Potential antidepressant effects of Traditional Chinese botanical drug formula Chaihu-Shugan-San and its active ingredients.

Background: Depression is a severe mental disorder that poses a significant threat to both the physical and mental wellbeing of individuals. Currently, there are various methods for treating depression, including traditional Chinese herbal formulations like Chaihu-Shugan-San (CSS), which have shown effective antidepressant effects in both clinical and animal research. Objective: This review aims to provide a comprehensive synthesis of evidence related to CSS, considering both preclinical and clinical studies, to uncover its potential multi-level, multi-pathway, and multi-target mechanisms for treating depression and identify its active ingredients. Methods: A thorough search was conducted in electronic databases, including PubMed, MEDLINE, Web of Science, Google Scholar, CNKI, and Wanfang, using keywords such as "Chaihu Shugan" and "depression" to retrieve relevant literature on CSS and its active ingredients. The review process adhered to the PRISMA guidelines. Results: This review consolidates the mechanisms underlying antidepressant effects of CSS and its active ingredients. It emphasizes its involvement in the regulation of monoaminergic neurotransmitter systems, synaptic plasticity, and the hypothalamic-pituitary-adrenal axis, among other aspects. Conclusion: CSS exerts a pivotal role in treating depression through various pathways, including the monoaminergic neurotransmitter system, the hypothalamic-pituitary-adrenal axis, synaptic plasticity, inflammation, brain-derived neurotrophic factor levels, and the brain-gut axis. This review facilitates a comprehensive understanding of the current state of CSS research, fostering an in-depth exploration of the etiological mechanisms of depression and the potential discovery of novel antidepressant drugs.

A Serum Pharmacochemistry and Network Pharmacology-based Approach to Study the Anti-depressant Effect of Chaihu-Shugan San.

AIMS: The aim of this study is to explore the anti-depressant mechanism of Chaihu- Shugan San based on serum medicinal chemistry and network pharmacology methods. BACKGROUND: Depression lacks effective treatments, with current anti-depressants ineffective in 40% of patients. Chaihu-Shugan San (CHSGS) is a well-known traditional Chinese medicine compound to treat depression. However, the chemical components and the underlying mechanisms targeting the liver and brain in the anti-depressant effects of CHSGS need to be elucidated. METHODS: The chemical components of CHSGS in most current network pharmacology studies are screened from TCMSP and TCMID databases. In this study, we investigated the mechanism and material basis of soothing the liver and relieving depression in the treatment of depression by CHSGS based on serum pharmacochemistry. The anti-depressant mechanism of CHSGS was further verified by proteomics and high-throughput data. RESULTS: Through serum medicinal chemistry, we obtained 9 bioactive substances of CHSGS. These ingredients have good human oral bioavailability and are non-toxic. Based on liver ChIPseq data, CHSGS acts on 8 targets specifically localized in the liver, such as FGA, FGB, and FGG. The main contributors to CHSGS soothing the liver qi targets are hesperetin, nobiletin, ferulic acid, naringin and albiflorin. In addition, network pharmacology analysis identified 9 blood components of CHSGS that corresponded to 63 anti-depressant targets in the brain. Among them, nobiletin has the largest number of anti-depressant targets, followed by glycyrrhizic acid, ferulic acid, albiflorin and hesperetin. We also validated the anti-depressant mechanism of CHSGS based on hippocampal proteomics. CHSGS exerts anti-depressant effects on synaptic structure and neuronal function by targeting multiple synapse related proteins. CONCLUSION: This study not only provides a theoretical basis for further expanding the clinical application of CHSGS, but also provides a series of potential lead compounds for the development of depression drugs.

Effects of Chaihu Shugan San on Brain Functional Network Connectivity in the Hippocampus of a Perimenopausal Depression Rat Model.

