Xiaoyao Pills, a Chinese patent medicine, treats mild and moderate depression: A randomized clinical trial combined with DNA methylation analysis.

BACKGROUND: Xiaoyao pills (XYP) is a commercial Chinese patent medicine used in the treatment of depression. However, the mechanisms underlying its therapeutic effects, as well as the patients who can benefit from XYP, have not been evaluated so far. OBJECTIVES: To this end, we conducted a double-blinded, random, and placebo-controlled clinical trial of orally administered XYP in patients with depression. METHODS: The 17-item Hamilton Depression Rating Scale (HAMD-17) scores were recorded at baseline, and every 2 weeks after the start of treatment. To further elucidate the epigenetic mechanism of XYP, we performed mRNA sequencing and genome-wide DNA methylation sequencing using peripheral blood leukocytes of patients and healthy. RESULTS: XYP effectively alleviated the symptoms in patients with mild or moderate depressive disorders, particularly that of psychomotor retardation. XYP restored aberrant gene expression and DNA methylation patterns associated with depression, and the normalization of DNA methylation correlated with downregulation of several genes. In addition, altered DNA methylation levels in the XYP-treated samples were attributed to increased expression of the DNA methyltransferase DNMT1. CONCLUSIONS: Our study provides new insights into the epigenetic mechanism underlying depression and the therapeutic effects of XYP, along with an experimental basis for using XYP in the treatment of depression. TRIAL REGISTRATION INFORMATION: The name of the registry and number: U.S. CLINICAL TRIALS REGISTRY: The link to the registration: ClinicalTrials.gov ISRCTN12746343 (https://www.isrctn.com/ISRCTN12746343). The name of the trial register is "Efficacy and safety of the Xiaoyao pill for improving the clinical symptoms of stagnation of liver qi (chi) and spleen deficiency". The clinical trial registration number is ISRCTN12746343.

Gene expression insights: Chronic stress and bipolar disorder: A bioinformatics investigation.

Bipolar disorder (BD) is a psychiatric disorder that affects an increasing number of people worldwide. The mechanisms of BD are unclear, but some studies have suggested that it may be related to genetic factors with high heritability. Moreover, research has shown that chronic stress can contribute to the development of major illnesses. In this paper, we used bioinformatics methods to analyze the possible mechanisms of chronic stress affecting BD through various aspects. We obtained gene expression data from postmortem brains of BD patients and healthy controls in datasets GSE12649 and GSE53987, and we identified 11 chronic stress-related genes (CSRGs) that were differentially expressed in BD. Then, we screened five biomarkers (IGFBP6, ALOX5AP, MAOA, AIF1 and TRPM3) using machine learning models. We further validated the expression and diagnostic value of the biomarkers in other datasets (GSE5388 and GSE78936) and performed functional enrichment analysis, regulatory network analysis and drug prediction based on the biomarkers. Our bioinformatics analysis revealed that chronic stress can affect the occurrence and development of BD through many aspects, including monoamine oxidase production and decomposition, neuroinflammation, ion permeability, pain perception and others. In this paper, we confirm the importance of studying the genetic influences of chronic stress on BD and other psychiatric disorders and suggested that biomarkers related to chronic stress may be potential diagnostic tools and therapeutic targets for BD.

Comparison of volatile and nonvolatile metabolites in green tea under hot-air drying and four heat-conduction drying patterns using widely targeted metabolomics.

Hot-air and heat-conduction drying are the most common drying patterns in green tea production. However, the differences between them in terms of the resulting green tea chemical compounds have not been illustrated systematically. In this study, 515 volatile and 204 nonvolatile metabolites were selected to compare the differences between hot-air drying green tea (HAGT) and four heat-conduction drying green teas (HCDGTs) using widely targeted metabolomics. The results showed notable changes in volatile compounds; for example, two kinds of HCDGTs preferred to form chestnut-like and caramel-like key odorants. In addition, 14 flavonol glycosides, 10 catechins, 9 phenolic acids, 8 amino acids, 7 flavonols, and 3 sugars were significantly changed between HAGT and HCDGTs (p < 0.05), presenting a tremendous discrepancy in the transformation of nonvolatile compounds. Our results provide clear guidance for the precise manufacturing of green tea by four common heat-drying patterns and hot air-drying patterns.

