Metabonomics Approach to Assessing the Modulatory Effects of St

Article pubs.acs.org/jpr

Metabonomics Approach to Assessing the Modulatory Effects of St John’s Wort, Ginsenosides, and Clomipramine in Experimental Depression Xiaoyan Wang,† Chuiyu Zeng,‡ Jingchao Lin,† Tianlu Chen,† Tie Zhao,† Zhiying Jia,† Xie Xie,† Yunping Qiu,§ Mingming Su,§ Tao Jiang,∥ Mingmei Zhou,∥ Aihua Zhao,† and Wei Jia*,†,§ †

Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China ‡ Shanghai New Asiatic Pharmaceuticals Minhang Co., Ltd, Shanghai, P. R. China § Department of Nutrition, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, North Carolina 28081, United States ∥ Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, P. R. China S Supporting Information *

ABSTRACT: The protective effects of St John’s Wort extract (SJ), ginsenosides (GS), and clomipramine (CPM) on chronic unpredictable mild stress (CUMS)induced depression in rats were investigated by using a combination of behavioral assessments and metabonomics. Metabonomic analyses were performed using gas chromatography/mass spectrometry in conjunction with multivariate and univariate statistical analyses. During and at the end point of the chronic stress experiment, food consumption, body weight, adrenal gland, thymus and spleen indices, behavior scores, sucrose consumption, and stress hormone levels were measured. Changes in these parameters reflected characteristic phenotypes of depression in rats. Metabonomic analysis of serum, urine, and brain tissue revealed that CPM and SJ mainly attenuated the alteration of monoamine neurotransmitter metabolites, while GS affected both excitatory/inhibitory amino acids and monoamine neurotransmitter metabolites. GS also attenuated the stress-induced alterations in cerebrum and peripheral metabolites to a greater extent than CPM and SJ. These results provide important mechanistic insights into the protective effects of GS against CUMS-induced depression and metabolic dysfunction. KEYWORDS: metabonomics, chronic unpredictable mild stress, ginsenoside, St John’s Wort, clomipramine, serum, urine, brain, gas chromatography/mass spectrometry

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INTRODUCTION Depression, a psychological and mental illness caused by anger, emotional suppression, pessimism, and/or frustration, exists in modern, high-pressure populations worldwide. Depression can impact every aspect of a person’s daily life and drive those with chronic serious depression to suicide. A significant number of patients with depression need antidepressants to manage this mood disorder. Although many synthetic antidepressants are used in the clinic, natural and herbal sources, such as St John’s Wort (Hypericum perforatum) and other types of complementary and alternative medicine (CAM), are thought to have antidepressant qualities with fewer side effects compared to synthetic antidepressants.1 However, there is an insufficient number of studies addressing the efficacy of CAM as well as the differences between synthetic antidepressants and CAM in treating depression.2 In this study, we compared the effects of three antidepression agents in an animal model of depression using behavioral, pharmacological, and biochemical indices combined with a metabonomics approach. The three agents © 2012 American Chemical Society

we tested were St John’s Wort extract, Panax ginseng C. A. Mey root extract (a ginsenoside), and clomipramine (a synthetic, tricyclic antidepressant, TCA, that acts primarily as serotoninnorepinephrine reuptake inhibitors (SNRIs) by blocking the serotonin transporter (SERT) and the norepinephrine transporter (NET)). St John’s Wort extract, used for centuries in the western world, is a preferred alternative medicine for the treatment of mild-to-moderate depression because of its longterm safety.3 Panax ginseng is an herbal medicine used to increase physical and mental well-being. On the basis of their antistress properties, ginseng extracts, especially ginsenosides, are used to treat fatigue, weakness, and mild depression, especially in Traditional Chinese Medicine.4 Clomipramine has been used in the clinic for the treatment of depression for nearly half a century and is recognized as a monoamine oxidase inhibitor (MAOI), having especially an effect on serotonin (5-HT) levels. Received: September 21, 2012 Published: October 30, 2012 6223

dx.doi.org/10.1021/pr300891v | J. Proteome Res. 2012, 11, 6223−6230

Journal of Proteome Research

Article

Our recent metabonomic profiling approach revealed that the contents of systemic metabolites significantly changed when chronic unpredictable mild stress (CUMS)-induced depression rat model underwent a series of mild stressors.5,6 Therefore, we hypothesized that the treatment of rats with CUMS-induced depression using antidepressants will attenuate the stressinduced variations in metabolites, readily identifiable by the analysis of metabolite profiles. To test this hypothesis, we evaluated therapeutic effects of antidepressants comprehensively in the CUMS-induced depression model by means of metabolite profiling along with classic pharmacological and biochemical indices.