In this study, we used Chaihu Shugan San (CSS), a traditional Chinese herbal formula, as a probe to investigate the involvement of brain functional network connectivity and hippocampus energy metabolism in perimenopausal depression. A network pharmacology approach was performed to discover the underlying mechanisms of CSS in improving perimenopausal depression, which were verified in perimenopausal depression rat models. Network pharmacology analysis indicated that complex mechanisms of energy metabolism, neurotransmitter metabolism, inflammation, and hormone metabolic processes were closely associated with the anti-depressive effects of CSS. Thus, the serum concentrations of estradiol (E2), glutamate (Glu), and 5-hydroxytryptamine (5-HT) were detected by ELISA. The brain functional network connectivity between the hippocampus and adjacent brain regions was evaluated using resting-state functional magnetic resonance imaging (fMRI). A targeted metabolomic analysis of the hippocampal tricarboxylic acid cycle was also performed to measure the changes in hippocampal energy metabolism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). CSS treatment significantly improved the behavioral performance, decreased the serum Glu levels, and increased the serum 5-HT levels of PMS + CUMS rats. The brain functional connectivity between the hippocampus and other brain regions was significantly changed by PMS + CUMS processes but improved by CSS treatment. Moreover, among the metabolites in the hippocampal tricarboxylic acid cycle, the concentrations of citrate and the upregulation of isocitrate and downregulation of guanosine triphosphate (GTP) in PMS + CUMS rats could be significantly improved by CSS treatment. A brain functional network connectivity mechanism may be involved in perimenopausal depression, wherein the hippocampal tricarboxylic acid cycle plays a vital role.

Reporting quality and risk of bias assessment of animal research on Chaihu-Shugan-San for depression: A systematic review.

Objective: Chaihu-Shugan-San (CSS) is a traditional Chinese medicine formula employed to treat depression. We aim to conduct a reporting quality assessment and risk of bias evaluation of animal research on CSS for depression. Methods: To acquire eligible studies, two reviewers searched plentiful databases from inception to October 23rd, 2021. Reporting quality assessment and risk of bias assessment of the included animal studies were evaluated by using Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines and the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool, respectively. Results: The initial search identified 720 records, while only 30 studies were included. The result of the reporting quality assessment was inferior, items 17 and 19 were not reported at all. The details of five items (items 3, 6, 7, 10, and 18) were not reported. The outcome of the risk of bias assessment suggested that half of the entries (5/10) displayed an unclear risk of bias and a high risk of bias. Blinding with regard to performance bias and detection bias revealed an unclear risk of bias (100%), followed by baseline characteristics (76.67%) and sequence generation (60%). Random outcome assessment showed a high risk of bias (100%). Conclusion: The included animal studies exhibited methodological defects and imprecise reporting. Hence, the ARRIVE guidelines and SYRCLE's RoB tool should be disseminated among basic medical researchers examining CSS for depression to publish studies with low risk of bias and sufficient reporting so that the animal research can promptly be transformed into clinical research.

Chaihu-Shugan-San inhibits neuroinflammation in the treatment of post-stroke depression through the JAK/STAT3-GSK3ß/PTEN/Akt pathway.

Post-stroke depression (PSD) is one of the most common neuropsychiatric consequence of stroke, affecting cognitive function, recovery of somatic function, and patient survival. The aim of this study was to evaluate whether Chaihu-Shugan-San, a traditional Chinese medicine formula used clinically to treat depression, could improve symptoms in a rat model for PSD, to investigate the potential mechanisms, and to validate the findings in an in vitro oxygen and glucose deprivation (OGD) model. Male rats were subjected to middle cerebral artery occlusion (MCAO) and to chronic unpredictable mild stress (CUMS). The rats were then allocated to experimental groups (n = 15) that were treated with Chaihu-Shugan-San, a JAK-STAT3 inhibitor, a GSK3ß overexpressing virus, or an empty virus (control). The subjects allocated to each group, as well as those that received no treatment and rats that did not undergo MCAO/CUMS, were then subjected to forced swimming, tail suspension, and sugar water preference tests, and their neurological deficit score was determined. Inflammatory factor levels and the expression of proteins related to the JAK/STAT3-GSK3ß/PTEN/Akt pathway were measured, and the synaptic ultrastructure was observed using transmission electron microscopy. Flow cytometry showed microglia polarization towards the M1 phenotype in an in vitro PSD model, which was reversed after treatment with a GSK3ß overexpression virus, Chaihu-Shugan-San, or a JAK-STAT3 inhibitor. The results showed that Chaihu-Shugan-San has a therapeutic effect on an in vivo model for PSD and can regulate microglia polarization through the activation of the JAK/STAT3-GSK3ß/PTEN/Akt pathway, suggesting that it exerts its effect via the inhibition of neuroinflammation.

