Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression

Author’s Accepted Manuscript Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression Kuo Zhang, Meiyao He, Dongmei Su, Xing Pan, Yuting Li, Haotian Zhang, Jingyu Yang, Chunfu Wu www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(18)31260-1 https://doi.org/10.1016/j.jep.2018.11.020 JEP11601

To appear in: Journal of Ethnopharmacology Received date: 9 April 2018 Revised date: 26 July 2018 Accepted date: 12 November 2018 Cite this article as: Kuo Zhang, Meiyao He, Dongmei Su, Xing Pan, Yuting Li, Haotian Zhang, Jingyu Yang and Chunfu Wu, Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.11.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Quantitative

proteomics

reveal

antidepressant

potential

protein

targets of

xiaochaihutang in corticosterone induced model of depression

Kuo Zhang, Meiyao He, Dongmei Su, Xing Pan, Yuting Li, Haotian Zhang, Jingyu Yang, Chunfu Wu*

a

Department of Pharmacology, Shenyang Pharmaceutical University, 110016,

Shenyang, PR China *Corresponding author at: Department of Pharmacology, Shenyang Pharmaceutical University, Box 31, 103 Wenhua Road, 110016, Shenyang, PR China. Tel. & Fax: +86 24 23986282. E-mail address: [email protected]

Abstract Ethnopharmacological relevance: Xiaochaihutang (XCHT), one of famous Chinese herbal prescription for treating Shaoyang symptom, has been used successfully in depressive disorders for many years. Our laboratory has demonstrated that XCHT remarkably alleviated various depressive behaviors induced by several depressive animal models, but previous studies only focused on one or several protein targets, lacked dynamic change and interrelation of proteins. Therefore, potential protein targets and mechanisms are required further systematic investigation. Aim of the study: 1

To discover and assess the differentially expressed proteins (DEPs) of hippocampus after oral administration of XCHT in corticosterone (CORT) induced model of depression by using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Materials and Methods: The antidepressant effects of XCHT were assessed by two behavioral despair models (forced swimming test and tail suspension test) in CORT induced model of depression. The DEPs of hippocampus after XCHT treatment were investigated by iTRAQ analysis. Potential protein targets and mechanisms were assessed by gene ontology (GO), Kyoto encyclopedia of gene and genomes (KEGG) and protein-protein interaction (PPI) network. Results: Our data demonstrated XCHT could significantly improve depressive behaviors. A total of 241 DEPs were identified after XCHT treatment, including 68 up regulation and 173 down regulation proteins. GO enrichment results indicated that XCHT mainly regulated intracellular structural proteins involved in cellular response to stress, transferase activity and steroid hormone. KEGG enrichment analysis showed that endocytosis might be the principal pathway of XCHT on depression. PPI analysis predicted cell division cycle and apoptosis regulator protein 1 (Ccar1) and Calretinin (Calb2) might play the central roles in XCHT’s antidepressant network. Conclusion: Our results indicate that XCHT plays the important roles in antidepressant action by 2

restoring DEPs, which results in the dysregulation of hippocampal neurogenesis, neurotransmitter and steroid hormone. The current results wish to provide novel perspectives for revealing the potential protein targets of XCHT on depression. Graphical abstract

Abbreviations Xiaochaihutang, XCHT; isobaric tags for relative and absolute quantification, iTRAQ; protein-protein interaction, PPI; Kyoto encyclopedia of gene and genomes, KEGG; gene ontology, GO; gonadotrophin releasing hormone, GnRH; division cycle and apoptosis regulator protein 1, Ccar1; Calretinin, Calb2; Ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS; phospholipase D2, Pld2; tyrosine-protein

kinase

Lyn,

Lyn;

corticosterone,

CORT;

estradiol,

E2;

follicle-stimulating hormone, FSH; differentially expressed proteins, DEPs; brain derived neurotrophic factor, BDNF; nerve growth factor, NGF; tyrosine receptor 3

kinase B, TrkB; tyrosine receptor kinase A, TrkA

Keywords Xiaochaihutang; proteomics; antidepressant; differentially expressed protein