Ginsenoside Rg1 can reverse fatigue behavior in CFS rats by regulating EGFR and affecting Taurine and Mannose 6-phosphate metabolism.

Background: Chronic fatigue syndrome (CFS) is characterized by significant and persistent fatigue. Ginseng is a traditional anti-fatigue Chinese medicine with a long history in Asia, as demonstrated by clinical and experimental studies. Ginsenoside Rg1 is mainly derived from ginseng, and its anti-fatigue metabolic mechanism has not been thoroughly explored. Methods: We performed non-targeted metabolomics of rat serum using LC-MS and multivariate data analysis to identify potential biomarkers and metabolic pathways. In addition, we implemented network pharmacological analysis to reveal the potential target of ginsenoside Rg1 in CFS rats. The expression levels of target proteins were measured by PCR and Western blotting. Results: Metabolomics analysis confirmed metabolic disorders in the serum of CFS rats. Ginsenoside Rg1 can regulate metabolic pathways to reverse metabolic biases in CFS rats. We found a total of 34 biomarkers, including key markers Taurine and Mannose 6-phosphate. AKT1, VEGFA and EGFR were identified as anti-fatigue targets of ginsenoside Rg1 using network pharmacological analysis. Finally, biological analysis showed that ginsenoside Rg1 was able to down-regulate the expression of EGFR. Conclusion: Our results suggest ginsenoside Rg1 has an anti-fatigue effect, impacting the metabolism of Taurine and Mannose 6-phosphate through EGFR regulation. This demonstrates ginsenoside Rg1 is a promising alternative treatment for patients presenting with chronic fatigue syndrome.

Comparison of Volatile and Nonvolatile Metabolites in Black Tea under Four Second-Drying Methods Using Widely Targeted Metabolomics.

Second-drying has an impact on the development of flavor and aroma in black tea. However, the effect of the shape changes of the tea leaves during second-drying on the quality of black tea has yet to be evaluated. In this study, GC-TOFMS and UPLC-HRMS identified 411 volatile metabolites and 253 nonvolatile metabolites. Additionally, 107 nonvolatile compounds and 21 different volatiles were screened. Significant alterations (p < 0.01) were found in 18 amino acid derivatives, 17 carbohydrates, 20 catechins, 19 flavonoids, 13 phenolic acids, and 4 organic acids. The content of certain amino acids and carbohydrates correlated with the shape of black tea. Furthermore, sweet aroma compound formation was facilitated by hot-air second-drying while the remaining second-drying approaches encouraged the formation of the fruity aroma compound. The results of the study provide a theoretical basis and technical instructions for the accurate and precise processing of premium black tea.

Xiaoyaosan Ameliorates Chronic Restraint Stress-Induced Depression-Like Phenotype by Suppressing A2AR Signaling in the Rat Striatum.

Depression is a common mental disorder characterized by pessimism and world-weariness. In our previous study, we found that Xiaoyaosan (XYS) could have antidepressive effects, however the underlying mechanisms remain unclear. Several studies have shown that adenosine A (2 A) receptor (A2AR) in the brain is a key point in the treatment of depression. Our present study aimed to investigate the effects of XYS on A2AR signaling in the striatum of rats exposed to chronic restraint stress (CRS). Ninety-six male Sprague-Dawley rats were randomly divided into 8 groups (control, model, negative control, XYS, A2AR antagonist, A2AR antagonist + XYS, A2AR agonist, A2AR agonist + XYS). The rats in the model group, XYS group, A2AR antagonist group and A2AR antagonist + XYS group were subjected to CRS for 3 h a day. The XYS decoction [2.224 g/(kg·d)] was intragastrical administered by oral gavage to the rats in the negative control group, XYS group, A2AR antagonist + XYS group, and A2AR agonist + XYS group. The rats in the A2AR antagonist group and A2AR antagonist + XYS group were treated with SCH 58261 [0.05 mg/(kg·d)], and the rats in the A2AR agonist and A2AR agonist + XYS group were treated with CGS 21680 [0.1 mg/(kg·d)]. These procedures were performed for 21 consecutive days. Behavioral studies including the open field test, elevated plus maze test, sucrose preference test and forced swimming test, were performed to examine depression-like phenotypes. Then, the effects of XYS on CRS- or A2AR agonist-induced striatal subcellular damage, microglial activation and A2AR signaling changes in the striatum were examined. Here, we report that XYS ameliorates depression-like phenotypes (such as body weight loss as well as depression- and anxiety-like behaviors) and improves synaptic survival and growth in the stratum of the CRS rats. Moreover, XYS reduces A2AR activity and suppresses hyper-activation of striatal microglia. The tissue and cellular effects of XYS were similar to those of the known A2AR antagonists. In conclusion, XYS alleviates depression in the CRS rats via inhibiting A2AR in the striatum.