Chaihu-Shugan-San ameliorates tumor growth in prostate cancer promoted by depression via modulating sphingolipid and glycerinphospholipid metabolism.

Background: Psychologic depression is a pivotal pathological characteristic and has been shown to promote prostate cancer (PCa) progression. Chaihu-Shugan-San (CSS), a well-known Chinese herbal decoction, exhibits efficacy in the treatment of stress-accelerated PCa. However, the underlying mechanism of CSS in resisting PCa growth is still unknown, and further study is needed. Objective: To evaluate the effects of CSS on stress-accelerated PCa in a BALB/C nude mice model and to investigate the underlying mechanisms. Methods: PC-3 cells were implanted into BALB/C nude mice, and the stressed mice were exposed to chronic unpredictable mild stress (CUMS) to study the effects of CSS. The PCa growth were evaluated by tumor volume and tumor weight. Analyses of depression-like behaviors were evaluated by sucrose consumption test, tail suspension test and open field test. Network pharmacology was used to analyze the potential targets and signaling pathways of CSS against PCa. Untargeted lipidomics were used to analyze the serum lipid profiles and further elucidate the possible mechanism. Results: In the CUMS stressed PCa mice, CSS can restrain tumor growth with reduced tumor volume and tumor weight, and depression-like behaviors with increased sucrose consumption, reduced immobility duration, and increased total distance and center distance. Network pharmacology suggested that the lipid metabolism-related pathways are the most likely potential targets of CSS against PCa. Using untargeted lipidomics analysis, 62 lipids were found to have significant changes in PCa mice under CUMS treatment. The levels of glycerophospholipids containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylglycerol (PG), except PC (18:0_22:6) and PC (18:0_20:4), were significantly increased. Likewise, the levels of all sphingolipids (including sphingomyelin (SM), ceramides (Cer) and hexosyl-1-ceramide (Hex1Cer)) and diglyceride (DG) (32:1e) were significantly increased. CSS water extract was found to contribute to restore 32 lipids including 6 sphingolipids, 25 glycerophospholipids and 1 glyceride. Conclusion: This study is the first to delineate the lipid profile of stressed PCa BALB/C nude mice using untargeted lipidomics analysis. CSS restrained tumor growth and ameliorated depression-like behaviors by reprogramming lipid metabolism. Intervention of lipid metabolism could be a preventive and therapeutic approach for PCa patients with depression.

SIRT1/FOXO1 Axis-Mediated Hippocampal Angiogenesis is Involved in the Antidepressant Effect of Chaihu Shugan San.

Objective: Chaihu Shugan San (CSS) has a long history for treating major depressive disorder (MDD), which has been verified effectively and safely in clinical studies. Deficient angiogenesis plays important roles in MDD. However, the underlying mechanisms of CSS on angiogenesis remain poorly understood. Methods: Network pharmacology analysis was applied to explore the potential angiogenic targets and pathways between CSS and MDD. These targets would be validated in chronic unpredictable mild stress (CUMS)-induced depressive-like mice by Western blots, immunofluorescence, and immunohistochemistry. Then, the underlying molecular mechanisms were further investigated in brain microvascular endothelial cells (BMVECs) with CSS-containing serum by Western blots and immunofluorescence. Results: Network pharmacology analysis showed that the antidepressant role of CSS was closely associated with Silent information regulator protein 1 (SIRT1)/Forkhead box O1 (FOXO1) axis-mediated angiogenesis. This prediction was confirmed in the following experiments. CSS induced angiogenesis, increased SIRT1 expression, and decreased FOXO1 expression in the hippocampus of CUMS mice. Five percent CSS-containing serum produced a significant increase in BMVECs proliferation, migration, and tube formation, but these effects were reduced by SIRT1 silencing. CSS serum could also promote FOXO1 translocation to the cytoplasm through SIRT1 signaling, which triggered FOXO1 protein degradation. What is more, CSS upregulated VEGFA and BDNF expressions not only in the hippocampus of depressive mice but also in BMVECs supernatants. Of note, these trophic factors play important roles in promoting neurogenesis. Conclusion: The study showed that CSS could promote angiogenesis and neurogenesis in the hippocampus of CUMS-induced mice. The underlying molecular mechanism involves the SIRT1/FOXO1 axis and subsequent regulation of VEGFA and BDNF. These findings provide novel insight into CSS drug development, and targeting the SIRT1/FOXO1 axis might be a promising strategy to treat MDD.