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Table 1. CUMS Schedule stressors

days of CUMS experiments

Forced swimming (5 min) Ambient temperature 40 °C for 5 min Water deprivation (24 h) Food deprivation (24 h) Squeeze tail for 1 min Electric shock (10 s) Day and night reversal Ambient temperature −10 °C for 30 min Behavior restriction (2 h)

1 2 3 4 5 6 7 8 9

16 15 12 10 11 14 13 17 18

25 21 24 23 22 20 19 26 27

MATERIALS AND METHODS

Animal Handling and Sampling

Behavioral Assessment of CUMS Group

This study was conducted in accordance with the Chinese national legislation and local guidelines, and was performed at the Centre of Laboratory Animals, Shanghai University of Traditional Chinese Medicine, Shanghai, P. R. China. Eightweek-old male Sprague−Dawley(SD) rats with stable metabolic status (200 ± 20 g) were purchased from the Shanghai Laboratory Animal Co. (SLAC, Shanghai, China), housed individually in stainless steel wire mesh cages, and provided with certified standard rat chow and tap water ad libitum. Room temperature and humidity were regulated at 24 ± 1 °C and 45 ± 15%, respectively. A 12 h on/off light cycle was used with lights coming on at 8 a.m. After 2 weeks of acclimatization in metabolic cages, rats were grouped for experiments. Rats were randomly divided into the following five groups (n = 6): CUMS model group (Model), St John’s Wort extract group (SJ), ginsenosides group (GS), clomipramine group (CPM), and control group (Control). A twenty-four-hour baseline urine sample was collected from each animal on day 0. St John’s Wort standard extract (containing hyperforine >3%, cyclosan >0.3%) and ginsenosides (>95%) were purchased from Hangzhou Greensky Biological Tech. Co. (China). Clomipramine-hydrochloride tablets were purchased from Beijing Novartis Pharma (China). All three treatments were resuspended in saline solution. SJ, GS and CPM group rats received SJ (1000 mg/ kg), GS (100 mg/kg) and CPM (50 mg/kg), equivalent to medium- to high- doses used clinically, respectively, while the other rats received the same volume of saline solution once a day from day 1 to day 25. Animals in the Model, SJ, GS, and CPM groups were exposed to chronic mild stress from day 1 through day 27, while the Control group was not exposed to stress. Stressors consisted of forced swimming at 40 °C for 5 min, water deprivation for 24 h, food deprivation for 24 h, tail squeezing for 1 min, electric shock for 10 s, reversal of day and night, exposure to −10 °C for 30 min, and 2 h behavior restriction.7,8 The CUMS group was exposed to one type of stress daily between 9:30 and 11:30 a.m. according to the random-design schedule in Table 1. Body weight, food consumption, sucrose intake, and results of an open-field test were recorded for each rat on days 0, 9, 18, and 27. A 24-h urine sample from each animal was collected on day 27, and centrifuged at 6000× g for 10 min at room temperature to remove particle contaminants. The resulting supernatants were stored at −80 °C pending metabonomic analysis. Plasma, serum, and brains of all animals were collected at the end of the experiment on day 27. The weights of the adrenal gland, thymus, and spleen were recorded and their ratios to bodyweight values were calculated as the adrenal, thymus, and spleen indices.

An open-field test was conducted in a quiet room at 1−3 p.m. Each animal was placed in the central square of a rectangular arena (80 × 80 cm with 40-cm-high side walls, the floor marked with a grid divided into 25 equal-size squares) and observed for 5 min. A record was kept of the amount of time spent grooming and rearing (defined as standing upright on hind legs) and the number of grid lines crossed with at least three paws. Every incidence of grooming or rearing counted as one point, every grid crossed counted as one point, and the behavioral score was the total number of points. The apparatus was cleaned between each test. Sucrose Preference Test

Rats were trained to sweet taste as described previously.6 Briefly, single housed rats were introduced to 1% sucrose solution supplied in two drinking bottles 48 h prior to the test. Then the sucrose preference test was carried out by providing rats with two bottles for 1 hone bottle filled with 1% sucrose and the other with plain water. Food and water were withheld from rats for 14 h before the sucrose preference test. Food Consumption and Body Weight

Rat chow was added to the feed bucket of each rat at 5 p.m. After 24 h, the uneaten food was removed, weighed, and the weight recorded. At the same time, the body weight of each rat was recorded. Stress Hormone Determination

After scarification, a trunk blood sample from each rat was collected and divided into two containers, one of which was coated with heparin and the other without heparin. Blood in the heparin container was centrifuged at 800× g for 10 min, the supernatant was removed and stored at −80 °C until analysis. Blood in the uncoated container was allowed to clot at room temperature before centrifuging and freezing. The plasma adrenocorticotropic hormone (ACTH) concentration was measured using the Adrenocorticotropic Hormone Radioimmunoassay Kit D14 PGA (Beijing, North Institute of Biological Technology Co.) The blood serum corticosterone (CORT) concentration was measured using the Rat Corticosterone RIA Kit DSL-80100 (Diagnostic Systems Laboratories, USA). Statistical Analysis

Data from the behavioral investigation and the stress hormone determination were expressed as mean ± standard deviation (S.D.) Differences between the means of the treatment and control groups were analyzed using one-way analysis of variance (ANOVA). The critical p-value was set at 0.05. 6224



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GC/MS Sample Preparation, Derivatization, and Spectral Acquisition