Chemical compounds studied in this article Liquiritin (PubChem CID: 503737); Baicalin (PubChem CID: 64982); Baicalein (PubChem CID: 5281605); Wogonin (PubChem CID: 5281703); Wogonoside (PubChem CID: 29927693); Diphenhydramine (PubChem CID: 3100); Saikosaponin a (PubChem CID: 167928); Verapamil (PubChem CID: 2520); Ginsenoside Rg1 (PubChem CID: 441923) 1. Introduction Depression is the psychiatry disease characterized by low mood, suicidality, sleep or wake imbalance, psychomotor retardation, especially hopelessness and anhedonia, which producing a significant social and economic burden (Pena et al., 2017). However, specific pathophysiology underlying the development of depression remains unclear. Current medical therapies for depression are invalid in many patients and usually cause much intolerable side effects (Duman and Aghajanian, 2012). Therefore, it is urgent to clarify the pathogenesis of depression and find new therapeutic targets. In China, some famous Chinese herbal prescriptions had been used for thousand 4

years based on high security and effective therapeutic effect. For instance, xiaochaihutang (XCHT) had been first recorded in “Shang Han Lun” about A.D. 219 and consists of Bupleurum chinense DC. (Chaihu), Scutellaria baicalensis Georgi (Huangqin), Panax ginseng C.A. Mey. (Renshen), Pinellia ternate (Thunb.) Breit. (Banxia), Glycyrrhiza uralensis Fisch. (Gancao), Zingiber officinale Rosc. (Shengjiang) and Ziziphus jujubeMill. (Dazao) (Su et al., 2014b). In ancient times, XCHT was applied for ‘Shaoyang syndrome’, which similar to depressive symptoms such as dysphoria and anepithymia. Now, XCHT had been successfully used to treat depressive disorders in clinical practice (Jia et al., 2009; Li and Gao, 1996). Based on above facts, our laboratory has focused on antidepressant effect of XCHT for many years. Our previous studies had demonstrated that XCHT could significantly improve various depression-like behaviors induced by several depressive animal models, including chronic unpredictable mild stress, chronic social isolation stress, chronic CORT and so on. Moreover, previous results also demonstrated that XCHT could increase monoamine neurotransmitters levels, enhance the expression of monoamine neurotransmitter synthesis enzymes in social isolation reared mice, improve the levels of BDNF, NGF, TrkB and TrkA in chronic unpredictable mild stress model, increased hippocampal neurogenesis and regulated steroid hormone in CORT induced model of depression (Ma et al., 2017; Su et al., 2014a; Su et al., 2014b; Zhang, K. et al., 2016b). However, like almost traditional Chinese medicine, XCHT also faces severe challenges due to the lack of scientific and systematic approaches. The previous studies only focused on one or several protein targets, lacked the interrelation of each 5

protein, especially the dynamic change of proteins. Proteomics has been considered as the important tool on exploring the targets and mechanisms of drug, which is already widely used to large amount of fields of life sciences (Zhang, J. et al., 2016). Moreover, proteomics also provides the high-throughput approach to analyze differentially expressed proteins (DEPs), and powerfully promotes traditional Chinese medicine more modernization and internationalization. Hence, to reveal antidepressant potential protein targets of XCHT, we assess the DEPs of hippocampus after XCHT treatment in CORT induced model of depression by iTRAQ. Moreover, the functions and mechanisms of protein targets are assessed by gene ontology (GO), Kyoto encyclopedia of gene and genomes (KEGG) and protein-protein interaction (PPI) network. The current results hope to provide novel perspectives for understanding the potential protein targets of XCHT on depression, obtaining the systematic antidepressant mechanisms of XCHT for treating depression.

2. Material and methods 2.1 Drug and quality control XCHT was composed of seven traditional herbs, comprised of Bupleurum chinense DC. (12 g, no. 1012002) root, Scutellaria baicalensis Georgi (9 g, no. 1101002) root, Panax ginseng C.A. Mey. (9 g, no. 20111001) root, Pinellia ternate (Thunb.) Breit. (9 g, no. 110303) stem, Glycyrrhiza uralensis Fisch. (6 g, no. 1101002) root, Zingiber officinale Rosc. (6 g, no. 2880240) root and Ziziphus jujube Mill (9 g, no.1010B2) fruits. The botanical raw materials were crushed into pieces 6