Differential Gene Analysis of Trastuzumab in Breast Cancer Based on Network Pharmacology and Medical Images.

The purpose of this study was to use network pharmacology, biomedical images and molecular docking technology in the treatment of breast cancer to investigate the feasible therapeutic targets and mechanisms of trastuzumab. In the first place, we applied pubchem swisstarget (http://www.swisstargetprediction.ch/), (https://pubchem.ncbi.nlm.nih.gov/) pharmmapper (http://lilab-ecust.cn/pharmmapper/), and the batman-tcm (http://bionet.ncpsb.org.cn/batman-tcm/) database to collect the trastuzumab targets. Then, in NCBI-GEO, breast cancer target genes were chosen (https://www.ncbi.nlm.nih.gov/geo/). The intersection regions of drug and disease target genes were used to draw a Venn diagram. Through Cytoscape 3.7.2 software, and the STRING database, we then formed a protein-protein interaction (PPI) network. Besides, we concluded KEGG pathway analysis and Geen Ontology analysis by using ClueGO in Cytospace. Finally, the top 5 target proteins in the PPI network to dock with trastuzumab were selected. After screening trastuzumab and breast cancer in databases separately, we got 521 target genes of the drug and 1,464 target genes of breast cancer. The number of overlapping genes was 54. PPI network core genes include GAPDH, MMP9, CCNA2, RRM2, CHEK1, etc. GO analysis indicated that trastuzumab treats breast cancer through abundant biological processes, especially positive regulation of phospholipase activity, linoleic acid metabolic process, and negative regulation of endothelial cell proliferation. The molecular function is NADP binding and the cellular component is tertiary granule lumen. The results of KEGG enrichment analysis exhibited four pathways related to the formation and cure of breast cancer, containing Drug metabolism, Glutathione metabolism, Pyrimidine metabolism and PPAR signaling pathway. Molecular docking showed that trastuzumab has good binding abilities with five core target proteins (GAPDH, MMP9, CCNA2, RRM2, CHEK1). This study, through network pharmacology and molecular docking, provides new pieces of evidence and ideas to understand how trastuzumab treats breast cancer at the gene level.

Integrating Metabolomics and Network Analysis for Exploring the Mechanism Underlying the Antidepressant Activity of Paeoniflorin in Rats With CUMS-Induced Depression.

Background: Paeoniflorin (PF) represents the major bioactive constituent of the traditional Chinese medicine plant Paeonia suffruticosa (Ranunculaceae), which has a long history as a folk medicine in Asian. Paeoniflorin, a bitter pinene monoterpene glycoside, has antidepressant effects, but its potential therapeutic mechanism has not been thoroughly explored. Methods: Experimental depression in rats was established by the chronic unpredictable mild stress (CUMS) combined with orphan method, and the efficacy of paeoniflorin on depression was evaluated by the sucrose preference test and open field test. The antidepressant mechanism of paeoniflorin was investigated by metabolomic and network pharmacology. The relevant pathways of biomarkers highlighted in metabolomics were explored, and the possible targets of paeoniflorin in the treatment of depression were further revealed through network analysis. The binding activity of paeoniflorin to key targets was verified by molecular docking. Results: Metabolomics showed that rats with CUMS-induced depression had urine metabolic disorders, which were reversed by paeoniflorin through the regulation of metabolic pathways. Metabolites that play a key role in the function of paeoniflorin include citric acid, thiamine monophosphate, gluconolactone, 5-hydroxyindoleacetic acid and stachyose. Key predicted targets are SLC6A4, TNF, IL6 and SLC6A3. An important metabolic pathway is the Citrate cycle (TCA cycle). Conclusion: Network integrative analysis in this study showed that paeoniflorin could improve depressive-like symptoms in model rats with CUMS-induced depression and overall correct the disordered metabolic profile through multiple metabolic pathways.

