant drug-metabolizing enzymes were performed to profile the biotransformations of RB-2/RB-4 in vitro and in vivo. Oxidation should be the major metabolic pathways for them in phase I, while CYP2C9 and CYP2E1 was the major contributor. In phase II, conjugational groups usually combined with the metabolites from phase I. This study provides an important reference basis for the safety evaluation and rational application of RB-2/RB-4.

[Advances in neurobiological mechanisms of comorbid depression and gastrointestinal disease].

Depression is a common mood disorder, which is harmful to public health critically. Gastrointestinal diseases are a series of diseases with both dynamic changes and organic disease, including functional gastrointestinal disease, gastroesophageal reflux disease, gastritis, and gastric ulcer. In recent years, the phenomena of comorbid depression and gastrointestinal disease have become common, however, most patients were diagnosed as unilateral depression or gastrointestinal disease in the clinical treatment process, resulting in delayed treatment or even invalid. The present review focuses on some of the clinical symptoms of comorbid depression and gastrointestinal disease, and begins to explore the possible pathogenesis, so as to find out the potential neurobiological pathways of comorbidity. Consequently, the more attention on comorbid depression and gastrointestinal disease will be paid, and the clinical and basic research of comorbidity and the drug development will be provided.

[Research progress in pathogenesis of hemolytic anemia and its treatment with traditional Chinese medicine].

Hemolytic anemia is a common clinical disease with diverse pathogenesis. In recent years, the incidence of hemolytic anemia is increasing dramatically. The present clinical treatment of immunosuppressive agents or splenectomy is effective to some extent; however, the accompanied clinical adverse reactions are also significant. Traditional Chinese medicine (TCM) has beneficial therapeutic effect on hemolytic anemia, with the obvious advantages including curative effect, less adverse reactions, and low price. The pathogenesis of hemolytic anemia as well as the pharmacological effects and mechanisms of the compound, single herb, and monomer composition of TCM in the treatment of hemolytic anemia were reviewed, aiming to provide the basis for the clinical treatment of hemolytic anemia and the modern research on the mechanism of TCM.

[Comparative study of the corticosterone and glutamate induced PC12 cells depression model by 1H NMR metabolomics].

This study was designed to analyze the change of metabolites in the PC12 cells and its medium induced by corticosterone (CORT) and glutamate (Glu) by proton nuclear magnetic resonance ( 1H NMR) metabolomics. The multivariate statistical analysis was employed to identify the difference between control groups and induced groups, respectively. In addition, metabolite pathway analysis was performed to explore the characteristic of CORT-induced and or Glu-induced PC12 cells depression model, and to provide the references for the selection of in vitro depression models as well as the further understanding of the mechanism on depressive disorders. We found 36 differential metabolites in CORT-induced PC12 cells and medium and 42 in Glu-induced PC12 cells. Furthermore, correlation analysis results show that serine and 2-oxoisoleucine were associated with most differential metabolites in CORT-induced PC12 cells. Lactate and glutathione were significantly correlated to the vast majority of differential metabolites in Glu-induced PC12 cells. We speculated that CORT-induced PC12 cell models may affect the fatty acid metabolism and cell membrane structure, and Glu-induced PC12 cell models may have a difference in the glycolysis and antioxidants.

[Intervention mechanism of psychological sub-health by Baihe Dihuang Tang based on network pharmacology].

This study was designed to screen the targets of bioactive ingredients of Baihe Dihuang Tang, and investigate the "multi-components, multi-targets and multi-pathways" intervention mechanism of Baihe Dihuang Tang on psychological sub-health. The ADME/T calculation method was used to screen the active ingredient of Baihe Dihuang Tang, and then using ADME/T calculation method to filtrate the active components of Baihe Dihuang Tang, then Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), Pharm Mapper database and Medical Subject Headings (MeSH) were combined to forecast and filtrate the targets of the main active ingredients. In addition, the predicted targets were verified by the Surflex-dock in Sybyl. The Cytoscape software was used to construct the Baihe Dihuang Tang ingredients-targets-disease network, while Clue GO software was used to analyze the molecular function and biological process of the targets. There are total 11 active ingredients and 21 targets in Baihe Dihuang Tang. A good interaction between them was supported by the score. The 21 targets were mainly involved in gamma-aminobutyric acid signaling pathway, c AMP metabolic process and monoamine transport relevant biological processes. Thus, Baihe Dihuang Tang may play a role in the intervention of psychological sub-health by regulating the activity of G-protein coupled amine receptor and the expression of monoamine neurotransmitter, which reflects the features of traditional Chinese medicine multi-components, multi-targets and multi-pathways. This research provides evidences on the pharmacological mechanism of Baihe Dihuang Tang effect on psychological sub-health.