Exploring mechanisms of Chaihu-Shugan-San against liver fibrosis by integrated multi-omics and network pharmacology approach.

Chaihu-Shugan-San (CHSGS), a noted traditional Chinese medicine formula, has been used as a complementary and alternative therapy for liver fibrosis. However, the antifibrotic mechanisms of CHSGS still remain unclear. Thus, we used network pharmacology approach in combination with single cell and bulk transcriptomics to elucidate the antifibrotic mechanisms of CHSGS. We first screened out 134 bioactive ingredients of CHSGS through the defined criteria. Then, 1150 genes were predicted to be targets for CHSGS, while 625 liver fibrosis-associated genes were identified by single cell transcriptomics analysis. Next, 71 intersecting genes of CHSGS and liver fibrosis were defined as the therapeutic targets in CHSGS against liver fibrosis. Further, 21 core targets and 12 core ingredients of CHSGS against liver fibrosis were also identified. Meanwhile, enrichment analyses of core targets highlighted that the key mechanisms of CHSGS against liver fibrosis include modulation of inflammation responses, inhibition of angiogenesis, and regulation of ECM remodeling, of which the most important mechanism was the regulation of ECM remodeling. The molecular docking simulation validated strong binding affinity between the core targets and core ingredients. Furthermore, 62-gene signature may be used for determining the prognosis in cirrhotic patients based on the results of ssGSEA-Cox analysis. In conclusion, the present study revealed the multiple pharmacological targets and therapeutic mechanisms of CHSGS against liver fibrosis, which may thus serve as an effective antifibrotic therapy. Meanwhile, CHSGS may improve survival of patients with liver cirrhosis by the interaction of 62-gene signature.

Exploration of a Brain-Liver-Communication-Related Mechanism Involved in the Experimental Perimenopausal Depression Rat Model using Chaihu-Shugan-San.

Existing research suggests the involvement of a brain-liver-communication-related mechanism in the occurrence of depression. In this study, we selected Chaihu-Shugan-San (CSS), a traditional Chinese herbal medicine that can simultaneously affect liver and depression, as a probe to investigate the involvement of the brain-liver-communication-related mechanism in perimenopausal depression. A total of 50 experimental perimenopausal depression rat models were established by ovariectomy surgery (PMS) followed by chronic unpredictable mild stress (CUMS) processes. Animals underwent CSS treatment or treatments with CSS + Ly294002, an inhibitor of the PI3K/Akt signalling pathway. We observed the behavioural performances of depression and anxiety, serum concentrations of biochemical indices, serum estrogen two levels, hippocampal 5-HT and NE levels and the morphological changes in liver tissues. The protein and mRNA expressions of PI3K and Akt were also evaluated. CSS treatment significantly ameliorated the behavioural performance, partial biochemical indices and the morphological changes in the liver tissues of PMS + CUMS rats. Ly294002 partially inhibited the CSS effects. The expressions of PI3K and Akt were significantly downregulated by PMS + CUMS processes but upregulated by CSS treatment, which could be significantly suppressed by Ly294002. A brain-liver-communication-related mechanism may be involved in perimenopausal depression, where the PI3K/Akt signalling pathway plays a vital role.

Network Pharmacology and Experimental Evidence: PI3K/AKT Signaling Pathway is Involved in the Antidepressive Roles of Chaihu Shugan San.