Saikosaponin D exerts antidepressant effect by regulating Homer1-mGluR5 and mTOR signaling in a rat model of chronic unpredictable mild stress.

BACKGROUND: Many studies about depression have focused on the dysfunctional synaptic signaling in the hippocampus that drives the pathophysiology of depression. Radix Bupleuri has been used in China for over 2000 years to regulate liver-qi. Extracted from Radix Bupleuri, Saikosaponin D (SSD) is a pharmacologically active substance that has antidepressant effects. However, its underlying mechanism remains unknown. MATERIALS AND METHODS: A chronic unpredictable mild stress (CUMS) paradigm was used as a rat model of depression. SD rats were randomly assigned to a normal control (NC) group or one exposed to a CUMS paradigm. Of the latter group, rats were assigned to four subgroups: no treatment (CUMS), fluoxetine-treated (FLU), high-dose and low-dose SSD-treated (SSDH and SSDL). SSD was orally administrated of 1.50 mg/kg and 0.75 mg/kg/days for three weeks in the SSDH and SSDL groups, respectively. Fluoxetine was administrated at a dose of 2.0 mg/kg/days. SSD's antidepressant effects were assessed using the open field test, forced swim test, and sucrose preference test. Glutamate levels were quantified by ELISA. Western blot and immunochemical analyses were conducted to quantify proteins in the Homer protein homolog 1 (Homer1)-metabotropic glutamate receptor 5 (mGluR5) and mammalian target of rapamycin (mTOR) pathways in the hippocampal CA1 region. To measure related gene expression, RT-qPCR was employed. RESULTS: CUMS-exposed rats treated with SSD exhibited increases in food intake, body weight, and improvements in the time spent in the central are and total distance traveled in the OFT, and less pronounced pleasure-deprivation behaviors. SSD also decreased glutamate levels in CA1. In CA1 region of CUMS-exposed rats, SSD treatment increased mGluR5 expression while decreasing Homer1 expression. SSD also increased expressions of postsynaptic density protein 95 (PSD95) and synapsin I (SYP), and the ratios of p-mTOR/mTOR, p-p70S6k/p70S6k, and p-4E-BP1/4E-BP1 in the CA1 region in CUMS-exposed rats. CONCLUSIONS: SSD treatment reduces glutamate levels in the CA1 region and promotes the expression of the synaptic proteins PSD-95 and SYP via the regulation of the Homer1-mGluR5 and downstream mTOR signaling pathways. These findings suggest that SSD could act as a natural neuroprotective agent in the prevention of depression.

Extracellular Vesicles: Emerging Roles in Developing Therapeutic Approach and Delivery Tool of Chinese Herbal Medicine for the Treatment of Depressive Disorder.

Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by cells, which play an essential role in intercellular communication by delivering cellular components including DNA, RNA, lipids, metabolites, cytoplasm, and cell surface proteins into recipient cells. EVs play a vital role in the pathogenesis of depression by transporting miRNA and effector molecules such as BDNF, IL34. Considering that some herbal therapies exhibit antidepressant effects, EVs might be a practical delivery approach for herbal medicine. Since EVs can cross the blood-brain barrier (BBB), one of the advantages of EV-mediated herbal drug delivery for treating depression with Chinese herbal medicine (CHM) is that EVs can transfer herbal medicine into the brain cells. This review focuses on discussing the roles of EVs in the pathophysiology of depression and outlines the emerging application of EVs in delivering CHM for the treatment of depression.

Corrigendum: Xiaoyaosan alleviates hippocampal glutamate-induced toxicity in the CUMS rats via NR2B and PI3K/Akt signaling pathway.

[This corrects the article DOI: 10.3389/fphar.2021.586788.].

Antidepressant Mechanism of Traditional Chinese Medicine Formula Xiaoyaosan in CUMS-Induced Depressed Mouse Model via RIPK1-RIPK3-MLKL Mediated Necroptosis Based on Network Pharmacology Analysis.