[Progress of new antidepressant drugs development].

Depression becomes more and more serious and has been greatly do harm to human's physical and mental health. WHO predicts that depression will become the second leading cause of abnormal death and disability by 2020. There is still the drug treatment as the first-line antidepressive therapy, but the effect of listed antidepressant drugs is not ideal. Therefore, discovery of new antidepressant drug of sunique, higher curative effect, less adverse reaction is the pursuit of pharmaceutical. The traditional Chinese medicine (TCM) of anti-depression and natural antidepressant in currently listed, drug development and drug discovery phase are summarized in this paper, aiming to provide reference for new antidepressant drug research.

[Study on TLC identification and UPLC determination method of atractylenolide in Atractylodes macrocephala].

This research is to establish TLC and UPLC methods for simultaneous determination of 3 atractylenolides in Atractylodes macrocephala. Silica gel GF254 plate was used for identification of A. macrocephala, and UPLC-PDA gradient elution method was used to simultaneously determine atractylenolide Ⅰ, Ⅱ and Ⅲ. The Waters BEH C18 column（2.1 mm×100 mm,1.7 µm）with acetonitrile-water as mobile phase and the wavelength of UV detector of 235 nm were performed. The quality control study showed that the characteristic for identification by TLC was distinct and highly specific. The method of content determination was in accordance with the regulations. The quantitative evaluation of atractylenolide Ⅰ,Ⅱ and Ⅲ was in good linear range（r>0.999 9）, and the average recovery was 93.48%（RSD 1.4%）,94.97%（RSD 1.6%）,92.71%（RSD 1.2%）,respectively. TLC identification was in good specificity and repeatability, and the UPLC-PDA method for the simultaneous determination of 3 atractylenolides was simple and reliable for the quality control of A.macrocephala.

[Quantitative analysis of eight polyacetylenes derived from Bupleuri Radix by ultra-performance liquid chromatography coupled with photodiode array detector].

To establish quantitative methods for determination of polyacetylenes in Bupleuri Radix, an ultra-performance liquid chromatography method coupled with photodiode array detector (UPLC-PDA) was developed. The analysis was performed on a Waters BEH C18 column (2.1 mm×100 mm, 1.7 µm) using a gradient system of methanol and water. The flow rate was 0.3 mL•min⁻¹ and the detection wavelength was 315 nm. Eight polyacetylenes were prepared using traditional extraction and isolation method, of which compounds 7 and 8 were two new polyacetylenes. All calibration curves showed good linearity (r>0.999 0) within the concentration range. Both the intra- and inter-day precisions for eight analytes were less than 1.9%, respectively, with the mean recovery at the range of 93.21%-108.4%. Meanwhile, 17 bupleurum samples were examined with this process. The results showed a variety either the chemotaxonomic or content of polyacetylenes. The method indicated good linearity, limit of detection and quantification, precision, accuracy and recovery. The developed method allows quantitative assessment and quality control of polyacetylenes, and might be a good alternative according to detection levels in polyacetylenes from Bupleurum Radix.

[Anti-depression mechanism of supercritical CO(2) extract from Compound Chaigui Fang based on network pharmacology].

The study was designed to clarify the antidepressant mechanism of supercritical CO(2) extract from Compound Chaigui Fang. We used TCMSP, HIT, Pharm GKB and Gene Cards bioinformatics software to predict and analyze the drug/disease targets and their common targets of compounds in the supercritical CO(2) extract of Compound Chaigui Fang for depression. Chronic unpredictable mild stress（CUMS）was used to induce depressed model in rats. Hippocampus and serum were collected after supercritical CO(2) extract treatment to detect the potential antidepressant targets with enzyme-linked immunosorbent assay（ELISA）. The results predicate that there are 22 chemical compounds and 78 potential therapeutic drug targets for depression. There are 177 disease markers for depression, and 14 common targets for drug intervention of depression, which includes the neurotransmitter transports/metabolic enzyme/receptors, hormone of hypothalamus-pituitary-adrenal/ thyroid/gonad axis, immune-associated factor, etc. ELISA results suggest that depression is associated with the low level of phosphate c AMP responsive element-binding protein in hippocampus and the high levels of corticosterone and interleukin 6 in serum with CUMS rat. Those were restored to normal levels by supercritical CO(2) extract of Compound Chaigui Fang. The study provides an antidepressant mechanism of supercritical CO(2) extract of Compound Chaigui Fang based on the network pharmacology, and a new strategy in the study of the effective extracts of compound Chinese traditional medicine.