Article

RESULTS

Behavioral Assessment Results

Serum samples were prepared according to published methods with minor modifications.9 Briefly, a 100 μL serum sample and standard solutions (10 μL L-2-chlorophenylalanine in water, 0.3 mg/mL; 10 μL heptadecanoic acid in methanol, 1 mg/mL) were vortexed for 10 s. The mixed solution was extracted with 300 μL of methanol/chloroform (3:1) and vortexed for 30 s. After 10 min at −20 °C, it was centrifuged at 12 000× g for 10 min and an aliquot of the resulting 300-μL supernatant was dried in a vacuum dryer at room temperature. The residue was derivatized using a two-step procedure: 80 μL methoxyamine (15 mg/mL in pyridine) was added to the vial and incubated at 30 °C for 90 min, followed by an incubation with 80 μL BSTFA (1% TMCS) at 70 °C for 60 min. Each 1-μL aliquot of the derivatized solution was injected in splitless mode into an Agilent 6890N g chromatograph coupled with a Pegasus HT time-of-flight mass spectrometer (GC/TOF, Leco Corp., St. Joseph, MI). Separation was achieved on a DB-5 ms capillary column (30 m × 250 μm i.d., 0.25 μm film, (5%-phenyl)-methylpol-ysiloxane bonded and cross-linked; Agilent J&W Scientific, Folsom, CA) with helium as the carrier gas at a constant flow rate of 1.0 mL/ min. The temperature of injection, transfer interface, and ion source was set to 270, 260, and 200 °C, respectively. The GC temperature programming was set to 2 min isothermal heating at 80 °C, followed by 10 °C/min oven temperature ramp to 180 °C, 5 °C/min to 240 °C, and 25 °C/min to 290 °C, and a final 9 min maintained temperature at 290 °C. Electron impact ionization (70 eV) at full scan mode (m/z 30−600) was used with an acquisition rate of 20 spectra/s in the TOFMS setting. Urine and brain tissue samples were prepared for GC−MS and relevant spectral acquisitions were performed according to previously published methods with minor modifications.6,10 A 1μL aliquot of analyte was injected into a DB-5MS capillary column coated with 5% diphenyl-cross-linked 95% dimethylpolysiloxane (30 m × 250 μm i.d., 0.25 μm film; Agilent J&W Scientific, Folsom, CA) and GC/MS was conducted using a hyphenated Perkin-Elmer gas chromatograph and TurboMass coupled to an Autosystem XL mass spectrometer (PerkinElmer, USA).

The open-field test scores of Model rats were decreased by the chronic stressors used compared to those of Control rats, particularly on day 27, as shown in Figure 1A. Crossing reflected

Figure 1. (A) Behavior scores and (B) sucrose consumption (%) of rats during chronic stress on days 0, 9, 18, and 27. * indicates p < 0.05 and ** indicates p < 0.01 compared to the Control group; △ indicates p < 0.05 and △△ indicates p < 0.01, compared to the CUMS Model group; 0, 9, 18, 27 refer to experimental days.

Data Reduction and Pattern Recognition

The peak information from the GC/TOF and GC/MS data was converted and prepared prior to the multivariate analysis, according previously published data pretreatment methods.5,6 Partial least-squares-discriminant analysis (PLS-DA) validated the PCA model and identified the brain tissue metabolites differentially produced by chronic stress exposure and the three treatments. A correlation coefficient (Corr(t,X)) of ±0.7, with a significance level of 0.05, was adopted as a cutoff value to select the most important variables for PLS-DA models of the integrated MS data. Fold changes of the arithmetic mean values (each group/ control group) of CUMS and p-values of the Kruskal−Wallis test of all the differentially expressed metabolites, identified by chromatogram-MS and standard compounds, were calculated. In addition to this nonparametric test, classical one-way analysis of variance (ANOVA) was also used to judge the statistical significance of the results. The critical p-value of both tests was set to 0.05 for this study.

the degree of animal activity, rearing reflected the degree of curiosity to the novel surroundings. The reduced activity and curiosity of Model animals fits the clinical psychomotor symptoms of depression.8 Interestingly, while GS significantly increased the frequency of animal actions after chronic stress, SJ and CPM had only a mild effect. Sucrose consumption reflects the rat’s preference for sweets, a rewarding and pleasurable stimulus. When faced with the choice between plain water and sucrose solution, rats normally prefer to drink the sucrose solution. Loss of interest in such rewarding and pleasurable stimuli is called anhedonia, which is a core symptom of clinical depression. Sucrose intake tests reflected the animal’s response to rewards, and therefore it is well accepted as a criterion for evaluating depression in this rat model.11,12 In this study, sucrose consumption began to decrease by day 9 in the CUMS group, and further decreased at day 27, indicating 6225



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Table 2. Food Consumption and Body Weight of Rats during the Period of Chronic Stressa days

items

control

model

CPM

SJ

GS

0

FC BW FC BW FC BW FC BW

30.8 ± 3.5 204.4 ± 8.8 31.5 ± 4.2 225.5 ± 11 32.8 ± 3.5 255.7 ± 11.8 33.4 ± 3.7 282.6 ± 12.8

32.5 ± 2.9 206.6 ± 8.2 21.7 ± 5.1* 217.6 ± 10.5* 24.8 ± 5.7* 238.2 ± 10.4* 27.1 ± 4.4 252.4 ± 10.5*

33.6 ± 3.7 206.3 ± 9.5 22.5 ± 3.8* 216.3 ± 10.1* 23.1 ± 4.3* 234.8 ± 12.3* 24.5 ± 4.3* 241.9 ± 13.0*

30.7 ± 3.4 205.0 ± 8.7 23.7 ± 3.6* 219.9 ± 11.2* 24.2 ± 3.8* 225.6 ± 9.5** 23.8 ± 5.1* 234.3 ± 9.4**