Background: Depression is a stress-related disorder that seriously threatens people's physical and mental health. Xiaoyaosan is a classical traditional Chinese medicine formula, which has been used to treat mental depression since ancient times. More and more notice has been given to the relationship between the occurrence of necroptosis and the pathogenesis of mental disorders. Objective: The purpose of present study is to explore the potential mechanism of Xiaoyaosan for the treatment of depression using network pharmacology and experimental research, and identify the potential targets of necroptosis underlying the antidepressant mechanism of Xiaoyaosan. Methods: The mice model of depression was induced by chronic unpredictable mild stress (CUMS) for 6 weeks. Adult C57BL/6 mice were randomly divided into five groups, including control group, chronic unpredictable mild stress group, Xiaoyaosan treatment group, necrostatin-1 (Nec-1) group and solvent group. Drug intervention performed from 4th to 6th week of modeling. The mice in Xiaoyaosan treatment group received Xiaoyaosan by intragastric administration (0.254 g/kg/d), and mice in CUMS group received 0.5 ml physiological saline. Meanwhile, the mice in Nec-1 group were injected intraperitoneally (i.p.) with Nec-1 (10 mg/kg/d), and the equivalent volume of DMSO/PBS (8.3%) was injected into solvent group mice. The behavior tests such as sucrose preference test, forced swimming test and novelty-suppressed feeding test were measured to evaluate depressive-like behaviors of model mice. Then, the active ingredients in Xiaoyaosan and the related targets of depression and necroptosis were compiled through appropriate databases, while the "botanical drugs-active ingredients-target genes" network was constructed by network pharmacology analysis. The expressions of RIPK1, RIPK3, MLKL, p-MLKL were detected as critical target genes of necroptosis and the potential therapeutic target compounds of Xiaoyaosan. Furthermore, the levels of neuroinflammation and microglial activation of hippocampus were measured by detecting the expressions of IL-1ß, Lipocalin-2 and IBA1, and the hematoxylin and eosin (H&E) stained was used to observe the morphology in hippocampus sections. Results: After 6-weeks of modeling, the behavioral data showed that mice in CUMS group and solvent group had obvious depressive-like behaviors, and the medication of Xiaoyaosan or Nec-1 could improve these behavioral changes. A total of 96 active ingredients in Xiaoyaosan which could regulate the 23 key target genes were selected from databases. Xiaoyaosan could alleviate the core target genes in necroptosis and improve the hippocampal function and neuroinflammation in depressed mice. Conclusion: The activation of necroptosis existed in the hippocampus of CUMS-induced mice, which was closely related to the pathogenesis of depression. The antidepressant mechanism of Xiaoyaosan included the regulation of multiple targets in necroptosis. It also suggested that necroptosis could be a new potential target for the treatment of depression.

Traditional Chinese Formula Xiaoyaosan Alleviates Depressive-Like Behavior in CUMS Mice by Regulating PEBP1-GPX4-Mediated Ferroptosis in the Hippocampus.

BACKGROUND: At present, the pathogenesis of depression is not fully understood, and nearly half of depression patients experience no obvious effects during treatment. This study aimed to establish a depression mouse model to explore the possible role of ferroptosis in the pathogenesis of depression, and observe the effects of Xiaoyaosan on PEBP1-GPX4-mediated ferroptosis in the hippocampus. METHODS: Forty-eight male C57BL/6 mice were randomly divided into a control group, CUMS group, Xiaoyaosan group and fluoxetine group, and the model was established by chronic unpredictable mild stress (CUMS) for a successive 6 weeks. The medication procedure was performed from the 4th to the 6th week of modeling. The behavioral evaluations were measured to evaluate depressive-like behaviors. The expressions of GPX4, FTH1, ACSL4 and COX2 were detected as ferroptosis-related indicators. Then, the total iron and ferrous content in the hippocampus were measured. The levels of PEBP1 and ERK1/2 were observed, and the expressions of GFAP and IBA1 were also detected to measure the functions of astrocytes and microglia in the hippocampus. RESULTS: Eight herbs of Xiaoyaosan had 133 active ingredients which could regulate the 43 ferroptosis-related genes in depression. After 6 weeks of modeling, the data showed that mice in the CUMS group had obvious depressive-like behaviors, and medication with Xiaoyaosan or fluoxetine could significantly improve the behavioral changes. The expressions of GPX4, FTH1, ACSL4, COX2, PEBP1, ERK1/2, GFAP and IBA1 changed in the CUMS group mice, while the total iron and ferrous content also changed. Xiaoyaosan and fluoxetine had obvious curative effects that could significantly alleviate the above changes in the hippocampus. CONCLUSION: Our results revealed that the activation of ferroptosis might exist in the hippocampi of CUMS-induced mice. The PEBP1-GPX4-mediated ferroptosis could be involved in the antidepressant mechanism of Xiaoyaosan. It also implied that ferroptosis could become a new target for research into the depression mechanism and antidepressant drugs.