and mixed in the ratio of 4:3:3:3:2:2:3 (w/w). All herbs were obtained from Decaotang traditional Chinese pharmacy Co. Ltd. (Shenyang, China) and affirmed by associate professor Jiuzhi Yuan (Shenyang Pharmaceutical University) in accordance with the Pharmacopeia of China. The voucher specimen of seven traditional herbs has been deposited in the herbarium of Traditional Chinese Medicine (Shenyang Pharmaceutical University, International Herbarium Code: SYPC). The preparation and manufacturing process of herbs followed standard operating procedures according to our previous studies (Su et al., 2014b; Zhang et al., 2015). In brief, the botanical raw materials were crushed into pieces and mixed in distilled water for 40 min, then boiling three times (100 g/1000 mL; 100 g/400 mL; 100 g/400 mL) for 30 min each time. The three decoctions were mixed and filtered, then lyophilized and stored in a desiccator. 15.2 g of extract was equal to 60 g total weight of botanical raw materials. Moreover, our laboratory also has established the strict quality control method of XCHT (Bo et al., 2017; Wang et al., 2015; Zhang et al., 2015), and UPLC-PDA-MS/MS chromatograms of XCHT have showed in Figure 1. The major compounds of XCHT were quantify, and content of Ginsenoside Rg1 is 87.8 µg/g, Saikosaponin a is 68.1 µg/g, Liquiritin is 159 µg/g, Baicalin is 1210 µg/g, Wogonoside is 623 µg/g, Baicalein is 0.906 µg/g, Wogonin is 58.6 µg/g.

7

Figure 1

2.2 Animals and treatment Thirty six 8-week-old male C57BL/6J mice (weight, 18-22 g; SCXK 2014-0004) were obtained from HFK Bioscience Co. Ltd. (Beijing, China). All the mice were fed at 23 ± 2 °C under 12 h light/dark cycle in constant environmental conditions with free access to food and water. All experiments were performed according to the institutional animal care policies, and the operation was made to minimize suffering. In brief, mice were randomly selected into three groups: control group (n=12), corticosterone (CORT) model group (n=12) and CORT combined with XCHT group 8

(n=12). In CORT induced model groups, mice were subcutaneously injected once daily with CORT (40 mg/kg, Tokyo Chemical Industry, Japan) dissolved in 0.1% dimethylsulfoxide and 0.1% Tween-80 between 8:00 a.m. and 10:00 a.m.for eight weeks (Zhang, K. et al., 2016a). After four weeks of the CORT protocol, XCHT was given by gastric gavages once daily for four weeks. All procedures were shown in Figure 2. The selected doses for XCHT administration were based on our previous studies (Zhang et al., 2015; Zhang, K. et al., 2016b). In previous studies, we tested four doses of XCHT (0.8, 2.3, 7 and 21 g/kg, botanical raw materials extracted by distilled water) in several depressive animal models and confirmed that 7 g/kg was the best effective dose of XCHT on depression (Ma et al., 2017; Zhang et al., 2015; Zhang, K. et al., 2016b). As a consequence, we chose the best effective dose 7 g/kg of XCHT to analyze quantitative proteomics by iTRAQ.

Figure 2 2.3 Behavioral tests At the end of XCHT treatment, behavioral tests were executed. During the behavioral tests period, animals continued to receive XCHT treatment and CORT 9

protocol. Tail suspension test (TST) was performed as described formerly (Steru et al., 1985). In brief, the mice were individually suspended by tails using adhesive tape. A total of test processes lasted for 6 min, and mice’s behavior was recorded by using high-definition camera. The videos were analyzed by motor tracking system (Ethovision, Wageningen, The Netherlands), and duration of immobility time was cumulatively calculated during the last 4 min. According to the method previously described, forced swim test (FST) was established (Porsolt et al., 1978). Briefly, place mice individually in plastic cylinders (40 cm in height × 12 cm in diameter) filled with 10 cm depth of water (24 ± 1 °C) for 6 min. Mice’s behavior was recorded by using high-definition camera, and the immobility time was measured within the final 4 min by motor tracking system.