31.0 ± 4.2 205.9 ± 8.5 34.2 ± 5.1Δ 230.6 ± 9.3 35.9 ± 4.2Δ 279.8 ± 13.0Δ 33.0 ± 3.9 316.4 ± 12.7

9 18 27

* indicates p < 0.05; ** indicates p < 0.01 compared to the Control group; Δ indicates p < 0.05 compared to the CUMS Model group. Measurements are expressed in mean ± standard deviation. Control, control group; model, CUMS model group; SJ: St John’s Wort extract group; GS: ginsenosides group, CPM: clomipramine group; FC, food consumption; BW, body weight.

a

Table 3. Adrenal Gland, Thymus, and Spleen Weights and Indices of Rats during the Period of Chronic Stressa adrenal gland

thymus

spleen

group

mg

index

mg

index

mg

index

Control Model CPM SJ GS

58.2 ± 7.2 62.4 ± 8.9 58.7 ± 11.4 57.5 ± 9.6 65.7 ± 10.7

0.206 ± 0.015 0.247 ± 0.023* 0.243 ± 0.018 0.245 ± 0.022 0.208 ± 0.018Δ

25.7 ± 3.6 16.4 ± 4.2 17.6 ± 4.3 16.4 ± 3.7 28.4 ± 3.9

0.909 ± 0.194 0.648 ± 0.206* 0.728 ± 0.195 0.699 ± 0.217 0.896 ± 0.216Δ

645.4 ± 64.2 523.4 ± 58.8 528.1 ± 61.7 520.3 ± 61.5 698.4 ± 67.6

2.28 ± 0.18 2.07 ± 0.22* 2.18 ± 0.16 2.22 ± 0.19 2.21 ± 0.20

a * indicates p < 0.05, compared to the control group; Δ indicates p < 0.05, compared to the CUMS Model group. Control, control group; Model, CUMS model group; SJ, St John’s Wort extract group; GS, ginsenosides group; CPM, clomipramine group.

CUMS−induced anhedonia (Figure 1B). SJ, GS, and CPM were all effective in reversing the decreased in sucrose consumption induced by CUMS at different time points; however, rats in the GS group had the highest food intake and gained the most body weight. As expected, chronic stress also resulted in reduced food consumption and loss of body weight, similar to the loss of appetite experienced by patients with depression.13 We found that food consumption of rats decreased initially, and then recovered as the CUMS continued, resulting in a moderate increase in body weight over time (Table 2). The results indicate that only GS increased both food consumption and body weight significantly in rats exposed to chronic stress, while SJ and CPM reduced food intake further, and hence induced greater weight loss. Adrenal, thymus and spleen indices are regarded as an indication of healthy immune system.14 In fact, CUMS significantly impacted these indices, whereas GS significantly alleviated the reduction of adrenal and thymus indices (Table 3).

Table 4. Plasma ACTH and Serum CORT Values in Rat Blooda group

ACTH (pg/mL)

CORT (ng/mL)

Control Model CPM SJ GS

31.8 ± 9.4 16.3 ± 5.6* 20.4 ± 8.2 22.3 ± 8.6 43.5 ± 11.4ΔΔ

1017.1 ± 117.1 749.3 ± 98.5** 914.3 ± 89.4Δ 952.1 ± 84.4ΔΔ 1196.0 ± 97.5ΔΔ

* indicates p < 0.05; ** indicates p < 0.01 compared to the Control group; Δ indicates p < 0.05; ΔΔ indicates p < 0.01 compared to the CUMS Model group. Control, control group; model, CUMS model group; SJ: St John’s Wort extract group; GS: ginsenosides group, CPM: clomipramine group. a

metabolites between the CUMS model and SJ, GS, and CPM groups were identified (Figure 2A). Nineteen differentially expressed brain metabolites between Control and CUMS model were also identified (Figure 2B). Furthermore, as we previously reported,6 16 urinary metabolites contributed to the differences in metabolic profiles between the Control and CUMS model (Figure 2C). This metabolite analysis was carried out using ANOVA and Kruskal−Wallis tests with the threshold for significance set at p = 0.05. Fold changes of the arithmetic mean values for these metabolites in each group was calculated and compared to those in the Control group (Figure 2). The average p-values of the ANOVA and Kruskal−Wallis tests are shown in Supporting Information Tables 1−3 for serum, urine, and brain. A clear separation of metabolic states between different groups after 27 days of chronic stress was observed (Supporting Information Figure 1), suggesting that exposure to unpredictable chronic stress may lead to metabolic variations in rat brains. On the other hand, SJ, GS, and CPM affected the metabolite variations in different directions.

Stress Hormone Variation

In the CUMS model group, both ACTH and CORT decreased on day 27 as compared to the control group, indicating CUMS− induced hypoadrenocorticism. SJ, GS, and CPM were all effective in elevating CORT levels, but only GS significantly increased ACTH levels (Table 4). Chronic stress undoubtedly affects the HPA axis, but it is unclear whether it causes an increase or decrease in glucocorticoids (e.g., ACTH, CORT), depending on the intensity of the stress stimulation or even the transient variation at different sampling times.6−8 A single incidence of acute stress induces the secretion of stress hormones,15 but longterm stress may reduce stress hormones levels, probably because of hypoadrenocorticism.16 Metabolic Variation Induced by CUMS

Thirty-two differentially expressed metabolites between the Control and CUMS model groups, as well as differential 6226



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Figure 2. Fold changes of the arithmetic mean values (experimental group/control group) of CUMS-induced changes in (A) serum, (B) brain, and (C) urine metabolites by GS, SJ, and CPM administration at the study end point. (M, Model/Control; C, CPM/Control; S, SJ/Control; G, GS/Control).