Xiaoyaosan Alleviates Hippocampal Glutamate-Induced Toxicity in the CUMS Rats via NR2B and PI3K/Akt Signaling Pathway.

Purpose: It is revealed that Xiaoyaosan could reduce glutamate level in the hippocampus of depressed rats, whose metabolism leads to the pathophysiology of depression. However, the underlying mechanism remains unclear. This study aims to explore the effect of Xiaoyaosan on glutamate metabolism, and how to regulate the excitatory injury caused by glutamate. Methods: Rats were induced by chronic unpredictable mild stress, then divided into control, vehicle (distilled water), Xiaoyaosan, fluoxetine, vehicle (DMSO), Xiaoyaosan + Ly294002 and Ly294002 groups. Ly294002 was microinjected into the lateral ventricular catheterization at 5 mM. Xiaoyaosan (2.224 g/kg) and fluoxetine (2.0 mg/kg) were orally administered for three weeks. The open field test (OFT), forced swimming test (FST), and sucrose preference test (SPT) were used to assess depressive behavior. The glutamate and corticosterone (CORT) levels were detected by ELISA. Western blot, immunochemistry or immunofluorescence were used to detect the expressions of NR2B, MAP2, PI3K and P-AKT/Akt in the hippocampal CA1 region. The mRNA level of MAP2, NR2B and PI3K were detected by RT-qPCR. Results: Compared to the rats in control group, body weight and food intake of CUMS rats was decreased. CUMS rats also showed depression-like behavior as well as down regulate the NR2B and PI3K/Akt signaling pathway. Xiaoyaosan treatments could increase food intake and body weight as well as improved time spent in the central area, total distance traveled in the OFT. Xiaoyaosan could also decrease the immobility time as well as increase the sucrose preference in SPT. Moreover, xiaoyaosan decreased the level of glutamate in the hippocampal CA1 region and serum CORT in CUMS rats. Furthermore, xiaoyaosan improved the expression of MAP2 as well as increased the expression of NR2B, PI3K and the P-AKT/AKT ratio in the hippocampal CA1 region in the CUMS rats. Conclusion: Xiaoyaosan treatment can exert the antidepressant effect by rescuing hippocampal neurons loss induced by the glutamate-mediated excitotoxicity in CUMS rats. The underlying pathway maybe through NR2B and PI3K/Akt signaling pathways. These results may suggest the potential of Xiaoyaosan in preventing the development of depression.

Influence of progestational stress on BDNF and NMDARs in the hippocampus of male offspring and amelioration by Chaihu Shugan San.

BACKGROUND: Progestational stress has been proven to be a risk for the neural development of offspring, especially in the hippocampus. However, whether Chaihu Shugan San (CSS) can ameliorate hippocampal neural development via the regulation of brain-derived neurotrophic factor (BDNF), and N-methyl-D-aspartate receptors (NMDAR) 2A (NR2A) and 2B (NR2B), and the mechanism of such action remains unclear. METHODS: Thirty-six female rats were randomly allocated into control, chronic immobilization stress (CIS) and CSS groups according to the random number table, respectively. The male offspring were fed for 21 days after birth then randomly divided into the same three groups (6 rats/group) as the female rats. Female rats, except for the control group, underwent 21-day CIS to established a progestational stress anxiety-like model which was evaluated by body weight, the elevated plus-maze (EPM) test and serum dopamine (DA) measured using an enzyme-linked immunosorbent assay (ELISA). The expression levels of estrogen receptors (ERα/ERß) and progesterone receptor (PR) in female rat ovaries were quantified by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. The hippocampal tissue in the 21-day offspring was observed by hematoxylin-eosin (HE) staining. The concentration of BDNF, NR2A, and NR2B were measured by RT-qPCR and immunohistochemistry in the CA3 and dentate gyrus (DG) regions of offsprings' hippocampus. RESULTS: Compared with the female control group, significant differences in body weight, EPM test and DA concentration were observed in the CIS group, meanwhile, the concentration of ERα (P < 0.05), PR (P < 0.05) and ERß in the ovaries were decreased. In the offsprings' hippocampus of the CIS group, the chromatin of the nucleus was edge set and with condensed and irregular morphology nucleus, and the cytoplasm was unevenly stained with spaces around the cells, moreover, the expression levels of BDNF, NR2A, and NR2B were also declined (P < 0.05). However, Chaihu Shugan San reversed these changes, especially the BDNF in the DG region (P < 0.05), and NR2A and NR2B in the CA3 and DG region (P < 0.05). CONCLUSIONS: CSS could ameliorate the neural development of the hippocampus in offspring damaged by anxiety-like progestational stress in female rats via regulating the expression levels of ERα, ERß, and PR in female rat ovaries and BDNF, NR2A, and NR2B in the hippocampus of their offspring.

