Stable Isotope Tracer Technique and Network Pharmacology to Reveal Antidepressant Targets and Active Components of Xiaoyao San.

In recent years, the research of mitochondrial dysfunction in depression has drawn the focus of researchers. Our research group previously found that Xiaoyao San (XYS) has improved the mitochondrial structure and the blocked tricarboxylic acid cycle (TCA cycle) in the hippocampal tissue of chronic unpredictable mild stress (CUMS) rats. However, the specific targets and active components of XYS remain unclear, and the potential to improve hippocampal mitochondrial TCA cycle disorder was also unexplored. In this research, a strategy to combine stable isotope-resolved metabolomics (SIRM), network pharmacology and transmission electron microscopy (TEM) was used to explore the potential, targets of action, and active components of XYS to improve hippocampal mitochondrial TCA cycle disorder of CUMS rats. The results of TEM showed that the ultrastructure of hippocampal mitochondria could be improved by XYS. A combination of SIRM and molecular docking showed that pyruvate carboxylase (PC), ATP citrate lyase (ACLK), glutamate dehydrogenase (GLDH), glutamate oxaloacetate transaminase (GOT) and pyruvate dehydrogenase (PDH) were targets of XYS to improve TCA cycle disorder. In addition, troxerutin was found to be the most potential active component of XYS to improve TCA cycle disorder. The above research results can provide new insights for the development of antidepressant drugs.

[Mechanism of Xiaoyao San in treatment of depression,breast hyperplasia,and functional dyspepsia based on network pharmacology].

This study aimed to explore the mechanism of Xiaoyao San(XYS) in the treatment of three diseases of liver depression and spleen deficiency, ie, depression, breast hyperplasia, and functional dyspepsia, and to provide a theoretical basis for the interpretation of the scientific connotation of "treating different diseases with the same method" of traditional Chinese medicines. Traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP) was used to screen the active components of XYS which underwent principal component analysis(PCA) with the available drugs for these three diseases to determine the corresponding biological activities. The targets of XYS on depression, breast hyperplasia, and functional dyspepsia were obtained from GeneCards, TTD, CTD, and DrugBank databases. Cytoscape was used to plot the "individual herbal medicine-active components-potential targets" network. The resulting key targets were subjected to Kyoto encyclopedia of genes and genomes(KEGG) pathway analysis and gene ontology(GO) enrichment analysis. A total of 121 active components of XYS and 38 common targets in the treatment of depression, breast hyperplasia, and functional dyspepsia were collected. The key biological pathways were identified, including advanced glycation and products(AGEs)-receptor for advanced glycation and products(RAGE) signaling pathway in diabetic complications, HIF-1 signaling pathway, and cancer-related pathways. The key targets of XYS in the treatment of depression, breast hyperplasia, and functional dyspepsia included IL6, IL4, and TNF, and the key components were kaempferol, quercetin, aloe-emodin, etc. As revealed by the molecular docking, a strong affinity was observed between the key components and the key targets, which confirmed the results. The therapeutic efficacy of XYS in the treatment of diseases of liver depression and spleen deficiency was presumedly achieved by reducing the inflammatory reactions. The current findings are expected to provide novel research ideas and approaches to classify the scientific connotation of "treating different diseases with the same method" of Chinese medicines, as well as a theoretical basis for understanding the mechanism of XYS and exploring its clinical applications.

[Effect of Xiaoyao San on OVX combined with CUS anxiety and depression model rats based on hippocampal microglia M1 polarization].