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DISCUSSION

value of the ACTH, CORT, and adrenal index were observed, indicating alterations in adrenal cortex function. The thymus and spleen index demonstrated an inhibition of the immune system after long-term stress. The treatment results indicated that GS markedly ameliorated all stress-induced changes whereas CPM and SJ only partially improved sucrose consumption and CORT.

Chronic unpredictable mild stress induced depression rat models successfully simulated the symptoms of depression, including the reduction in exploratory behavior, food consumption, body weight, and a loss of responsiveness to pleasant stimuli (e.g., Figure 1B).12 As a consequence of chronic stress, changes in the 6227



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glycine and histamine (HSM), were significantly reduced compared to the brains of the Control group rats. While in serum, except for N-acetylaspartate, N-acetylglutamate, and Nacetylglutamine, excitatory/inhibitory neurotransmitters and metabolites, including inhibitory compounds and their metabolites (e.g., taurine, hypotaurine, histidine, and 4-hydroxybutyrate (GHB)( were significantly downregulated by CUMS. In addition, only glutamate and glutamine increased in urine excretion by CUMS. Glutamine is a main precursor of the excitatory neurotransmitter glutamate, and previous studies reveal that both of them are sensitive to chronic stress.20,21 NAcetylated amino acids are the important forms of some amino acids in vivo. For example, N-acetylaspartate, the second highest concentrated molecule in the brain after glutamate, acts as a precursor for the synthesis of the important neuronal dipeptide, N-acetylaspartylglutamate, and is involved in energy production from the amino acid glutamate in mitochondria.22 Our results suggest that chronic stress may induce the acetylation of some amino acids. Monoamine neurotransmitter metabolites, such as 5-HTrelated compounds and dopamine, were detected mostly at a lower level in the CUMS model group, except for 3-indolelactate and tyramine. The downregulated monoamine metabolites indicated a biochemical depression status for the CUMS model rats.23 Tryptophan is metabolized by two major pathways in humans with the help of gastrointestinal microflora, either through kynurenine or through a series of indoles, where 3indolelactate, together with 5-HIAA, are the main tryptophan metabolites of indoles (Figure 3).24 Our results suggest that pathways downstream of tyrosine and tyrptophan are impacted by chronic stress, as these metabolite levels were reported varied in previous studies.25 TCA cycle was reported being affected by mental or mood disorders such as stress, depression and anxiety.20,26 In agreement with previous findings, TCA cycle metabolites in serum and in the brain were affected by CUMS. Similar to decrease in citrate levels in urine, the levels of fumarate, succinate and nicotinamide(NADP precursor) were decreased in serum (Figure 2). However, unlike the reduced excretion of citrate in urine, citrate levels increased notably in the brain, suggesting that the changes in glutamate and aspartate levels may have resulted from the disturbed TCA cycle.27 In addition to neurotransmitters, most other amino acids were decreased by CUMS in 27 d. For example, a decrease in proline levels in all the three samples was observed, due to the susceptibility of proline dehydrogenase (PRODH) genes in mental disorders.28 The variations in methionine and valine levels may indicate a brain-derived neurotrophic factor (BDNF) valine-66−methionine (Val66Met) genotype that is considered as major risk factor for depression, anxiety, and other psychiatric disorders,29 as S-adenosyl-L-methionine, the active form of methionine and a major methyl group donor, has been effective in depression therapy.30 Furthermore, low lysine levels were found in patients with anxiety disorder and depression, and lysine therapy is associated with improvement of some patients with these conditions.31 It was interesting to find that two polyunsaturated fatty acids, arachidonic acid (ARA) and docosahexaenoic acid (DHA), were also downregulated by CUMS. It is believed ARA and DHA contribute to maintaining the function of the cerebrum and retina. It was reported that ARA and DHA decrease in chronic fatigue syndrome patients, and that the ratio of the two can predict the suicide tendency of depression patients.32 Therefore,

Interestingly, SJ significantly reduced food consumption and body weight, suggesting that it is an appetite suppressor.17 Several encephalic regions, such as the frontal lobe, thalamus, and hippocampus, are associated with depression.18 For this reason, we analyzed whole brain tissue together with serum and urine samples using metabonomics to compare the systemic therapeutic effects of SJ, GS, and CPM. We allowed for a 2 d washout period for the major ingredients of the treatments to be eliminated from the body. In addition, the preliminary experiment indicated that the metabolites of exogenous medicines including ginsenosides, hypericin, flavonoids, and clomipramine are not detectable by GC−MS analysis, because these molecules are excluded in the chemical derivatization process.19 Therefore, the contamination from exogenous chemical compounds and metabolites of endogenous metabolites is minimal in GC−MS analysis. The relatively small sample size (n = 6 per group) used in the experiment is a limitation of this study, which may result in the lack of statistical representation of the observed metabolic changes. However, the present study with limited sample sizes was nevertheless able to detect many differences among stress model group, treatment groups and controls in metabolic profiles from different pathways, lending deeper insights and a novel approach for detection of stress-related metabolic states, Meanwhile, many observations presented here are consistent with what is known about stress and antidepressants We characterized differentially expressed metabolites associated with CUMS, including excitatory/inhibitory amino acid neurotransmitters (e.g., glycine, glutamate, aspartate), monoamine neurotransmitters (e.g., 5-HT, catecholamines/dopamine), metabolites of the TCA cycle and organic acid pathways using our metabonomics approach.6 The identified metabolites in serum, urine, and brain were grouped into several types according to the variation induced by CUMS and SJ, GS, and CPM (Figures 2 and 3). Because of the blood-brain barrier, the