OBJECTIVE: Chaihu Shugan San (CSS) is a common antidepressant prescription in traditional Chinese medicines. However, its active ingredients and mechanisms are unknown. The aim of this study was to explore the potential active ingredients and pharmacological mechanisms of CSS for the treatment of major depressive disorder (MDD). METHODS: Active compounds in CSS were screened using the Traditional Chinese Medicine Systems Pharmacology database. Compound-related targets were retrieved using the SwissTargetPrediction database. MDD-related targets were determined using DisGeNET, Therapeutic Target Database and DrugBank databases. The common targets of active compounds in CSS and MDD were retained to construct a compound-MDD target network. Then, functional enrichment analysis and protein-protein interaction analysis were performed to identify hub targets and explore the underlying molecular mechanisms. Finally, hub-targeted genes and pathways were validated by Western blotting and immunofluorescence using chronic unpredictable mild stress (CUMS) mice with or without CSS treatment. The affinities between the active compounds in CSS and hub-targeted genes were evaluated by molecular docking. RESULTS: Network pharmacology analysis revealed 24 potential targets for treatment of MDD by CSS. Functional enrichment analysis showed that PI3K/AKT signaling pathway was likely to be evidently affected by CSS in the treatment of MDD. In vivo experiments showed that CSS could improve depressive-like behaviors and promote neurogenesis in CUMS mice. Furthermore, CSS could increase phosphorylated (p) PI3K/PI3K and pAKT/AKT levels and decrease the pGSK3ß/GSK3ß level in the hippocampus of CUMS mice. The active compounds mainly included quercetin and luteolin, which showed good docking scores targeting the PI3K protein. CONCLUSION: This network pharmacological and experimental study highlights that the PI3K/AKT pathway is the potential mechanism by which CSS is involved in MDD treatment. Quercetin, luteolin, and kaempferol are probable active compounds in CSS, and these results might provide valuable guidance for further studies of MDD treatment.

Chaihu-Shugan-San (Shihosogansan) alleviates restraint stress-generated anxiety and depression in mice by regulating NF-κB-mediated BDNF expression through the modulation of gut microbiota.

BACKGROUND: Chaihu-Shugan-San (CSS, named Shihosogansan in Korean), a Chinese traditional medicine, is frequently used to treat anxiety and depression. Psychiatric disorders including depression are associated with gut dysbiosis. Therefore, to comprehend gut microbiota-involved anti-depressive effect of CSS, we examined its effect on restraint stress (RS)-induced depression and gut dysbiosis in mice METHODS: CSS was extracted with water in boiling water bath and freeze-dried. Anxiety and depression was induced in C57BL/6 mice by exposure to RS. Anxiety- and depression-like behaviors were measured in the light/dark transition and elevated plus maze tasks, forced swimming test, and tail suspension test. Biomarkers were assayed by using the enzyme-linked immunosorbent assay and immunoblotting. The gut microbiota composition was analyzed by Illumina iSeq sequencer. RESULTS: CSS significantly reduced the RS-induced anxiety- and depression-like behaviors in mice. CSS suppressed the RS-induced activation of NF-κB and expression of interleukin (IL)-6 and increased the RS-suppressed expression of brain-derived neurotrophic factor (BDNF). Furthermore, CSS suppressed the RS-induced IL-6 and corticosterone level in the blood and IL-6 expression and myeloperoxidase activity in the colon. CSS decreased the RS-induced γ-Proteobacteria population in gut microbiota, while the RS-suppressed Lactobacillaceae, Prevotellaceae, and AC160630_f populations increased. Fecal transplantation of vehicle-treated control or RS/CSS-treated mice into RS-exposed mice significantly mitigated RS-induced anxity- and depression-like behaviors, suppressed the NF-κB activation in the hippocampus and colon, and reduced the IL-6 and corticosterone levels in the blood. These fecal microbiota transplantations suppressed RS-induced Desulfovibrionaceae and γ-Proteobacteria populations and increased RS-suppressed Lactobacillaceae and Prevotellaceae poulation in the gut microbiota. CONCLUSIONS: CSS alleviated anxiety and depression by inducing NF-κB-involved BDNF expression through the regulation of gut inflammation and microbiota.

Mechanism underlying effect of Chaihu Shugan San on major depressive disorder: a network pharmacology-based study.

OBJECTIVE: To investigate the mechanism underpinning the effect of Chaihu Shugan San ( CHSGS) on major depressive disorder (MDD). METHODS: We searched the compound components of from seven herbal ingredients of CHSGS from TCMSP, SymMap, ETCM, NPASS databases, and the chemical structure of the compound from PubChem, and collected the compound targets from TCMSP and TargetNet databases, and MDD-related targets from DiseaseGene Network. We established protein-protein interaction in the STRING database. Through gene mapping, topology analysis and enrichment analysis, the core targets and pathways of CHSGS for MDD, and the involved biological processes (BP), cell components (CC), and molecular functions (MF) were predicted. RESULTS: We collected a total of 1135 CHSGS compounds. After screening by ADME standards and the five rules of Ribinski, we obtained 99 different chemical components with different chemical structures, and related targets of 183 different CHSGS compounds. In the DiseaseGene Network, a total of 740 relevant targets for MDD were collected. Through gene mapping and topological analysis, 62 related targets of CHSGS for MDD, 24 targets with topological Chinese herbal medicine were obtained. Through enrichment analysis, 10 relevant pathways and 3 core pathways were obtained with the involvement of 127 BP, 27 CC, and 43 MF. CONCLUSION: There are multiple targets and signaling pathways are involved in the action of CHSGS in the treatment of MDD.

