A combination of cecum microbiome and metabolome in CUMS depressed rats reveals the antidepressant mechanism of traditional Chinese medicines: A case study of Xiaoyaosan

doi:10.1016/j.jep.2021.114167

Journal of Ethnopharmacology

Volume 276, 10 August 2021, 114167

https://doi.org/10.1016/j.jep.2021.114167 Get rights and content

Highlights

•
A combination of cecum microbiome and metabolome was used on CUMS depression rats.
•
Both 16S rRNA gene sequencing and ¹H NMR based metabolomics were applied.
•
XYS reversed the diversity and the composition of cecum microbiota of CUMS rats.
•
Changes of cecal microbiota were correlated with potential metabolites regulated by XYS.

Abstract

Ethnopharmacological relevance

Xiaoyaosan (XYS), a representative and classic prescription in traditional Chinese medicines (TCMs), has been used for thousands of years for treating depression. The anti-depression effect of XYS has been demonstrated both clinically and experimentally. However, it is still unclear that whether XYS could regulate the abnormalities of gut microbiota and metabolites of cecum induced by depression, and in which way. This study aimed to explore the underlying mechanism of the anti-depressant effects of XYS from the perspective of cecal microbiota and metabolites.

Materials and methods

Chronic unpredictable mild stress (CUMS)-induced depression-like rats were used as the depression animal model. Various classic behavioral tests were performed to assess the anti-depressant effects of XYS. Additionally, the composition, the richness, and the diversity of the cecum microbiota were assessed by 16S rRNA gene sequencing technology. Besides, the metabolic profiling of cecum samples was analyzed by ¹H-NMR metabolomics. Multivariate data analysis was then applied to screen the differential metabolites and to characterize the changes in cecum metabolites. Moreover, a correlation analysis between differential metabolites and crucial microbiota was conducted.

Results

XYS significantly improved depressive behaviors and the abnormal diversity of cecum microbiota induced by CUMS. At the phylum level, XYS could significantly increase the abundance of Firmicutes while decrease the abundance of Actinobacteria in depressed rats. XYS significantly regulated the abundances of 9 out of 13 potential microbial biomarkers at the genus level. Cecal metabolomics showed that XYS could also regulate the abnormal levels of alanine, proline, lactate, and valine of depression rats.

Conclusions

This study revealed, for the first time, from the perspectives of microbiota and cecum metabolites, the anti-depression mechanisms of XYS. This study is of significance for not only comprehensively understanding the anti-depression effects and mechanisms of XYS, but also for providing a research approach for revealing the underlying mechanisms of action of TCMs, i.e. to apply a combination of 16S rRNA gene sequencing and metabolomics.

Graphical abstract

Introduction

The occurrence of depression is a complete result of the multi-dimensional and multi-leveled interactions between genes and the environment, whereas its pathogenesis is still rather vague. Up to the present, the established mechanisms of depression mainly involve the changes of neurotransmitters, neural circuits, immune and endocrine functions (Makris et al., 2021), but no single perspective has been able to satisfactorily explain all aspects of depression. Accumulating evidence has indicated that an imbalance of the gut microbiota, known as dysbiosis, may also influence brain functions and behaviors, and may play notable roles in the development of depression (Cryan and Dinan, 2012; Foster and McVey Neufeld, 2013). For instance, the changes of microbial community structure may be related to translocation of gut microbiota, activation of immune system, and hyperactivity of the hypothalamic-pituitary-adrenal axis in depression. Meanwhile, it can also influence the secretion of neurotransmitters, the production of microbial metabolites, and the levels of host metabolites (Chen et al., 2017).

Traditional Chinese medicines (TCMs), implying profound philosophical wisdom and gathering Chinese wisdom, have been used for thousands of years. Nowadays, based on their confirmed efficacies and fewer side effects, TCMs are attracting more and more attention. Xiaoyaosan (XYS), originated from Taiping Huimin Heji Jufang (Song Dynasty, 960-1127 A.D), consists of Radix Bupleuri, Radix Angelicae Sinensis, Radix Paeoniae Alba, Rhizoma Atractylodis Macrocephalae, Poria, Radix Glycyrrhizae, Herba Menthae, and Rhizoma Zingiberis Recens. The anti-depressive mechanisms of XYS have been demonstrated to be involved in regulating neural pathways, neurotransmitters, synaptic plasticity, neuroendocrine system, and immune system (Ma et al., 2019). Yet, due to its unique characteristics, i.e., multi-herbs, multi-targets, and multi-components, it is needed to reveal the mechanisms of XYS comprehensively and multi-dimensionally, for example, from the perspective of regulating gut microflora.