2.4 Protein extraction, digestion and iTRAQ labeling After finish the experiment, all mice were sacrificed and the tissues of hippocampus were quickly isolated on glass plate. Before the protein processing, each 4 individual hippocampus samples were mixed equally into 1 specimen. Lysis buffer was added to the hippocampus samples in homogenizer on ice. Later the lysate was centrifuged at 14000 g for 40 min to get the supernatant. Then, the sample was stored at -80 °C. On the grounds of the filter-aided sample preparation procedure, protein digestion was executed. In brief, sample was diluted with UA buffer (200 μL) and then centrifuged by twice. Then, samples were incubated with iodoacetamide for 30 min away from light and protein was digested overnight in a 40 μL dissolution 10

buffer using trypsin at 37 °C. The iTRAQ reagent (AB SCIEX, Framingham, MA) was used to label the peptide mixture according to the manufacturer's instructions. Control group, model group, XCHT group and internal standard were labeled with different isobaric tags 113, 114, 117 and 119, respectively.

2.5 Strong cation exchange (SCX) fractionation and LC-MS/MS analysis The peptide mixture was fractionated by SCX chromatography (AKTA Purifier, GE), and eluted by gradient at the flow rate (1 ml/min) with buffer A (10 mM KH2PO4 in 25% of acetonitrile, pH 3.0) and 0%-8% buffer B (10 mM NaH2PO4, 500 mM KCl in 25% of acetonitrile, pH 3.0) for 22 min, 8-52% buffer B for 22-47 min, 52%-100% buffer B for 47-50 min, 100% buffer B for 50-58 min. Then, 15 fractions were collected by vacuum-dried. LC-MS/MS analysis was operated by Q Exactive mass spectrometer (Thermo Fisher Scientific, CA) which coupled to Easy nLC for 240 min. The positive ion mode was applied in mass spectrometer. Specific parameters were showed as follows: survey scan (300-1800 m/z), automatic gain control target (3e6), normalized collision energy (30 eV), maximum inject time (10 ms), dynamic exclusion duration (40.0 s). The peptide recognition mode was chose to run by instrument. The data of MS/MS spectra

were

contrasted

and

searched

in

the

uniprot

protein

database

(uniprot_mouse_80417_20160308.fasta) by Proteome Discoverer (version: 1.4) combined with MASCOT engine (Matrix Science, London, UK; version 2.2). Only proteins compared to CORT group displayed the changes (ratios with fold change > 11

1.2 or < 0.83) and P values < 0.05) were considered to be significant.

2.6 Bioinformatics analysis GO annotation and KEGG pathway were applied to analyze the DEPs. In GO annotation, proteins have three ontologies which are defined as biological process, molecular function and cellular component, respectively. Furthermore, GO enrichment and KEGG pathway enrichment analyses were executed according to the Fisher’ exact test. Cluster 3.0 and the Java Treeview software were used. Euclidean distance algorithm for similarity measure and average linkage clustering algorithm for clustering were selected when performing hierarchical clustering. PPI information of the DEPs was obtained from STRING software. Furthermore, the importance of the protein in the PPI network was assessed by calculating the degree of every protein.

2.7 Statistical analysis In LC-MS/MS analysis, P value was calculated between XCHT group and CORT group followed by independent samples t-test. P < 0.05 was considered to be statistically significant. In behavioral test, data were reported as the mean ± standard error of mean (SEM) and analyzed by one-way analysis of variance followed by Fisher’s LSD post hoc. Statistical analysis was performed with SPSS 20.0 (SPSS, Armonk, New York, USA). P < 0.05 was considered to be statistically significant.

12

3. Results 3.1 XCHT ameliorated CORT induced depressive behaviors TST and FST are two classical behavioral despair models, and have been widely applied to assess drug antidepressant activity (Porsolt et al., 1978; Steru et al., 1985). Therefore, we firstly detected the antidepressant effects of XCHT by TST and FST. The result showed that XCHT significantly decrease the immobility time both in TST (Figure 3A, P < 0.05) and FST (Figure 3B, P < 0.05), which indicated that XCHT could ameliorate CORT induced depressive behaviors.

Figure 3 3.2 iTRAQ quantitative of DEPs after XCHT treatment Aim to excavate potential antidepressant protein targets of XCHT in CORT induced model of depression, iTRAQ-based proteomic was applied to assess DEPs of hippocampus between model group and XCHT group. The experimental data in our study were validated using three times biological. Our results showed that 5996 proteins were recognized and identified with high reliability, which correspond to 1% FDR. Further analysis confirmed 241 DEPs when model group compared with XCHT 13

group, including 68 up regulation and 173 down regulation proteins in XCHT group by a strict cutoff value of a 1.2-fold change. The information of DEPs was showed in Table S1.