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.

Proteomics Study Reveals the Anti-Depressive Mechanisms and the Compatibility Advantage of Chaihu-Shugan-San in a Rat Model of Chronic Unpredictable Mild Stress.

Background: Chaihu-Shugan-San is a classical prescription to treat depression. According to the traditional Chinese medicine (TCM) principle, the 2 decomposed recipes in Chaihu-Shugan-San exert synergistic effects, including Shu Gan (stagnated Gan-Qi dispersion) and Rou Gan (Gan nourishment to alleviate pain). However, the specific mechanism of Chaihu-Shugan-San on depression and its compatibility rule remain to be explored. Objective: We aimed to explore the anti-depression mechanisms and analyze the advantage of TCM compatibility of Chaihu-Shugan-San. Methods: The chronic unpredictable mild stress (CUMS) rat model was established. Antidepressant effects were evaluated by sucrose preference test (SPT), and forced swimming test (FST). Tandem Mass Tag (TMT)-based quantitative proteomics of the hippocampus was used to obtain differentially expressed proteins (DEPs). Bioinformatics analysis including Gene Ontology (GO), pathway enrichment, and protein-protein interaction (PPI) networks was utilized to study the DEPs connections. At last, the achieved key targets were verified by western blotting. Results: Chaihu-Shugan-San increased weight gain and food intake, as well as exhibited better therapeutic effects including enhanced sucrose preference and extended immobility time when compared with its decomposed recipes. Proteomics showed Chaihu-Shugan-San, Shu Gan, and Rou Gan regulated 110, 12, and 407 DEPs, respectively. Compared with Shu Gan or Rou Gan alone, the expression of 22 proteins was additionally changed by Chaihu-Shugan-San treatment, whereas the expression of 323 proteins whose expression was changed by Shu Gan or Rou Gan alone were not changed by Chaihu-Shugan-San treatment. Bioinformatics analysis demonstrated that Chaihu-Shugan-San affected neurotransmitter's release and transmission cycle (e.g., γ-aminobutyric acid (GABA), glutamate, serotonin, norepinephrine, dopamine, and acetylcholine). GABA release pathway is also targeted by the 22 DEPs. Unexpectedly, only 2 pathways were enriched by the 323 DEPs: Metabolism and Cellular responses to external stimuli. Lastly, the expression of Gad2, Vamp2, and Pde2a was verified by western blotting. Conclusions: Chaihu-Shugan-San treats depression via multiple targets and pathways, which may include regulations of 110 DEPs and some neurotransmitter's transmission cycle. Compared with Shu Gan and Rou Gan, the 22 Chaihu-Shugan-San advanced proteins and the affected GABA pathway may be the advantages of Chaihu-Shugan-San compatibility. This research offers data and theory support for the clinical application of Chaihu-Shugan-San.

Simultaneous quantification of nine components in the plasma of depressed rats after oral administration of Chaihu-Shugan-San by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry and its application to pharmacokinetic studies.

Chaihu-Shugan-San (CSS), a classic Chinese formula, has long been used to treat depression. For a better and rational use of this formula, here, we investigated the comprehensive pharmacokinetic features of multiple ingredients from CSS using ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS) in depressed rats. Force swimming experiments were conducted to establish a rat model of depression. Prolonged immobility time, increased plasma adrenocorticotropic hormone, and decreased plasma 5-hydroxytryptamine (5-HT) and dopamine (DA) were confirmed in this model. Nine compounds from CSS, including ferulic acid, naringin, hesperidin, meranzin hydrate, glycyrrhizic acid, saikosaponin A, nobiletin, and hesperetin, were simultaneously determined in plasma samples. Sulfamethoxazole and schisandrin were used as internal standards. The separation was performed on a C18 column with gradient elution over 10 min, and detection was executed using multiple reaction monitoring (MRM) in the positive ionization mode. The optimized MRM transitions showed no interference in rat plasma. Validation parameters were all in accordance with the current criterion. The established method was successfully applied to the pharmacokinetic study of these nine components after the oral administration of CSS to depressed rats. This study provides a chemical basis for the clinical application of this formula.