To investigate the anti-anxiety and anti-depression effect and mechanism of Xiaoyao San on rats with ovariectomy(OVX) combined with chronic unpredictable stress(CUS) model. The model of perimenopausal depression was established by OVX and CUS; the level of anxiety and depression was evaluated by open field test; the levels of interleukin-1ß(IL-1ß) and interleukin-6(IL-6) mRNA in rat hippocampus were detected by Real-time qPCR; double staining immunofluorescence was used to detect the expression of ionized calcium binding adaptor molecule-1(Iba-1) and inducible nitric oxide synthase(iNOS) in microglia of rat dentate gyrus(DG); Western blot was used to detect the protein expression of Iba-1 and iNOS of microglia in DG region of rat hippocampus. The results showed that in the model group, the number of horizontal movement, the number of vertical movement and central residence time were significantly reduced, and the grooming time was significantly prolonged(P<0.05 or P<0.01); the levels of IL-1ß and IL-6 in hippocampus increased significantly(P<0.05); the number of positive cells with co-expression of Iba-1/iNOS of microglia cells in DG region of hippocampus increased; the expression levels of Iba-1 and iNOS protein in hippocampus were significantly increased(P<0.01), suggesting that microglia in DG region of hippocampus was activated and polarized toward M1 type in rats with stress. The high dose group of Xiaoyao San significantly increased the number of horizontal movement, vertical movement and central residence time of model rats(P<0.05 or P<0.01), and significantly down-regulated the levels of inflammatory factors IL-1ß and IL-6(P<0.05). Meanwhile, it reversed the activation and quantity change of microglia in hippocampus. Although the Xiaoyao San low dose group had no significant effect on the behavioral indicators in the open field test and the levels of IL-1ß and IL-6, they all showed a trend of improvement. Low dose Xiaoyao San significantly decreased iNOS protein level(P<0.05), and high dose Xiaoyao San significantly down-regulated the protein expression of Iba-1 and iNOS in hippocampus microglia(P<0.05 or P<0.01). In conclusion, Xiaoyao San can improve anxiety and depression-like behavior in OVX combined with CUS model rats, and its mechanism is related to its anti-inflammatory effect by inhibiting M1 polarization of hippocampal microglia.

[Protective effect and mechanism of Xiaoyao San on lipopolysaccharide-induced hippocampal neurons injury].

To investigate the relationship between anti-depressant effect and hippocampal nerve growth of Xiaoyao San,the inflammatory model of hippocampal neuron was induced by lipopolysaccharide( LPS). The effect of Xiaoyao San serum( final concentration of4%,8%) on the cell proliferation activity was detected by immunofluorescence,the levels of BDNF and ß-NGF in the supernatant of hippocampal neurons were detected by ELISA,and the expressions of BDNF,NGF,Trk B,Trk A and CREB mRNA in cell lysate of hippocampal neuron were detected by PCR. Western blot was used to detect the expressions of Trk B,CREB,p-CREB and SYP protein in cell lysate of hippocampal neuron,and to reveal the neuroprotective effect and mechanism of Xiaoyao San. The results showed that8% Xiaoyao San serum could significantly increase in Brdu/Neu N ratio( P<0. 01). 4%,8% Xiaoyao San serum could significantly improve the levels of BDNF and ß-NGF in supernatant( P<0. 05 or P<0. 01),up-regulate the expression of BDNF,NGF,Trk B,Trk A,CREB mRNA and Trk B,p-CREB,SYP protein in cell lysate( P< 0. 05 or P< 0. 01). 8% Xiaoyao San serum could significantly increase CREB protein in cell lysate( P<0. 05),and elevate in p-CREB/CREB ratio( P<0. 01). All the above results indicate that Xiaoyao San has a certain protective effect on LPS induced hippocampal neuron injury,which suggests that the protective effect of Xiaoyao San is related to the promotion of hippocampal nerve growth,which is one of its antidepressant mechanisms.

The regulatory effect of Xiaoyao San on glucocorticoid receptors under the condition of chronic stress.

In modern society, fierce competitions cause yearly increase of depression and anxiety. Xiaoyao San is a traditional Chinese medicine which relieves depression and nourishes liver. The active ingredients contain saikoside A, saikoside C, saikoside D, ferulic acid, ligustilide, Atractylenolide I, Atractylenolide II, Atractylenolide Ⅲ, paeoniflorin, Albiflorin, liquiritin, glycyrrhizic acid and pachymic acid. In stress condition, glucocorticoid receptors participate in the hypothalamus-pituitarium-adrenal gland (HPA) axis to regulate the balance of organism. In response to stress, the HPA axis (hypothalamus-pituitarium-adrenal gland) is activated and the levels of glucocorticoid (GC) and catecholamine (CA) are increased to enhance neuroendocrine reactions. Chronic stress activates HPA axis and sustaining increase of GC, reduces the expression amount of GR and inhibits the mechanism of negative feedback on HPA. The lower negative feedback on HPA could lead to ketonemia. Several active ingredients of Xiaoyao San can raise the expression of GR and recover the negative feedback of HPA axis to relieve depression and illness state. In spite of the poor understanding of the current effective components in Xiaoyao San, this will be the focus of our further research. The study of Xiaoyao San could help us better understand its anti-depression mechanism and cure the patients.