Effects of Xiaoyaosan on Depressive-Like Behaviors in Rats With Chronic Unpredictable Mild Stress Through HPA Axis Induced Astrocytic Activities.

ABSTRACT: Astrocytes in the hippocampus are immediately relevant to depressive-like behavior. By regulating their activities, Xiaoyaosan (XYS), a traditional Chinese medicine compound, works in the treatment of depression. OBJECTIVE: Chronic unpredictable mild stress (CUMS) rat model was established to observe the regulation of XYS. We investigated the behavioral changes of CUMS, the expression of corticosterone (CORT) of the hypothalamo-pituitary-adrenal (HPA) axis, the expression of Glu-NMDA receptor and astrocytes glial fibrillary acidic protein (GFAP) in the hippocampus. We also investigated whether these changes were linked to XYS. METHODS: 80 adult SD rats were randomly divided into four groups, control group, CUMS group, XYS group, and fluoxetine group. The rats in the control group and the CUMS group received 0.5 ml of deionized water once a day by intragastrically administration. Rats in the two treatment groups received XYS (2.224g/kg/d) and fluoxetine (2.0mg/kg/d) once a day, respectively. Rat hippocampus GFAP and Glu-NMDA receptor were respectively detected by real-time fluorescent quantitative PCR and western blot. The CORT of HPA axis was detected by Elisa. Body weight, food intake, and behavioral tests, such as open field tests, the sucrose preference test, and exhaustive swimming test, were used to assess depressive-like behavior in rats. RESULTS: In this work, significant behavioral changes and differences in expression of the CORT of HPA axis and hippocampal GFAP and Glu-NMDA receptor were presented in CUMS-exposed rats. Like fluoxetine, XYS improved CUMS-induced rat's body weight, food intake, and depressive-like behavior. The study also proved that XYS could reverse the CUMS-induced changes of the CORT of HPA axis and affect the astrocytic activities and down-regulate the NR2B subunit of NMDA receptor (NR2B) level in the hippocampus. CONCLUSION: Changes in the hippocampus GFAP and Glu-NMDA receptor may be an essential mechanism of depression. Besides, XYS may be critical to the treatment of depression by intervention the HPA axis, GFAP and Glu-NMDA receptor.

Free Wanderer Powder regulates AMPA receptor homeostasis in chronic restraint stress-induced rat model of depression with liver-depression and spleen-deficiency syndrome.

Free Wanderer Powder (FWP) is a classic formula for depression with digestive dysfunctions, i.e., liver-depression and spleen-deficiency syndrome (LDSDS) in Chinese Medicine. But its protective mechanism has not been fully clarified. Here a chronic restraint stress (CRS) induced rat model showed depression with LDSDS in food intake, metabolism, and behaviour tests. Then 75 rats were randomly divided, and received CRS and different treatment with behaviour tests. Expressions of c-Fos and AMPA-type glutamate receptor subunits GluR1-3 in hippocampus CA1, CA3, DG and amygdala BLA were detected by immunohistochemistry, western blot and RT-PCR, respectively. In CRS rats, FWP alleviated depressive behaviour and c-Fos expression. FWP suppressed the increasement of GluR1 in CA1 and DG, p-GluR1 in CA1, and p-GluR2 and GluR3 in BLA. FWP also blocked the decrease of GluR1 and Glur2/3 in CA3, p-GluR1 in CA3, and p-GluR2 in CA1 and CA3. Furthermore, constituents of FWP and their potential targets were explored using UHPLC-MS and systematic bioinformatics analysis. There were 23 constituents identified in FWP, 9 of which regulated glutamatergic synapse. Together, these results suggest that FWP contains effective constituents and alleviates depression with LDSDS by regulating AMPA-type glutamate receptor homeostasis in amygdala and hippocampus.