3.3 Bioinformatics analysis of DEPs after XCHT treatment In order to further investigate the functions and mechanisms of protein targets, we firstly evaluated DEPs by means of hierarchical clustering analysis (Figure 4). Clustering data showed that DEPs in model group and XCHT group were remarkably separated into two obvious distinguishing branches. This result demonstrated that feature expressed proteins of hippocampus after XCHT treatment are obvious distinct from model group.

14

Figure 4 Then, Blast2GO was used to assess the functional annotations of DEPs (Figure 5). In biological process, DEPs mainly took part in cellular process (73.0%), single-organism process (56.4%), biological regulation (49.8%) and metabolic 15

process (48.9%). About molecular function, the largest proportion of DEPs was in binding (66.4%), followed by catalytic activity (31.5%). In cellular component, cell (75.5%), organelle (53.9%) and membrane (31.1%) were the top three parts of cellular localization for DEPs. Moreover, GO enrichment data indicated that the top five categories DEPs were significantly involved in cellular response to stress (6.2%), regulation of transferase activity (4.6%) and autophagy (2.5%) in biological process, kinase binding (3.3%) in molecular function, and nucleus (21.2%) in cellular component (Figure 6).

Figure 5

16

Figure 6 In order to analyze biological pathways which were involved in antidepressant effect of XCHT, DEPs were conducted based on KEGG (Figure 7). Our results showed that many pathways related to the DEPs were enriched, the top four pathways were endocytosis (4.6%, 11 proteins), pathways in cancer (3.7%, 9 proteins), RNA transport (3.3%, 8 proteins), and Ras signaling pathway (3.3%, 8 proteins).

Figure 7 PPI among the DEPs were analyzed by the STRING network. According to 17

calculate the degree of proteins, degree data indicated that several proteins could play the important roles in the network (Figure 8), such as cell division cycle and apoptosis regulator protein 1 (Ccar1), Calretinin (Calb2), phospholipase D2 (Pld2), CAD protein (Cad), tyrosine-protein kinase Lyn (Lyn) and so on.

Figure 8 4. Discussion Depression is one of quite common recurrent psychiatric disorders in the world, which severely affects the normal functions of many brain regions. Although the 18

pathogenesis of depression is still unknown, more and more researches indicate hypothalamus, amygdala, habenula nucleus, especially hippocampus, play the key role in depression (McEwen et al., 2015). Many evidences show that hippocampus has the critical role in regulating of the HPA axis, the stress response and emotion (Snyder et al., 2011). Furthermore, more and more researches pay attention to hippocampus due to the important phenomenon that most antidepressants exert antidepressant effect by the neurogenesis-dependent manner (Santarelli et al., 2003). Like above phenomenon, our studies also found CORT could impair hippocampal neurogenesis. In contrast, XCHT treatment not only increased hippocampal neurogenesis, but also restored negative feedback loop of HPA axis (Zhang, K. et al., 2016b). However, the precise targets and molecular mechanisms of XCHT on regulating hippocampal neurogenesis and HPA axis still need to be clarified. Therefore, quantitative proteomics analysis was used to identify the DEPs of hippocampus between XCHT group and model group in CORT induced model of depression. In present study, iTRAQ analysis is applied to investigate the DEPs of hippocampus after XCHT treatment. It is worthy that the high throughout quantitative proteomics approach is the first applied to assess antidepressant mechanisms of XCHT. Our results found 241 DEPs (Table S1) after XCHT treatment compared with model group, including 68 up regulation and 173 down regulation proteins. Among above DEPs, many proteins could act the important roles in depression. For example, Cell cycle exit and neuronal differentiation protein 1 (NO.14, Cend1, in Table S1) 19