Effects of Xiaoyao San on exercise capacity and liver mitochondrial metabolomics in rat depression model.

Objective: This study aimed to investigate the therapeutic effects of Xiaoyao San (XYS), a herbal medicine formula, on exercise capacity and liver mitochondrial metabolomics in a rat model of depression induced by chronic unpredictable mild stress (CUMS). Methods: A total of 24 male SD rats were randomly divided into four groups: control group (C), CUMS control group (M), Venlafaxine positive treatment group (V), and XYS treatment group (X). Depressive behaviour and exercise capacity of rats were assessed by body weight, sugar-water preference test, open field test, pole test, and rotarod test. The liver mitochondria metabolomics were analyzed by using liquid chromatography-mass spectrometry (LC-MS) method. TCMSP database and GeneCards database were used to screen XYS for potential targets for depression, and GO and KEGG enrichment analyses were performed. Results: Compared with C group, rats in M group showed significantly lower body weight, sugar water preference rate, number of crossing and rearing in the open field test, climbing down time in the pole test, and retention time on the rotarod test (P < 0.01). The above behaviors and exercise capacity indices were significantly modulated in rats in V and X groups compared with M group (P < 0.05, 0.01). Compared with C group, a total of 18 different metabolites were changed in the liver mitochondria of rats in M group. Nine different metabolites and six metabolic pathways were regulated in the liver mitochondria of rats in X group compared with M group. The results of network pharmacology showed that 88 intersecting targets for depression and XYS were obtained, among which 15 key targets such as IL-1ß, IL-6, and TNF were predicted to be the main differential targets for the treatment of depression. Additionally, a total of 1 553 GO signaling pathways and 181 KEGG signaling pathways were identified, and the main biological pathways were AGE-RAGE signaling pathway, HIF-1 signaling pathway, and calcium signaling pathway. Conclusion: XYS treatment could improve depressive symptoms, enhance exercise capacity, positively regulate the changes of mitochondrial metabolites and improve energy metabolism in the liver of depressed rats. These findings suggest that XYS exerts antidepressant effects through multi-target and multi-pathway.

Mechanism Study of Xiaoyao San against Nonalcoholic Steatohepatitis-Related Liver Fibrosis Based on a Combined Strategy of Transcriptome Analysis and Network Pharmacology.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of liver disease worldwide. Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD. The livers of patients with NASH are more likely to develop fibrosis. Xiaoyao San (XYS) is a classic traditional Chinese medicine (TCM) formula that has been widely used in treating liver diseases. In this study, we elucidated the effects and mechanism of XYS in treating NASH-related liver fibrosis by combining high-throughput sequencing-based high-throughput screening with network pharmacology analysis. Our work revealed that XYS may play a role in preventing NASH-related liver fibrosis by regulating biological functions related to the extracellular matrix (ECM), inflammation, and metabolism. Additionally, Bupleuri Radix, Poria, Zingiberis Rhizoma Recens, and Paeoniae Radix Alba are the key herbs of XYS that could partially represent the functions of XYS. These regulatory effects are mediated by targeting signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NFκB), and peroxisome proliferator-activated receptor gamma (PPARγ) signaling. Narcissin, casuarictin, and γ-sitosterol were identified as representative active compounds in XYS targeting STAT3, NFκB, and PPARγ, respectively. Taken together, our findings provide a novel strategy for investigating the pharmacological effects and biological mechanisms of a TCM formula.

A multi-module structure labelled molecular network orients the chemical profiles of traditional Chinese medicine prescriptions: Xiaoyao San, as an example.

Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) technology has emerged as a crucial tool for identifying components in traditional Chinese medicine (TCM). However, the characterization of the chemical profiles of TCM prescriptions (TCMPs) which often consist of multiple herbal medicines and contain diverse structural types, presents several challenges, such as component overlapping and time-consuming. In this study, a novel strategy known as the multi-module structure labelled molecular network (MSLMN), which integrates molecular networking, database annotation, and cluster analysis techniques, has been successfully proposed, which facilitates the identification of chemical constituents by leveraging a high-structural similarity ion list derived from the MSLMN. It has been effectively applied to analyze the chemical profile of Xiaoyao San (XYS), a classical TCMP. Through the MSLMN method, a total of 302 chemical constituents were identified, covering nine structural types in XYS. Furthermore, a validated and quantitative analytical method using UHPLC-QqQ-MS/MS technology was developed for 31 identified chemicals, encompassing all eight herbal medicines present in XYS, and the developed analytical approach was applied to investigate the content distribution across 40 different batches of commercially available XYS. In total, the proposed strategy has practical significance for improving the insight into the chemical profile of XYS and serves as a valuable approach for handling complex system data based on UHPLC-MS, particularly for TCMPs.

Preclinical evidence evaluation of Xiaoyao san in treating chronic unpredictable mild stress model of depression based on meta-analysis.

BACKGROUND: In view of the current challenges in the treatment of depression, in order to improve the efficacy and avoid adverse reactions, people pay attention to the treatment of traditional Chinese medicine. Xiaoyao san is a classic prescription commonly used in the treatment of depression, with the role of harmonizing liver and spleen, and shows great potential in the treatment of depression. PURPOSE: The purpose of this study is to comprehensively evaluate the efficacy and specific mechanism of Xiaoyao san in the treatment of chronic unpredictable mild stress (CUMS) model of depression through systematic evaluation and meta-analysis, so as to provide strong preclinical evidence for the clinical treatment of Xiaoyao san and provide a new strategy for the development of antidepressants. METHODS: The preclinical literature published before March 2023 was searched in Cochrane Library, PubMed, Web of Science, EMbase, CNKI, VMIS, Wan-Fang, and CBM and other database systems. Stata15 was used for overall effect analysis and subgroup analysis, and summarized the potential mechanism of action. RESULTS: A total of 25 studies were included, involving 569 animals. The average score of methodological quality was 7.48/10. Meta-analysis shows that Xiaoyao san can effectively improve food intake and body weight, restore sucrose consumption, reduce the immobility time in forced swimming, and increase the total exercise distance, grid crossing and upright times in the open field experiment. Its therapeutic effect is closely related to improving the abnormal activation of Hypothalamic-pituitary-adrenal axis and inhibiting the expression of glutamate. CONCLUSION: To sum up, in CUMS animal model, Xiaoyao san can significantly improve the symptoms of depression, restore the lost pleasure behavior and curiosity about the new environment, relieve tension and anxiety, and improve the occurrence of depression in many ways, which may be a new treatment strategy for CUMS model of depression.

Jiawei Xiaoyao San in treatment of anxiety disorder and anxiety: A review.

Jiawei Xiaoyao San (JWXYS) has shown excellent clinical efficacy in anxiety disorder, but has not yet attracted widespread attention. The animal experiments, clinical trials and mechanism studies of JWXYS were reviewed in this article, which may provide a reference for developing new anxiolytic drugs based on this prescription. The literature was searched in PubMed and CNKI and the documents written in English or with English abstracts were selected. JWXYS could reduce the anxiety symptoms of patients alone and reduce the adverse reactions when it is used in combination with other drugs in the clinic. In preclinical studies, JWXYS also showed therapeutic effects in reducing anxiety-like behavior. The mechanisms may include improving the hypothalamic-pituitaryadrenal (HPA) axis and hormone disorders, increasing neurotransmitter content, neurogenesis, and regulating the synthesis of related enzymes. This article shows that JWXYS could effectively treat anxiety disorders by regulating the central nervous system. In the future, with the participation of more researchers, it is expected to develop innovative drugs for the treatment of anxiety disorders based on JWXYS.