Effects of Chaihu-Shugan-San on Small Intestinal Interstitial Cells of Cajal in Mice.

Chaihu-Shugan-San (CSS) has been widely used as an alternative treatment for gastrointestinal (GI) diseases in East Asia. Interstitial cells of Cajal (ICCs) are pacemakers in the GI tract. In the present study, we examined the action of CSS on pacemaker potentials in cultured ICCs from the mouse small intestine in vitro and on GI motility in vivo. We used the electrophysiological methods to measure the pacemaker potentials in ICCs. GI motility was investigated by measuring intestinal transit rates (ITR). CSS inhibited the pacemaker potentials in a dose-dependent manner. The capsazepine did not block the effect of CSS. However, the effects of CSS were blocked by glibenclamide. In addition, NG-nitro-L-arginine methyl ester (L-NAME) also blocked the CSS-induced effects. Pretreatment with SQ-22536 or with KT-5720 did not suppress the effects of CSS; however, pretreatment with ODQ or KT-5823 did. Furthermore, CSS significantly suppressed murine ITR enhancement by neostigmine in vivo. These results suggest that CSS exerts inhibitory effects on the pacemaker potentials of ICCs via nitric oxide (NO)/cGMP and ATP-sensitive K+ channel dependent and transient receptor potential vanilloid 1 (TRPV1) channel independent pathways. Accordingly, CSS could provide the basis for the development of new treatments for GI motility dysfunction.

A combined molecular biology and network pharmacology approach to investigate the multi-target mechanisms of Chaihu Shugan San on Alzheimer's disease.

Chaihu Shugan San (CSS) is a well-known herbal formula used to nourish liver and blood, promote blood circulation and Qi flow in Traditional Chinese Medicine. Modern pharmacological studies and clinical uses showed that CSS could ameliorate cognitive dysfunction of Alzheimer's disease (AD). The present study aimed to elucidate the multi-target mechanisms of CSS on AD using network pharmacology analysis and verify its effect by biological experiments. Firstly, a total of 152 active compounds in CSS, 520 predicted biological targets and 160 AD-related targets were identified. Subsequently, the networks including herb-compound-target network, AD-target network, and CSS potential target-AD target network were constructed. 60 key targets highly responsible for the beneficial effect of CSS on AD were identified by central network topological analysis. They were significantly characterized as nuclear or cytoplasmic proteins with molecular function of protein binding. They were also enriched in various biological processes through PI3K-Akt signaling pathway, MAPK signaling pathway and HIF signaling pathway by GO function and KEGG pathway enrichment analysis. Pretreatment with CSS ameliorated Aß-induced neural cell death and reduced the number of apoptotic cells in differentiated PC12 cells. Moreover, increased phosphorylation of Akt accompanied with decreased Bax expression was found after CSS pretreatment, suggesting that Akt signaling pathway was involved in the protective effect of CSS against neural cells death. The present study systematically revealed the multi-target mechanisms of CSS on AD using network pharmacology approach, as well as validated the protective effect of CSS against Aß-induced neural cells death through Akt signaling pathway. It provided indications for further mechanistic studies and also for the development of CSS as a potential treatment for AD patients.

Chaihu-Shugan-San and absorbed meranzin hydrate induce anti-atherosclerosis and behavioral improvements in high-fat diet ApoE-/- mice via anti-inflammatory and BDNF-TrkB pathway.