Figure 3. Changes in metabolites in the brain, serum, and urine with chronic stress compared to the metabolite levels in the Control group.

metabolism of the brain is independent of peripheral circulation, and hence the metabolic variances induced by CUMS were different among serum, urine, and brain. In brains of CUMS Model rats, excitatory amino acid neurotransmitters and metabolites involving aspartate, glutamate, glutamine, and Nacetylaspartate were increased, while inhibitory amino acids, 6228



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well as by the fact that GS restored cerebellar and peripheral metabolites to a greater extent than SJ and CPM. In conclusion, both the classic pathophysiological indices and the metabonomic results reveal the protective effects of CPM, SJ, and GS in an experimental depression rat model. GS produced the greatest improvement in body weight, immune system health, independent behavior, pleasant stimulus response, and stress hormone levels, compared to SJ and CPM. These metabonomic results suggest that CPM and SJ attenuated the alteration of monoamine neurotransmitter metabolites, while GS affected both excitatory/inhibitory amino acids and monoamine neurotransmitters with the highest amount of restoration of cerebellar and peripheral metabolites. Collectively, our results indicate that GS possesses the best protective effect against CUMS-induced depression symptoms and metabolic disturbances.

the decrease in ARA and DHA may be associated with reduced autonomic activities (behavior score) of CUMS rats. The three antidepressants tested in this study exhibited various clinical effects; GS ameliorated almost all the stress reactions induced by CUMS, whereas SJ and CPM improved only some. The effects of these different treatments cannot be entirely captured with classic behavioral tests and pharmacological biochemical indices; instead, our metabolic analysis reveals a more detailed picture of the antidepression effects of these treatments. CPM, or chlorimiramine, is a tricycle antidepressant with both antidepressant and antiobsessional properties. Like other tricyclics, CPM inhibits norepinephrine and serotonin uptake in central nerve terminals, possibly by blocking the neuronal pumps, thereby increasing the concentration of transmitter monoamines at receptor sites. CPM’s capacity to inhibit norepinephrine and serotonin reuptake is reflected by the retrieved catecholamines metabolites including: tyrosine, tyramine, homovanillate, phenylalanine, and 5-HT metabolites, such as tryptophan and 5-HIAA. Except for hexanoate and valine, CUMS-induced metabolites in urine were all significantly reversed by CPM. In serum, CPM increased some neurotransmitter metabolites including aspartate, glutamate, pyroglutamate, taurine,hypotaurine and phenylalanine. It also increased most of other amino acids and most of the glycometabolism metabolites including aerobic and anaerobic glycolysis. But in brain tissue, only three metabolites, phenylalanine, alanine and histamine, were regulated to normal levels. As a natural antidepressant, St John’s Wort is thought to inhibit the synaptosomal uptake of several neurotransmitters, such as serotonin, dopamine and serotonin, norepinephrine, dopamine, GABA, and L-glutamate.33 It was reported that SJ leads to a down-regulation of beta-adrenergic receptors and an upregulation of serotonin 5-HT(2) receptors in the rat frontal cortex, causing changes in neurotransmitter concentrations in brain areas that are implicated in depression.34 Our results provide evidence that SJ can resist the CUMS-induced changes in catecholamine metabolites, such as tyrosine, phenylalanine, and homovanillate (HVA), and 5-HT metabolites, including tryptophan and 5-HIAA in peripheral blood. Furthermore, the changes in the levels of cholesta-4, 6-dien-3-ol, ARA, DHA, and pimelate in CNS are restored by SJ. Similar to CPM, SJ does not change some cerebral neurotransmitters and their metabolites, including glycine, N-acetylaspartate, and glutamine; however, it can reverse histamine and aspartate level changes while restoring glutamate ratios. In serum, the metabolic regulation efficacy of SJ did not surpass that of CPM or GS. The antidepression activities of ginsenosides are thought to be associated with monoamine neurotransmitters, including catecholamines and 5-HT metabolites.35 Our results show that, unlike CPM and SJ, GS reversed the changes in both the amino acid neurotransmitter metabolites and the monoamine neurotransmitter metabolites. It was reported that ginsenosides can also regulate excitatory and inhibitory neurotransmitters, such as glutamate and GABA, and prevent injury induced by glutamate.36 Interestingly, we found that, in serum, GS remarkably increased several TCA cycle and saccharide metabolites, and decreased some fatty acids, even some that were not changed by CUMS (Figure 2), indicating the possible ginsenoside targets. Our results highlighted the therapeutic potential of ginseng for CUMS-induced depression symptoms, as evidenced by the improved biochemical and behavioral results in the GS group as

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ASSOCIATED CONTENT

* Supporting Information S

Supplemental tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (704) 250-5803. Fax: (704) 2505809. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant 30901997), SJTU-SMC Morningstar Young Scholars Program 2010 (B), National Basic Research Program of China (2007CB914700 and 2012CB910102), and the National Science and Technology Major Project (2009ZX10005-020).