[Discovering potential biomarkers of depression and drug intervention of paroxetine based on (1)H NMR metabolomics].

The purpose of this study was to find potential biomarkers in the serum of patients with depression and provide the basis for clinical diagnosis of depression. Sixteen patients of severe depression were selected according to the inclusion criteria and 16 healthy people were used in the control group. The depression patients took paroxetine for two weeks. The serum was collected from the patients and healthy control group before and after paroxetine treatment. The samples were analyzed by (1)H NMR based metabolomics to determine the changes in profiles of endogenous metabolites and metabolites with significant differences were selected in analysis. Related pathways and receiver operating characteristic (ROC) were examined in analysis of the correlation between the potential biomarkers and depression. Their feasibility and reliability was determined for the clinical practice. Significant differences were observed in the metabolic profile of serum of the patients and the healthy controls. Depression had an effect on metabolism for an increase in leucine, isoleucine and alanine, glutamate, glutamine and N-acetyl-glycoprotein and a decrease in glucose. Those may be considered as potential biomarkers of depression. Clinical application of the biomarkers may improve the objectivity of the diagnosis and treatment of depression by antidepressant drugs. The metabolomics approach is an effective tool in the investigation of biomarkers.

[An exploration in the action targets for antidepressant bioactive components of Xiaoyaosan based on network pharmacology].

The present study aims to predict the action targets of antidepressant active ingredients of Xiaoyaosan to understand the "multi-components, multi-targets and multi-pathways" mechanism. Using network pharmacology, the reported antidepressant active ingredients in Xiaoyaosan (saikosaponin A, saikosaponin C, saikosaponin D, ferulic acid, Z-ligustilide, atractylenolide I, atractylenolide II, atractylenolide III, paeoniflorin, albiflorin, liquiritin, glycyrrhizic acid and pachymic acid), were used to predict the targets of main active ingredients of Xiaoyaosan according to reversed pharmacophore matching method. The prediction was made via screening of the antidepressive drug targets approved by FDA in the DrugBank database and annotating the information of targets with the aid of MAS 3.0 biological molecular function software. The Cytoscape software was used to construct the Xiaoyaosan ingredients-targets-pathways network. The network analysis indicates that the active ingredients in Xiaoyaosan involve 25 targets in the energy metabolism-immune-signal transmutation relevant biological processes. The antidepressant effect of Xiaoyaosan reflects the features of traditional Chinese medicine in multi-components, multi-targets and multi-pathways. This research provides a scientific basis for elucidation of the antidepressant pharmacological mechanism of Xiaoyaosan.

[Anti-depressive mechanism of Fufang Chaigui prescription based on neuroendocrine hormone and metabolomic correlation analysis].

To elucidate the anti-depressive effect of Fufang Chaigui prescription and its mechanism and investigate its effect on neuroendocrine hormone, rats were included into a chronic unpredictable mild stress (CUMS) model for 28 d, and drugs were administered at the same time. During the period, rats' behaviors were observed and the blood was collected by using ELISA to determine representative hormone concentrations of HPAA, HPTA and HPGA. The changes in endogenous metabolites were analyzed by using H NMR metabolomics to seek the potential biomarkers. Results showed Fufang Chaigui prescription could improve the behaviors of CUMS rats obviously, increase contents of ACTH, CORT, T3and decrease contents of TSH and TESTO and regulate the levels of lactate, α-glucose, choline, N-acetylglycoprotein, trimethylamine oxide and leucine to get closer to the contents of control group. The results of correlation analysis indicated that HPTA was associated with glycometabolism, amino acid metabolism and choline metabolism. And HPAA was related to glycometabolism and amino acid metabolism. However, HPGA was only correlated with glycometabolism. In conclusion, Fufang Chaigui prescription could show an obvious anti-depressive effect and its underlying mechanism might involve regulations of neuroendocrine function and pathways of glycometabolism, amino acid metabolism and choline metabolism.

[1H NMR based metabolomics study of bu-zhong-yi-qi-tang in the spleen-qi deficiency rat model].