belongs to neuron-specific protein, which plays the critical role in regulation of neural precursor cells (Katsimpardi et al., 2008). Previous study has demonstrated that over-express Cend1 could promote neuronal differentiation (Katsimpardi et al., 2008). As we known, hippocampal neurogenesis derived from neural stem cells, and neural precursor cells is one of important steps from neural stem cells to hippocampal neurogenesis. Base on the functions of Cend1, hippocampal neurogenesis possibly is influenced by Cend1. Interestingly, quantitative proteomics data showed the level of Cend1 was significantly increased after XCHT treatment. This result also further explained our previous studies which XCHT could promote hippocampal neurogenesis (Zhang, K. et al., 2016b), and reminded Cend1 could be potential protein target of XCHT on hippocampal neurogenesis. According to iTRAQ data analysis, GO annotation of DEPs was comparative analyzed to reveal underlying molecular mechanisms of XCHT. The largest proportion of GO items showed DEPs were significantly involved in nucleus of cellular component, reminded that the proteins which located in nucleus or exerted functions in nucleus must be worthily focused. Moreover, our previous study showed that baicalin, which chief representative component of XCHT, could normalize CORT induced the imbalance of glucocorticoid receptor (GR) nuclear translocation and GR phosphorylation (Zhang, K. et al., 2016a). As we know, GR belongs to nuclear translocation receptor, and exerts the functions when translocate from cytoplasm to nucleus (Guidotti et al., 2013). The effects of baicalin on GR were in line with our GO annotation data, which DEPs were significantly involved in nucleus 20

of cellular component. Moreover, GO annotation also showed that DEPs participated in cellular response to stress and estrogen stimulus. In line with this result, our previous study found that CORT could damage the function HPA axis, but XCHT treatment restored the abnormal change (Zhang, K. et al., 2016b). On the other hand, the effects and mechanisms of XCHT on estrogen are still unknown, and we will answer this question in another paper. In general, these findings suggested that XCHT could exert antidepressant action in hippocampus by influencing the proteins, which locate in nuclear region, regulate cellular environment signals recognition/response and steroid hormone. Another important bioinformatics analysis of DEPs was biological pathways and protein-protein interaction. In KEGG pathways, our results showed that the largest proportion pathway was endocytosis. It is well known that the desensitization of receptor was closely related to endocytosis. The numbers of synapses usually keep homeostatic balance under physiological conditions. Some researchers found that stress could destroy synaptic homeostatic by promoting the internalization of GluA1 resulted in decreasing the number of GluA1 receptors, but rapid antidepressants ketamine can increase glutamate transmission and synaptogenesis (Duman and Aghajanian, 2012). In line with previous study, our results demonstrated that the level of 5-HT1A receptor was decreased in chronic social isolation stress model, which might be the result of endocytosis (Ma et al., 2016). After XCHT treatment for six weeks, the level of 5-HT1A receptor was restored. In addition, based on DEPs data, potential protein targets of XCHT on depression were predicted by STRING network. 21

For instance, Ccar1, one of our potential protein targets, has exerted the inhibited effect on cell growth and promoted effect on apoptosis (Chang et al., 2017). So far, Ccar1 has been mainly reported in cancer researches (Ha et al., 2016), and the roles of Ccar1 in central nervous system are still vague. Interestingly, Ccar1 has proven to be essential for estrogen induced gene expression (Kim et al., 2008), again reminds that XCHT may have regulative effect on estrogen. Calretinin (Calb2), a calcium-binding protein, has the neuroprotection against excitotoxicity in neuron (Altobelli et al., 2015). Previous study reported Calretinin could improve the beta amyloid induced model of Alzheimer‫׳‬s disease (Altobelli et al., 2015). Taken together, above DEPs of hippocampus involved in signal transduction and regulation appear to result in the dysregulation of hippocampal neurogenesis, neurotransmitter and steroid hormone in CORT induced model of depression. However, the present data were mainly derived from mass-spectrometric technique and bioinformation databases, and must have the limitation. Our future studies will verify these DEPs by western blot and answer these questions.

5. Conclusion Current study demonstrated that XCHT could remarkably improve depressive behaviors induced by CORT, and then identified 241 DEPs, including 68 up regulation and 173 down regulation proteins. Bioinformatics analysis demonstrated that XCHT could exert antidepressant action by restoring DEPs involved in signal transduction and regulation, which result in the dysregulation of hippocampal 22

neurogenesis, neurotransmitter and steroid hormone. Current results wish to provide novel perspectives for revealing the potential protein targets of XCHT on depression and facilitating its clinical usage.