Study on urinary metabolomics of premenstrual dysphoric disorder patients with liver-qi depression syndrome treated with Xiaoyaosan: Study Protocol Clinical Trial (SPIRIT Compliant).

INTRODUCTION: Premenstrual dysphoric disorder (PMDD) is a serious form of premenstrual syndrome with mental symptoms as its main manifestation, which seriously affects women's health and daily life. Some basic research and clinical studies have shown that the Chinese herbal medicine of Xiaoyaosan can relieve the symptoms of mental disorders with few side effects. The aim of this study is to evaluate the clinical efficacy of Xiaoyaosan for treating PMDD with liver-qi depression syndrome. In addition, metabonomics and small molecular marker compounds closely related to the pathogenesis of PMDD are expected to be found, and mechanism of Xiaoyaosan is further explored from the metabolic level. METHODS AND ANALYSIS: This study is a clinical pilot trial. Thirty PMDD patients with liver-qi depression syndrome and thirty healthy participants will be recruited. Study participants will be assigned in a 1:1 ratio to 2 groups: a normal control group and Xiaoyaosan treatment group. The treatment group will receive the Chinese patent medicine of Xiaoyaosan for 3 menstrual cycles. The primary outcome is the syndrome change in the Daily Record of Severity of Problems (DRSP). The secondary outcome is improvement in TCM syndrome, which will be measured with TCM symptom score scale. Urine metabolism profiles of participants by liquid chromatograph-mass spectrometer (LC-MS) method will be measured to explore the mechanism of PMDD pathogenesis and action of Xiaoyaosan on PMDD. DISCUSSION: This trial will evaluate the effectiveness and the therapeutic mechanism from the metabolomics level of Xiaoyaosan in individuals with PMDD. If successful, the outcome of this trial will provide a viable treatment option for PMDD patients and objective evidence on the efficacy of Xiaoyaosan for PMDD. ETHICS AND DISSEMINATION: The trial has been approved by the Institutional Ethics Committee of Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine (file number: DZMEC-KY-2019-73). Written informed consent will be obtained from all participants. The results of the study will be published in peer-reviewed journals or communicated via yearly reports to funding bodies. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1900026296.

Hydroxysafflor yellow A induces autophagy in human liver cancer cells by regulating Beclin 1 and ERK expression.

Hydroxysafflor yellow A (HSYA) is a water-soluble component of the safflower (Carthamus tinctorius), and research has revealed that HSYA exhibits antitumor effects. In the present study, the effects of HSYA on the autophagy of a Hep-G2 liver cancer cell line, as well as the underlying mechanisms, were investigated. Hep-G2 cells were treated with HSYA and the viability of cells was measured using an MTT assay. Western blotting and immunofluorescence assays were performed to determine the expression of light chain 3 II (LC3-II) and p62, as well as the autophagy regulators Beclin 1 and ERK1/2. Transmission electron microscopy was performed to observe the formation of autophagosomes. The combined effects of HSYA and the autophagy inhibitor chloroquine (CQ) were also determined. The results revealed that the viability of Hep-G2 cells decreased with increasing concentrations of HSYA. Furthermore, LC3-II expression increased significantly and the level of p62 decreased significantly in the HYSA group compared with the control group. Additionally, an increase in Beclin 1 expression and a decrease in phosphorylated-ERK1/2 expression was observed in Hep-G2 cells treated with HYSA. Following treatment with CQ and HSYA, a significant increase in the viability of Hep-G2 cells was observed compared with the HSYA group. Collectively, the results indicated that HSYA induced autophagy by promoting the expression of Beclin 1 and inhibiting the phosphorylation of ERK in liver cancer cells. Therefore, HSYA may serve as a potential therapeutic agent for liver cancer.