The present study aimed to investigate the effect and the mechanisms of Bu-zhong-yi-qi-tang (BZYQ) on Spleen-Qi deficiency rat's model using nuclear magnetic resonance (NMR) metabolomics and multivariate statistical analysis methods. The rat Spleen-Qi deficiency model was established as follows: oral administration of Radix Rhei extract, loaded swimming and starvation for 24 h. The body weight and motor behavior of the rats were measured and recorded once a week. BZYQ could significantly improve body weight and behavioral of Spleen-Qi deficiency model rats compared with the model group (P < 0.05, 0.01). After drug administration, the changes in the levels of endogenous metabolites in the spleen including decreasing lactate, taurine and hypoxanthine, increasing glutamate and scyllo-inositol compared with the model group. The metabolomics approach is an effective tool for the investigation of the pharmacologic mechanism of BZYQ and it is helpful to further research.

[1H NMR based metabonomics study on the antidepressant effect of genipin in rat hippocampus].

The purpose of this study is to explore depression metabolic markers in rat hippocampus and to investigate the anti-depressant effect of genipin and its mechanisms using nuclear magnetic resonance (NMR) metabonomics. Chronic unpredictable mild stress (CUMS) procedure was conducted to establish the depressive rat model. At the beginning of the third week, genipin low dose (25 mg x kg(-1)), middle dose (50 mg x kg(-1)), high dose (100 mg x kg(-1)), and venlafaxine (50 mg x kg(-1)) were given to the CUMS rats separately once daily for two weeks except control and model groups. Rat hippocampus was analyzed by 1H NMR based metabonomics after drug administration for 2 weeks. Significant differences in the metabolic profile of rat hippocampus of the CUMS treated group and the control group were observed with metabolic effects of CUMS including decreasing in glycine and N-acetylaspartate, increasing in inositol, glutamate, lactate, glutamine, taurine and alanine. Genipin showed ideal antidepressive effects at a dose of 50 mg x kg(-1) in rats, decrease of inositol, glutamate, lactate, alanine were observed, while glycine and N-acetylaspartate were increased. Important influence has been found on normal nervous system function of these significant changed metabolites, which suggests that the antidepressant effect of genipin may be played by enhancing the activity of neurons in hippocampus, repairing and improving the function of the neuron. The metabonomics approach is an effective tool for the investigation of the anti-depressant effect and pharmacologic mechanisms of genipin.

[Study on supercritical CO2 extraction of xiaoyaosan and its GC-MS fingerprint].

To determine the optimum conditions of supercritical CO2 extraction of Xiaoyaosan, and establish its fingerprint by gas chromatography-mass spectrometry (GC-MS), the yield of extract were investigated, an orthogonal test was used to quantify the effects of extraction temperature, pressure, CO2 flow rate and time, and fingerprint analysis of different batches of extracts were by GC-MS. The optimal extraction conditions were determined as follows: extraction pressure 20 MPa, extraction temperature 50 degrees C, CO2 flow rate 25 kg x h(-1), extraction time 3 h, and average yield 2.2%. The GC-MS fingerprint was established and 27 common peaks were found, whose contents add up to 81.89% of the total peak area. Among them, 21 compounds were identified, accounting for 53.20% of the total extract. The extraction process is reasonable and favorable for industrial production. The GC-MS method is accurate, reliable, reproducible, and can be used for quality control of supercritical CO2 extract from Xiaoyaosan.

[Anti-depressant effect and mechanism of supercritical CO2 extract from Compound Chaigui Fang].

The tail suspension test (TST), forced swimming test (FST) and chronic unpredictable mild stress (CUMS) model were used to evaluate the anti-depressant effect of supercritical CO2 extract from Compound Chaigui Fang (FFCGF). A nuclear magnetic resonance (NMR)-based metabonomics combined with multivariate statistical analysis was performed to explore the mechanism of FFCGF. Rats were conducted by CUMS procedure for 28 days and drugs were administrated at the same time. The body weight, sucrose preference, crossings and rearings in open-field tests were evaluated and the urine was collected simultaneously. The metabonomic profiles of rats' urine were analyzed by NMR and potential biomarkers were searched by multivariate statistical analysis. The results showed that administration of FFCGF significantly decreasing the immobility time in FST and TST and improving rats' body weight, sucrose preference, crossings and rearings in CUMS, which were indication that the anti-depressant effect of FFCGF was abvious. Significant differences in the metabolic profile of the CUMS treated group and the control group were observed, which were consistent with the results of behavioral tests. Decreased levels of acetic acid, succinic acid, 2-oxidation glutaric acid and citric acid and increased glycine and pyruvic acid in urine were significantly affected by the CUMS procedure and the 6 biomarkers were reversed evidently after administration of FFCGF. These changes were suggestion that the anti-depressant mechanism of FFCGF was associated with energy metabolism, lipid metabolism and amino acid metabolism.