The previous study has found that depression induced by a chronic unpredictable mild stress (CUMS) model, a typical animal model of depression, negatively affected the cecum functions of rats, while XYS significantly repaired the damage of cecum structure and tissue and reduced the expression of genes that related to inflammatory responses in the cecum (Chen et al., 2015). Therefore, the mechanisms of anti-depressant effects of XYS need to be further verified from the point of view of the cecum, a part of the immune system.

In the present study, an integrative approach of 16S rRNA gene sequencing and NMR-based cecum metabolomics was performed on CUMS depression rats with and without XYS treatment to comprehensively reveal the interactions between cecum microbiota and depression, and the anti-depression effects of XYS, in terms of analyzing both structural and functional changes of cecum microbiota induced by CUMS, the changes of cecum metabolites, as well as the correlations between gut microbiota and specific metabolites. The current results will lay the foundation for a comprehensive understanding of the pathogenesis of CUMS-induced depression, and the anti-depression mechanisms of XYS.

Section snippets

Drugs and reagents

The composition of XYS was as follows: Radix Bupleuri (Bupleurum chinense DC.) (batch number: 2019–0327001), Radix Angelicae Sinensis (Angelica sinensis (Oliv.) Diels) (batch number: 2019–0327002), Radix Paeoniae Alba (Paeonia lactiflora Pall.) (batch number: 2019–0327003), Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala Koidz.) (batch number: 2019–0327004), Poria (Poria cocos (Schw.) Wolf) (batch number: 2019–0327005), Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch.) (batch

XYS improves the behavioral abnormalities of the CUMS depression rats

Before CUMS modeling, there were no significant differences in body weights and the levels of sucrose preference rates among the NC, MS, XYS, and VLF groups (Fig. 1A and B). At the end of modeling, the body weights and the levels of sucrose preference rates of MS were significantly decreased than that of NC (P < 0.01), all the treatments significantly increased these two indexes as compared with MS (P < 0.05, P < 0.01).

On the day 28, after CUMS modeling, compared with the NC, the total

Discussion

This study revealed, for the first time, from the perspectives of microbiota and metabolites of the cecum, the mechanisms of the anti-depression effects of XYS. In recent years, a large number of studies have demonstrated that the occurrence and the development of depression are closely related to gut microbiota. Gut microbiota may play an important role in the prevention and the treatment of depression by acting on the neuroendocrine system, autonomic nervous system, and immune pathways (Guo

Conclusion

In conclusion, both 16S rRNA gene sequencing and ¹H-NMR based metabolomics were applied to explore the composition and the abundance of cecal microbiota and metabolic changes of cecal contents induced by CUMS and the effects of XYS. CUMS depression rats exhibited obvious disorders of cecal microbiota and the abnormality of the metabolic profiling of cecal contents. The anti-depression effects of XYS may be related to the regulation of alpha and beta diversity of microbiota, the reduction of the

Author contributions statement

ML carried out the animal experiments, behavioral tests, collected the data, performed the analysis, and wrote the manuscript. YW made contributions to animal experiments and behavioral tests. PQ, SL, ZY, and XQ reviewed the manuscript. XL conceived the experiments and reviewed the manuscript.

Declaration of competing interest

All of the authors declared there is no potential conflict of interests for this manuscript.

Acknowledgments

This research was financially supported by National Natural Science Foundation of China [No. 81803962], Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province [20200013], National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Twelfth Five-year Plan Period of China [2017ZX09301047], and Key Laboratory of Effective Substances Research and Utilization in TCM of Shanxi Province [202105D121009

References (29)

A. Abildgaard et al.
Probiotic treatment reduces depressive-like behaviour in rats independently of diet
Psychoneuroendocrinology
(2017)
G. Akkasheh et al.
Clinical and metabolic response to probiotic administration in patients with major depressive disorder: a randomized, double-blind, placebo-controlled trial
Nutrition
(2016)
Q. An et al.
Scaffold hopping of agomelatine leads to enhanced antidepressant effects by modulation of gut microbiota and host immune responses
Pharmacol. Biochem. Behav.
(2020)
J.L. Chen et al.
Effect of Xiaoyaosan on caecum inflammatory response related genes expression in chronic unpredictable mild stress model rats
Chin. J. Pharmacol. Toxicol.
(2015)
M. Chen et al.
Intake of Ganoderma lucidum polysaccharides reverses the disturbed gut microbiota and metabolism in type 2 diabetic rats
Int. J. Biol. Macromol.
(2020)
Y. Feng et al.
Multi-omics reveals the mechanisms of antidepressant-like effects of the low polarity fraction of Bupleuri Radix
J. Ethnopharmacol.
(2020)
J.A. Foster et al.
Gut-brain axis: how the microbiome influences anxiety and depression
Trends Neurosci.
(2013)
T.L. Guo et al.
Gut microbiome in neuroendocrine and neuroimmune interactions: the case of genistein
Toxicol. Appl. Pharmacol.
(2020)
H. Jiang et al.
Altered fecal microbiota composition in patients with major depressive disorder
Brain Behav. Immun.
(2015)
Y.W. Liu et al.
Psychotropic effects of Lactobacillus plantarum PS128 in early life-stressed and naïve adult mice
Brain Res.
(2016)

Y. Liu et al.

Xiaoyaosan exerts antidepressant-like effects by regulating the functions of astrocytes and EAATs in the prefrontal cortex of mice

BMC Compl. Alternative Med.