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ABBREVIATIONS: Asp, aspartate; Leu, leucine; Trp, tryptophan; Met, methionine; Pyr, pyruvate; Lact, lactate; TCP, tocopherol; Gln, glutamine; HSM, histamine; Phe, phenylalanine; HpTau, hypotaurine; Pglu, pyroglutamate; HVA, homovanillate; ETA, ethanolamine; Glu, glutamate; His, histidine; Ala, alanine; Pro, proline; Ser, serine; Ly, lysine; Xyl, xylitol; Gly, glycine; Tau, taurine; Tyr, tyrosine; Ty, tyramine; Val, valine; Cys, cysine; Ara, arabinose; NAcAsp, N-acetylaspartate; NAcGlu, N-acetylglutamate; NAcGln, Nacetylglutamine; GHB, 4-hydroxybutyrate; 3-ILA, 3-indolelactate; HYP-, 4-hydroxyproline; 2-HBA, 2-hydroxybutyrate

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REFERENCES

(1) Freeman, M. P. Complementary and alternative medicine for perinatal depression. J. Affect Disord. 2009, 112 (1−3), 1−10. (2) van der Watt, G.; Laugharne, J.; Janca, A. Complementary and alternative medicine in the treatment of anxiety and depression. Curr. Opin. Psychiatry 2008, 21 (1), 37−42. (3) Brattstrom, A. Long-term effects of St. John’s wort (Hypericum perforatum) treatment: a 1-year safety study in mild to moderate depression. Phytomedicine 2009, 16 (4), 277−83. (4) Dang, H.; Chen, Y.; Liu, X.; Wang, Q.; Wang, L.; Jia, W.; Wang, Y. Antidepressant effects of ginseng total saponins in the forced swimming test and chronic mild stress models of depression. Prog. Neuropsychopharmacol. Biol. Psychiatry 2009, 33 (8), 1417−24.

6229



Article

(5) Ni, Y.; Su, M.; Lin, J.; Wang, X.; Qiu, Y.; Zhao, A.; Chen, T.; Jia, W. Metabolic profiling reveals disorder of amino acid metabolism in four brain regions from a rat model of chronic unpredictable mild stress. FEBS Lett. 2008, 582 (17), 2627−36. (6) Wang, X.; Zhao, T.; Qiu, Y.; Su, M.; Jiang, T.; Zhou, M.; Zhao, A.; Jia, W. Metabonomics approach to understanding acute and chronic stress in rat models. J. Proteome Res. 2009, 8 (5), 2511−8. (7) Willner, P.; Towell, A.; Sampson, D.; Sophokleous, S.; Muscat, R. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology (Berlin) 1987, 93 (3), 358−64. (8) Katz, R. J.; Roth, K. A.; Carroll, B. J. Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci. Biobehav. Rev. 1981, 5 (2), 247−51. (9) Chen, T.; Xie, G.; Wang, X.; Fan, J.; Qiu, Y.; Zheng, X.; Qi, X.; Cao, Y.; Su, M.; Xu, L. X.; Yen, Y.; Liu, P.; Jia, W. Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma. Mol. Cell. Proteomics 2011, 10 (7), M110 004945. (10) Lin, S.; Su, M.; Wang, X.; Qiu, Y.; Li, H.; Hao, J.; Yang, H.; Zhou, M.; Yan, C.; Jia, W. Multiparametric analysis of amino acids and organic acids in rat brain tissues using GC/MS. J. Sep. Sci. 2008, 31 (15), 2831− 8. (11) Baker, S. L.; Kentner, A. C.; Konkle, A. T.; Santa-Maria Barbagallo, L.; Bielajew, C. Behavioral and physiological effects of chronic mild stress in female rats. Physiol. Behav. 2006, 87 (2), 314−22. (12) Yadid, G.; Nakash, R.; Deri, I.; Tamar, G.; Kinor, N.; Gispan, I.; Zangen, A. Elucidation of the neurobiology of depression: insights from a novel genetic animal model. Prog. Neurobiol. 2000, 62 (4), 353−78. (13) Sohlberg, S. Personality, life stress and the course of eating disorders. Acta Psychiatr. Scand. Suppl. 1990, 361, 29−33. (14) Archana, R.; Namasivayam, A. Acute noise-induced alterations in the immune status of albino rats. Indian J. Physiol. Pharmacol. 2000, 44 (1), 105−8. (15) Wang, X.; Su, M.; Qiu, Y.; Ni, Y.; Zhao, T.; Zhou, M.; Zhao, A.; Yang, S.; Zhao, L.; Jia, W. Metabolic regulatory network alterations in response to acute cold stress and ginsenoside intervention. J. Proteome Res. 2007, 6 (9), 3449−55. (16) Armario, A.; Castellanos, J. M.; Balasch, J. Dissociation between corticosterone and growth hormone adaptation to chronic stress in the rat. Horm. Metab. Res. 1984, 16 (3), 142−5. (17) Nachtigal, M. C.; Patterson, R. E.; Stratton, K. L.; Adams, L. A.; Shattuck, A. L.; White, E. Dietary supplements and weight control in a middle-age population. J. Altern. Complement. Med. 2005, 11 (5), 909− 15. (18) Johnstone, T.; van Reekum, C. M.; Urry, H. L.; Kalin, N. H.; Davidson, R. J. Failure to regulate: counterproductive recruitment of top-down prefrontal-subcortical circuitry in major depression. J. Neurosci. 2007, 27 (33), 8877−84. (19) Husek, P.; Liebich, H. M. Organic acid profiling by direct treatment of deproteinized plasma with ethyl chloroformate. J. Chromatogr., B: Biomed. Appl. 1994, 656 (1), 37−43. (20) Li, Z. Y.; Zheng, X. Y.; Gao, X. X.; Zhou, Y. Z.; Sun, H. F.; Zhang, L. Z.; Guo, X. Q.; Du, G. H.; Qin, X. M. Study of plasma metabolic profiling and biomarkers of chronic unpredictable mild stress rats based on gas chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 2010, 24 (24), 3539−46. (21) Liu, X. J.; Li, Z. Y.; Li, Z. F.; Gao, X. X.; Zhou, Y. Z.; Sun, H. F.; Zhang, L. Z.; Guo, X. Q.; Du, G. H.; Qin, X. M. Urinary metabonomic study using a CUMS rat model of depression. Magn. Reson. Chem. 2012, 50 (3), 187−92. (22) Kishimoto, H.; Yamada, K.; Iseki, E.; Kosaka, K.; Okoshi, T. Brain imaging of affective disorders and schizophrenia. Psychiatry Clin. Neurosci. 1998, 52 (Suppl), S212−4. (23) Maes, M.; Lin, A. H.; Verkerk, R.; Delmeire, L.; Van Gastel, A.; Van der Planken, M.; Scharpe, S. Serotonergic and noradrenergic markers of post-traumatic stress disorder with and without major depression. Neuropsychopharmacology 1999, 20 (2), 188−97.