The comorbidity of coronary heart disease (CHD) and depression in patients is extremely prevalent, with a rate from 20 to 50%, while depression-like behaviors exist in a larger percentage of patients. Therefore, the study of comorbidities is particularly urgent. Chaihu-Shugan-San (CSS), a classical traditional Chinese medicine formula, has been used to treat CHD with depression for hundreds of years. However, the mechanism of its action on comorbidities remains unclear. Here, we focused on the behavioral changes in ApoE-/- mice fed a high-fat diet (HFD) and elucidated the mechanism of CSS and its main absorbed component, meranzin hydrate (MH), as an anti-atherosclerosis and anti-depression agent. In the present study, mice were fed an HFD for 16 weeks and were intragastrically administered high and low doses of CSS and MH. Depressive-like behaviors were evaluated by the sucrose preference test, the open-field test, the light-dark test and the tail-suspension test, after which atherosclerotic plaques, plasma lipids, inflammatory cytokine levels and the expression of BDNF/TrkB were measured. We demonstrated that the atherosclerosis model group exhibited significant depressant behaviors. Moreover, CSS inhibited depressive-like behavioral changes, reduced atherosclerotic plaque areas, plasma total cholesterol, triglycerides, LDL-cholesterol levels and inflammatory cytokines including TNF-α, IL-1ß, and IL-6 in plasma and hippocampi, increased the protein and mRNA expression of BDNF and TrkB in the aorta and the hippocampus. Interestingly, MH, the main component in CSS that is absorbed in the plasma and hippocampus, exerted effects similar to those of CSS. In addition, MH increased the protein and mRNA expression of BDNF and TrkB in human umbilical vein endothelial cells (HUVECs) induced by LPS. Collectively, these results suggest that MH represents the CSS decoction, induces anti-atherosclerosis effects and improves depression-like behaviors in HFD-fed ApoE-/- mice. Moreover, the regulation of proinflammatory factors and BDNF-TrkB signaling are possibly involved in this process. Our findings indicate that MH is a potential phytochemical compound for the prevention of the comorbidity of atherosclerosis and depression.

Clinical Efficacy of Xiaoai Jiedu Formula Combined with FEC Chemotherapy on Breast Cancer Patients / 中国实验方剂学杂志

Objective:To observe effect of Xiaoai Jiedu formula combined with fluorouracil (5-FU)+epirubicin (EPI)+cyclophosphamide (CTX) (FEC) chemotherapy regimen on immune function, tumor index, traditional Chinese medicine (TCM) symptom scale score and adverse reactions of patients with breast cancer. Method:A total of 60 patients with breast cancer were randomly divided into study group and control group. FEC (CTX 0.6 g·m-2,d1,EPI 100 mg·m-2,d1,5-FU 0.5 g·m-2,d1) regimen was used in study group, and Xiaoai Jiedu formula recipe was used for two consecutive cycles. FEC regimen was used in control group only. After 2 cycles, immunological changes, tumor index, TCM symptom score and adverse reactions were analyzed and compared between two groups. Result:There was not significant difference in immune indexes between two groups before treatment, but statistically significant differences after treatment (PPPPConclusion:Xiaoai Jiedu formula combined with FEC chemotherapy can improve clinical efficacy and alleviate clinical symptoms of patients.

Chaihu-Shugan-San exerts an antidepressive effect by downregulating miR-124 and releasing inhibition of the MAPK14 and Gria3 signaling pathways.

Dysregulation of miR-124 has been reported to be involved in the pathophysiology of depression. Chaihu-Shugan-San, a traditional Chinese medicine, has antidepressive activity; however, the underlying mechanisms remain unclear. In this study, to generate a rodent model of depression, rats were subjected to a combination of solitary confinement and chronic unpredictable mild stress for 28 days. Rats were intragastrically administered Chaihu-Shugan-San (2.835 mL/kg/d) for 4 weeks, once a day. Real-time reverse-transcription quantitative polymerase chain reaction, miRNA microarray, western blot assay and transmission electron microscopy demonstrated that Chaihu-Shugan-San downregulated miR-124 expression and upregulated the mRNA and protein levels of mitogen-activated protein kinase 14 (MAPK14) and glutamate receptor subunit 3 (Gria3). Chaihu-Shugan-San also promoted synapse formation in the hippocampus. The open field test, sucrose consumption test and forced swimming test were used to assess depression-like behavior. After intragastric administration of Chaihu-Shugan-San, sucrose consumption increased, while the depressive behaviors were substantially reduced. Together, these findings suggest that Chaihu-Shugan-San exerts an antidepressant-like effect by downregulating miR-124 expression and by releasing the inhibition of the MAPK14 and Gria3 signaling pathways.