(2019)

X. Liu et al.

Deciphering the compatibility rules of traditional Chinese medicine prescriptions based on NMR metabolomics: a case study of Xiaoyaosan

J. Ethnopharmacol.

(2020)

Q. Xiao et al.

Crocin-I alleviates the depression-like behaviors probably via modulating "microbiota-gut-brain" axis in mice exposed to chronic restraint stress

J. Affect. Disord.

(2020)

M. Yu et al.

Variations in gut microbiota and fecal metabolic phenotype associated with depression by 16S rRNA gene sequencing and LC/MS-based metabolomics

J. Pharmaceut. Biomed. Anal.

(2017)

Cited by (16)

Preclinical evidence evaluation of Xiaoyao san in treating chronic unpredictable mild stress model of depression based on meta-analysis
2023, Phytomedicine
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.
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.
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.
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.
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.
Ginsenoside Rh4 remodels the periphery microenvironment by targeting the brain-gut axis to alleviate depression-like behaviors
2023, Food Chemistry
Citation Excerpt :
These microbes interact with the body to maintain a homeostatic environment for the immune system. Recently, several studies have revealed the importance of the microbiota in depression (Bercik et al., 2011; Lv et al., 2021). Bidirectional communication between the central nervous system (CNS) and the gut microbiota involves the brain-gut axis and various signaling pathways, including neurons, the neuroendocrine system, and neuroimmune pathways.
Depression is a neuropsychiatric disease that threatens the physical and mental health of humans worldwide. This study explored the potential anti-depressant effects of ginsenoside Rh4 and its mechanisms of action. The results showed that Rh4 could significantly inhibit depression-like behavior in the depression mouse model and alleviate neuronal damage and hypothalamic–pituitary–adrenal axis disorder. Concurrently, Rh4 inhibits hippocampal neuronal apoptosis and synaptic structural damage due to the overexpression of proinflammatory cytokines and overactivation of microglia and astrocytes by inhibiting the immune-inflammatory response and signaling molecular interaction pathways. Rh4 can also improve intestinal flora and increase the short-chain fatty-acids content. The correlation analysis indicated that the Rh4-inhibited LPS/NLRP3/caspase-1/IL-1β signaling pathway plays a key role in ameliorating depression. Therefore, this study provides valuable insights into the mode of action of Rh4 on the brain-gut axis in depression, suggesting that Rh4 may be a promising clinical drug for the treatment of depression.
The therapeutic potential of traditional Chinese medicine in depression: Targeting adult hippocampal neurogenesis
2022, Phytomedicine
Citation Excerpt :
XYS significantly improved depression-like behavior in CRS-induced depressed rats by regulating the GM and its metabolite short-chain fatty acids (SCFAs) (Zhu et al., 2019). In addition, XYS also improved depression-like behavior in CUMS-induced depressed rats by modulating the GM (Lv et al., 2021). The total iridoids of Valeriana jatamansi (TIV) improved depression-like symptoms in CUMS-induced mice by regulating the GM structure, which was attributed to the possible involvement brain neurotransmitters (Wang et al., 2020).
Depression is a common mental disorder characterized by persistent sadness and lack of interest or pleasure in previously rewarding or enjoyable activities. Understandably, the causes of depression are complex. Nevertheless, the understanding of depression pathophysiology has progressed considerably and numerous studies indicate that hippocampal neurogenesis plays a pivotal role. However, no drugs specifically targeting hippocampal neurogenesis yet exist. Meanwhile, the effects of traditional Chinese medicine (TCM) on hippocampal neurogenesis have received increasing attention in the field of antidepressant treatment because of its multi-ingredient, multi-target, and holistic view. However, the effects and mechanisms of TCM on hippocampal neurogenesis in clinical trials and pharmaceutical studies remain to be comprehensively delineated.
To summarize the importance of hippocampal neurogenesis in depression and illustrate the targets and mechanisms of hippocampal neurogenesis regulation that underlie the antidepressant effects of TCM.
A systematic review of clinical trials and studies ending by January 2022 was performed across eight electronic databases (Web of Science, PubMed, SciFinder, Research Gate, ScienceDirect, Google Scholar, Scopus and China Knowledge Infrastructure) according to the PRISMA criteria, using the search terms ‘traditional Chinese medicine’ “AND” ‘depression’ “OR” ‘hippocampal neurogenesis’ “OR” ‘multi-ingredient’ “OR” ‘multi-target’.