(24) Mohammed, N.; Onodera, R.; Or-Rashid, M. M. Degradation of tryptophan and related indolic compounds by ruminal bacteria, protozoa and their mixture in vitro. Amino Acids 2003, 24 (1−2), 73−80. (25) Zheng, S.; Yu, M.; Lu, X.; Huo, T.; Ge, L.; Yang, J.; Wu, C.; Li, F. Urinary metabonomic study on biochemical changes in chronic unpredictable mild stress model of depression. Clin. Chim. Acta 2010, 411 (3−4), 204−9. (26) Zhang, Y.; Filiou, M. D.; Reckow, S.; Gormanns, P.; Maccarrone, G.; Kessler, M. S.; Frank, E.; Hambsch, B.; Holsboer, F.; Landgraf, R.; Turck, C. W. Proteomic and metabolomic profiling of a trait anxiety mouse model implicate affected pathways. Mol. Cell. Proteomics 2011, 10 (12), M111 008110. (27) Waagepetersen, H. S.; Qu, H.; Sonnewald, U.; Shimamoto, K.; Schousboe, A. Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons. Neurochem. Int. 2005, 47 (1−2), 92−102. (28) Tunbridge, E.; Burnet, P. W.; Sodhi, M. S.; Harrison, P. J. Catechol-o-methyltransferase (COMT) and proline dehydrogenase (PRODH) mRNAs in the dorsolateral prefrontal cortex in schizophrenia, bipolar disorder, and major depression. Synapse 2004, 51 (2), 112−8. (29) Gatt, J. M.; Nemeroff, C. B.; Schofield, P. R.; Paul, R. H.; Clark, C. R.; Gordon, E.; Williams, L. M. Early life stress combined with serotonin 3A receptor and brain-derived neurotrophic factor valine 66 to methionine genotypes impacts emotional brain and arousal correlates of risk for depression. Biol. Psychiatry 2010, 68 (9), 818−24. (30) Papakostas, G. I. Evidence for S-adenosyl-L-methionine (SAM-e) for the treatment of major depressive disorder. J. Clin. Psychiatry 2009, 70 (Suppl 5), 18−22. (31) Lakhan, S. E.; Vieira, K. F. Nutritional and herbal supplements for anxiety and anxiety-related disorders: systematic review. Nutr. J. 2010, 9, 42. (32) Sublette, M. E.; Hibbeln, J. R.; Galfalvy, H.; Oquendo, M. A.; Mann, J. J. Omega-3 polyunsaturated essential fatty acid status as a predictor of future suicide risk. Am. J. Psychiatry 2006, 163 (6), 1100−2. (33) Wonnemann, M.; Singer, A.; Muller, W. E. Inhibition of synaptosomal uptake of 3H-L-glutamate and 3H-GABA by hyperforin, a major constituent of St. John’s Wort: the role of amiloride sensitive sodium conductive pathways. Neuropsychopharmacology 2000, 23 (2), 188−97. (34) Butterweck, V. Mechanism of action of St John’s wort in depression: what is known? CNS Drugs 2003, 17 (8), 539−62. (35) Teige, M.; Scheikl, E.; Eulgem, T.; Doczi, R.; Ichimura, K.; Shinozaki, K.; Dangl, J. L.; Hirt, H. The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol. Cell 2004, 15 (1), 141−52. (36) Liao, B.; Newmark, H.; Zhou, R. Neuroprotective effects of ginseng total saponin and ginsenosides Rb1 and Rg1 on spinal cord neurons in vitro. Exp. Neurol. 2002, 173 (2), 224−34.

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