Author Contributions Statement Experimental design: Kuo Zhang ([email protected]), Jingyu Yang ([email protected]) and Chunfu Wu ([email protected]). Experimental operation:

Kuo

Zhang,

([email protected]),

Meiyao

He

Xing

Pan

([email protected]),

Dongmei

([email protected]).

Su

Provided

reagents/materials/analysis tools: Yuting Li ([email protected]) and Haotian Zhang ([email protected]). All authors reviewed the manuscript.

Conflict of interest The authors declare that they have no competing financial interests.

Acknowledgments We thank Lijuan Wang for providing the chemical profile. This work was supported by the Doctoral Scientific Research Foundation of Liaoning province (20170520193), young and middle-aged teachers’ career development project of Shenyang Pharmaceutical University (ZQN2016022) and the Key Project of the National Natural Science Foundation of China, P.R. China (81130071).

23

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Xiaochaihutang

prevents

depressive-like behaviour in rodents by enhancing the serotonergic system. The Journal of pharmacy and pharmacology 66(6), 823-834. Wang, L., Wu, C., Zhao, L., Lu, X., Wang, F., Yang, J., Xiong, Z., 2015. An Ultra-Performance Liquid Chromatography Photodiode Array Detection Tandem Mass Spectrometric Method for Simultaneous Determination of Seven Major Bioactive Constituents in Xiaochaihutang and Its Application to Fourteen Compatibilities Study. Journal of chromatographic science 53(9), 1570-1576. Zhang, J., Yang, M.K., Zeng, H., Ge, F., 2016. GAPP: A Proteogenomic Software for Genome Annotation and Global Profiling of Post-translational Modifications in Prokaryotes. Molecular & cellular proteomics : MCP 15(11), 3529-3539. Zhang, K., Pan, X., Wang, F., Ma, J., Su, G., Dong, Y., Yang, J., Wu, C., 2016a. Baicalin promotes hippocampal neurogenesis via SGK1- and FKBP5-mediated glucocorticoid receptor phosphorylation in a neuroendocrine mouse model of anxiety/depression. Scientific reports 6, 30951. Zhang, K., Wang, F., Yang, J.Y., Wang, L.J., Pang, H.H., Su, G.Y., Ma, J., Song, S.J., Xiong, Z.L., Wu, C.F., 2015. Analysis of main constituents and mechanisms underlying antidepressant-like effects of Xiaochaihutang in mice. Journal of ethnopharmacology 175, 48-57. 27

Zhang, K., Yang, J., Wang, F., Pan, X., Liu, J., Wang, L., Su, G., Ma, J., Dong, Y., Xiong, Z., Wu, C., 2016b. Antidepressant-like effects of Xiaochaihutang in a neuroendocrine

mouse

model

of

anxiety/depression.

Journal

of

ethnopharmacology 194, 674-683.

Figure captions Figure 1 UPLC-PDA-MS/MS chromatograms of seven bioactive constituents in (A) mixed reference substances and (B) XCHT sample. 1: Baicalin 2: Diphenhydramine (IS) 3: Wogonoside 4: Wogonin 5: Saikosaponin a 6: Verapamil (IS) 7: Baicalein 8: Ginsenoside Rg1 9: Liquiritin Figure 2 Schematic representation of the experimental procedure. Figure 3 Effects of chronic CORT and XCHT treatment (7 g/kg) on tail suspension test (A), forced swimming test (B). Data were expressed as means ± SEM (n=12/group). *P < 0.05 vs vehicle. #P < 0.05 vs CORT model. Figure 4 Hierarchical clustering of DEPs in hippocampus. The log2-transformed expression values were shown in red-green color scale. Red represents a higher expression and green represents a lower expression. Gene names corresponding to proteins were listed on the right side of each row. Figure 5 The functional annotations of DEPs were analyzed by GO category. Proteins were classified in terms of their role in biological process, molecular function and cellular component. Figure 6 Significantly enriched GO terms in DEPs identified through GO enrichment 28

analysis. Figure 7 Significantly enriched pathways in DEPs identified through KEGG pathway enrichment analysis. Figure 8 The network of DEPs obtained by STRING analysis.

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