Numerous studies show that hippocampal neurogenesis is attenuated in depression, and that antidepressants act by enhancing hippocampal neurogenesis. Moreover, compound Chinese medicine (CCM), Chinese meteria medica (CMM), and major bioactive components (MBCs) can promote hippocampal neurogenesis exerting antidepressant effects through modulation of neurotransmitters and receptors, neurotrophins, the hypothalamic-pituitary-adrenal axis, inflammatory factors, autophagy, and gut microbiota.
We have comprehensively summarized the effect and mechanism of TCM on hippocampal neurogenesis in depression providing a unique perspective on the use of TCM in the antidepressant field. TCM has the characteristics and advantages of multiple targets and high efficacy, showing great potential in the field of depression treatment.
Jia Wei Xiao Yao San ameliorates chronic stress-induced depression-like behaviors in mice by regulating the gut microbiome and brain metabolome in relation to purine metabolism
2022, Phytomedicine
Citation Excerpt :
An integrated pharmacology-based analysis revealed that neurotoxicity and neuroprotective effects coexist in complex components of XYS, and a few antidepressant signaling pathways were selected to conduct cellular and molecular evaluations (Yuan et al., 2020). Another case study attempted to use an integrated approach of 16S rRNA sequencing and 1H NMR to explore some mechanisms of gut microbiome at genus level altered by XYS in depression-associated mice, despite taxonomic annotation to genus level and lack of potential function profiling (Lv et al., 2021). In the present study, we found that JWXYS improved the behavioral symptoms of depression-like mice in a nondose-dependent manner.
The pathogenesis of depression remains largely unknown. Accumulating evidence demonstrates the existence of a complex relationship between gut microbiome composition and brain functions. Jia Wei Xiao Yao San (JWXYS) is considered a potential antidepressant. However, the pharmacological mechanisms of JWXYS have not yet been clarified.
This study aimed to explore the effects of JWXYS on chronic stress-induced depression-like behaviors in mice.
A chronic restraint stress mouse model of depression was established. JWXYS was administered, and the responses of these mice to treatment were evaluated through several behavioral tests. The activity of astrocytes and microglia was detected by specific fluorescent labels. Inflammatory cytokines were quantified in intestinal and cerebral tissues. An integrated approach with full-length 16S rRNA sequencing and different types of untargeted metabolomics was conducted to investigate the relationship between the gut microbiome at the species level, metabolic brain functions, and JWXYS.
We found that behavioral symptoms were associated with the relative abundance of Lactobacillus animalis. After JWXYS treatment, the relative abundance of Ileibacterium valens with enzymes potentially involved in purine metabolism was also described. The activation of astrocytes and microglia was negatively correlated with the relative abundance of L. animalis. Combined with network pharmacological analysis, several targets predicted based on JWXYS treatment focused on purine metabolism, which was also enriched from cerebral metabolites regulated by JWXYS.
Our study suggests that L. animalis is involved in depression-like behaviors in mice. JWXYS increases the abundance of I. valens with potential enzymes in relation to cerebral purine metabolism, which is positively correlated with the activation of astrocytes in the amygdala.
Research strategies and applications of functional metabolomics in anti-depressive mechanisms of traditional Chinese medicine
2023, Yaoxue Xuebao
Reshaped Gut Microbial Composition and Functions Associated with the Antifatigue Effect of Salidroside in Exercise Mice
2023, Molecular Nutrition and Food Research

View all citing articles on Scopus

View full text

A combination of cecum microbiome and metabolome in CUMS depressed rats reveals the antidepressant mechanism of traditional Chinese medicines: A case study of Xiaoyaosan

Highlights

Abstract

Ethnopharmacological relevance

Materials and methods

Results

Conclusions

Graphical abstract

Introduction

Section snippets

Drugs and reagents

XYS improves the behavioral abnormalities of the CUMS depression rats

Discussion

Conclusion

Author contributions statement

Declaration of competing interest

Acknowledgments

Psychoneuroendocrinology

Nutrition

Pharmacol. Biochem. Behav.

Chin. J. Pharmacol. Toxicol.

Int. J. Biol. Macromol.

J. Ethnopharmacol.

Trends Neurosci.

Toxicol. Appl. Pharmacol.

Brain Behav. Immun.

Brain Res.

BMC Compl. Alternative Med.

J. Ethnopharmacol.

J. Affect. Disord.

J. Pharmaceut. Biomed. Anal.